2,3-Butanediol (2,3-BD) is a platform chemical with extensive industrial applications since it can be converted into other valuable chemicals by dehydration, dehydrogenation, ketalization and esterification. Especially, 1,3-butadiene, a dehydration product of 2,3-BD, is a main substance used for producing synthetic rubber. Most microbial fermentations for 2,3-BD production have been focused on pathogenic bacteria, which makes large-scale fermentations difficult in terms of safety and industrialization. As an alternative, 2,3-BD production by a GRAS (Generally Regarded As Safe) microorganism Saccharomyces cerevisiae would be suitable. Also, production of 2,3-BD from marine biomass is one of the key issues for economic viability of bio-based chemicals. Among various sugars, glucose and galactose are of special interest because they are abundant in marine biomass. One of the most important concerns in mixed sugar fermentations is the catabolite repression that represses the enzyme expression to utilize sugars other than glucose.Pyruvate decarboxylase(Pdc)-deficient S. cerevisiae (SOS2) was constructed to eliminate ethanol production. Then, the evolved SOS2 strain (SOS4) was obtained by serial cultivation in excess glucose medium for cell growth on glucose medium. Among the chromosomal mutations of the SOS4 strain, a single nucleotide polymorphism was found on the MTH1 gene which functions as a glucose signal regulator.Efficient 2,3-BD production using engineered strains capable of effective fermentation from glucose and galactose is the objective in this study.First, the 2,3-BD-producing Pdc-deficient BD4 strain with the MTH1 mutation was tested for a performance of 2,3-BD production in a batch fermentation, resulting in simultaneous utilization of glucose and galactose. Therefore, in order to evaluate if the MTH1 mutation affects the fermentation performances in a mixture of glucose and galactose, the evolved pdc-deficient strain (SOS2) and engineered strain by a point mutation on the MTH1 gene (SOS2_Mth1) were used. The SOS2 and SOS2_Mth1 strains containing the alsS gene encoding α-acetolactate synthase and the alsD gene encoding α-acetolactate decarboxylase both from Bacillus subtilis and overexpression of the endogenous BDH1 gene coding for 2,3-BD dehydrogenase were constructed (BD2 and BD2M) and tested for a performance of 2,3-BD production in batch fermentation under oxygen-limited conditions. The BD2M strain which is the MTH1 mutant was able to co-ferment both sugars, resulting in increased 2,3-BD productivity by 41% compared to the control strain, BD2 strain. To evaluate the fermentation aspects of the BD2M strain in a bioreactor, fed-batch fermentation was carried out to obtain 75.5 g/L of 2,3-BD by simultaneous consumption of glucose and galactose. However, in the rest of the fermentation, galactose consumption rate was substantially reduced compared with glucose consumption rate.Second, to increase galactose uptake rate in 2,3-BD production, the GAL10, GAL1, GAL7, PGM1 and PGM2 genes involved in the Leloir pathway were overexpressed into the BD2M strain. These five consutructed strains were tested for a performance of galactose consumption in batch fermentation under oxygen-limited conditions. Only the strain for overexpression of the PGM2 gene, the BD2M_PGM2 strain obtained 13% increased galactose uptake rate. Also, the BD2M_PGM2 strain resulted in 42% increased galactose uptake rate and 39% improved 2,3-BD productivity compared to the control strain, BD2M strain in a fed-batch fermentation.Finally, to decrease the accumulation of glycerol and to solve the C2-dependent growth in 2,3-BD-producing Pdc-deficient S. cerevisiae, the NADH oxidase (noxE) gene from Lactococcus lactis and the pyruvate decarboxylase 1 (pdc1) gene from Candida tropicalis were expressed in the BD2M_PGM2 strain. To test for a performance of 2,3-BD production, batch and fed-batch fermentation was carried out. Unlike other conditions of fermentations in this study, this cultivation was conducted without addition of ethanol due to expression of the pdc1 gene. The resulting strain (BD2M_PGM2_Ctnox) obtained 19% reduced yield of glycerol and was able to grow without addition of ethanol in a batch fermentation. Also, in a fed-batch fermentation, the resulting strain produced 116 g/L of 2,3-BD from glucose and galactose with a low glycerol yield (0.08 gglyerol/gsugars) with 0.47 g2,3-BD/gsugars of yield and 1.75 g/L/h of productivity in a fed-batch fermentation. These results suggested that the BD2M_PGM2_Ctnox strain is suitable for producing 2,3-BD from marine biomass for industrial applications Keywords : 2,3-butanediol, pyruvate decarboxylase (Pdc), galactose, marine biomass, mixed sugar fermentation, mth1, phosphoglucomutase (Pgm2), catabolite repression, redox balance
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Production of 2,3-butanediol from glucose and galactose in engineered Saccharomyces cerevisiae
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