Improvement of 2,3-butanediol production from xylose in engineered Saccharomyces cerevisiae

Abstract

2,3-Butanediol (2,3-BD) is a chemical compound with many industrial applications as a precursor of synthetic rubber and moistening and softening agents. Most of microbial production of 2,3-BD have been based on pathogenic bacteria, making difficult large-scale fermentations for 2,3-BD production. Therefore, GRAS (Generally Regarded As Safe) microorganisms including Saccharomyces cerevisiae would be desirable. Production of 2,3-BD from cellulosic biomass is needed for sustainable development. Among various sugars, glucose and xylose are of special interest because they are abundant in cellulosic biomass. This thesis is aimed at production of 2,3-BD by using engineered S. cerevisiae capable of fermenting xylose. To eliminate ethanol production, pyruvate decarboxylase (Pdc)-deficient S. cerevisiae (SOS5) was constructed by deleting the PDC 1, 5 and 6 genes. The XYL1, XYL2 and XYL3 genes coding for xylose assimilating enzymes derived from Scheffersomyces stipitis were introduced into the SOS5 strain for xylose utilization. Then, the alsS gene encoding α-acetolactate synthase and the alsD gene encoding α-acetolactate decarboxylase both from Bacillus subtilis and endogenous BDH1 gene were overexpressed (BD5X). First, the 2,3-BD-producing Pdc-deficient BD5X and BD4X strains were compared for a performance of 2,3-BD production in a batch fermentation. The BD4X strain was constructed through deleting the PDC1 and PDC5 genes and had mutation on the MTH1 gene, encoding a negative regulator of the glucose-sensing signal transduction pathway. BD5X produced 11.5 g/L of 2,3-BD from xylose with a yield of 0.34 g2,3-BD/gxylose. The titer of 2,3-BD increased 16% higher than the BD4X strain. Second, to enhance xylose uptake rate in 2,3-BD production, the TAL1 gene encoding transaldolase and the mutant XR gene encoding xylose reductase from S.sitipitis were expressed in the BD5X strain. The resulting strain, BD5X-TA1-mXR was tested for a performance of xylose consumption in batch fermentation under oxygen-limited conditions with BD5X as a control. BD5X-TA1-mXR produced 10.7 g/L of 2,3-BD from xylose with a productivity of 0.45 g/L/h. The productivity increased 66% higher in the BD5X-TAL1-mXR strain compare to the control strain BD5X strain. Also, the BD5X-TAL1 strain showed 1.85-fold higher specific activity of transaldolase than the control strain (0.24 versus 0.13 U/mg-protein) and 2.43-fold higher specific activity of xylose reductase than the control strain, BD5X-TAL1 (2.28 versus 0.42 U/mg-protein). Finally, to solve defects about accumulation of glycerol and C2-dependent growth in the Pdc-deficient strain, the noxE gene encoding NADH oxidase from Lactococcus lactis and the PDC1 gene encoding pyruvate decarboxylase 1 from Candida tropicalis were expressed in BD5X-TAL1-mXR. To test for a performance of 2,3-BD production, batch and fed-batch fermentations were carried out. With expression of L. lactis noxE, the yield of glycerol decreased by 40.1 % and the yield of 2,3-BD increased by 23.3% in BD5X-TAL1-mXR-nox compared with the control strain, BD5X-TAL1-mXR. With expression of PDC from C. tropicalis, xylose uptake rate increased by 33 % and productivity of 2,3-BD increased by 20.5 % in the BD5X-TAL1-mXR-nox-pdc compared with the BD5X-TAL1-mXR-nox strain. Also, to test for C2-independent growth, a batch fermentation was conducted without addition of ethanol. The BD5X-TAL1-mXR-nox-pdc strain was able to grow without addition of ethanol in a batch fermentation, as expected. In a fed-batch fermentation, the resulting strain produced 69.2 g/L of 2,3-BD from xylsoe with a low glycerol yield, 0.08 gglyerol/gxylose, and the high yield of 2,3-BD of 0.38 g2,3-BD/gxylose. The titer of 2,3-BD produced from xylose in the BD5X-TAL1-mXR-nox-pdc strain was 58% higher than that obtained with the BD4X strain in a mixture of glucose and xylose under similar conditions. These results suggested that the BD5X-TAL1-mXR-nox-pdc strain is suitable for producing 2,3-BD from cellulosic biomass.