Top-Down, Knowledge-Based Genetic Reduction of Yeast Central Carbon Metabolism.

Saccharomyces cerevisiae, whose evolutionary past includes a whole-genome duplication event, is characterized by a mosaic genome configuration with substantial apparent genetic redundancy. This apparent redundancy raises questions about the evolutionary driving force for genomic fixation of "minor" paralogs and complicates modular and combinatorial metabolic engineering strategies. While isoenzymes might be important in specific environments, they could be dispensable in controlled laboratory or industrial contexts. The present study explores the extent to which the genetic complexity of the central carbon metabolism (CCM) in S. cerevisiae, here defined as the combination of glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and a limited number of related pathways and reactions, can be reduced by elimination of (iso)enzymes without major negative impacts on strain physiology. Cas9-mediated, groupwise deletion of 35 of the 111 genes yielded a "minimal CCM" strain which, despite the elimination of 32% of CCM-related proteins, showed only a minimal change in phenotype on glucose-containing synthetic medium in controlled bioreactor cultures relative to a congenic reference strain. Analysis under a wide range of other growth and stress conditions revealed remarkably few phenotypic changes from the reduction of genetic complexity. Still, a well-documented context-dependent role of GPD1 in osmotolerance was confirmed. The minimal CCM strain provides a model system for further research into genetic redundancy of yeast genes and a platform for strategies aimed at large-scale, combinatorial remodeling of yeast CCM. IMPORTANCE Fundamental questions regarding the minimal requirements for life have prompted scientists to embark on top-down efforts to reduce microbial genomes to the minimum set of genes and proteins necessary to sustain cell survival and division. While these efforts are generally focused on small, prokaryotic genomes, Saccharomyces cerevisiae, a popular industrial and model organism, has a typical eukaryotic genome characterized by a high genetic redundancy. The cellular function of redundant genes is generally poorly understood and is often investigated at the scale of a few genes. In this study, we explore genetic redundancy at large scale, encompassing the ~100 genes involved in central carbon metabolism, a part of metabolism essential for life and highly conserved among eukaryotes. This study reveals the remarkable resilience of this model eukaryote, as it was hardly affected, under a broad range of conditions, by a 32% reduction of its central carbon metabolism.

Stress-induced expression is enriched for evolutionarily young genes in diverse budding yeasts.

The Saccharomycotina subphylum (budding yeasts) spans 400 million years of evolution and includes species that thrive in diverse environments. To study niche-adaptation, we identify changes in gene expression in three divergent yeasts grown in the presence of various stressors. Duplicated and non-conserved genes are significantly more likely to respond to stress than genes that are conserved as single-copy orthologs. Next, we develop a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis reveals that genes that emerged in recent evolutionary time are enriched amongst stress-responsive genes for each species. This gene expression pattern suggests that budding yeasts share a stress adaptation mechanism, whereby selective pressure leads to functionalization of young genes to improve growth in adverse conditions. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology.

Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae.

The organic compound 2-phenylethanol (2PE) has a pleasant floral scent and is intensively used in the cosmetic and food industries. Microbial production of 2PE by phenylalanine bioconversion or de novo biosynthesis from sugar offer sustainable, reliable and natural production processes compared to chemical synthesis. Despite the ability of Saccharomyces cerevisiae to naturally synthesize 2PE, de novo synthesis in high concentration and yield remains a metabolic engineering challenge. Here, we demonstrate that improving phosphoenolpyruvate supply by expressing pyruvate kinase variants and eliminating the formation of p-hydroxy-phenylethanol without creating tyrosine auxotrophy significantly contributed to improve 2PE production in S. cerevisiae. In combination with the engineering of the aromatic amino acid biosynthesis and Ehrlich pathway, these mutations enabled better connection between glycolysis and pentose phosphate pathway optimizing carbon flux towards 2PE. However, attempts to further connect these two parts of central carbon metabolism by redirecting fructose-6P towards erythrose-4P by expressing a phosphoketolase-phosphotransacetylase pathway did not result in improved performance. The best performing strains were capable of producing 13mM of 2PE at a yield of 0.113 mol mol-1, which represents the highest yield for de novo produced 2PE in S. cerevisiae and other yeast species.

Engineering de novo anthocyanin production in Saccharomyces cerevisiae.

BACKGROUND: Anthocyanins are polyphenolic pigments which provide pink to blue colours in fruits and flowers. There is an increasing demand for anthocyanins, as food colorants and as health-promoting substances. Plant production of anthocyanins is often seasonal and cannot always meet demand due to low productivity and the complexity of the plant extracts. Therefore, a system of on-demand supply is useful. While a number of other (simpler) plant polyphenols have been successfully produced in the yeast Saccharomyces cerevisiae, production of anthocyanins has not yet been reported. RESULTS: Saccharomyces cerevisiae was engineered to produce pelargonidin 3-O-glucoside starting from glucose. Specific anthocyanin biosynthetic genes from Arabidopsis thaliana and Gerbera hybrida were introduced in a S. cerevisiae strain producing naringenin, the flavonoid precursor of anthocyanins. Upon culturing, pelargonidin and its 3-O-glucoside were detected inside the yeast cells, albeit at low concentrations. A number of related intermediates and side-products were much more abundant and were secreted into the culture medium. To optimize titers of pelargonidin 3-O-glucoside further, biosynthetic genes were stably integrated into the yeast genome, and formation of a major side-product, phloretic acid, was prevented by engineering the yeast chassis. Further engineering, by removing two glucosidases which are known to degrade pelargonidin 3-O-glucoside, did not result in higher yields of glycosylated pelargonidin. In aerated, pH controlled batch reactors, intracellular pelargonidin accumulation reached 0.01 µmol/gCDW, while kaempferol and dihydrokaempferol were effectively exported to reach extracellular concentration of 20 µM [5 mg/L] and 150 µM [44 mg/L], respectively. CONCLUSION: The results reported in this study demonstrate the proof-of-concept that S. cerevisiae is capable of de novo production of the anthocyanin pelargonidin 3-O-glucoside. Furthermore, while current conversion efficiencies are low, a number of clear bottlenecks have already been identified which, when overcome, have huge potential to enhance anthocyanin production efficiency. These results bode very well for the development of fermentation-based production systems for specific and individual anthocyanin molecules. Such systems have both great scientific value for identifying and characterising anthocyanin decorating enzymes as well as significant commercial potential for the production of, on-demand, pure bioactive compounds to be used in the food, health and even pharma industries.

CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus.

BACKGROUND: The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae. RESULTS: This study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design. CONCLUSIONS: These results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus.