Final Report Summary - LAPTOP (LAntibiotic Production: Technology, Optimization and improved Process)
Executive Summary:
The LAPTOP objective was the development of a robust and economically feasible production process for NAI-107, with the final objective of producing Good Laboratory Practice-grade compound. The lantibiotic NAI-107, produced by the actinomycete Microbispora sp, is a new antibiotic with the potential to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. It is active against all Gram-positive pathogens including multidrug-resistant isolates, with good potency and efficacy in sophisticated experimental models of infection, such as rat endocarditis caused by methicillin-resistant Staphylococcus aureus. NAI-107 is currently undergoing formal toxicology studies. Because of its complex chemical structure, NAI-107 cannot be produced by chemical means. Thus, the supply of sufficient amounts of product for future clinical studies requires robust fermentation and recovery processes to deliver a high-quality compound at reasonable cost. This is particularly relevant for NAI-107 because of the lack of precedents: no lantibiotic is produced at an industrial scale as a drug for human use and no Microbispora strain is used for industrial production. Due to limited knowledge of the Microbispora strain, the initial objectives were the development of tools instrumental for analyzing the physiology and genetics of the producing strain and for getting insights into NAI-107’s mechanism of action. Next, knowledge generated during the previous reporting period was translated into the generation of improved strains, the design of optimized media and recovery procedures. A gene transfer system based on conjugation between E. coli and Microbispora was developed and applied to generate knockout and over-expression mutants. From a draft genome sequence of Microbispora, consisting of about 8,000 coding sequences, key components of the N-regulon and P-regulon were identified and a 2D-protein map was constructed during primary and secondary metabolism, establishing differentially abundant proteins. After the identification of chemically defined media suitable for NAI-107 production, carbon flux analysis established that C-assimilation occurs mostly through the pentose phosphate pathway. Since production of an antibiotic is often limited by the self-resistance mechanism in the producer strain, it was important to investigate in detail the mechanism of action of NAI-107 in model strains, as well as establishing key features in self-resistance, transport and cell wall composition in Microbispora. In whole cell and cell-free experiments, NAI-107 was demonstrated to inhibit cell wall biosynthesis by binding to Lipid II with high affinity and inhibiting all enzymes utilizing this key biosynthetic intermediate. Pore formation, as observed with some Lipid-II binding lantibiotics, did not occur with NAI-107 in vitro. Analysis of the Microbispora cell wall indicates that it contains stem peptides with either glycine or alanine at the first position and it contains a direct linkage between peptide chains. Differential proteome analyses established that the proteins associated with NAI-107 production are, among others, those required for amino sugar metabolism, cell wall biosynthesis, lantibiotic resistance, and nitrogen metabolism. Previous work on a different Microbispora strain had identified key roles for the regulatory genes, which are also present in the NAI-107 gene cluster: mibX encoding a sigma factor, mibW encoding the cognate anti-sigma factor, and mibR encoding a likely DNA-binding protein. Critical parameters for Microbispora growth and NAI-107 production have been delineated and new seed and production media have been designed. These have resulted in production titers up to 10 fold higher than in the starting production process. A simple recovery and purification process has been designed, which allows purification of NAI-107 with few, industrially scalable steps. The minor components present in the NAI-107 complex have been purified, chemically characterized and evaluated for their antimicrobial activity. The fermentation process was successfully transferred to a different laboratory, with satisfactory production levels at both the 15 and the 250-L scale.
Project Context and Objectives:
Due to limited knowledge of the Microbispora producer strain, the objectives of the first half of the project were the development of the tools necessary for analyzing the physiology and genetics of the producing strain, understanding the elements contributing to NAI-107 production, as well as getting insights into the details of its mechanism of action. In the second part of the project, that knowledge has been translated into the generation of improved strains, the design of optimized media and recovery procedures, to achieve the overall objective of the proposal the scale-up and industrialization of the production process, with the production of GLP-grade NAI-107. As the molecular mechanism of antibiotic activity and resistance are often intimately linked, another objective of LAPTOP was to understand the mechanism of action of and resistance to NAI-107 in the producing strain. As self-resistance mechanisms have evolved to cope only with levels of antibiotic production found in nature, therefore any attempt to improve production levels must include appropriate measures to increase the level of self-protection.
The specific objectives of this project were:
- Development of tools and technology to allow familiarization with Microbispora;
- Identification of the NAI-107 target in susceptible bacteria and determine the molecular details of NAI-107’s mode of action;
- Investigation of the resistance (immunity) mechanism in the producer strain;
- Identification and biological characterization of the components of NAI-107-complex;
- Isolate antibiotic resistant mutants of Microbispora producing increased levels of NAI-107;
- Define precursor uptake and product excretion in the producer strain;
- Identify and manipulate components of regulatory networks leading to NAI-107 production;
- Analyze and control nitrogen regulation and its impact on antibiotic production;
- Characterize aspects of primary metabolism required for optimized production;
- Design an improved production medium;
- Design an efficient recovery process.
The Laptop project has benefited substantially from groundwork performed by one of the partners in the consortium on a related strain (designated hereafter as Microbispora corallina) that produces a lantibiotic with structural features similar to those of NAI-107, although a full comparison of the two antibiotic complexes had not performed before the start of the project. This work has established the roles of most of the genes present in the mib cluster in Microbispora corallina, facilitating studies on the corresponding mlb genes in Microbispora sp.
The development of genetic tools for analyzing and manipulating the producer strain and the production process has been achieved, in particular:
• a gene transfer system based on conjugation between E. coli and Microbispora has been developed. The protocol has been successfully applied by different Partners for generating knockout and over-expression mutants.
• a draft genome sequence of Microbispora has been generated. The annotated genome (~ 8000 CDSs) will be soon available in public databases. The sequence information has been used in the course of the project for several studies.
• a 2D-protein map of Microbispora during primary and secondary metabolism has been constructed. Differentially abundant protein spots, identified by mass spectrometry, are available to investigate relationships between biochemical pathways and regulatory networks along with NAI-107 production. In addition, an interactive Microbispora proteome web site will soon be available.
• a chemically defined medium allowing NAI-107 production and suitable for metabolic flux analysis has been designed. C-flux methodology has been optimized and applied to Microbispora.
The interaction of NAI-107 with its target has been characterized.
• Using whole-cell experiments, NAI-107 was identified as a cell wall biosynthesis inhibitor; in vitro assays with purified cell wall biosynthesis enzymes and substrates identified the molecular target of the lantibiotic, the lipid-bound cell wall building block Lipid II. NAI-107 was found to bind to Lipid II with high affinity; pore formation, as observed with some Lipid-II binding lantibiotics, does not occur with NAI-107 in vitro.
• Analysis of the proteomic response profile of Bacillus subtilis, as compared to the profile induced by Lipid II binding antibiotics, showed the highest degree of similarity with gallidermin, another non-pore forming lantibiotic.
The mechanism of self-resistance in the producer strain has been investigated.
• The cell wall precursors of Microbispora have been analyzed, and key proteins involved in NAI-107 immunity in Microbispora have been identified.
• Work towards understanding the producer self-protection mechanisms identified a putative ABC transporter and a putative lipoprotein MlbQ which could meditate self-protection. Functional analysis suggests a specific role of the membrane-localized MlbQ in resistance to NAI-107, while NMR studies revealed an unusual structure. Analysis of the cell wall building block of the producer does not indicate specific alterations in cell wall composition.
• Analysis of the Microbispora cell wall indicates that it contains stem peptides with either glycine or alanine at the first position. The cell wall does not contain an interbridge but a direct linkage between peptide chains. These features were also observed in the knockout mutant of Microbispora that does not produce any NAI-107.
Key components of the N-regulon and P-regulon have been identified.
• Key components of the N-regulon and P-regulon with the response regulators GlnR and PhoP respectively, were identified in the Microbispora genome and their expression analyzed.
• It has been demonstrated that nitrogen excess positively controls NAI-107 production and the biomass accumulation, and increased expression of mlbA was observed in defined medium supplemented with ammonia or nitrate; The results revealed that Pi positively influences biomass accumulation and NAI-107 production but not productivity (amount of NAI-107 per biomass amount). In particular, Pi limitation does not exert a beneficial effect on NAI-107 production as reported in the case of other antibiotics produced by actinomycete strains.
• Comparative proteome analyses revealed proteins associated with NAI-107 production: mostly, those involved in nitrogen metabolism regulation, sulphur metabolism, phosphate metabolism, cell wall biosynthesis, oxidative and general stress response factors, tricarboxylic acid cycle, protein folding and modification, fatty acid and cell membrane metabolism, proteins regulating physiological differentiation, stringent response key factors, NAI-107 biosynthetic gene cluster products, multidrug resistance ABC transporters.
Key enzymes involved in carbon metabolism have been identified.
• The C-flux data for the wild type strain have been generated and compared to existing data from other actinomycetes to gain insights into Microbispora central carbon metabolism and its links to secondary metabolism. The highest metabolic flux of C-assimilation was observed through the pentose phosphate pathway; fluxes of C-assimilation through the tricarboxylic acid cycle and Embden-Meyerhof-Parnas pathway were comparatively low.
Characterization of the NAI-107 complex has been completed.
• The minor congeners (F0, F1, F2, A0, B1 and B2) have been purified, structurally characterized and assayed for their antibiotic activity in comparison with the main congeners A1 and A2.
The isolation of improved strains has been achieved.
• antibiotic resistant mutants producing increased levels of NAI-107 have been isolated. The streptomycin-resistant mutant S13 of Microbispora gave consistent results, and was selected for all subsequent tests of the productive media at bioreactor scale.
The general regulatory mechanisms that influence NAI-107 biosynthesis.
• The key regulatory genes, mlbR, mlbX and mlbW, were identified in the NAI-107 biosynthetic gene cluster. Over-expression of mlbR or mlbX resulted in higher levels of NAI-107 production under laboratory conditions.
• ppGpp synthesis was identified as a key intracellular signaling molecule in triggering NAI-107 production.
An improved production process and recovery procedures have been designed.
• Critical parameters for Microbispora growth and NAI-107 production have been delineated and new optimized seed and production media have been designed. These have resulted in production titers up to 10 fold higher than those available at the start of the project.
• A simple recovery and purification process has been designed, which allows purification of NAI-107 with few, industrially scalable steps.
The scale-up and industrialization of the production process have been achieved
• The fermentation process was successfully transferred to a pilot plant, with satisfactory production levels at the 15-L scale.
• A successful fermentation at the 250-liter scale has been performed.
Thus we succeeded to achieve the ultimate aim of the project, namely the design of an improved production process for NAI-107. The process has been successfully transferred to an industrial facility and production of a Good Manufacturing Practice-grade batch is underway.
Project Results:
S&T results/foregrounds
1. Background information
The increasing frequency of nosocomial infections attributable to multi-drug resistant bacterial pathogens exerts a significant toll in the industrialized world, especially when infections are associated with a decline in immune defenses due to age, invasive surgical procedures or other ailments. For example, in the US more people die every year from hospital infections than from all accidental deaths. The UK National Health Service has estimated that hospital-acquired infections cost approximately 1.5 billion € and cause 5000 deaths per annum in the UK alone. Similar statistics exist for Germany. The availability of new and more effective antibiotics is thus a major challenge for the well being of EU citizens.
Despite this increasing medical need, the number of new antibiotics approved for human use has been steadily declining, with only two new chemical classes approved for systemic use during the last three decades. This shortage of new antibiotics has resulted from a diminished interest of large pharmaceutical companies to invest in discovery and development of antibacterial agents, favoring instead drugs for chronic use. Consequently, the antibacterial field has been left almost entirely to biotech companies, where advancement through costly clinical trials is dependent on limited financial resources. To exacerbate the problem, the FDA has in the past few years denied approval of biotech-derived improved versions of existing antibiotics, confirming the view of the medical community that we need new chemical classes with new mechanisms of action to keep pace with the increasing number of drug-resistant bacterial pathogens. However, new antibiotic classes have proven extremely hard to come by.
A critical issue for a new antibiotic is how fast and effectively it can be developed and brought to market. This is particularly critical for the competitiveness of EU industries, since a new drug requires many years of development while having limited time of patent protection. The general aim of this proposal is to generate, through an integrated and interdisciplinary approach, detailed knowledge and efficient tools for the development of an economically viable production process for the new antibiotic NAI-107, a promising new injectable antibiotic with the potential to treat life-threatening infections caused by multi-resistant Gram-positive pathogens.
NAI-107 belongs to the class of lantibiotics and is produced by fermentation of the actinomycete Microbispora sp. It exhibits a relatively wide spectrum, covering all Gram-positive pathogens including multidrug-resistant isolates; it shows good potency, with MIC values for most strains ≤1 μg/ml; it shows good efficacy at 10 mg/Kg in sophisticated experimental models of infection, such as rat endocarditis caused by methicillin-resistant Staphylococcus aureus; its pharmacokinetics in animals suggest the feasibility of once-a-day administration in humans; and it has shown so far a favorable safety profile. All these properties make NAI-107 a promising new antibiotic, suggesting that it will be a safe, once-a-day injectable antibiotic for severe hospital infections.
Lantibiotics have been found to be unique in terms of biological activities. Besides essential functions in quorum sensing and self-regulation of production, several combine two or more antibiotic activities in one molecule resulting in high potency. The majority of lantibiotics target the bacterial cell wall precursor Lipid II, and two different groups of conserved structural motifs involved in Lipid II binding have been identified. The first group is contained within the widely used food preservative nisin, while peptides with a more globular shape often contain the second binding motif, with mersacidin being a well-characterized example. The formation of stoichiometric complexes with Lipid II leads to sequestration of the precursor and blocks its incorporation into the growing cell wall. Therefore, these lantibiotics are inhibitors of cell wall biosynthesis following a mechanism similar to that of the glycopeptide antibiotics; however, the lantibiotic binding site on Lipid II involves the sugar-pyrophosphate rather than the D-Ala-D-Ala moiety, resulting therefore in lack of cross-resistance. Binding to Lipid II then provokes additional activities for some lantibiotics: e.g. for nisin, Lipid II acts as an anchor molecule for the formation of a defined and stable pore consisting of Lipid II and nisin molecules.
NAI-107 has a nisin-like Lipid II binding-motif and also shares features of the non-pore forming lantibiotic mersacidin. Furthermore, NAI-107 has only one positive charge at the free N-terminus and thus is less positively charged than most other lantibiotics. The positive charges have been implicated in the poor pharmacokinetic properties of nisin and of cationic antimicrobial peptides in general. Also, mersacidin and the related compound actagardine (both are neutrally charged at physiological pH) were found to be as effective as vancomycin in vivo in spite of in vitro inferiority.
