Large scale microfluidic CRISPR screening for increased amylase secretion in yeast.

Key to our ability to increase recombinant protein production through secretion is a better understanding of the pathways that interact to translate, process and export mature proteins to the surrounding environment, including the supporting cellular machinery that supplies necessary energy and building blocks. By combining droplet microfluidic screening with large-scale CRISPR libraries that perturb the expression of the majority of coding and non-coding genes in S. cerevisiae, we identified 345 genes for which an increase or decrease in gene expression resulted in increased secretion of α-amylase. Our results show that modulating the expression of genes involved in the trafficking of vesicles, endosome to Golgi transport, the phagophore assembly site, the cell cycle and energy supply improve α-amylase secretion. Besides protein-coding genes, we also find multiple long non-coding RNAs enriched in the vicinity of genes associated with endosomal, Golgi and vacuolar processes. We validated our results by overexpressing or deleting selected genes, which resulted in significant improvements in α-amylase secretion. The advantages, in terms of precision and speed, inherent to CRISPR based perturbations, enables iterative testing of new strains for increased protein secretion.

Recombination in a sterile polyploid hybrid yeast upon meiotic Return-To-Growth.

The sustainable future of food industry and consumer demands meet the need to generate out-performing new yeast variants. This is addressed by using the natural yeast diversity and breeding via sexual reproduction but the recovery of recombined spores in many industrial strains is limited. To circumvent this drawback, we examined whether or not the process of meiotic Return to Growth (RTG) that allows S. cerevisiae diploid cells to initiate meiotic recombination genome-wide and then re-enter into mitosis, will be effective to generate recombinants in a sterile and polyploid baking yeast strain (CNCM). We proceeded in four steps. First, whole genome sequencing of the CNCM strain revealed that it was an unbalanced polymorphic triploid. Second, we annotated a panel of genes likely involved in the success of the RTG process. Third, we examined the strain progression into sporulation and fourth, we developed an elutriation and reiterative RTG protocol that allowed to generate extensive libraries of recombinant RTGs, enriched up to 70 %. Altogether, the genome analysis of 122 RTG cells demonstrated that they were bona fide RTG recombinants since the vast majority retained the parental ploidy and exhibited allelic variations involving 1-60 recombined regions per cell with a length of ∼0.4-400 kb. Thus, beyond diploid laboratory strains, we demonstrated the proficiency of this natural non-GM and marker-free process to recombine a sterile and polyploid hybrid yeast, thus providing an unprecedented resource to screen improved traits.

Metabolic engineering of malolactic wine yeast.

Malolactic fermentation is essential for the deacidification of high acid grape must. We have constructed a genetically stable industrial strain of Saccharomyces cerevisiae by integrating a linear cassette containing the Schizosaccharomyces pombe malate permease gene (mae1) and the Oenococcus oeni malolactic gene (mleA) under control of the S. cerevisiae PGK1 promoter and terminator sequences into the URA3 locus of an industrial wine yeast. The malolactic yeast strain, ML01, fully decarboxylated 5.5 g/l of malate in Chardonnay grape must during the alcoholic fermentation. Analysis of the phenotype, genotype, transcriptome, and proteome revealed that the ML01 yeast is substantially equivalent to the parental industrial wine yeast. The ML01 yeast enjoys 'Generally Regarded As Safe' status from the FDA and is the first genetically enhanced yeast that has been commercialized. Its application will prevent the formation of noxious biogenic amines produced by lactic acid bacteria in wine.

Aquaporin-mediated improvement of freeze tolerance of Saccharomyces cerevisiae is restricted to rapid freezing conditions.

Previous observations that aquaporin overexpression increases the freeze tolerance of baker's yeast (Saccharomyces cerevisiae) without negatively affecting the growth or fermentation characteristics held promise for the development of commercial baker's yeast strains used in frozen dough applications. In this study we found that overexpression of the aquaporin-encoding genes AQY1-1 and AQY2-1 improves the freeze tolerance of industrial strain AT25, but only in small doughs under laboratory conditions and not in large doughs under industrial conditions. We found that the difference in the freezing rate is apparently responsible for the difference in the results. We tested six different cooling rates and found that at high cooling rates aquaporin overexpression significantly improved the survival of yeast cells, while at low cooling rates there was no significant effect. Differences in the cultivation conditions and in the thawing rate did not influence the freeze tolerance under the conditions tested. Survival after freezing is determined mainly by two factors, cellular dehydration and intracellular ice crystal formation, which depend in an inverse manner on the cooling velocity. In accordance with this so-called two-factor hypothesis of freezing injury, we suggest that water permeability is limiting, and therefore that aquaporin function is advantageous, only under rapid freezing conditions. If this hypothesis is correct, then aquaporin overexpression is not expected to affect the leavening capacity of yeast cells in large, industrial frozen doughs, which do not freeze rapidly. Our results imply that aquaporin-overexpressing strains have less potential for use in frozen doughs than originally thought.