Periodic Reporting for period 4 - ELOXY (Eliminating Oxygen Requirements in Yeasts)
Reporting period: 2021-03-01 to 2021-08-31
Products of yeasts range from beer to insulin and from fine wines to car fuels. Most of the thousands of yeast species that are currently known can make ethanol from sugars and, thereby, obtain energy for keeping themselves alive and for making more yeast cells. Since ethanol production does not require oxygen, one might intuitively assume that yeasts that make ethanol can also grow in the absence of oxygen. This assumption, however, turns out to be almost entirely wrong. Only very few yeasts, including bakers' yeast, can grow without any oxygen and even bakers' yeast needs some special nutrients to do so. Understanding why yeasts need oxygen is fundamentally interesting, as it will help us understand how microbes grow in natural environments that do not contain oxygen. Additionally, at the outset of ELOXY, a lack of understanding of the oxygen requirements of yeasts precluded use of industrially highly interesting yeast, such as yeasts capable of growing at high temperatures, in large-scale biofuels production. The overall objectives of ELOXY were tto elucidate oxygen requirements of yeasts, subsequently, eliminate them by targeted genetic modification. This challenge was addressed by a combination of physiology (measuring metabolism in growing yeast cultures), genomics (reading the DNA of yeast cells and measuring which yeast genes are active) and metabolic engineering (introduction of targeted changes in the DNA to understand and improve metabolism in industrial yeasts).
During the 5-year ELOXY project, a large effort was made to improve experimental procedures for oxygen-free cultivation of yeasts in laboratory systems. Using the resulting optimized systems, we established that, in contrast to a long-standing 'dogma' in yeast research, S. cerevisiae (bakers' yeast) is able to grow in the absence of oxygen without supplementation of unsaturated fatty acids. An absolute oxygen requirement of S. cerevisiae for synthesis of sterols and for the cofactors NAD and coenzyme A was confirmed and we discovered that synthesis of biotin by yeasts also requires oxygen.
Novel strategies to eliminate oxygen requirements in bakers' yeast were inspired by a fundamental research line on Neocallimastigomycetes ('Neo's'), a group of fungi that can grow in the complete absence of oxygen. During their evolution, Neo's acquired the ability to 'bypass' reactions that in other fungi and yeasts require oxygen. ELOXY focused on the mechanisms by which Neo's are able to synthesize the cofactors NAD and CoA in the absence of oxygen. Studying these processes by expressing candidate genes from these fungi in bakers' yeast was shown to be a powerful approach to understand evolutionary adaptations of Neo's, which are not yet accessible to genetic modification, to growth in oxygen-free environments.
Based on analysis of the genomes of Neo's, we were able to eliminate oxygen requirements of bakers' yeast for synthesis of the cofactors NAD and CoA by genetic modification. Evolutionary adaptations of Neo's to oxygen-free environments also provided the inspiration for a successful metabolic engineering strategy to bypass the oxygen requirements for sterol synthesis in baker's yeast by introducing a gene that enables production of tetrahymanol, a 'sterol surrogate' whose synthesis does not require oxygen. Introduction of a set of bacterial genes eliminated oxygen requirements for biotin synthesis. While these results provide clear proofs of principle for elimination of the main oxygen requirements of bakers' yeast under laboratory conditions, further research is required to translate them to robust industrial strains.
Research on sterol surrogates led to an unexpected discovery. The yeast Schizosaccharomyces japonicus was found to grow very fast in the absence of oxygen, sterols and unsaturated fatty acids. By a combination of genome analysis, chemical analysis of lipids in cells of this yeast and by expression of a single gene in bakers' yeast, it was shown that, during its evolution, Sch. japonicus acquired bacterial gene that enables it to make hopenes as sterol surrogates. The vigorous growth of Sch. japonicus in oxygen- and sterol-free media makes it a highly interesting model for further research on how membrane composition affects growth and robustness. Another unexpected finding concerned the synthesis of pyrimidines. In most yeasts and fungi, synthesis of these building blocks for DNA and RNA requires oxygen and only bakers' yeast and closely related yeasts were known to bypass this requirement. In the ELOXY project we showed that, during their evolution, Neo's, Schizosaccharomyces japonicus and the yeast Dekkera bruxellensis have, independently acquired another way to anaerobically synthesize pyrimidines. These results illustrate the flexibility of fungal evolution in natural environments.
A major effort was invested in analysing the oxygen requirements of two yeast species, Ogataea parapolymorpha and Kluyveromyces marxianus that, in contrast to bakers' yeast, can grow at temperatures of up to 50 degrees C, but cannot grow without oxygen. Early in the project, protocols were developed for CRISPR-Cas9-mediated genetic modification of these two yeasts. From a detailed quantitative analysis in bioreactor cultures, we found that O. parapolymorpha requires much more oxygen than K. marxianus. Analysis of product formation, combined with analysis of gene expression, revealed that the large oxygen requirements of O. parapolymorpha were related to multiple cellular processes, with regeneration of NAD as a major contributor. While genetic modification led to a reduction of the oxygen requirements, we were not yet able to completely eliminate them. Intensive studies on K. marxianus, encompassing bioreactor cultivation, genome and gene expression analysis and sterol-uptake studies strongly suggested that absence of a functional sterol uptake system was a key contribution to its inability to grow anaerobically. Inspired by our research on S. cerevisiae, we engineered K. marxianus for synthesis of the sterol surrogate tetrahymanol. This strategy, combined with laboratory evolution, yielded K. marxianus strains that grew in the absence of oxygen at a temperature of 45 degrees C. We hope to build on this breakthrough result to eventually enable the construction of robust K. marxianus strains that can be applied in high-temperature, cost-effective fermentation processes.