Before the start of the project, the process for producing NAI-107 was barely sufficient to generate ~0.5 kg of compound necessary for formal toxicology tests. Hence, the production process needed to be optimized and made economically feasible to supply, in a reliable manner, adequate amounts of compound for clinical trials and, in case of favorable results, to launch a product on the market at a price similar to those of comparable life-saving injectable antibiotics. Thus, one of the challenges in advancing a new antibiotic into clinical development is to devise a production process that can deliver a high-quality compound at reasonable costs. This was particularly relevant for NAI-107 because of the lack of precedents: no lantibiotic is currently produced at an industrial scale as a drug for human use and currently no Microbispora strain is used for industrial production.
Actinomycetes such as Microbispora are bacteria that are capable of efficiently utilizing a wide range of substrates and can deal with many toxic compounds present in feedstocks because their genomes contain a large number of hydrolase and oxygenase genes. One of the main issues in the development of a new drug is an efficient supply of compound. For microbial natural products, this is normally achieved through improvement of the producing strain, the fermentation process and/or the recovery of the active compound by a combination of empirical and rational approaches. The latter rely on a detailed understanding of the physiology of the producing strain, particularly with respect to precursor supply. Genomic data allow comparisons with other actinomycetes, which in turn provide new insights into the physiology of the producer strain. Consequently, metabolic flux analysis provides a powerful tool for the overall objective of improving productivity and characterizing different production strains. Since one of the limiting factors in antibiotic production is usually self-resistance in the producer strain, it is also necessary to understand the mechanism(s) of antibiotic export and resistance, which in turn requires a detailed understanding of the antibiotic’s mode of action in model systems.
The identification of metabolic and bioprocess parameters crucial for NAI-107 biosynthesis is necessary for the development of a robust and economically feasible production process, thereby providing a new biotechnological process for a high added-value compound, which has the added benefit of being a potential life-saving antibiotic. Because of its complex chemical structure, NAI-107 cannot be produced by chemical means, and requires robust fermentation processes with optimized strains, media and recovery procedures.
The development of a robust and economically feasible production process for NAI-107 requires the integration of basic knowledge of the physiology of the strain, which can be best obtained by a combination of classical and post-genomic approaches, with a detailed knowledge of the production process and its scalability to industrial level.
The aim of the project, the development of a robust and economically feasible production process for the lantibiotic NAI-107, has been achieved through the integration of basic knowledge of the physiology of the strain, obtained by a combination of classical and post-genomic approaches, with a detailed knowledge of the production process and its scalability to industrial level. This has required combining the knowledge of specialists from different fields, including microbiology, molecular biology, genetics, fermentation, enzymology, metabolic flux analysis and process scale-up.
NAI-107 is a 24-amino acid long lantibiotic containing 5 lanthionine and methyllanthionine rings, and one halogenated tryptophan residue. It is produced as a complex consisting mainly of two congeners differing in the extent of hydroxylation of a proline residue (mono- or di-hydroxylated). The genes responsible for NAI-107 biosynthesis, organized in a cluster designated mlb, have been isolated and sequenced from two Microbispora sp.. The two mlb clusters are highly related, span approximately 20 kb and contain 18 genes encoding: MlbA, the precursor peptide of NAI-107; MlbB (dehydratase) and MlbC (cyclase), involved in post-translational modification of MlbA to produce the thioether bridges; MlbD, involved in the oxidative decarboxylation of the C-terminal Cys to yield the S-[(Z)-2-aminovinyl]-(3S)-3-methyl-D-cysteine residue; MlbH, the tryptophan halogenase; MlbP, a cytochrome P450 hydroxylase likely responsible for proline mono- and di-hydroxylation; five transporters of the ABC type, and one Na/K ion-antiporter, likely involved in exporting lantibiotic intermediate(s) or the finished products out of the cytoplasm and in conferring resistance to the producer cell; and three likely regulatory proteins. No obvious protease or protease domain could be identified as the product of either mlb cluster.
The biosynthesis of many different antibiotics and secondary metabolites is regulated by phosphate. Recently, insights into the molecular mechanisms through which phosphate controls global antibiotic production have started to emerge: in Streptomyces coelicolor and Streptomyces lividans phosphate control of antibiotic biosynthesis is mediated by the two-component system PhoR-PhoP, with PhoP binding to promoters of phosphate-regulated genes. Moreover, another gene (ppk, encoding polyphosphate kinase) was shown to play a key role in the regulation of antibiotic biosynthesis in S. lividans, stressing the important role played by polyphosphate storage. Key enzymes involved in carbon metabolism have also a strong impact on antibiotic production. Whereas the EMP pathway is the central pathway for glucose utilization in many streptomycetes, the Entner-Doudoroff (ED) pathway is critical for Nonomuraea and its presence has implications on the reducing cofactor biosynthesis and energy metabolism. It is well known from industrial fermentations that N-limitation can trigger the onset of antibiotic production. The molecular mechanisms, however, are still unknown. Recent analysis of nitrogen assimilation and its regulation in actinomycetes revealed the presence of a central transcriptional regulator (GlnR) and a number of signal transmitters such as GlnK and GlnD.
The biosynthesis of secondary metabolites is generally dependent on growth phase but also is influenced by a wide array of environmental and physiological signals and nutritional stress. In liquid-grown cultures it generally coincides with stationary phase and it is frequently postulated to result from nutrient limitation. The expression of secondary metabolic gene cluster is regulated by pathway-specific activators, and at least some regulatory networks, for example the phosphate-responsive cascade, are believed to regulate these pathway-specific activator genes.
All gene clusters coding for lantibiotics that target Lipid II contain a dedicated ABC transporter, also present in the mlb cluster, which has been phenotypically implicated in producer immunity. Mechanistically, it is poorly understood how this transporter works. Strains in which the epidermin and the mersacidin transporters had been knocked out accumulate considerably more radiolabeled lantibiotic on the cell surface, indicating that these transporters act by pumping extracellular antibiotic away from the target membrane and may not be able to pump out internal antibiotic to the outside. Moreover, Lipid II targeting lantibiotics do not appear to be proteolytically activated inside the cell such that the dedicated biosynthesis-associated exporter translocates only the modified but unprocessed prepeptide; activation then occurs in the course of, or after transport, either by a transporter with an N-terminal Cys-protease domain, or by a signal protease located on the outside of the cytoplasmic membrane. Such safety measures apparently need to be taken because Lipid II, which is primarily targeted from the outside, is also present on the inside of the cell membrane. Moreover, the precursor to Lipid II, Lipid I, is also targeted by lantibiotics. Thus, a Lipid I/II targeting lantibiotic that is activated on the inside of the cell would likely be inhibitory to the producing strain.
Recent analysis of the physiological responses of cells confronted with increasing concentrations of lantibiotics and adapted to yield a 50 fold decrease in susceptibility indicate that the most effective defense strategy includes significant changes in the architecture of the cell envelope. This includes changes in the overall surface charge through modification of cell wall teichoic acids as well as modulation of expression profiles of penicillin binding proteins.
The molecular mechanisms of antibiotic activity and resistance are often intimately linked. In addition to physiological adaptations which down-modulate susceptibility, antibiotic producing strains usually develop dedicated self-protection (immunity) systems which, on the molecular level, also tightly reflect the specific activity of the antibiotic itself. However, protection and self-resistance systems usually can only cope with levels of antibiotic occurring under natural growth conditions. Therefore, any attempts to raise production levels must include appropriate measures to increase self-protection capabilities.
A limiting factor in antibiotic production can be export of the product from the cell. Specific transporters for antibiotics are encoded by a number of biosynthetic gene clusters; most of them belong to the ABC-transporter family. For glycopeptide antibiotics it was shown that inactivation of the transporter gene leads to a dramatic decrease of the antibiotic in the fermentation broth whereas an intracellular accumulation was observed. Over-expression studies of the transporter gene indicated that yield improvement may be obtained by such an approach.
Antibiotics are usually produced as a complex of related molecules differing for one or more substitutions. NAI-107 is produced as a complex consisting mainly of two factors differing in the extent of hydroxylation of a proline residue (mono- or di-hydroxylated), as well as other minor, as yet uncharacterized factors.
2. Main results
2a. Tools for Microbispora
Due to limited knowledge of the Microbispora producer strain, the objectives of the initial period of the project were the development of the tools necessary for analyzing the physiology and genetics of the producing strain and for understanding the elements contributing to NAI-107 production.
Highlights
• a gene transfer system based on conjugation between E. coli and Microbispora has been developed. The protocol has been successfully applied by different Partners for generating knockout and over-expression mutants.
• a draft genome sequence of Microbispora has been generated. The annotated genome (~ 8000 CDSs) will be soon available in public databases. The sequence information has been used in the course of the project for several studies.
• a 2D-protein map of Microbispora during primary and secondary metabolism has been constructed. Differentially abundant protein spots, identified by mass spectrometry, are available to investigate relationships between biochemical pathways and regulatory networks along with NAI-107 production. In addition, an interactive Microbispora proteome web site will soon be available
• a chemically defined medium allowing NAI-107 production and suitable for metabolic flux analysis has been designed. C-flux methodology has been optimized and applied to Microbispora
Main results
A gene transfer system based on conjugation has been developed for Microbispora. While published protocols for conjugation from Escherichia coli to actinomycetes have generally used a methylation-defective E. coli donor strain, the use of such strains with Microbispora has resulted in a very low frequency of exconjugants. After testing alternative donors, Escherichia coli S17-1 was established as a suitable donor strain. Further adjustments were made to the antibiotics used to select against E. coli and to select for Microbispora exconjugants. We have applied the optimized conjugation protocol to develop expression systems with GFP and mibR, a key regulator from the mib cluster. To facilitate the construction of knockout mutants (i.e. clones where a double cross over event had occurred), we developed a strategy based on the gusA reporter system, which allows to distinguish between plasmid-carrying colonies (blue) and colorless colonies that have lost the plasmid (double cross-over).
The Microbispora draft genome sequence was obtained combining different sequence methodologies: the 454 technology, which initially yielded 8 scaffolds for a total of 8,091,813 bases; and the Illumina technology, which led to a single scaffold of 8576389 bp. The Microbispora genome shares extensive synteny with the complete genome of Streptosporangium roseum, a strain belonging to the same Actinobacteria family as Microbispora. The sequence information has been instrumental to generate the 2D protein maps of Microbispora (see below), as well as to identify key genes involved in nutrient assimilation.
2D-differential gel electrophoresis was used to perform comparative analysis of Microbispora protein expression patterns before and during NAI-107 production, collecting biomass samples at several times before and during NAI-107 production from parallel cultures grown in triplicate. Comparative proteome analysis was carried out between samples collected before and during NAI107-production during rapid growth, and between samples collected during NAI-107 production at early, mid and late decline stages. Identification, by mass spectrometry procedures, of 533 protein spots differentially abundant revealed by global (327 spots) and membrane (206 spots) proteome analyses was accomplished using biomass samples collected at rapid growth stages before and during NAI-107 production. Identification of 310 differentially abundant proteins revealed by global proteome analysis was accomplished using biomass samples collected during NAI-107 production at early-, mid- and late-decline stages.
These proteins were used to construct an interactive Microbispora ATCC-PTA-5024 reference 2D-map that will soon be available. In order to have a medium useful for the C, N and P assimilation studies and for flux analyses, a number of different defined and minimal media were investigated, monitoring Microbispora growth and NAI-107 production. Furthermore, batch cultivations in well-controlled reactors were carried out in a few complex and defined media. Maximum specific growth rates in the range of 0.07-0.09 were seen.
2b. Mechanism of action, resistance and transport
Due to the fact that production of an antibiotic is often limited by the self-resistance mechanism in the producer strain, it was important to investigate in detail the mechanism of action of NAI-107, a cell wall inhibitor, in model strains, as well as establishing key features in self-resistance, transport and cell wall composition in Microbispora.
Highlights
• Using whole-cell experiments, NAI-107 was identified as a cell wall biosynthesis inhibitor; in vitro assays with purified cell wall biosynthesis enzymes and substrates identified the molecular target of the lantibiotic, the lipid-bound cell wall building block Lipid II. NAI-107 was found to bind to Lipid II with high affinity; pore formation, as observed with some Lipid-II binding lantibiotics, did not occur with NAI-107 in vitro.
• Analysis of the proteomic response profile of Bacillus subtilis, as compared to the profile induced by Lipid II binding antibiotics, showed the highest degree of similarity with gallidermin, another non-pore forming lantibiotic.
• Work towards understanding the producer self-protection mechanisms identified a putative ABC transporter and a putative lipoprotein MlbQ which could meditate self-protection. Functional analysis suggests a specific role of the membrane-localized MlbQ in resistance to NAI-107, while NMR studies revealed an unusual structure. Analysis of the cell wall building block of the producer does not indicate specific alterations of the cell wall composition.
• Analysis of the Microbispora cell wall indicates that it contains stem peptides with either glycine or alanine at the first position. The cell wall does not contain an interbridge but a direct linkage between peptide chains. These features were also observed in the knockout mutant of Microbispora that does not produce any NAI-107.
Main results
Our results demonstrate that NAI-107 is a potent inhibitor of cell wall biosynthesis with lipid II as a specific target. We studied the mode of action of NAI-107 using whole cells, isolated membranes and purified enzymes. We found that the lantibiotic very efficiently interferes with late stages of cell wall biosynthesis in intact bacterial cells as demonstrated by inhibition of N-acetylglucosamine incorporation into polymeric cell wall and the accumulation of the ultimate soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide in the cytoplasm. Using membrane preparations and the complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we show that NAI-107 forms complexes with undecaprenyl pyrophosphate- N-acetylmuramic acid(pentapeptide)-N-acetylglucosamine (lipid II), which is readily accessible at the outside of the cell and which forms a complex with the antibiotic in a 1:2 molar stoichiometry. Furthermore, we found that pore-formation does not seem to be a major component of the antibiotic activity as seen by whole cell assays measuring the membrane potential and potassium efflux as well as CF-efflux from artificial liposomes.
We compared the proteomic response of B. subtilis after treatment with the lantibiotics nisin, mersacidin, gallidermin and NAI-107 and an untreated control. Proteins found to be induced more than 2-fold in three independent biological replicates are reported as marker proteins. NAI-107 treatment led to upregulation of 20 protein spots, 15 of which could be identified by mass spectrometry: among them, YceC and YceH are known cell envelope stress markers; LiaH, YtrB, YtrE, and YpuA are specific markers for inhibition of membrane-bound steps of cell wall biosynthesis; PspA and NadE are membrane stress markers. NAI-107 elicits a typical dual cell wall biosynthesis inhibition and membrane stress response. The proteome response strongly overlaps with that of gallidermin suggesting high mechanistic similarity between these two lantibiotics. High similarity of the proteome response profile of NAI-107 was also observed with MP196 and gramicidin S, both of which integrate into the cytoplasmic membrane and disturb membrane architecture. Less overlap was observed with nisin indicating that membrane interaction of NAI-107 is not characterized by pore formation but rather “unorganized” integration and interference with the membrane architecture.
