Production of 2,3-butanediol from glucose and cassava hydrolysates by metabolically engineered industrial polyploid Saccharomyces cerevisiae

Abstract

Background 2,3-Butanediol (2,3-BDO) is a valuable chemical for industrial applications. Bacteria can produce 2,3-BDO with a high productivity, though most of their classification as pathogens makes them undesirable for the industrial-scale production. Though Saccharomyces cerevisiae (GRAS microorganism) was engineered to produce 2,3-BDO efficiently in the previous studies, their 2,3-BDO productivity, yield, and titer were still uncompetitive compared to those of bacteria production. Thus, we propose an industrial polyploid S. cerevisiae as a host for efficient production of 2,3-BDO with high growth rate, rapid sugar consumption rate, and resistance to harsh conditions. Genetic manipulation tools for polyploid yeast had been limited; therefore, we engineered an industrial polyploid S. cerevisiae strain based on the CRISPR-Cas9 genome-editing system to produce 2,3-BDO instead of ethanol. Results Endogenous genes coding for pyruvate decarboxylase and alcohol dehydrogenase were partially disrupted to prevent declined growth rate and C 2 -compound limitation. A bacterial 2,3-BDO-producing pathway was also introduced in engineered polyploid S. cerevisiae . A fatal redox imbalance was controlled through the heterologous NADH oxidase from Lactococcus lactis during the 2,3-BDO production. The resulting strain (YG01_SDBN) still retained the beneficial traits as polyploid strains for the large-scale fermentation. The combination of partially disrupted PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase) did not cause the severe growth defects typically found in all pdc- or adh-deficient yeast. The YG01_SDBN strain produced 178 g/L of 2,3-BDO from glucose with an impressive productivity (2.64 g/L h). When a cassava hydrolysate was used as a sole carbon source, this strain produced 132 g/L of 2,3-BDO with a productivity of 1.92 g/L h. Conclusions The microbial production of 2,3-BDO has been limited to bacteria and haploid laboratorial S. cerevisiae strains. This study suggests that an industrial polyploid S. cerevisiae (YG01_SDBN) can produce high concentration of 2,3-BDO with various advantages. Integration of metabolic engineering of the industrial yeast at the gene level with optimization of fed-batch fermentation at the process scale resulted in a remarkable achievement of 2,3-BDO production at 178 g/L of 2,3-BDO concentration and 2.64 g/L h of productivity. Furthermore, this strain could make a bioconversion of a cassava hydrolysate to 2,3-BDO with economic and environmental benefits. The engineered industrial polyploid strain could be applicable to production of biofuels and biochemicals in large-scale fermentations particularly when using modified CRISPR-Cas9 tools.

Increasing ATP turnover boosts productivity of 2,3-butanediol synthesis in Escherichia coli

“…One possible way to achieve strong coupling of ATP and 2,3-BDO synthesis is to use microaerobic instead of fully aerobic conditions where, on the one hand, the NADH supply for 2,3-BDO synthesis increases due to limited oxygen availability, while the amount of supplied oxygen exactly balances the remaining surplus of NADH when producing 2,3-BDO from glucose. For this reason, microaerobic cultivations have frequently been used for 2,3-BDO synthesis [ 20 , 24 , 29 , 30 ] and we therefore tested the effect of ATP wasting also for this regime. First of all, applying microaerobic conditions under growth arrest indeed doubled the 2,3-BDO yield in the ATPase strain to 87 % of the maximum yield, which is the highest yield ever reported for E. coli on minimal glucose medium.…”

“…Thus, since the specific productivity governs the volumetric rate of growth-decoupled production phases, superior volumetric productivities can be expected for the ATPase strain in the three-stage process if comparable biomasses concentrations are used. Finally, the proposed three-stage process as well as the strategy of enforced ATP wasting in general could also be used to further enhance other developed production hosts and/or processes for 2,3-BDO synthesis published in the literature [ 24 , 29 , 30 ].…”

“…Therefore, other organisms have been genetically engineered for 2,3-BDO production, e.g. E. coli [ 20 , 24 , 25 ], Vibrio natriegens [ 26 ], L. lactis [ 27 ], S. cerevisiae [ 28 , 29 ], Bacillus subtilis [ 30 ], and Zymomonas mobilis [ 31 ].…”

Technological development of the bio‐based 2,3‐butanediol process

“…Several microorganisms can produce 2,3‐BDO, including GRAS (generally recognized as safe) bacteria such as Bacillus and Paenibacillu s, risk class 2 bacteria such as Klebsiella , Enterobacter , and Serratia , 1 and some engineered yeasts such as Saccharomyces cerevisiae 3 . The best 2,3‐BDO titers were achieved using metabolically engineered strains such as S. cerevisiae (178 g L −1 ), 3 E. cloacae (152 g L −1 ), 4 and K. oxytoca (130 g L −1 ), 5 and highly productive wild strains such as K. pneumonia (150 g L −1 ), 6 and P. polymyxa (111 g L −1 ) 7 …”

Effect of Dissolved Oxygen and Acid on the Yield of 2,3-butanediol by Saccharomyces Cerevisiae W141

“…However, the 2,3-BDO output obtained using S. cerevisiae is extremely low, which has become a bottleneck limiting large-scale industrial yields. At present, the primary strategies to increase 2,3-BDO yields from S. cerevisiae can be divided into four categories: (1) reducing the formation of other by-products in S. cerevisiae [8]; (2) modifying the levels of relevant cofactors in the 2,3-BDO metabolic pathway [9]; (3) enhancing glycolysis and the 2,3-BDO biosynthetic pathway [10]; and (4) exogenously expressing the 2,3-BDO synthesis pathway [11]. Among these approaches, the elimination of ethanol and glycerol yields and rebalancing cellular redox are the key factors for increase the yield of 2,3-BDO from yeast [8,9].…”