Metabolic Engineering Of Bacillus Subtilis Chassis Cells For Efficient Acetoin Production

Acetoin is a high-value-added biobased platform chemical widely used in industries such as agriculture,chemical,and food.With the depletion of global fossil resources and the increasing problem of environmental pollution,people have turned their attention to the use of biological fermentation to produce acetoin.In this study,the engineering model strain Bacillus subtilis 168was used as the starting strain,and a series of rational metabolic engineering modifications were carried out using modern molecular biology techniques.Acetoin fermentation production was conducted using glucose and sugarcane bagasse hydrolysate as fermentation substrates.The specific research results are as follows:（1）Enhancing the continued metabolic production capacity of the chassis cell:In order to reduce the autolysis rate during the fermentation of Bacillus subtilis,this study knocked out different types of autolysis genes.The results showed that the key gene lyt C encoding peptidoglycan hydrolase could increase cell biomass,thereby enhancing the fermentation density of cells during production.To further reduce cell biomass and energy loss during acetoin production,the gene spo IIE,which regulates cell sporulation,was knocked out.The recombinant strain QC-3showed a 23%increase in maximum biomass compared to the original strain B.subtilis 168,and acetoin production was carried out using only 100 g·L^-1glucose as the sole carbon source for shake-flask fermentation.The acetoin yield increased from 29.6 g·L^-1to 33.7 g·L^-1,a 13.9%improvement over the B.subtilis 168.（2）Optimizing cofactor levels to enhance acetoin yield:In this study,a dual cofactor regulation strategy was adopted to solve the problem of insufficient intracellular reducing power（NAD⁺/NADH）and the prevalence of coenzyme-dependent competitive byproducts during the fermentation process of Bacillus subtilis,aiming to improve the intracellular reducing power level.First,the key gene ydi H encoding the oxidoreduction-responsive global regulator Rex protein was knocked out,while the NADH oxidase-encoding gene yod C was overexpressed to construct an NAD⁺coenzyme regeneration system.By optimizing the intracellular cofactor levels,the fermentation cycle of acetoin was shortened by nearly 12 hours.To further improve the yield of acetoin,the metabolic pathway of competitive byproducts in the acetoin synthesis pathway during fermentation was blocked.The recombinant strain QR-1 was subjected to shake-flask fermentation,and the yield of acetoin reached 37.9 g·L^-1,with a production efficiency of 0.45 g·L^-1·h^-1.（3）Improving acetoin synthesis efficiency by enhancing metabolic flux:In order to further enhance the metabolic carbon flux of the key pathway for acetoin synthesis and improve the cell efficiency in producing acetoin,this study combined previous research by the research group and matched the stage-specific promoter P_{srf A}with different ribosome binding site（RBS）sequences to screen for the optimal combination.The enzyme activities of ALS（α-acetolactate synthase）and ALDC（α-acetolactate decarboxylase）,key enzymes in the acetoin synthesis pathway,were moderately increased to strengthen the metabolic flux from pyruvic acid to acetoin synthesis pathway and improve the efficiency of acetoin synthesis.The acetoin yield of the recombinant strain QRCR-2 in shake flasks reached 44.5 g·L^-1.Subsequently,the recombinant strain QRCR-2was subjected to batch-feeding fermentation in a 5 L fermenter,and the maximum acetoin yield reached 81.6 g·L^-1,which was about 1.97 times higher than that of the wild-type strain,with a production efficiency of 0.97 g·L^-1·h^-1.（4）Using low-cost biomass resources to produce acetoin:It is an important research direction to use low-cost biomass resources as raw materials for fermentation to produce high-value-added chemicals.In order to achieve simultaneous utilization of xylose and glucose in the hydrolysate of sugarcane bagasse lignocellulose,this study overexpressed the xylose transport protein gene ara E and the assimilation metabolic pathway encoding genes xyl A and xyl B.It was found that after overexpression of the xylose assimilation pathway encoding genes xyl A and xyl B,the carbon decomposition blockade effect that existed in Bacillus subtilis was relieved,and the strain achieved simultaneous utilization of xylose and glucose in the hydrolysate of sugarcane bagasse.The results showed that using the hydrolysate of sugarcane bagasse lignocellulose as the fermentation substrate for batch-feeding fermentation in a 5 L fermenter,the recombinant strain QRCRT-2 had significantly improved ability and tolerance to utilize crude substrates compared to the wild-type strain,and the maximum yield of acetoin reached 71.8 g·L^-1,which was 2.09 times higher than the wild-type strain.