Interference with membrane-bound steps of cell wall biosynthesis was confirmed by microscopic inspection of the cell shape (Fig 1). The impact of NAI-107 on the cytoplasmic membrane was investigated by studying the localization of a GFP fusion to the cell division protein MinD, which normally localizes at the cell poles and in the cell division plane. In a depolarized cell, MinD delocalizes appearing as irregularly distributed GFP clusters. After treatment with NAI-107, complete GFP-MinD delocalization was observed suggesting efficient membrane depolarization. NAI-107 did not efficiently facilitate penetration of propidium iodide into B. subtilis suggesting that no large pores are formed. Thus, NAI-107 seems to interfere with membrane-bound steps of cell wall biosynthesis and with the architecture of the cytoplasmic membrane.
Fig. 1. Microscopy of B. subtilis cells treated with NAI-107 or nisin. (A) Examination of the cell shape after acetic acid/methanol fixation. (B) Depolarization assays using GFP-MinD. (C) Light microscopy of cells in (B). (D) Pore formation monitoring using the live/deadBacLight bacterial viability kit.
Lantibiotics are synthesized as precursor peptides which undergo posttranslational modifications and proteolysis to give the final product. Lantibiotic biosynthetic gene clusters encode dedicated ATP-binding cassette (ABC) transporters to export the modified peptide from the producing cells. Bacterial ABC transporters consist of 2 transmembrane domains and 2 nucleotide binding domains which utilize ATP hydrolysis as a source of energy for the transport. The biosynthetic gene cluster of NAI-107 codes for three ABC transporters: MlbY-MlbZ, MlbT-MlbU and MlbE-MlbF (Fig. 2). In order to obtain a strain with optimized excretion of NAI-107, each ABC transporter was overexpressed in Microbispora. However, there were no significative differences between Microbispora wild type and each of the ABC-overexpressing strains, suggesting that excretion is not a limiting step in NAI-107 production. A sensitivity assay using spores of the M. corallina mibEF deletion mutant and the wild type strain shows that the mutant is 40-fold more sensitive to NAI-107 than the wild-type strain. When mlbEF were overexpressed in Streptomyces coelicolor, a two-fold increase in resistance towards NAI-107 was observed.
The putative lipoprotein MlbQ may be involved in immunity. When expressed in S. coelicolor, where it localizes to the membrane, MlbQ confers a slight increase in resistance to NAI-107 compared to the wild type. However, we could not yet demonstrate a binding of the lipoprotein MlbQ to NAI-107. The MlbQ structure, as solved by NMR (collaboration with the Max Planck Institute, Tübingen), does not share homology with the protein SpaI (conferring immunity to subtilin), the only immunity protein whose structure has been so far solved by NMR. Up to now, interaction studies between MlbQ and NAI-107 could not be performed by NMR because of NAI-107 limited solubility in the protein buffer.
Fig. 2 NAI-107 gene cluster. The genes encoding the three ABC transporters are framed.
The Microbispora mutant strain RP0, resulting from a single crossover within the mlb cluster, was constructed. This mutant does not produce NAI-107 as shown by bioassay and HPLC-MS. This mutant has been used in further studies as described in the following sections.
To elucidate the cell wall composition of Microbispora, we analyzed in parallel cell wall precursors and murein from S. coelicolor. The latter strains contains two major cell wall precursors: UDP-MurNAc-L-Ala-D-Glu-LL-Dap-D-Ala-D-Ala; and one with a Gly linked to LL-DAP. In contrast, Microbispora contains only the former precursor. Furthermore, Microbispora does not possess a monoglycine interpeptide bridge as reported for S. coelicolor and we suspect a direct linkage between peptide chains. There was no alteration in the cell wall of the non-producer strain Microbispora RP0 as compared to that of the wild type. However, both strains contain also Gly and Ser in the first position.
Undecaprenylphosphate represents the central-membrane-bound lipid carrier involved in the synthesis of diverse bacterial cell envelope polymers and polysaccharides, including not only peptidoglycan but also whole teichoic acid (WTA) and capsule biosynthesis. Besides with lipid II, nisin and related lantibiotics interact also with lipid I in vitro; additionally, complex formation with bactoprenolpyrophosphate (C55-PP) has been described for nisin. These findings suggest that nisin and related lantibiotics containing the lipid II binding motif may interact with further cell envelope precursors, attached to C55-P as a carrier molecule, such as those for capsule or WTA biosynthesis. In S. aureus, WTA biosynthesis involves at least 9 enzymatic steps (Fig. 3). We have set up in vitro assays for the first part of the WTA reactions and were already able to prove the interaction of nisin with WTA precursors. We also observed that, in the presence of NAI-107 in a 2-fold molar ratio, almost no conversion of Lipid III to Lipid IV was detectable. This proves that also NAI-107 is able to interact with WTA precursors in vitro.
Fig. 3. WTA-biosynthesis in S. aureus
2c. Carbon, nitrogen and phosphate assimilation
Understanding the major pathways for substrate assimilation and mapping metabolic fluxes are required for understanding the major changes in the proteome associated with NAI-107 production. This information can then be used to design improved production strains and processes.
Highlights
• Key components of the N-regulon and P-regulon with the response regulators GlnR and PhoP respectively, were identified in the Microbispora genome and their expression analyzed.
• Nitrogen excess positively controls NAI-107 production yield and the biomass accumulation; phosphate limitation positively regulates biomass accumulation but has no influence on NAI-107 production.
• Comparative proteome analyses revealed that proteins involved in nitrogen metabolism regulation, sulphur metabolism, phosphate metabolism, cell wall biosynthesis, oxidative and general stress response factors, TCA cycle, protein folding and modification, fatty acid and cell membrane metabolism, proteins regulating physiological differentiation, stringent response key factors, NAI-107 biosynthetic gene cluster products, multidrug resistance ABC transporters are associated with NAI-107 production.
• The highest metabolic flux of C-assimilation was observed through the pentose phosphate (PP) pathway; fluxes of C-assimilation through the tricarboxylic acid (TCA) cycle and Embden-Meyerhof-Parnas (EMP) pathway were comparatively low.
Main results
In multiple species of actinomycetes, inorganic phosphate (Pi) negatively regulates antibiotic production. Phosphate control of antibiotic biosynthesis in Streptomyces is mediated by the two-component PhoR/PhoP system, where PhoR is a membrane sensor kinase and PhoP is a DNA-binding response regulator. In response to phosphate starvation, PhoP activates expression of the pho regulon by binding to consensus phosphate boxes in the corresponding promoter regions (PHO boxes). Key components of the P-regulon were found in Microbispora. In a chemically defined medium containing different Pi concentrations (0, 0.1 0.5 5, 15 and 30 mM), biomass yields of Microbispora increased up to 5 mM Pi. However, NAI-107 productivity did not change significantly from 0.1 to 30 mM Pi. Quantitative RT-PCR of pho regulon genes (phoR, phoP, ppk and pstS) was performed revealing that these genes were upregulated under low Pi, as expected.
Actinomycetes are able to produce a diverse range of secondary metabolites during differentiation, which require precursors from primary metabolism. The production of secondary metabolites and cellular differentiation is finely tuned by various intra- and extracellular signals such as N limitation. Nitrogen assimilation involves the uptake and incorporation of nitrogen sources and results in the synthesis of glutamate and glutamine, which are the key intracellular nitrogen donors. Due to environmental limitation and variation in nitrogen availability, bacteria have evolved complex regulatory networks for the transcriptional control of genes involved in nitrogen metabolism. In S. coelicolor, GlnR, an orphan response regulator, controls nitrogen assimilation by transcriptional regulation of 13 genes, of which six are directly involved in nitrogen metabolism. The glnR gene was also found in Microbispora genome. Other key components of the N-regulon (glnR, glnRII, glnA, glnII, gdh, amtB operon, nnaR, nasA, nirBD) were identified. When Microbispora was grown in minimal medium supplemented with various nitrogen sources, we observed that nitrogen positively controls NAI-107 production.
To analyze the expression pattern of the genes of central N-metabolism, Microbispora was grown in minimal medium supplemented with various N sources and in complex media. Overall, the expression profiles of 18 genes relevant for N-metabolism in either S. coelicolor or M. smegmatis were followed, as well as 4 mlb genes. Expression of glnR (global nitrogen regulator) was induced only under N-limiting conditions, while no expression was detected In N-rich media. Microbispora GlnR is likely to be regulated post-translationally in response to nitrogen changes in the medium. The expression pattern of GlnR target genes in Microbispora seems to be similar to that of S. coelicolor. Concerning the mlb genes, the expression of MlbX and MlbW was elevated in all complex and defined media in an N-independent manner. The expression of the structural gene mlbA increased in complex media during exponential growth, and clearly showed N-dependence in minimal media.
Since the generation of glnR deletion mutants resulted problematic, we concentrated our efforts on over-expressing glnR, nnaR and glnK. The biomass formation in all three overexpression mutants was lower compare to the wild type and RP0. All three overexpression mutants showed almost no differences in the biomass formation between each other. A slight increase in NAI-107 productivity over wild type was observed in the glnR¬- and nnaR-overexpression strains. Thus, high ammonium concentrations positively regulate NAI-107 production independently of the phosphate concentrations in the medium. Under ammonium excess glutamate dehydrogenase is a key enzyme for ammonia assimilation, which is repressed by GlnR only under nitrogen limitation. Regulation of the nitrogen assimilation in Microbispora and its influence on NAI-107 production does not only depend on nitrogen concentration but also on the nitrogen source.
To investigate the central carbon metabolism, Microbispora was grown in minimal medium supplemented with 13C-labeled glucose. Amino acid analysis after biomass hydrolysis, summed fractional labelings were calculated and used for calculating metabolic fluxes. Strikingly, Microbispora seems to have a high flux through the pentose phosphate (PP) pathway. The observed value is the highest of all analyzed Actinobacteria, including Corynebacterium glutamicum, which is known to have a high PP flux. Furthermore, no activity through the Entner-Doudoroff (ED) pathway was observed under the conditions used and fluxes through the tricarboxylic acid (TCA) cycle and Embden-Meyerhof-Parnas (EMP) pathway were comparatively low.
Microbispora proteome analysis was carried during growth in complex medium. Microbispora growth kinetics revealed initial rapid growth, sustained by glucose utilization, followed by a second stage of decline and cell death. NAI-107 production started at mid rapid growth stage and reached its maximum at early decline stage. To understand the molecular processes and metabolic pathways associated with lantibiotic production, differential analyses were performed between samples collected before and during NAI-107 production in the rapid growth phase, and between samples collected at early-, mid- and late-decline stages. In the first case, comparative global proteome analysis revealed 533 protein spots differentially abundant revealed by global (327 spots) and membrane (206 spots) proteome analyses. Positively associated with NAI-107 production were proteins required for amino sugar metabolism, cell wall biosynthesis, lantibiotic resistance, nitrogen metabolism, oxidative stress response, protein folding and modification, and pleiotropic regulators. In the second case, a total of 310 differentially abundant proteins were identified, mostly involved in lantibiotic resistance, nitrogen metabolism, cell wall biosynthesis and pleiotropic regulators are accumulated at the early decline stage. Combining the results of both analyses, a list of key proteins associated with NAI-107 production was obtained. Some of the corresponding genes have been targeted for over-expression and knockout experiments in Microbispora.
2d. Regulation
Production of an antibiotic is invariably controlled by cluster-specific regulators and/or by the general regulatory networks of the producer strain. The ultimate aim of these studies is to understand the pathway-specific and ultimately the general regulatory mechanisms that influence NAI-107 biosynthesis, and to use the resulting knowledge to increase the level of NAI-107 production. Previous work on a different Microbispora strain had identified key roles for three regulatory genes: mibX encoding an ECF-sigma factor, mibW encoding the cognate anti-sigma factor and mibR encoding a likely DNA-binding protein. MibX and MibR were shown to be transcriptional activators of the biosynthetic gene cluster, and MibW to interact with MibX, likely inhibiting the latter’s activity. Homologues of each of these genes are found in the NAI-107 biosynthetic gene cluster (Fig. 2).
Highlights
• Key regulatory genes identified in the NAI-107 biosynthetic gene cluster – mlbR, mlbX and mlbW.
• Over-expression of mibR resulted in over-production of NAI-107.
• ppGpp synthesis identified as a key intracellular signaling molecule in triggering NAI-107 production.
Main results
To understand the regulation of the mlb cluster in Microbispora, the mlbA, mlbE, mlbJ, mlbQ, mlbR and mlbX promoter regions were cloned upstream of a reporter gene and introduced into S. coelicolor strains containing either mlbR or mlbX under the control of a strong promoter. The results show that mlbA is regulated directly by the transcriptional activator MlbR whilst the other genes are regulated directly by the sigma factor MlbX.
An experiment in which the wild type Microbispora is grown next to different concentrations of purified NAI-107 has shown that NAI-107 itself can induce precocious production in the wild type strain (Fig. 4). Several antibiotics, including other cell-wall inhibitors, have then been tested and so far no other compound has been able to induce NAI-107 production in the wild type. This suggests that NAI-107 is specifically able to act as an autoinducer of its own production, likely interacting directly with MlbW.
Fig. 4. NAI-107 triggers precocious production of NAI-107 in the wild-type Microbispora sp. strain. The strain was streaked vertically (two streaks) and either NAI-107 (left) or water (right) were spotted onto sterile filter paper discs that were placed adjacent to the two streaks. The shape of the zone of inhibition indicates auto-induction of NAI-107 production.
The model for the regulation of NAI-107 production suggested that a key event in triggering biosynthesis of the lantibiotic occurred through activation of transcription of mlbR from a second thus far unidentified MlbX-independent promoter. A second promoter, mlbRp2, identified by RT-PCR in Microbispora, was used to direct transcription of a reporter gene in S. coelicolor wild type and its congenic ppGpp synthetase null mutant. Measurement of GusA activity indicated that transcription from mlbRp2 was dependent on synthesis of ppGpp, as a key intracellular signaling molecule involved in the activation of transcription of pathway-specific regulatory genes in other actinomycetes. This result is important since it links nutrient limitation to activation of transcription of mlbR, which results in the production of small amounts of a NAI-107 congener, which then functions in a feed-forward mechanism to release MlbX from MlbW, resulting in high levels of transcription of the entire gene cluster, and production of the main NAI-107 congeners.
2e. Metabolic engineering
The objectives of these studies were directly aimed at designing an improved and scalable production process. They included the engineering of improved production strains, the design of an effective production process and the implementation of a robust recovery procedure.