Novel strategies to eliminate oxygen requirements in bakers' yeast were inspired by a fundamental research line on Neocallimastigomycetes ('Neo's'), a group of fungi that can grow in the complete absence of oxygen. During their evolution, Neo's acquired the ability to 'bypass' reactions that in other fungi and yeasts require oxygen. ELOXY focused on the mechanisms by which Neo's are able to synthesize the cofactors NAD and CoA in the absence of oxygen. Studying these processes by expressing candidate genes from these fungi in bakers' yeast was shown to be a powerful approach to understand evolutionary adaptations of Neo's, which are not yet accessible to genetic modification, to growth in oxygen-free environments.
Based on analysis of the genomes of Neo's, we were able to eliminate oxygen requirements of bakers' yeast for synthesis of the cofactors NAD and CoA by genetic modification. Evolutionary adaptations of Neo's to oxygen-free environments also provided the inspiration for a successful metabolic engineering strategy to bypass the oxygen requirements for sterol synthesis in baker's yeast by introducing a gene that enables production of tetrahymanol, a 'sterol surrogate' whose synthesis does not require oxygen. Introduction of a set of bacterial genes eliminated oxygen requirements for biotin synthesis. While these results provide clear proofs of principle for elimination of the main oxygen requirements of bakers' yeast under laboratory conditions, further research is required to translate them to robust industrial strains.
Research on sterol surrogates led to an unexpected discovery. The yeast Schizosaccharomyces japonicus was found to grow very fast in the absence of oxygen, sterols and unsaturated fatty acids. By a combination of genome analysis, chemical analysis of lipids in cells of this yeast and by expression of a single gene in bakers' yeast, it was shown that, during its evolution, Sch. japonicus acquired bacterial gene that enables it to make hopenes as sterol surrogates. The vigorous growth of Sch. japonicus in oxygen- and sterol-free media makes it a highly interesting model for further research on how membrane composition affects growth and robustness. Another unexpected finding concerned the synthesis of pyrimidines. In most yeasts and fungi, synthesis of these building blocks for DNA and RNA requires oxygen and only bakers' yeast and closely related yeasts were known to bypass this requirement. In the ELOXY project we showed that, during their evolution, Neo's, Schizosaccharomyces japonicus and the yeast Dekkera bruxellensis have, independently acquired another way to anaerobically synthesize pyrimidines. These results illustrate the flexibility of fungal evolution in natural environments.
A major effort was invested in analysing the oxygen requirements of two yeast species, Ogataea parapolymorpha and Kluyveromyces marxianus that, in contrast to bakers' yeast, can grow at temperatures of up to 50 degrees C, but cannot grow without oxygen. Early in the project, protocols were developed for CRISPR-Cas9-mediated genetic modification of these two yeasts. From a detailed quantitative analysis in bioreactor cultures, we found that O. parapolymorpha requires much more oxygen than K. marxianus. Analysis of product formation, combined with analysis of gene expression, revealed that the large oxygen requirements of O. parapolymorpha were related to multiple cellular processes, with regeneration of NAD as a major contributor. While genetic modification led to a reduction of the oxygen requirements, we were not yet able to completely eliminate them. Intensive studies on K. marxianus, encompassing bioreactor cultivation, genome and gene expression analysis and sterol-uptake studies strongly suggested that absence of a functional sterol uptake system was a key contribution to its inability to grow anaerobically. Inspired by our research on S. cerevisiae, we engineered K. marxianus for synthesis of the sterol surrogate tetrahymanol. This strategy, combined with laboratory evolution, yielded K. marxianus strains that grew in the absence of oxygen at a temperature of 45 degrees C. We hope to build on this breakthrough result to eventually enable the construction of robust K. marxianus strains that can be applied in high-temperature, cost-effective fermentation processes.
Key elements of progress beyond state of the art as achieved in the ELOXY project include:
- discovery of oxygen requirement for biotin synthesis in yeast
- elimination of 'dogma' on strict requirements of anaerobic yeast cultures for unsaturated fatty acids
- discovery of novel eukaryotic adaptation to anaerobic, sterol-free growth
- use of S. cerevisiae as experimental platform to study evolutionary adaptations in genetically non-tractable, deep-branching fungi
- strategies of elimination of all currently known biosynthetic oxygen requirements of Saccharomyces cerevisiae
- demonstration of anaerobic growth of an engineered thermotolerant yeast strain
- evidence for convergent evolution in three eukaryotic lineages to enable anaerobic pyrimidine synthesis
Although the project term of ELOXY has ended, key research lines are continued.
- discovery of oxygen requirement for biotin synthesis in yeast
- elimination of 'dogma' on strict requirements of anaerobic yeast cultures for unsaturated fatty acids
- discovery of novel eukaryotic adaptation to anaerobic, sterol-free growth
- use of S. cerevisiae as experimental platform to study evolutionary adaptations in genetically non-tractable, deep-branching fungi
- strategies of elimination of all currently known biosynthetic oxygen requirements of Saccharomyces cerevisiae
- demonstration of anaerobic growth of an engineered thermotolerant yeast strain
- evidence for convergent evolution in three eukaryotic lineages to enable anaerobic pyrimidine synthesis
Although the project term of ELOXY has ended, key research lines are continued.