Application of novel metabolic engineering tools for engineering of the complex trait of glycerol yield in Saccharomyces cerevisiae

The yeast, Saccharomyces cerevisiae is still the prime species used in ethanol production. Traditionally, ethanol has been produced from raw materials such as sugar cane or sugar beet and starch from corn or other grains. Future expansion of ethanol production are planed its use as a biofuel requiring the utilization new non- food resources, particularly lignocelluloses. In the present and future ethanol production process, the expenditure for the raw material is a significant cost factor. Although industrial yeast strains are very efficient in producing ethanol, the current ethanol yield (90 - 93% of the theoretical maximum) could in principle be further improved. Particularly, the synthesis of glycerol, a major by-product of alcoholic fermentation, has been regarded as a wasteful process. The overall goal of this project was to develop novel industrial yeast strains, which are able to produce more ethanol from the traditional and future raw materials by reducing the yield of the undesirable by-product glycerol. The challenge herein is that glycerol serves different important physiological functions such as redox balancing and osmotic stress tolerance. In this project, two alternative avenues were used to identify a genetic configuration for a low glycerol producer strain. The first approach was a rational engineering strategy aiming to finely tune the activity of the rate-limiting enzyme of glycerol synthesis, the glycerol 3-phosphate dehydrogenase. The basic idea here was to gradually reduce the expression of the corresponding genes, GPD1 and GPD2, by replacing their native promoters through mutated low strength TEF1 promoter versions. After the promoter exchange, the mutants were characterized for their residual glycerol formation, fermentation and growth. In the second approach, the genetic configuration of wild-type yeast with low glycerol yields were analyzed. Its genetic determinants, which cause the low glycerol phenotype were identified, using an advanced genetic mapping methodology base on next-generation pooled segregants whole-genome sequence analysis. The four genes, GPD1 SSK1 SMP1 and HOT1 were identified, which harbored mutations linked to the 'low glycerol' phenotype. The SSK1 allele of the superior low producing glycerol strain, ssk1E330N...K356N was subsequently used as novel gene tool for targeted genetic improvement of the ethanol yield of a frequently used ethanol production strain with minimal risk of affecting its other industrially important traits.status: publishe