Highlights
• Among the antibiotic resistant mutants of Microbispora, two streptomycin-resistant mutants (S13 and S22) perform better than the wild type for NAI-107 production.
• The minor congeners (F0, F1, F2, A0, B1 and B2) have been purified, structurally characterized and assayed for their antibiotic activity in comparison with the main congeners A1 and A2.
• An improved production process has been designed. Critical parameters for Microbispora growth and NAI-107 production have been delineated and new seed and production media have been designed.
• Over-expression of mlbR or mlbX resulted in higher levels of NAI-107 production under laboratory conditions.
• A simple recovery and purification process has been designed, which allows purification of NAI-107 with few, industrially scalable steps.
Main results
In actinomycetes, mutants resistant to streptomycin (Str), rifamycin (Rif) and/or gentamicin (Gen), have been reported to be deregulated in antibiotic production, allowing an easy selection of high-producing strains. In Microbispora mutants, while RifR mutations result in lower productivity, there was a 20-30% increase in productivity in selected StrR mutants affecting either ribosomal protein S12 or RsmG.
NAI-107 is produced as a complex of related molecules, with the most abundant congeners A1 and A2 differing for the presence at position 14 of a hydroxy-proline (A2) or a di-hydroxy-proline (A1). Additional congeners were purified and structurally characterized: A0 lacks altogether hydroxyls at Pro14; F1 and F2 correspond to A1 and A2, respectively, but are devoid of the chlorine atom on Trp4; and B1 and B2 correspond to A1 and A2, respectively, but carry a sulfoxide on the first thioether. When tested for antibacterial activity, the B and F forms were slightly less active than the A forms, while substitution at Pro14 had only a minor impact.
[start of CONFIDENTIAl section]
When mlbR and mlbX were placed downstream of strong or inducible promoters and separately introduced into Microbispora, the resulting strains not only produced higher levels of NAI-107, but also did so earlier than the wild type. When these strains were grown under the scalable, there was no statistically significant difference between the production levels observed with the engineered strains (600-800 mg/L) and those achieved with the wild type. An ad hoc process may need to be developed to enhance the performance of the engineered strains.
At the start of the project, the fermentation procedure for NAI-107 production was poorly defined, resulting in a highly variable process. Through a series of empirical observations, we noticed that a robust process depended on following three parameters at the time of crossover in vegetative medium: strain morphology as dispersed mycelium; 2-4 g/L residual glucose; and ≤10 mg/L NAI-107.
Varying carbon and nitrogen sources, we were able to design an improved production medium. In general, higher glucose concentrations adversely affected NAI-107 production, while complex nitrogen sources were correlated with higher NAI-107 yields (~0.25 g/L). Further improvements were obtained through the addition of micronutrients (zinc sulfate), olive oil and decreasing calcium carbonate, boosting NAI-107 production to ~1 g/L at flask level. Addition of glucose at the onset of NAI-107 production strongly reduced further antibiotic formation, suggesting glucose repression of NAI-107 production.
Under the optimized medium, the performance of different StrR mutants was also evaluated, as reported in Fig. 5. While there is no significant difference in the highest titers observed, mutants S22 and S24 reach ~1 g/L earlier (after about 14 days) compared to the 21 days necessary for S13.
Fig. 5. Time-course of NAI-107 production (sum of congeners A, B and F) in the optimized medium. Error bars represent standard deviation.
Before the start of this project, the first large-campaign of NAI-107 production (~0.4 kg) had provided the undesired presence of soyasaponins, carried over from the spent growth medium. We thus devised a scalable and effective downstream process that consists of: 1) separation of mycelium from the spent broth through centrifugation after acidification of the culture; 2) extraction of the mycelium with aqueous methanol and adsorption on an HP20 resin; 3) recovery of NAI-107 by methanol elution and concentration by evaporation; 4) cation exchange chromatography; and precipitation of NAI-107. This procedure afforded NAI-107 in satisfactory yield (32%, compared with the 15% of the original process) and acceptable purity, devoid of soyasaponins. Furthermore, it requires fewer chromatographic steps and less solvent volumes than the original process.
[end of CONFIDENTIAl section]
2f. Process scaleup
The objectives of these studies were to scale-up the production process from the laboratory to pilot plant, producing consistent amounts of NAI-107 in good purity and yield.
Highlights
• The fermentation process was successfully transferred to a different laboratory, with satisfactory production levels at both the 15 and the 250-L scale.
Potential Impact:
Socio-economic impact and societal implications
The objective of this proposal was to develop, through an integrated and interdisciplinary approach, an economically viable production process for the new antibiotic NAI-107. There is a need for novel and better antibiotics to fight infectious disease, especially for life-threatening infections. However, despite this increasing medical need, the number of new antibiotics approved for human use has been steadily declining. Therefore, promising new antibiotics, such as NAI-107, need not only to be discovered but also to be effectively developed and produced. One of the main issues in the development of a new drug is an efficient supply of compound. The outcome of this project was the development and application of industrial biotechnology for the production of high-value products such as the antibiotic NAI-107. Before the start of this project the process for producing NAI-107 was sufficient to generate the 0.5 kg batches of compound necessary for formal toxicology tests. However, the production process was complex, not sufficiently robust and required several chromatographic steps during compound purification. Therefore, the entire process required to be optimized and made economically feasible if it was to supply, in a reliable manner, adequate amounts of compound for clinical trials and, in the event of favorable results, to launch a product on the market at a price similar to those of comparable life-saving injectable antibiotics. Thus, one of the challenges in advancing a new antibiotic into clinical development is to devise a production process that can deliver a high-quality compound at reasonable costs. This is particularly relevant for NAI-107 because of the lack of precedents: no lantibiotic is currently produced at an industrial scale for use as a drug for human use and there are no examples of the use of Microbispora strains for industrial production. The solution of this problem has required diverse and specialized skills that could not reside solely within the limited resources of an SME, nor can they could be found within the boundaries of a single country. The present proposal has brought together experts in the physiology and genetics of actinomycetes, who have successfully applied basic knowledge from model organisms to less tractable actinomycete genera during FP5 and FP6 projects; experts in lantibiotic genetics and mode of action; and experts in the scale-up of fermentation processes. These skills were critical for achieving the ultimate goal of our project, namely the design of an improved and scalable production process for NAI-107. The projected outcomes of this project will therefore make an important contribution to the biotechnology industry in Europe with the added payback of providing a new drug that can improve the health and the quality of life of EU citizens.
In late 2009, while this project was still under negotiation with the Commission, the Coordinator of this project (NAICONS) and the owner of the IP rights on NAI-107, was able to transfer rights to NAI-107 and related lantibiotic technology to Sentinella Pharmaceuticals, a US-based company backed by Care Capital, a venture capital firm active in health care. This agreement supplied sufficient resources for initiating the preclinical studies necessary for obtaining an authorization for initiating studies in human volunteers. Furthermore, also because of the results achieved in the course of the LAPTOP project, NAICONS has contributed to the successful transfer of the production process to a facility in North America, where production of GMP-grade NAI-107 is under way. The importance of compounds such as NAI-107 has recently attracted the attention of specialized journals in biotechnology (see for example Nature Biotechnology 31, 379–382, 8 May 2013).
In conclusion, not only has the project reached its ultimate goal, the development of an improved production process for NAI-107, but the compound itself has attracted interest from third parties, demonstrating that SMEs are a vital part of the EU economy and can “export” technology and high-added value products.
In addition to industrial competitiveness, in the field of antibiotic research Europe has attained a leading role. Mainly due to the activities of several members of this consortium, Europe has caught up and overtaken the US. One aim of this project was to further improve this position in academic research, by strengthening the collaboration and technology transfer between academia and industry, thus helping foster the EU biotechnology industry. By combining industrial microbiology integrated with modern molecular techniques, high quality academic research has becomes tightly interconnected with industrial research and production processes. This connection is particularly relevant in the present work, since only one of the academic partner in the present consortium had previous experience with lantibiotic-producing strains. Nonetheless, the overall objective of the work could be achieved.
The successful completion of this project demonstrates the viability of the type of consortium at work during this project, and during previous projects funded under FP5 and FP6 on a different class of antibacterial compounds, i.e. glycopeptides. This bode well for the future of antibiotic research within the EU. The knowledge generated as a result of the proposed work and the techniques developed has also enabled the application of similar methodologies to other potentially valuable products, thereby further promoting a sustainable biotechnology industry. The progress in this area requires a multidisciplinary consortium of specialists in molecular genetics, microbiology, natural product chemistry, physiology, enzymology and fermentation technology. Since each of these specialists needs to have a broad knowledge of antibiotics, such a team cannot be put together from researchers of one country, but needs a European dimension. The LAPTOP project, which has involved five groups from academic institutions and two from industry, is an example in full agreement with statements from the European Commission to build a research and innovation structure equivalent to the “common market for goods and services” (Participating in European Research, October 2002).
Another important community objective addressed at the time of writing the proposal and followed during the course of this project has dealt with the future employment of EU scientists. During the last decade, many pharmaceutical companies have witnessed major mergers, acquisitions and reorganizations that have changed the face of the pharmaceutical industry. In order to maintain profitability, these changes have often resulted in the consolidation and centralization of research and development centers, with the concomitant loss of thousands of jobs. Apart from the direct impact on unemployment, this world-wide phenomenon has resulted in fewer opportunities for employment by large pharmaceutical firms for graduate and post-graduate scientists coming out of academic institutions. However, the existence of very large pharmaceutical firms has also created a wealth of opportunities for smaller "biotech" companies focused on providing specialized technologies and services to larger enterprises, and/or developing their own products. Indeed, the number of NDAs (new drug application for market authorization filed at regulatory agencies) by biotech industries has, in recent years, surpassed the number of applications filed by large pharmaceutical companies. However, few biotech companies have their own marketed products, so their revenues depend on contracts, financial investors and public grants. In the long term, biotech companies can become financially stable only if they have direct revenues from the sale of products.
In the course of the project, attention has been paid to contribute to the creation of an entrepreneurial attitude among academic researchers, that will hopefully lead to better employment levels for young scientists. This has also been achieved through a series of exchanges of young scientists at the start and during the execution of the project.
Exploitation and dissemination plan
The exploitation and dissemination plan has been highlighted throughout the project. In order to ensure that adequate IP protection of any foreground, an IP Committee was established in the course of the project. The IP Committee handled all matters relating to public disclosure of any information and proved to be very efficient in providing timely answers to requests for publication, which is extremely important for young scientists. All requests handled by the IP Committee were unanimously approved.
Because IP had already been secured before the start of the project (NAICONS IP on NAI-107, subsequently transferred to Sentinella Pharmaceuticals as mentioned above), exploitation of the main foreground of the project had already been achieved at project start. Nonetheless, as mentioned above, the results obtained during the course of the project were instrumental for transferring the production process to a facility based in North America, where production of GMP-grade NAI-107 is under way. Therefore, this is giving the long-term continuity necessary for the clinical development of a new drug, particularly a life-saving new antibacterial agent active against all multi-drug resistant pathogens.
The other industrial partner in the LAPTOP project, Gnosis, was interested in expanding its portfolio of fermentation products. Its participation in the current project has contributed in increasing its expertise in actinomycete fermentations, and now Gnosis is involved in the industrial production of natural and semi-synthetic glycopeptides (http://www.gnosis-bio.com/biopharma.php).
While data mining its strain collection for lantibiotics related to NAI-107, NAICONS has discovered a new lanthipeptide with marked antinociceptive activity. A patent application on the compound has been filed in July 2012.
Dissemination of the results, after having protected any patentable inventions, have been achieved through:
• Posting relevant findings on the LapTop web site
• Presentations at scientific conferences
• Publications in international journals
• Presentations at a mini-symposium
• Dissemination of leaflets and brochure(s)
During the project we were able to present our results in more than 30 conferences (national and international meetings), and several manuscripts have been submitted to high-ranking international journals or are in the final stages of revision for future submissions. At the time of writing this report, the lack of published results should not come as a surprise, since most of the Partners involved in the project had no direct experience with lantibiotics or lantibiotic-producing strains, and had therefore to develop the necessary knowledge and expertise before publishable results could be obtained.
The LAPTOP project was shortly introduced during a "Pupils Congress" on November 8, 2011, for pupils of Baden-Württemberg, Germany, interested in biotechnology/antibiotics. In total 80 pupils attended this meeting with 10 teachers. In addition, the German Research Association (DFG) has organized the touring exhibition “Mensch-Mikrobe” (humans-microbes), held in Tübingen for three months (in winter/spring 2012). Prof. Wolfgang Wohlleben (EKUT) gave an oral presentation on February 8, 2012, to about 200 citizens from Tübingen, Germany on antibiotics. In the course of the presentation, the objectives and importance of the LAPTOP project were highlighted.
To ensure proper dissemination of the results a final minisymposium was organized to present and to discuss the results of the LAPTOP project with the leading scientists in the field. The International Workshop on “Novel Developments in Lantibiotic Research” took place in Verona from June 16 to June 17, 2013. The aim of the minisymposium was to achieve public awareness of the LAPTOP project and to have leading scientists in the field not involved in the project critically evaluate our results. We invited scientists who contributed most to the progress in lantibiotic research during the last decade. We were really pleased with the outcome, since all eight contacted scientists accepted our invitation, including one coming specifically from North America. This clearly demonstrated the interest around the LAPTOP project and the broad expectation of a new lantibiotic advancing towards clinical development for treating hospital-acquired infections.
Each of the invited scientists presented their results. At the same time, the results of the LAPTOP projects were presented by the young scientists (PhD students and postdocs) from each partner in the consortium. The LAPTOP results were intensively discussed and positively evaluated by the external participants. The meeting has also led to new collaborations with some of the external scientists on specific topics related to NAI-107.
The overall results of the LAPTOP project were presented by the Coordinator at the Symposium on the Genetics of Industrial Microorganisms held in Cancun, Mexico, on June 23-29, 2013. This symposium, attended by about 250 scientists from all countries, represents the traditional venue for presenting advanced research on industrial microorganisms producing added-value compounds. Further dissemination activities are planned in the upcoming year, as the scientists who have participated in the LAPTOP project will continue presenting their results at national and international conferences.
