Identification of Sc-type ILV6 as a target to reduce diacetyl formation in lager brewers' yeast.

Diacetyl causes an unwanted buttery off-flavor in lager beer. It is spontaneously generated from α-acetolactate, an intermediate of yeast's valine biosynthesis released during the main beer fermentation. Green lager beer has to undergo a maturation process lasting two to three weeks in order to reduce the diacetyl level below its taste-threshold. Therefore, a reduction of yeast's α-acetolactate/diacetyl formation without negatively affecting other brewing relevant traits has been a long-term demand of brewing industry. Previous attempts to reduce diacetyl production by either traditional approaches or rational genetic engineering had different shortcomings. Here, three lager yeast strains with marked differences in diacetyl production were studied with regard to gene copy numbers as well as mRNA abundances under conditions relevant to industrial brewing. Evaluation of data for the genes directly involved in the valine biosynthetic pathway revealed a low expression level of Sc-ILV6 as a potential molecular determinant for low diacetyl formation. This hypothesis was verified by disrupting the two copies of Sc-ILV6 in a commercially used lager brewers' yeast strain, which resulted in 65% reduction of diacetyl concentration in green beer. The Sc-ILV6 deletions did not have any perceptible impact on beer taste. To our knowledge, this has been the first study exploiting natural diversity of lager brewers' yeast strains for strain optimization.

Engineering of Saccharomyces cerevisiae for the production of dihydroxyacetone (DHA) from sugars: a proof of concept.

Dihydroxyacetone (DHA) has numerous industrial applications. In this work, we pursue the idea to produce DHA from sugars in the yeast Saccharomyces cerevisiae, via glycerol as an intermediate. Firstly, three glycerol dehydrogenase (GDH) genes from different microbial sources were expressed in yeast. Among them, the NAD(+)-dependent GDH of Hansenula polymorpha showed the highest glycerol-oxidizing activity. DHA concentration in shake-flask experiments was roughly 100mg/lDHA from 20g/l glucose, i.e. five times the wild-type level. This level was achieved only when cultures were subjected to osmotic stress, known to enhance glycerol production and accumulation in S. cerevisiae. Secondly, DHA kinase activity was abolished to prevent conversion of DHA to dihydroxyacetone phosphate (DHAP). The dak1Deltadak2Delta double-deletion mutant overexpressing H. polymorpha gdh produced 700mg/l DHA under the same conditions. Although current DHA yield and titer still need to be optimized, our approach provides the proof of concept for producing DHA from sugars in yeast.

Fermentative production of L-glycerol 3-phosphate utilizing a Saccharomyces cerevisiae strain with an engineered glycerol biosynthetic pathway.

Interest in L-glycerol 3-phosphate (L-G3P) production via microbial fermentation is due to the compound's potential to replace the unstable substrate dihydroxyacetone phosphate (DHAP) in one-pot enzymatic carbohydrate syntheses. A Saccharomyces cerevisiae strain with deletions in both genes encoding specific L-G3Pases (GPP1 and GPP2) and multicopy overexpression of L-glycerol 3-phosphate dehydrogenase (GPD1) was studied via small-scale (100 mL) batch fermentations under quasi-anaerobic conditions. Intracellular accumulation of L-G3P reached extremely high levels (roughly 200 mM) but thereafter declined. Extracellular L-G3P was also detected and its concentration continuously increased throughout the fermentation, such that most of the total L-G3P was found outside the cells as fermentation concluded. Moreover, in spite of the complete elimination of specific L-G3Pase activity, the strain showed considerable glycerol formation suggesting unspecific dephosphorylation as a mechanism to relieve cells of intracellular L-G3P accumulation. Up-scaling the process employed fed-batch fermentation with repeated glucose feeding, plus an aerobic growth phase followed by an anaerobic product accumulation phase. This produced a final product titer of about 325 mg total L-G3P per liter of fermentation broth.

Osmoregulation and glycerol metabolism in the yeast Saccharomyces cerevisiae.

Glycerol is the main compatible solute in Saccharomyces cerevisiae. It is accumulated intracellularly when cells are exposed to decreased extracellular water activity. In general, increased intracellular accumulation of a solute may be caused by enhanced production, restricted dissimilation, increased retention by the plasma membrane and increased uptake from the medium. In this review, we evaluate current knowledge concerning mechanisms leading to the accumulation of glycerol in osmotically stressed cells of S. cerevisiae at the molecular and metabolic levels. An overview of glycerol metabolism in S. cerevisiae is provided.

Reduced pyruvate decarboxylase and increased glycerol-3-phosphate dehydrogenase [NAD+] levels enhance glycerol production in Saccharomyces cerevisiae.

This investigation deals with factors affecting the production of glycerol in Saccharomyces cerevisiae. In particular, the impact of reduced pyruvate-decarboxylase (PDC) and increased NAD-dependent glycerol-3-phosphate dehydrogenase (GPD) levels was studied. The glycerol yield was 4.7 times (a pdc mutant exhibiting 19% of normal PDC activity) and 6.5 times (a strain exhibiting 20-fold increased GPD activity resulting from overexpression of GPD1 gene) that of the wild type. In the strain carrying both enzyme activity alterations, the glycerol yield was 8.1 times higher than that of the wild type. In all cases, the substantial increase in glycerol yield was associated with a reduction in ethanol yield and a higher by-product formation. The rate of glycerol formation in the pdc mutant was, due to a slower rate of glucose catabolism, only twice that of the wild type, and was increased by GPD1 overexpression to three times that of the wild-type level. Overexpression of GPD1 in the wild-type background, however, led to a six- to seven-fold increase in the rate of glycerol formation. The experimental work clearly demonstrates the rate-limiting role of GPD in glycerol formation in S. cerevisiae.

Cells of the yeast Saccharomyces cerevisiae are transformable by DNA under non-artificial conditions.

Transformants of bakers' yeast (Saccharomyces cerevisiae) can be generated when non-growing cells metabolize sugars (without additional nutrients) in the presence of plasmid DNA. These results suggest that there is a mechanism by which DNA can naturally be taken up by the yeast cell. Natural transformation does not take place in common complete or minimal yeast culture media such as YPD and YNB. The starvation conditions used in our experiments thus seem to be an important prerequisite for such transformation events.

Engineering of Saccharomyces cerevisiae for the production of L-glycerol 3-phosphate.

L-glycerol 3-phosphate (L-G3P) was accumulated in Saccharomyces cerevisiae by pathway engineering. Intracellular concentration of this metabolic intermediate could be increased more than 20 times compared to the wild type by overexpressing GPD1 encoding the glycerol 3-phosphate dehydrogenase in a gpp1 Delta gpp2 Delta mutant which lacks both isoenzymes of glycerol 3-phosphatase. Investigation of cellular pattern of triacylglycerols and glycerophospholipids did not reveal considerable changes due to accumulation of their precursor L-G3P. Hyperosmotic stress did not affect the L-G3P pool in the gpp1 Delta gpp2 Delta mutant overexpressing GPD1 despite an about 4-fold increase of specific GPD activity. In contrast, oxygen limitation improved intracellular L-G3P concentration by enhancing the availability of cytosolic NADH. The reduction of pyruvate decarboxylase activity by deleting PDC2 led to an additional increase. In fact, the triple mutant gpp1 Delta gpp2 Delta pdc2 Delta overexpressing GPD1 accumulated 17 mg L-G3P/g dry weight during glucose batch fermentation under oxygen limitation. This value corresponds to an about 100-fold increase compared to that found in the wild type.