Metabolic Engineering Of Poly (3-hydroxybutyrate) And Acetoin Production From Sugar Mixture

Poly(3-hydroxybutyrate)(PHB),which is the best characterized member of polyhydroxyalkanoate(PHA),has broaden potential application prospects in many fields such as medicine,food and agriculture because of its biodegradablity and biocompatibility.Acetoin(3-hydroxy-2-butanone),which is also called acetyl methyl carbinol,is a kind of food flavor and platform chemical in many fields.Lignocellulose hydrolysate,which is mainly composed by glucose and xylose,has attracted many attentions because of its renewability for the production of many bio-products.Here,the engineered strains can use glucose-xylose mixture to produce PHB and acetoin through the introduction of exogenous xylose pathway to release carbon catabolite repression(CCR).In this study,the xylose isomerase,xylulokinase and the arabinose/xylose transport protein from Bacillus subtilis 168(encoded by xylA,xylB and araE respectively)were co-expressed in the ndh knockout JM109 strain(LJ03),with different plasmids owning copy numbers from low to high.The strains with low and moderate copy number plasmids can utilize xylose in higher rates(0.772g/(L·h)and 0.598g/(L·h))than the control strain(0.549g/(L·h),without the xylose pathway from Bacillus subtilis,in medium containing 15g/L xylose,indicating that the genes can express normally in LJ03.While the strain with high copy number plasmid utilizes xylose in a much slower rate.In the mixed glucose-xylose fermentation,the strains with low and moderate copy number plasmids produce 3.17g/L and 3.67g/L PHB.Then in the 5-L reactor,the strain with moderate copy number plasmid produced 21.0g/L PHB in 28 h,consuming 92.6g/L glucose and 40.2g/L xylose with a yield of 0.16g/g total sugar.We successfully constructed strains F31 and F34,which can utilize glucose and xylose simultaneously,basing on the construction of two strains producing acetoin in high level.In both strains xylA,xylB from E.coli and araE of Bacillus subtilis were overexpressed.In flask cultivation,strains F31 produced 1.58g/L acetoin and 2.92g/L meso-2,3-butanediol and F34 produced 5.36g/L acetoin after 48 h fermentation after medium optimization,temperature optimization.Then strain F31 produced 20.01g/L acetoin and 8.76g/L meso-2,3-butanediol after 65 h fermentation in 5-L bioreactor.Strain F34 produced 31.3g/L acetoin and 3.0g/L D-2,3-butanediol in the same time with a total yield of 0.319g/g.The results indicated that introducing an exogenous xylose pathway is a useful strategy to release CCR and consumed lignocellulose hydrolysate to produce bio-product.