List of Beneficiaries
Beneficiary Number * Beneficiary name Beneficiary short name Country
1. Margherita Sosio- NAICONS NAICONS Italy
2. Mervyn J Bibb - John Innes Centre JIC UK
3. Anna Eliasson Lantz- Technical University of Denmark DTU Denmark
4. Anna Maria Puglia- University of Palermo DBCS Italy
5. Hans-Georg Sahl - Universitaetsklinikum Bonn UKB Germany
6. Wolfgang Wohlleben Eberhard-Karls-Universität Tübingen EKUT Germany
7. Marco Berna Gnosis Gnosis Italy
The LAPTOP objective was the development of a robust and economically feasible production process for NAI-107, with the final objective of producing Good Laboratory Practice-grade compound. The lantibiotic NAI-107, produced by the actinomycete Microbispora sp, is a new antibiotic with the potential to treat life-threatening infections caused by multidrug-resistant Gram-positive pathogens. It is active against all Gram-positive pathogens including multidrug-resistant isolates, with good potency and efficacy in sophisticated experimental models of infection, such as rat endocarditis caused by methicillin-resistant Staphylococcus aureus. NAI-107 is currently undergoing formal toxicology studies. Because of its complex chemical structure, NAI-107 cannot be produced by chemical means. Thus, the supply of sufficient amounts of product for future clinical studies requires robust fermentation and recovery processes to deliver a high-quality compound at reasonable cost. This is particularly relevant for NAI-107 because of the lack of precedents: no lantibiotic is produced at an industrial scale as a drug for human use and no Microbispora strain is used for industrial production. Due to limited knowledge of the Microbispora strain, the initial objectives were the development of tools instrumental for analyzing the physiology and genetics of the producing strain and for getting insights into NAI-107’s mechanism of action. Next, knowledge generated during the previous reporting period was translated into the generation of improved strains, the design of optimized media and recovery procedures. A gene transfer system based on conjugation between E. coli and Microbispora was developed and applied to generate knockout and over-expression mutants. From a draft genome sequence of Microbispora, consisting of about 8,000 coding sequences, key components of the N-regulon and P-regulon were identified and a 2D-protein map was constructed during primary and secondary metabolism, establishing differentially abundant proteins. After the identification of chemically defined media suitable for NAI-107 production, carbon flux analysis established that C-assimilation occurs mostly through the pentose phosphate pathway. Since production of an antibiotic is often limited by the self-resistance mechanism in the producer strain, it was important to investigate in detail the mechanism of action of NAI-107 in model strains, as well as establishing key features in self-resistance, transport and cell wall composition in Microbispora. In whole cell and cell-free experiments, NAI-107 was demonstrated to inhibit cell wall biosynthesis by binding to Lipid II with high affinity and inhibiting all enzymes utilizing this key biosynthetic intermediate. Pore formation, as observed with some Lipid-II binding lantibiotics, did not occur with NAI-107 in vitro. Analysis of the Microbispora cell wall indicates that it contains stem peptides with either glycine or alanine at the first position and it contains a direct linkage between peptide chains. Differential proteome analyses established that the proteins associated with NAI-107 production are, among others, those required for amino sugar metabolism, cell wall biosynthesis, lantibiotic resistance, and nitrogen metabolism. Previous work on a different Microbispora strain had identified key roles for the regulatory genes, which are also present in the NAI-107 gene cluster: mibX encoding a sigma factor, mibW encoding the cognate anti-sigma factor, and mibR encoding a likely DNA-binding protein. Critical parameters for Microbispora growth and NAI-107 production have been delineated and new seed and production media have been designed. These have resulted in production titers up to 10 fold higher than in the starting production process. A simple recovery and purification process has been designed, which allows purification of NAI-107 with few, industrially scalable steps. The minor components present in the NAI-107 complex have been purified, chemically characterized and evaluated for their antimicrobial activity. The fermentation process was successfully transferred to a different laboratory, with satisfactory production levels at both the 15 and the 250-L scale.
Project Context and Objectives:
Due to limited knowledge of the Microbispora producer strain, the objectives of the first half of the project were the development of the tools necessary for analyzing the physiology and genetics of the producing strain, understanding the elements contributing to NAI-107 production, as well as getting insights into the details of its mechanism of action. In the second part of the project, that knowledge has been translated into the generation of improved strains, the design of optimized media and recovery procedures, to achieve the overall objective of the proposal the scale-up and industrialization of the production process, with the production of GLP-grade NAI-107. As the molecular mechanism of antibiotic activity and resistance are often intimately linked, another objective of LAPTOP was to understand the mechanism of action of and resistance to NAI-107 in the producing strain. As self-resistance mechanisms have evolved to cope only with levels of antibiotic production found in nature, therefore any attempt to improve production levels must include appropriate measures to increase the level of self-protection.
The specific objectives of this project were:
- Development of tools and technology to allow familiarization with Microbispora;
- Identification of the NAI-107 target in susceptible bacteria and determine the molecular details of NAI-107’s mode of action;
- Investigation of the resistance (immunity) mechanism in the producer strain;
- Identification and biological characterization of the components of NAI-107-complex;
- Isolate antibiotic resistant mutants of Microbispora producing increased levels of NAI-107;
- Define precursor uptake and product excretion in the producer strain;
- Identify and manipulate components of regulatory networks leading to NAI-107 production;
- Analyze and control nitrogen regulation and its impact on antibiotic production;
- Characterize aspects of primary metabolism required for optimized production;
- Design an improved production medium;
- Design an efficient recovery process.
The Laptop project has benefited substantially from groundwork performed by one of the partners in the consortium on a related strain (designated hereafter as Microbispora corallina) that produces a lantibiotic with structural features similar to those of NAI-107, although a full comparison of the two antibiotic complexes had not performed before the start of the project. This work has established the roles of most of the genes present in the mib cluster in Microbispora corallina, facilitating studies on the corresponding mlb genes in Microbispora sp.
The development of genetic tools for analyzing and manipulating the producer strain and the production process has been achieved, in particular:
• a gene transfer system based on conjugation between E. coli and Microbispora has been developed. The protocol has been successfully applied by different Partners for generating knockout and over-expression mutants.
• a draft genome sequence of Microbispora has been generated. The annotated genome (~ 8000 CDSs) will be soon available in public databases. The sequence information has been used in the course of the project for several studies.
• a 2D-protein map of Microbispora during primary and secondary metabolism has been constructed. Differentially abundant protein spots, identified by mass spectrometry, are available to investigate relationships between biochemical pathways and regulatory networks along with NAI-107 production. In addition, an interactive Microbispora proteome web site will soon be available.
• a chemically defined medium allowing NAI-107 production and suitable for metabolic flux analysis has been designed. C-flux methodology has been optimized and applied to Microbispora.
The interaction of NAI-107 with its target has been characterized.
• Using whole-cell experiments, NAI-107 was identified as a cell wall biosynthesis inhibitor; in vitro assays with purified cell wall biosynthesis enzymes and substrates identified the molecular target of the lantibiotic, the lipid-bound cell wall building block Lipid II. NAI-107 was found to bind to Lipid II with high affinity; pore formation, as observed with some Lipid-II binding lantibiotics, does not occur with NAI-107 in vitro.
• Analysis of the proteomic response profile of Bacillus subtilis, as compared to the profile induced by Lipid II binding antibiotics, showed the highest degree of similarity with gallidermin, another non-pore forming lantibiotic.
The mechanism of self-resistance in the producer strain has been investigated.
• The cell wall precursors of Microbispora have been analyzed, and key proteins involved in NAI-107 immunity in Microbispora have been identified.
• Work towards understanding the producer self-protection mechanisms identified a putative ABC transporter and a putative lipoprotein MlbQ which could meditate self-protection. Functional analysis suggests a specific role of the membrane-localized MlbQ in resistance to NAI-107, while NMR studies revealed an unusual structure. Analysis of the cell wall building block of the producer does not indicate specific alterations in cell wall composition.
• Analysis of the Microbispora cell wall indicates that it contains stem peptides with either glycine or alanine at the first position. The cell wall does not contain an interbridge but a direct linkage between peptide chains. These features were also observed in the knockout mutant of Microbispora that does not produce any NAI-107.
Key components of the N-regulon and P-regulon have been identified.
• Key components of the N-regulon and P-regulon with the response regulators GlnR and PhoP respectively, were identified in the Microbispora genome and their expression analyzed.
• It has been demonstrated that nitrogen excess positively controls NAI-107 production and the biomass accumulation, and increased expression of mlbA was observed in defined medium supplemented with ammonia or nitrate; The results revealed that Pi positively influences biomass accumulation and NAI-107 production but not productivity (amount of NAI-107 per biomass amount). In particular, Pi limitation does not exert a beneficial effect on NAI-107 production as reported in the case of other antibiotics produced by actinomycete strains.
• Comparative proteome analyses revealed proteins associated with NAI-107 production: mostly, those involved in nitrogen metabolism regulation, sulphur metabolism, phosphate metabolism, cell wall biosynthesis, oxidative and general stress response factors, tricarboxylic acid cycle, protein folding and modification, fatty acid and cell membrane metabolism, proteins regulating physiological differentiation, stringent response key factors, NAI-107 biosynthetic gene cluster products, multidrug resistance ABC transporters.
Key enzymes involved in carbon metabolism have been identified.
• The C-flux data for the wild type strain have been generated and compared to existing data from other actinomycetes to gain insights into Microbispora central carbon metabolism and its links to secondary metabolism. The highest metabolic flux of C-assimilation was observed through the pentose phosphate pathway; fluxes of C-assimilation through the tricarboxylic acid cycle and Embden-Meyerhof-Parnas pathway were comparatively low.
Characterization of the NAI-107 complex has been completed.
• The minor congeners (F0, F1, F2, A0, B1 and B2) have been purified, structurally characterized and assayed for their antibiotic activity in comparison with the main congeners A1 and A2.
The isolation of improved strains has been achieved.
• antibiotic resistant mutants producing increased levels of NAI-107 have been isolated. The streptomycin-resistant mutant S13 of Microbispora gave consistent results, and was selected for all subsequent tests of the productive media at bioreactor scale.
The general regulatory mechanisms that influence NAI-107 biosynthesis.
• The key regulatory genes, mlbR, mlbX and mlbW, were identified in the NAI-107 biosynthetic gene cluster. Over-expression of mlbR or mlbX resulted in higher levels of NAI-107 production under laboratory conditions.
• ppGpp synthesis was identified as a key intracellular signaling molecule in triggering NAI-107 production.
An improved production process and recovery procedures have been designed.
• Critical parameters for Microbispora growth and NAI-107 production have been delineated and new optimized seed and production media have been designed. These have resulted in production titers up to 10 fold higher than those available at the start of the project.
• A simple recovery and purification process has been designed, which allows purification of NAI-107 with few, industrially scalable steps.
The scale-up and industrialization of the production process have been achieved
• The fermentation process was successfully transferred to a pilot plant, with satisfactory production levels at the 15-L scale.
• A successful fermentation at the 250-liter scale has been performed.
Thus we succeeded to achieve the ultimate aim of the project, namely the design of an improved production process for NAI-107. The process has been successfully transferred to an industrial facility and production of a Good Manufacturing Practice-grade batch is underway.
Project Results:
S&T results/foregrounds
1. Background information
The increasing frequency of nosocomial infections attributable to multi-drug resistant bacterial pathogens exerts a significant toll in the industrialized world, especially when infections are associated with a decline in immune defenses due to age, invasive surgical procedures or other ailments. For example, in the US more people die every year from hospital infections than from all accidental deaths. The UK National Health Service has estimated that hospital-acquired infections cost approximately 1.5 billion € and cause 5000 deaths per annum in the UK alone. Similar statistics exist for Germany. The availability of new and more effective antibiotics is thus a major challenge for the well being of EU citizens.
Despite this increasing medical need, the number of new antibiotics approved for human use has been steadily declining, with only two new chemical classes approved for systemic use during the last three decades. This shortage of new antibiotics has resulted from a diminished interest of large pharmaceutical companies to invest in discovery and development of antibacterial agents, favoring instead drugs for chronic use. Consequently, the antibacterial field has been left almost entirely to biotech companies, where advancement through costly clinical trials is dependent on limited financial resources. To exacerbate the problem, the FDA has in the past few years denied approval of biotech-derived improved versions of existing antibiotics, confirming the view of the medical community that we need new chemical classes with new mechanisms of action to keep pace with the increasing number of drug-resistant bacterial pathogens. However, new antibiotic classes have proven extremely hard to come by.
A critical issue for a new antibiotic is how fast and effectively it can be developed and brought to market. This is particularly critical for the competitiveness of EU industries, since a new drug requires many years of development while having limited time of patent protection. The general aim of this proposal is to generate, through an integrated and interdisciplinary approach, detailed knowledge and efficient tools for the development of an economically viable production process for the new antibiotic NAI-107, a promising new injectable antibiotic with the potential to treat life-threatening infections caused by multi-resistant Gram-positive pathogens.
NAI-107 belongs to the class of lantibiotics and is produced by fermentation of the actinomycete Microbispora sp. It exhibits a relatively wide spectrum, covering all Gram-positive pathogens including multidrug-resistant isolates; it shows good potency, with MIC values for most strains ≤1 μg/ml; it shows good efficacy at 10 mg/Kg in sophisticated experimental models of infection, such as rat endocarditis caused by methicillin-resistant Staphylococcus aureus; its pharmacokinetics in animals suggest the feasibility of once-a-day administration in humans; and it has shown so far a favorable safety profile. All these properties make NAI-107 a promising new antibiotic, suggesting that it will be a safe, once-a-day injectable antibiotic for severe hospital infections.
Lantibiotics have been found to be unique in terms of biological activities. Besides essential functions in quorum sensing and self-regulation of production, several combine two or more antibiotic activities in one molecule resulting in high potency. The majority of lantibiotics target the bacterial cell wall precursor Lipid II, and two different groups of conserved structural motifs involved in Lipid II binding have been identified. The first group is contained within the widely used food preservative nisin, while peptides with a more globular shape often contain the second binding motif, with mersacidin being a well-characterized example. The formation of stoichiometric complexes with Lipid II leads to sequestration of the precursor and blocks its incorporation into the growing cell wall. Therefore, these lantibiotics are inhibitors of cell wall biosynthesis following a mechanism similar to that of the glycopeptide antibiotics; however, the lantibiotic binding site on Lipid II involves the sugar-pyrophosphate rather than the D-Ala-D-Ala moiety, resulting therefore in lack of cross-resistance. Binding to Lipid II then provokes additional activities for some lantibiotics: e.g. for nisin, Lipid II acts as an anchor molecule for the formation of a defined and stable pore consisting of Lipid II and nisin molecules.
NAI-107 has a nisin-like Lipid II binding-motif and also shares features of the non-pore forming lantibiotic mersacidin. Furthermore, NAI-107 has only one positive charge at the free N-terminus and thus is less positively charged than most other lantibiotics. The positive charges have been implicated in the poor pharmacokinetic properties of nisin and of cationic antimicrobial peptides in general. Also, mersacidin and the related compound actagardine (both are neutrally charged at physiological pH) were found to be as effective as vancomycin in vivo in spite of in vitro inferiority.
