Metabolic Engineering Of Bacillus Subtilis/Corynebacterium Glutamicum For Efficient Chiral 2,3-butanediol And Acetoin Production

In this study,firstly,we used the strategies of metabolic engineering,cofactors engineering and fermentation engineering to improve the metabolic capacity of B.subtilis for the production of D-(-)-or meso-2,3-butanediol.Secondly,we used the cofactors engineering strategy to solve the problem of redundancy of reducing power,and finally achieved a high acetoin titer and productivity.Finally,we introduced exogenous 2,3-butanediol synthetic pathway into C.glutamicum to obtain an engineered strain for D-(-)-2,3-butanediol production.Firstly,by literature comparison and experimental data analysis,we proposed a rational explanation for the result that B.subtilis168 produce only chiral D-(-)-2,3-butanediol in this study.To increase the D-(-)-2,3-butanediol titer and yield,the D-(-)-2,3-butanediol biosynthesis pathway,competitive by-product pathway,NADH availability and fermentation conditions optimization were systematically studied.In this study,D-(-)-2,3-BD production was abolished by deleting D-(-)-2,3-BD dehydrogenase coding gene bdhA,and acoA gene was knocked out to prevent the degradation of acetoin(AC),the immediate precursor of 2,3-BD.Both pta and ldh gene were deleted to decrease the accumulation of the byproduct acetate and L-lactate.The deletion of ldh and introduction of udhA gene(coding a soluble transhydrogenase)increased the NADH availability.By acetoin reductase activity assay and intracellular cofactor levels measurement,it was indicated that the high level of NADH availability,instead of high AR activity,contributed more to 2,3-BD production.NADH availability was further increased to facilitate the conversion of meso-2,3-BD from AC by low dissolved oxygen control during the cultivation.Under micro-aerobic oxygen conditions,the best strain BSF30 produced 101.2 g/L D-(-)-2,3-BD with a yield of 0.478 g/g glucose in the 5L batch fermenter.To achieve the efficient production of chiral meso-2,3-BD,we further introduced the meso-2,3-BD dehydrogenase coding gene budC from Klebsiella pneumoniae CICC10011,and the best engineered strain BSF9 G produced 103.7 g/L meso-2,3-butanediol in the 5L batch fermenter.Meanwhile,by relieving the regulation of the expression repression of arabinose transporter protein,and introducing the xylose isomerase and xylulose kinase coding gene from E.coli,the mutants for D-(-)-or meso-2,3-butanediol production using the mixture of glucose,xylose and arabinose were constructed.Secondly,through the cofactors engineering strategy,we used a high OD level to achieve satisfactory acetoin titer and productivity at a higher fermentation temperature.The best mutant BSF30 produce 63.2 g / L acetoin within 12 h,with a yield of 0.462 g/g glucost(94.5% of the theoretical maximum yield),and the productivity was 5.32 g/(L·h).And the acetoin titer could be increased to 67.6 g/L when the fermentation time was extended to 16 h.To the best of our knowledge,this was the highest productivity and yield among all the reported acetoin production strain.Finally,the non-pathogenic Corynebacterium glutamicum(could not naturally produce 2,3-butanediol)was engineered to produce chiral D-(-)-2,3-butanediol.With the purpose of D-(-)-2,3-butanediol production in C.glutamicum,its native butA gene(coding meso-2,3-butanediol dehydrogenase)was deleted,and an artificial gene cluster,encoding the 2,3-butanediol biosynthetic pathway from Bacillus subtilis was introduced.The resulted mutant CGF41 could produce 60.2 g/L D-(-)-butanediol in the 5-L fermentor.