Before the start of the project, the process for producing NAI-107 was barely sufficient to generate ~0.5 kg of compound necessary for formal toxicology tests. Hence, the production process needed to be optimized and made economically feasible to supply, in a reliable manner, adequate amounts of compound for clinical trials and, in case of favorable results, to launch a product on the market at a price similar to those of comparable life-saving injectable antibiotics. Thus, one of the challenges in advancing a new antibiotic into clinical development is to devise a production process that can deliver a high-quality compound at reasonable costs. This was particularly relevant for NAI-107 because of the lack of precedents: no lantibiotic is currently produced at an industrial scale as a drug for human use and currently no Microbispora strain is used for industrial production.
Actinomycetes such as Microbispora are bacteria that are capable of efficiently utilizing a wide range of substrates and can deal with many toxic compounds present in feedstocks because their genomes contain a large number of hydrolase and oxygenase genes. One of the main issues in the development of a new drug is an efficient supply of compound. For microbial natural products, this is normally achieved through improvement of the producing strain, the fermentation process and/or the recovery of the active compound by a combination of empirical and rational approaches. The latter rely on a detailed understanding of the physiology of the producing strain, particularly with respect to precursor supply. Genomic data allow comparisons with other actinomycetes, which in turn provide new insights into the physiology of the producer strain. Consequently, metabolic flux analysis provides a powerful tool for the overall objective of improving productivity and characterizing different production strains. Since one of the limiting factors in antibiotic production is usually self-resistance in the producer strain, it is also necessary to understand the mechanism(s) of antibiotic export and resistance, which in turn requires a detailed understanding of the antibiotic’s mode of action in model systems.
The identification of metabolic and bioprocess parameters crucial for NAI-107 biosynthesis is necessary for the development of a robust and economically feasible production process, thereby providing a new biotechnological process for a high added-value compound, which has the added benefit of being a potential life-saving antibiotic. Because of its complex chemical structure, NAI-107 cannot be produced by chemical means, and requires robust fermentation processes with optimized strains, media and recovery procedures.
The development of a robust and economically feasible production process for NAI-107 requires the integration of basic knowledge of the physiology of the strain, which can be best obtained by a combination of classical and post-genomic approaches, with a detailed knowledge of the production process and its scalability to industrial level.
The aim of the project, the development of a robust and economically feasible production process for the lantibiotic NAI-107, has been achieved through the integration of basic knowledge of the physiology of the strain, obtained by a combination of classical and post-genomic approaches, with a detailed knowledge of the production process and its scalability to industrial level. This has required combining the knowledge of specialists from different fields, including microbiology, molecular biology, genetics, fermentation, enzymology, metabolic flux analysis and process scale-up.
NAI-107 is a 24-amino acid long lantibiotic containing 5 lanthionine and methyllanthionine rings, and one halogenated tryptophan residue. It is produced as a complex consisting mainly of two congeners differing in the extent of hydroxylation of a proline residue (mono- or di-hydroxylated). The genes responsible for NAI-107 biosynthesis, organized in a cluster designated mlb, have been isolated and sequenced from two Microbispora sp.. The two mlb clusters are highly related, span approximately 20 kb and contain 18 genes encoding: MlbA, the precursor peptide of NAI-107; MlbB (dehydratase) and MlbC (cyclase), involved in post-translational modification of MlbA to produce the thioether bridges; MlbD, involved in the oxidative decarboxylation of the C-terminal Cys to yield the S-[(Z)-2-aminovinyl]-(3S)-3-methyl-D-cysteine residue; MlbH, the tryptophan halogenase; MlbP, a cytochrome P450 hydroxylase likely responsible for proline mono- and di-hydroxylation; five transporters of the ABC type, and one Na/K ion-antiporter, likely involved in exporting lantibiotic intermediate(s) or the finished products out of the cytoplasm and in conferring resistance to the producer cell; and three likely regulatory proteins. No obvious protease or protease domain could be identified as the product of either mlb cluster.
The biosynthesis of many different antibiotics and secondary metabolites is regulated by phosphate. Recently, insights into the molecular mechanisms through which phosphate controls global antibiotic production have started to emerge: in Streptomyces coelicolor and Streptomyces lividans phosphate control of antibiotic biosynthesis is mediated by the two-component system PhoR-PhoP, with PhoP binding to promoters of phosphate-regulated genes. Moreover, another gene (ppk, encoding polyphosphate kinase) was shown to play a key role in the regulation of antibiotic biosynthesis in S. lividans, stressing the important role played by polyphosphate storage. Key enzymes involved in carbon metabolism have also a strong impact on antibiotic production. Whereas the EMP pathway is the central pathway for glucose utilization in many streptomycetes, the Entner-Doudoroff (ED) pathway is critical for Nonomuraea and its presence has implications on the reducing cofactor biosynthesis and energy metabolism. It is well known from industrial fermentations that N-limitation can trigger the onset of antibiotic production. The molecular mechanisms, however, are still unknown. Recent analysis of nitrogen assimilation and its regulation in actinomycetes revealed the presence of a central transcriptional regulator (GlnR) and a number of signal transmitters such as GlnK and GlnD.
The biosynthesis of secondary metabolites is generally dependent on growth phase but also is influenced by a wide array of environmental and physiological signals and nutritional stress. In liquid-grown cultures it generally coincides with stationary phase and it is frequently postulated to result from nutrient limitation. The expression of secondary metabolic gene cluster is regulated by pathway-specific activators, and at least some regulatory networks, for example the phosphate-responsive cascade, are believed to regulate these pathway-specific activator genes.
All gene clusters coding for lantibiotics that target Lipid II contain a dedicated ABC transporter, also present in the mlb cluster, which has been phenotypically implicated in producer immunity. Mechanistically, it is poorly understood how this transporter works. Strains in which the epidermin and the mersacidin transporters had been knocked out accumulate considerably more radiolabeled lantibiotic on the cell surface, indicating that these transporters act by pumping extracellular antibiotic away from the target membrane and may not be able to pump out internal antibiotic to the outside. Moreover, Lipid II targeting lantibiotics do not appear to be proteolytically activated inside the cell such that the dedicated biosynthesis-associated exporter translocates only the modified but unprocessed prepeptide; activation then occurs in the course of, or after transport, either by a transporter with an N-terminal Cys-protease domain, or by a signal protease located on the outside of the cytoplasmic membrane. Such safety measures apparently need to be taken because Lipid II, which is primarily targeted from the outside, is also present on the inside of the cell membrane. Moreover, the precursor to Lipid II, Lipid I, is also targeted by lantibiotics. Thus, a Lipid I/II targeting lantibiotic that is activated on the inside of the cell would likely be inhibitory to the producing strain.
Recent analysis of the physiological responses of cells confronted with increasing concentrations of lantibiotics and adapted to yield a 50 fold decrease in susceptibility indicate that the most effective defense strategy includes significant changes in the architecture of the cell envelope. This includes changes in the overall surface charge through modification of cell wall teichoic acids as well as modulation of expression profiles of penicillin binding proteins.
The molecular mechanisms of antibiotic activity and resistance are often intimately linked. In addition to physiological adaptations which down-modulate susceptibility, antibiotic producing strains usually develop dedicated self-protection (immunity) systems which, on the molecular level, also tightly reflect the specific activity of the antibiotic itself. However, protection and self-resistance systems usually can only cope with levels of antibiotic occurring under natural growth conditions. Therefore, any attempts to raise production levels must include appropriate measures to increase self-protection capabilities.
A limiting factor in antibiotic production can be export of the product from the cell. Specific transporters for antibiotics are encoded by a number of biosynthetic gene clusters; most of them belong to the ABC-transporter family. For glycopeptide antibiotics it was shown that inactivation of the transporter gene leads to a dramatic decrease of the antibiotic in the fermentation broth whereas an intracellular accumulation was observed. Over-expression studies of the transporter gene indicated that yield improvement may be obtained by such an approach.
Antibiotics are usually produced as a complex of related molecules differing for one or more substitutions. NAI-107 is produced as a complex consisting mainly of two factors differing in the extent of hydroxylation of a proline residue (mono- or di-hydroxylated), as well as other minor, as yet uncharacterized factors.
2. Main results
2a. Tools for Microbispora
Due to limited knowledge of the Microbispora producer strain, the objectives of the initial period of the project were the development of the tools necessary for analyzing the physiology and genetics of the producing strain and for understanding the elements contributing to NAI-107 production.
Highlights
• a gene transfer system based on conjugation between E. coli and Microbispora has been developed. The protocol has been successfully applied by different Partners for generating knockout and over-expression mutants.
• a draft genome sequence of Microbispora has been generated. The annotated genome (~ 8000 CDSs) will be soon available in public databases. The sequence information has been used in the course of the project for several studies.
• a 2D-protein map of Microbispora during primary and secondary metabolism has been constructed. Differentially abundant protein spots, identified by mass spectrometry, are available to investigate relationships between biochemical pathways and regulatory networks along with NAI-107 production. In addition, an interactive Microbispora proteome web site will soon be available
• a chemically defined medium allowing NAI-107 production and suitable for metabolic flux analysis has been designed. C-flux methodology has been optimized and applied to Microbispora
Main results
A gene transfer system based on conjugation has been developed for Microbispora. While published protocols for conjugation from Escherichia coli to actinomycetes have generally used a methylation-defective E. coli donor strain, the use of such strains with Microbispora has resulted in a very low frequency of exconjugants. After testing alternative donors, Escherichia coli S17-1 was established as a suitable donor strain. Further adjustments were made to the antibiotics used to select against E. coli and to select for Microbispora exconjugants. We have applied the optimized conjugation protocol to develop expression systems with GFP and mibR, a key regulator from the mib cluster. To facilitate the construction of knockout mutants (i.e. clones where a double cross over event had occurred), we developed a strategy based on the gusA reporter system, which allows to distinguish between plasmid-carrying colonies (blue) and colorless colonies that have lost the plasmid (double cross-over).
The Microbispora draft genome sequence was obtained combining different sequence methodologies: the 454 technology, which initially yielded 8 scaffolds for a total of 8,091,813 bases; and the Illumina technology, which led to a single scaffold of 8576389 bp. The Microbispora genome shares extensive synteny with the complete genome of Streptosporangium roseum, a strain belonging to the same Actinobacteria family as Microbispora. The sequence information has been instrumental to generate the 2D protein maps of Microbispora (see below), as well as to identify key genes involved in nutrient assimilation.
2D-differential gel electrophoresis was used to perform comparative analysis of Microbispora protein expression patterns before and during NAI-107 production, collecting biomass samples at several times before and during NAI-107 production from parallel cultures grown in triplicate. Comparative proteome analysis was carried out between samples collected before and during NAI107-production during rapid growth, and between samples collected during NAI-107 production at early, mid and late decline stages. Identification, by mass spectrometry procedures, of 533 protein spots differentially abundant revealed by global (327 spots) and membrane (206 spots) proteome analyses was accomplished using biomass samples collected at rapid growth stages before and during NAI-107 production. Identification of 310 differentially abundant proteins revealed by global proteome analysis was accomplished using biomass samples collected during NAI-107 production at early-, mid- and late-decline stages.
These proteins were used to construct an interactive Microbispora ATCC-PTA-5024 reference 2D-map that will soon be available. In order to have a medium useful for the C, N and P assimilation studies and for flux analyses, a number of different defined and minimal media were investigated, monitoring Microbispora growth and NAI-107 production. Furthermore, batch cultivations in well-controlled reactors were carried out in a few complex and defined media. Maximum specific growth rates in the range of 0.07-0.09 were seen.
2b. Mechanism of action, resistance and transport
Due to the fact that production of an antibiotic is often limited by the self-resistance mechanism in the producer strain, it was important to investigate in detail the mechanism of action of NAI-107, a cell wall inhibitor, in model strains, as well as establishing key features in self-resistance, transport and cell wall composition in Microbispora.
Highlights
• Using whole-cell experiments, NAI-107 was identified as a cell wall biosynthesis inhibitor; in vitro assays with purified cell wall biosynthesis enzymes and substrates identified the molecular target of the lantibiotic, the lipid-bound cell wall building block Lipid II. NAI-107 was found to bind to Lipid II with high affinity; pore formation, as observed with some Lipid-II binding lantibiotics, did not occur with NAI-107 in vitro.
• Analysis of the proteomic response profile of Bacillus subtilis, as compared to the profile induced by Lipid II binding antibiotics, showed the highest degree of similarity with gallidermin, another non-pore forming lantibiotic.
• Work towards understanding the producer self-protection mechanisms identified a putative ABC transporter and a putative lipoprotein MlbQ which could meditate self-protection. Functional analysis suggests a specific role of the membrane-localized MlbQ in resistance to NAI-107, while NMR studies revealed an unusual structure. Analysis of the cell wall building block of the producer does not indicate specific alterations of the cell wall composition.
• Analysis of the Microbispora cell wall indicates that it contains stem peptides with either glycine or alanine at the first position. The cell wall does not contain an interbridge but a direct linkage between peptide chains. These features were also observed in the knockout mutant of Microbispora that does not produce any NAI-107.
Main results
Our results demonstrate that NAI-107 is a potent inhibitor of cell wall biosynthesis with lipid II as a specific target. We studied the mode of action of NAI-107 using whole cells, isolated membranes and purified enzymes. We found that the lantibiotic very efficiently interferes with late stages of cell wall biosynthesis in intact bacterial cells as demonstrated by inhibition of N-acetylglucosamine incorporation into polymeric cell wall and the accumulation of the ultimate soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide in the cytoplasm. Using membrane preparations and the complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we show that NAI-107 forms complexes with undecaprenyl pyrophosphate- N-acetylmuramic acid(pentapeptide)-N-acetylglucosamine (lipid II), which is readily accessible at the outside of the cell and which forms a complex with the antibiotic in a 1:2 molar stoichiometry. Furthermore, we found that pore-formation does not seem to be a major component of the antibiotic activity as seen by whole cell assays measuring the membrane potential and potassium efflux as well as CF-efflux from artificial liposomes.
We compared the proteomic response of B. subtilis after treatment with the lantibiotics nisin, mersacidin, gallidermin and NAI-107 and an untreated control. Proteins found to be induced more than 2-fold in three independent biological replicates are reported as marker proteins. NAI-107 treatment led to upregulation of 20 protein spots, 15 of which could be identified by mass spectrometry: among them, YceC and YceH are known cell envelope stress markers; LiaH, YtrB, YtrE, and YpuA are specific markers for inhibition of membrane-bound steps of cell wall biosynthesis; PspA and NadE are membrane stress markers. NAI-107 elicits a typical dual cell wall biosynthesis inhibition and membrane stress response. The proteome response strongly overlaps with that of gallidermin suggesting high mechanistic similarity between these two lantibiotics. High similarity of the proteome response profile of NAI-107 was also observed with MP196 and gramicidin S, both of which integrate into the cytoplasmic membrane and disturb membrane architecture. Less overlap was observed with nisin indicating that membrane interaction of NAI-107 is not characterized by pore formation but rather “unorganized” integration and interference with the membrane architecture.
Interference with membrane-bound steps of cell wall biosynthesis was confirmed by microscopic inspection of the cell shape (Fig 1). The impact of NAI-107 on the cytoplasmic membrane was investigated by studying the localization of a GFP fusion to the cell division protein MinD, which normally localizes at the cell poles and in the cell division plane. In a depolarized cell, MinD delocalizes appearing as irregularly distributed GFP clusters. After treatment with NAI-107, complete GFP-MinD delocalization was observed suggesting efficient membrane depolarization. NAI-107 did not efficiently facilitate penetration of propidium iodide into B. subtilis suggesting that no large pores are formed. Thus, NAI-107 seems to interfere with membrane-bound steps of cell wall biosynthesis and with the architecture of the cytoplasmic membrane.
Fig. 1. Microscopy of B. subtilis cells treated with NAI-107 or nisin. (A) Examination of the cell shape after acetic acid/methanol fixation. (B) Depolarization assays using GFP-MinD. (C) Light microscopy of cells in (B). (D) Pore formation monitoring using the live/deadBacLight bacterial viability kit.
Lantibiotics are synthesized as precursor peptides which undergo posttranslational modifications and proteolysis to give the final product. Lantibiotic biosynthetic gene clusters encode dedicated ATP-binding cassette (ABC) transporters to export the modified peptide from the producing cells. Bacterial ABC transporters consist of 2 transmembrane domains and 2 nucleotide binding domains which utilize ATP hydrolysis as a source of energy for the transport. The biosynthetic gene cluster of NAI-107 codes for three ABC transporters: MlbY-MlbZ, MlbT-MlbU and MlbE-MlbF (Fig. 2). In order to obtain a strain with optimized excretion of NAI-107, each ABC transporter was overexpressed in Microbispora. However, there were no significative differences between Microbispora wild type and each of the ABC-overexpressing strains, suggesting that excretion is not a limiting step in NAI-107 production. A sensitivity assay using spores of the M. corallina mibEF deletion mutant and the wild type strain shows that the mutant is 40-fold more sensitive to NAI-107 than the wild-type strain. When mlbEF were overexpressed in Streptomyces coelicolor, a two-fold increase in resistance towards NAI-107 was observed.
The putative lipoprotein MlbQ may be involved in immunity. When expressed in S. coelicolor, where it localizes to the membrane, MlbQ confers a slight increase in resistance to NAI-107 compared to the wild type. However, we could not yet demonstrate a binding of the lipoprotein MlbQ to NAI-107. The MlbQ structure, as solved by NMR (collaboration with the Max Planck Institute, Tübingen), does not share homology with the protein SpaI (conferring immunity to subtilin), the only immunity protein whose structure has been so far solved by NMR. Up to now, interaction studies between MlbQ and NAI-107 could not be performed by NMR because of NAI-107 limited solubility in the protein buffer.
Fig. 2 NAI-107 gene cluster. The genes encoding the three ABC transporters are framed.
The Microbispora mutant strain RP0, resulting from a single crossover within the mlb cluster, was constructed. This mutant does not produce NAI-107 as shown by bioassay and HPLC-MS. This mutant has been used in further studies as described in the following sections.
To elucidate the cell wall composition of Microbispora, we analyzed in parallel cell wall precursors and murein from S. coelicolor. The latter strains contains two major cell wall precursors: UDP-MurNAc-L-Ala-D-Glu-LL-Dap-D-Ala-D-Ala; and one with a Gly linked to LL-DAP. In contrast, Microbispora contains only the former precursor. Furthermore, Microbispora does not possess a monoglycine interpeptide bridge as reported for S. coelicolor and we suspect a direct linkage between peptide chains. There was no alteration in the cell wall of the non-producer strain Microbispora RP0 as compared to that of the wild type. However, both strains contain also Gly and Ser in the first position.
Undecaprenylphosphate represents the central-membrane-bound lipid carrier involved in the synthesis of diverse bacterial cell envelope polymers and polysaccharides, including not only peptidoglycan but also whole teichoic acid (WTA) and capsule biosynthesis. Besides with lipid II, nisin and related lantibiotics interact also with lipid I in vitro; additionally, complex formation with bactoprenolpyrophosphate (C55-PP) has been described for nisin. These findings suggest that nisin and related lantibiotics containing the lipid II binding motif may interact with further cell envelope precursors, attached to C55-P as a carrier molecule, such as those for capsule or WTA biosynthesis. In S. aureus, WTA biosynthesis involves at least 9 enzymatic steps (Fig. 3). We have set up in vitro assays for the first part of the WTA reactions and were already able to prove the interaction of nisin with WTA precursors. We also observed that, in the presence of NAI-107 in a 2-fold molar ratio, almost no conversion of Lipid III to Lipid IV was detectable. This proves that also NAI-107 is able to interact with WTA precursors in vitro.
Fig. 3. WTA-biosynthesis in S. aureus
2c. Carbon, nitrogen and phosphate assimilation
Understanding the major pathways for substrate assimilation and mapping metabolic fluxes are required for understanding the major changes in the proteome associated with NAI-107 production. This information can then be used to design improved production strains and processes.
Highlights
• Key components of the N-regulon and P-regulon with the response regulators GlnR and PhoP respectively, were identified in the Microbispora genome and their expression analyzed.
• Nitrogen excess positively controls NAI-107 production yield and the biomass accumulation; phosphate limitation positively regulates biomass accumulation but has no influence on NAI-107 production.
• Comparative proteome analyses revealed that proteins involved in nitrogen metabolism regulation, sulphur metabolism, phosphate metabolism, cell wall biosynthesis, oxidative and general stress response factors, TCA cycle, protein folding and modification, fatty acid and cell membrane metabolism, proteins regulating physiological differentiation, stringent response key factors, NAI-107 biosynthetic gene cluster products, multidrug resistance ABC transporters are associated with NAI-107 production.
• The highest metabolic flux of C-assimilation was observed through the pentose phosphate (PP) pathway; fluxes of C-assimilation through the tricarboxylic acid (TCA) cycle and Embden-Meyerhof-Parnas (EMP) pathway were comparatively low.
Main results
In multiple species of actinomycetes, inorganic phosphate (Pi) negatively regulates antibiotic production. Phosphate control of antibiotic biosynthesis in Streptomyces is mediated by the two-component PhoR/PhoP system, where PhoR is a membrane sensor kinase and PhoP is a DNA-binding response regulator. In response to phosphate starvation, PhoP activates expression of the pho regulon by binding to consensus phosphate boxes in the corresponding promoter regions (PHO boxes). Key components of the P-regulon were found in Microbispora. In a chemically defined medium containing different Pi concentrations (0, 0.1 0.5 5, 15 and 30 mM), biomass yields of Microbispora increased up to 5 mM Pi. However, NAI-107 productivity did not change significantly from 0.1 to 30 mM Pi. Quantitative RT-PCR of pho regulon genes (phoR, phoP, ppk and pstS) was performed revealing that these genes were upregulated under low Pi, as expected.
Actinomycetes are able to produce a diverse range of secondary metabolites during differentiation, which require precursors from primary metabolism. The production of secondary metabolites and cellular differentiation is finely tuned by various intra- and extracellular signals such as N limitation. Nitrogen assimilation involves the uptake and incorporation of nitrogen sources and results in the synthesis of glutamate and glutamine, which are the key intracellular nitrogen donors. Due to environmental limitation and variation in nitrogen availability, bacteria have evolved complex regulatory networks for the transcriptional control of genes involved in nitrogen metabolism. In S. coelicolor, GlnR, an orphan response regulator, controls nitrogen assimilation by transcriptional regulation of 13 genes, of which six are directly involved in nitrogen metabolism. The glnR gene was also found in Microbispora genome. Other key components of the N-regulon (glnR, glnRII, glnA, glnII, gdh, amtB operon, nnaR, nasA, nirBD) were identified. When Microbispora was grown in minimal medium supplemented with various nitrogen sources, we observed that nitrogen positively controls NAI-107 production.
To analyze the expression pattern of the genes of central N-metabolism, Microbispora was grown in minimal medium supplemented with various N sources and in complex media. Overall, the expression profiles of 18 genes relevant for N-metabolism in either S. coelicolor or M. smegmatis were followed, as well as 4 mlb genes. Expression of glnR (global nitrogen regulator) was induced only under N-limiting conditions, while no expression was detected In N-rich media. Microbispora GlnR is likely to be regulated post-translationally in response to nitrogen changes in the medium. The expression pattern of GlnR target genes in Microbispora seems to be similar to that of S. coelicolor. Concerning the mlb genes, the expression of MlbX and MlbW was elevated in all complex and defined media in an N-independent manner. The expression of the structural gene mlbA increased in complex media during exponential growth, and clearly showed N-dependence in minimal media.
Since the generation of glnR deletion mutants resulted problematic, we concentrated our efforts on over-expressing glnR, nnaR and glnK. The biomass formation in all three overexpression mutants was lower compare to the wild type and RP0. All three overexpression mutants showed almost no differences in the biomass formation between each other. A slight increase in NAI-107 productivity over wild type was observed in the glnR¬- and nnaR-overexpression strains. Thus, high ammonium concentrations positively regulate NAI-107 production independently of the phosphate concentrations in the medium. Under ammonium excess glutamate dehydrogenase is a key enzyme for ammonia assimilation, which is repressed by GlnR only under nitrogen limitation. Regulation of the nitrogen assimilation in Microbispora and its influence on NAI-107 production does not only depend on nitrogen concentration but also on the nitrogen source.
To investigate the central carbon metabolism, Microbispora was grown in minimal medium supplemented with 13C-labeled glucose. Amino acid analysis after biomass hydrolysis, summed fractional labelings were calculated and used for calculating metabolic fluxes. Strikingly, Microbispora seems to have a high flux through the pentose phosphate (PP) pathway. The observed value is the highest of all analyzed Actinobacteria, including Corynebacterium glutamicum, which is known to have a high PP flux. Furthermore, no activity through the Entner-Doudoroff (ED) pathway was observed under the conditions used and fluxes through the tricarboxylic acid (TCA) cycle and Embden-Meyerhof-Parnas (EMP) pathway were comparatively low.
Microbispora proteome analysis was carried during growth in complex medium. Microbispora growth kinetics revealed initial rapid growth, sustained by glucose utilization, followed by a second stage of decline and cell death. NAI-107 production started at mid rapid growth stage and reached its maximum at early decline stage. To understand the molecular processes and metabolic pathways associated with lantibiotic production, differential analyses were performed between samples collected before and during NAI-107 production in the rapid growth phase, and between samples collected at early-, mid- and late-decline stages. In the first case, comparative global proteome analysis revealed 533 protein spots differentially abundant revealed by global (327 spots) and membrane (206 spots) proteome analyses. Positively associated with NAI-107 production were proteins required for amino sugar metabolism, cell wall biosynthesis, lantibiotic resistance, nitrogen metabolism, oxidative stress response, protein folding and modification, and pleiotropic regulators. In the second case, a total of 310 differentially abundant proteins were identified, mostly involved in lantibiotic resistance, nitrogen metabolism, cell wall biosynthesis and pleiotropic regulators are accumulated at the early decline stage. Combining the results of both analyses, a list of key proteins associated with NAI-107 production was obtained. Some of the corresponding genes have been targeted for over-expression and knockout experiments in Microbispora.
2d. Regulation
Production of an antibiotic is invariably controlled by cluster-specific regulators and/or by the general regulatory networks of the producer strain. The ultimate aim of these studies is to understand the pathway-specific and ultimately the general regulatory mechanisms that influence NAI-107 biosynthesis, and to use the resulting knowledge to increase the level of NAI-107 production. Previous work on a different Microbispora strain had identified key roles for three regulatory genes: mibX encoding an ECF-sigma factor, mibW encoding the cognate anti-sigma factor and mibR encoding a likely DNA-binding protein. MibX and MibR were shown to be transcriptional activators of the biosynthetic gene cluster, and MibW to interact with MibX, likely inhibiting the latter’s activity. Homologues of each of these genes are found in the NAI-107 biosynthetic gene cluster (Fig. 2).
Highlights
• Key regulatory genes identified in the NAI-107 biosynthetic gene cluster – mlbR, mlbX and mlbW.
• Over-expression of mibR resulted in over-production of NAI-107.
• ppGpp synthesis identified as a key intracellular signaling molecule in triggering NAI-107 production.
Main results
To understand the regulation of the mlb cluster in Microbispora, the mlbA, mlbE, mlbJ, mlbQ, mlbR and mlbX promoter regions were cloned upstream of a reporter gene and introduced into S. coelicolor strains containing either mlbR or mlbX under the control of a strong promoter. The results show that mlbA is regulated directly by the transcriptional activator MlbR whilst the other genes are regulated directly by the sigma factor MlbX.
An experiment in which the wild type Microbispora is grown next to different concentrations of purified NAI-107 has shown that NAI-107 itself can induce precocious production in the wild type strain (Fig. 4). Several antibiotics, including other cell-wall inhibitors, have then been tested and so far no other compound has been able to induce NAI-107 production in the wild type. This suggests that NAI-107 is specifically able to act as an autoinducer of its own production, likely interacting directly with MlbW.
Fig. 4. NAI-107 triggers precocious production of NAI-107 in the wild-type Microbispora sp. strain. The strain was streaked vertically (two streaks) and either NAI-107 (left) or water (right) were spotted onto sterile filter paper discs that were placed adjacent to the two streaks. The shape of the zone of inhibition indicates auto-induction of NAI-107 production.
The model for the regulation of NAI-107 production suggested that a key event in triggering biosynthesis of the lantibiotic occurred through activation of transcription of mlbR from a second thus far unidentified MlbX-independent promoter. A second promoter, mlbRp2, identified by RT-PCR in Microbispora, was used to direct transcription of a reporter gene in S. coelicolor wild type and its congenic ppGpp synthetase null mutant. Measurement of GusA activity indicated that transcription from mlbRp2 was dependent on synthesis of ppGpp, as a key intracellular signaling molecule involved in the activation of transcription of pathway-specific regulatory genes in other actinomycetes. This result is important since it links nutrient limitation to activation of transcription of mlbR, which results in the production of small amounts of a NAI-107 congener, which then functions in a feed-forward mechanism to release MlbX from MlbW, resulting in high levels of transcription of the entire gene cluster, and production of the main NAI-107 congeners.
2e. Metabolic engineering
The objectives of these studies were directly aimed at designing an improved and scalable production process. They included the engineering of improved production strains, the design of an effective production process and the implementation of a robust recovery procedure.
Highlights
• Among the antibiotic resistant mutants of Microbispora, two streptomycin-resistant mutants (S13 and S22) perform better than the wild type for NAI-107 production.
• The minor congeners (F0, F1, F2, A0, B1 and B2) have been purified, structurally characterized and assayed for their antibiotic activity in comparison with the main congeners A1 and A2.
• An improved production process has been designed. Critical parameters for Microbispora growth and NAI-107 production have been delineated and new seed and production media have been designed.
• Over-expression of mlbR or mlbX resulted in higher levels of NAI-107 production under laboratory conditions.
• A simple recovery and purification process has been designed, which allows purification of NAI-107 with few, industrially scalable steps.
Main results
In actinomycetes, mutants resistant to streptomycin (Str), rifamycin (Rif) and/or gentamicin (Gen), have been reported to be deregulated in antibiotic production, allowing an easy selection of high-producing strains. In Microbispora mutants, while RifR mutations result in lower productivity, there was a 20-30% increase in productivity in selected StrR mutants affecting either ribosomal protein S12 or RsmG.
NAI-107 is produced as a complex of related molecules, with the most abundant congeners A1 and A2 differing for the presence at position 14 of a hydroxy-proline (A2) or a di-hydroxy-proline (A1). Additional congeners were purified and structurally characterized: A0 lacks altogether hydroxyls at Pro14; F1 and F2 correspond to A1 and A2, respectively, but are devoid of the chlorine atom on Trp4; and B1 and B2 correspond to A1 and A2, respectively, but carry a sulfoxide on the first thioether. When tested for antibacterial activity, the B and F forms were slightly less active than the A forms, while substitution at Pro14 had only a minor impact.
[start of CONFIDENTIAl section]
When mlbR and mlbX were placed downstream of strong or inducible promoters and separately introduced into Microbispora, the resulting strains not only produced higher levels of NAI-107, but also did so earlier than the wild type. When these strains were grown under the scalable, there was no statistically significant difference between the production levels observed with the engineered strains (600-800 mg/L) and those achieved with the wild type. An ad hoc process may need to be developed to enhance the performance of the engineered strains.
At the start of the project, the fermentation procedure for NAI-107 production was poorly defined, resulting in a highly variable process. Through a series of empirical observations, we noticed that a robust process depended on following three parameters at the time of crossover in vegetative medium: strain morphology as dispersed mycelium; 2-4 g/L residual glucose; and ≤10 mg/L NAI-107.
Varying carbon and nitrogen sources, we were able to design an improved production medium. In general, higher glucose concentrations adversely affected NAI-107 production, while complex nitrogen sources were correlated with higher NAI-107 yields (~0.25 g/L). Further improvements were obtained through the addition of micronutrients (zinc sulfate), olive oil and decreasing calcium carbonate, boosting NAI-107 production to ~1 g/L at flask level. Addition of glucose at the onset of NAI-107 production strongly reduced further antibiotic formation, suggesting glucose repression of NAI-107 production.
Under the optimized medium, the performance of different StrR mutants was also evaluated, as reported in Fig. 5. While there is no significant difference in the highest titers observed, mutants S22 and S24 reach ~1 g/L earlier (after about 14 days) compared to the 21 days necessary for S13.
Fig. 5. Time-course of NAI-107 production (sum of congeners A, B and F) in the optimized medium. Error bars represent standard deviation.
Before the start of this project, the first large-campaign of NAI-107 production (~0.4 kg) had provided the undesired presence of soyasaponins, carried over from the spent growth medium. We thus devised a scalable and effective downstream process that consists of: 1) separation of mycelium from the spent broth through centrifugation after acidification of the culture; 2) extraction of the mycelium with aqueous methanol and adsorption on an HP20 resin; 3) recovery of NAI-107 by methanol elution and concentration by evaporation; 4) cation exchange chromatography; and precipitation of NAI-107. This procedure afforded NAI-107 in satisfactory yield (32%, compared with the 15% of the original process) and acceptable purity, devoid of soyasaponins. Furthermore, it requires fewer chromatographic steps and less solvent volumes than the original process.
[end of CONFIDENTIAl section]
2f. Process scaleup
The objectives of these studies were to scale-up the production process from the laboratory to pilot plant, producing consistent amounts of NAI-107 in good purity and yield.
Highlights
• The fermentation process was successfully transferred to a different laboratory, with satisfactory production levels at both the 15 and the 250-L scale.
Potential Impact:
Socio-economic impact and societal implications
The objective of this proposal was to develop, through an integrated and interdisciplinary approach, an economically viable production process for the new antibiotic NAI-107. There is a need for novel and better antibiotics to fight infectious disease, especially for life-threatening infections. However, despite this increasing medical need, the number of new antibiotics approved for human use has been steadily declining. Therefore, promising new antibiotics, such as NAI-107, need not only to be discovered but also to be effectively developed and produced. One of the main issues in the development of a new drug is an efficient supply of compound. The outcome of this project was the development and application of industrial biotechnology for the production of high-value products such as the antibiotic NAI-107. Before the start of this project the process for producing NAI-107 was sufficient to generate the 0.5 kg batches of compound necessary for formal toxicology tests. However, the production process was complex, not sufficiently robust and required several chromatographic steps during compound purification. Therefore, the entire process required to be optimized and made economically feasible if it was to supply, in a reliable manner, adequate amounts of compound for clinical trials and, in the event of favorable results, to launch a product on the market at a price similar to those of comparable life-saving injectable antibiotics. Thus, one of the challenges in advancing a new antibiotic into clinical development is to devise a production process that can deliver a high-quality compound at reasonable costs. This is particularly relevant for NAI-107 because of the lack of precedents: no lantibiotic is currently produced at an industrial scale for use as a drug for human use and there are no examples of the use of Microbispora strains for industrial production. The solution of this problem has required diverse and specialized skills that could not reside solely within the limited resources of an SME, nor can they could be found within the boundaries of a single country. The present proposal has brought together experts in the physiology and genetics of actinomycetes, who have successfully applied basic knowledge from model organisms to less tractable actinomycete genera during FP5 and FP6 projects; experts in lantibiotic genetics and mode of action; and experts in the scale-up of fermentation processes. These skills were critical for achieving the ultimate goal of our project, namely the design of an improved and scalable production process for NAI-107. The projected outcomes of this project will therefore make an important contribution to the biotechnology industry in Europe with the added payback of providing a new drug that can improve the health and the quality of life of EU citizens.
In late 2009, while this project was still under negotiation with the Commission, the Coordinator of this project (NAICONS) and the owner of the IP rights on NAI-107, was able to transfer rights to NAI-107 and related lantibiotic technology to Sentinella Pharmaceuticals, a US-based company backed by Care Capital, a venture capital firm active in health care. This agreement supplied sufficient resources for initiating the preclinical studies necessary for obtaining an authorization for initiating studies in human volunteers. Furthermore, also because of the results achieved in the course of the LAPTOP project, NAICONS has contributed to the successful transfer of the production process to a facility in North America, where production of GMP-grade NAI-107 is under way. The importance of compounds such as NAI-107 has recently attracted the attention of specialized journals in biotechnology (see for example Nature Biotechnology 31, 379–382, 8 May 2013).
In conclusion, not only has the project reached its ultimate goal, the development of an improved production process for NAI-107, but the compound itself has attracted interest from third parties, demonstrating that SMEs are a vital part of the EU economy and can “export” technology and high-added value products.
In addition to industrial competitiveness, in the field of antibiotic research Europe has attained a leading role. Mainly due to the activities of several members of this consortium, Europe has caught up and overtaken the US. One aim of this project was to further improve this position in academic research, by strengthening the collaboration and technology transfer between academia and industry, thus helping foster the EU biotechnology industry. By combining industrial microbiology integrated with modern molecular techniques, high quality academic research has becomes tightly interconnected with industrial research and production processes. This connection is particularly relevant in the present work, since only one of the academic partner in the present consortium had previous experience with lantibiotic-producing strains. Nonetheless, the overall objective of the work could be achieved.
The successful completion of this project demonstrates the viability of the type of consortium at work during this project, and during previous projects funded under FP5 and FP6 on a different class of antibacterial compounds, i.e. glycopeptides. This bode well for the future of antibiotic research within the EU. The knowledge generated as a result of the proposed work and the techniques developed has also enabled the application of similar methodologies to other potentially valuable products, thereby further promoting a sustainable biotechnology industry. The progress in this area requires a multidisciplinary consortium of specialists in molecular genetics, microbiology, natural product chemistry, physiology, enzymology and fermentation technology. Since each of these specialists needs to have a broad knowledge of antibiotics, such a team cannot be put together from researchers of one country, but needs a European dimension. The LAPTOP project, which has involved five groups from academic institutions and two from industry, is an example in full agreement with statements from the European Commission to build a research and innovation structure equivalent to the “common market for goods and services” (Participating in European Research, October 2002).
Another important community objective addressed at the time of writing the proposal and followed during the course of this project has dealt with the future employment of EU scientists. During the last decade, many pharmaceutical companies have witnessed major mergers, acquisitions and reorganizations that have changed the face of the pharmaceutical industry. In order to maintain profitability, these changes have often resulted in the consolidation and centralization of research and development centers, with the concomitant loss of thousands of jobs. Apart from the direct impact on unemployment, this world-wide phenomenon has resulted in fewer opportunities for employment by large pharmaceutical firms for graduate and post-graduate scientists coming out of academic institutions. However, the existence of very large pharmaceutical firms has also created a wealth of opportunities for smaller "biotech" companies focused on providing specialized technologies and services to larger enterprises, and/or developing their own products. Indeed, the number of NDAs (new drug application for market authorization filed at regulatory agencies) by biotech industries has, in recent years, surpassed the number of applications filed by large pharmaceutical companies. However, few biotech companies have their own marketed products, so their revenues depend on contracts, financial investors and public grants. In the long term, biotech companies can become financially stable only if they have direct revenues from the sale of products.
In the course of the project, attention has been paid to contribute to the creation of an entrepreneurial attitude among academic researchers, that will hopefully lead to better employment levels for young scientists. This has also been achieved through a series of exchanges of young scientists at the start and during the execution of the project.
Exploitation and dissemination plan
The exploitation and dissemination plan has been highlighted throughout the project. In order to ensure that adequate IP protection of any foreground, an IP Committee was established in the course of the project. The IP Committee handled all matters relating to public disclosure of any information and proved to be very efficient in providing timely answers to requests for publication, which is extremely important for young scientists. All requests handled by the IP Committee were unanimously approved.
Because IP had already been secured before the start of the project (NAICONS IP on NAI-107, subsequently transferred to Sentinella Pharmaceuticals as mentioned above), exploitation of the main foreground of the project had already been achieved at project start. Nonetheless, as mentioned above, the results obtained during the course of the project were instrumental for transferring the production process to a facility based in North America, where production of GMP-grade NAI-107 is under way. Therefore, this is giving the long-term continuity necessary for the clinical development of a new drug, particularly a life-saving new antibacterial agent active against all multi-drug resistant pathogens.
The other industrial partner in the LAPTOP project, Gnosis, was interested in expanding its portfolio of fermentation products. Its participation in the current project has contributed in increasing its expertise in actinomycete fermentations, and now Gnosis is involved in the industrial production of natural and semi-synthetic glycopeptides (http://www.gnosis-bio.com/biopharma.php).
While data mining its strain collection for lantibiotics related to NAI-107, NAICONS has discovered a new lanthipeptide with marked antinociceptive activity. A patent application on the compound has been filed in July 2012.
Dissemination of the results, after having protected any patentable inventions, have been achieved through:
• Posting relevant findings on the LapTop web site
• Presentations at scientific conferences
• Publications in international journals
• Presentations at a mini-symposium
• Dissemination of leaflets and brochure(s)
During the project we were able to present our results in more than 30 conferences (national and international meetings), and several manuscripts have been submitted to high-ranking international journals or are in the final stages of revision for future submissions. At the time of writing this report, the lack of published results should not come as a surprise, since most of the Partners involved in the project had no direct experience with lantibiotics or lantibiotic-producing strains, and had therefore to develop the necessary knowledge and expertise before publishable results could be obtained.
The LAPTOP project was shortly introduced during a "Pupils Congress" on November 8, 2011, for pupils of Baden-Württemberg, Germany, interested in biotechnology/antibiotics. In total 80 pupils attended this meeting with 10 teachers. In addition, the German Research Association (DFG) has organized the touring exhibition “Mensch-Mikrobe” (humans-microbes), held in Tübingen for three months (in winter/spring 2012). Prof. Wolfgang Wohlleben (EKUT) gave an oral presentation on February 8, 2012, to about 200 citizens from Tübingen, Germany on antibiotics. In the course of the presentation, the objectives and importance of the LAPTOP project were highlighted.
To ensure proper dissemination of the results a final minisymposium was organized to present and to discuss the results of the LAPTOP project with the leading scientists in the field. The International Workshop on “Novel Developments in Lantibiotic Research” took place in Verona from June 16 to June 17, 2013. The aim of the minisymposium was to achieve public awareness of the LAPTOP project and to have leading scientists in the field not involved in the project critically evaluate our results. We invited scientists who contributed most to the progress in lantibiotic research during the last decade. We were really pleased with the outcome, since all eight contacted scientists accepted our invitation, including one coming specifically from North America. This clearly demonstrated the interest around the LAPTOP project and the broad expectation of a new lantibiotic advancing towards clinical development for treating hospital-acquired infections.
Each of the invited scientists presented their results. At the same time, the results of the LAPTOP projects were presented by the young scientists (PhD students and postdocs) from each partner in the consortium. The LAPTOP results were intensively discussed and positively evaluated by the external participants. The meeting has also led to new collaborations with some of the external scientists on specific topics related to NAI-107.
The overall results of the LAPTOP project were presented by the Coordinator at the Symposium on the Genetics of Industrial Microorganisms held in Cancun, Mexico, on June 23-29, 2013. This symposium, attended by about 250 scientists from all countries, represents the traditional venue for presenting advanced research on industrial microorganisms producing added-value compounds. Further dissemination activities are planned in the upcoming year, as the scientists who have participated in the LAPTOP project will continue presenting their results at national and international conferences.
List of Beneficiaries
Beneficiary Number * Beneficiary name Beneficiary short name Country
1. Margherita Sosio- NAICONS NAICONS Italy
2. Mervyn J Bibb - John Innes Centre JIC UK
3. Anna Eliasson Lantz- Technical University of Denmark DTU Denmark
4. Anna Maria Puglia- University of Palermo DBCS Italy
5. Hans-Georg Sahl - Universitaetsklinikum Bonn UKB Germany
6. Wolfgang Wohlleben Eberhard-Karls-Universität Tübingen EKUT Germany
7. Marco Berna Gnosis Gnosis Italy
