| 1,3-propanediol?1,3-PDO?and 1,2,4-butanetriol?BT?are high value-added chemical product with a wide range of applications.In the process of producing 1,3-PDO by glycerol and xylose co-substrate fermentation in Klebsiella pneumoniae,reducing power and yield were increased,but the problems of low xylose utilization and high substrate cost existed.The production of 1,3-PDO and BT by glycerol and xylose co-substrate fermentation could not only solve these problems effectively,but also increase production value and reduce fermentation cost.In this study,the co-production of 1,3-PDO and BT by K.pneumoniae was improved by constructing a BT synthesis pathway,bioengineering BT synthesis pathway and reducing carbon catabolite repression.To achieve the co-production of 1,3-PDO and BT,the BT synthesis pathway was first constructed by overexpressing xdh from Caulobacter crescentus,kivD from Lactococcus lactis,and yjhG from Escherichia coli in K.pneumoniae ZG25 which could yield 1,3-PDO.The fermentation results showed the BT yield was 1.8 g·L-1.Optimizing the fermentation conditions and media at shaker level,when the induction time was 2 h,the inoculation amount was 1%,the speed was 200 r·min-1,and the pH was controlled by adding 10 g·L-11 CaCO3,the concentration xylose and glucose were 30 g·L-1 and 10 g·L-1,respectively,and the base media was 1.5 times LB,The BT yield increased by 83%to 3.3 g·L-1.Further optimization by adding glycerol,inorganic salts and others,the co-production of 1,3-PDO and BT was achieved,and the 1,3-PDO and BT yield were 18.1 g·L-1 and 3.3 g·L-1,respectively.Further metabolic modifications enhance to product synthesis,strategies such as weakening xylose branch metabolism,blocking the by-product pathway in the BT synthesis pathway and weakening the carbon catabolite repression were used to knock out xylose isomerase genes xylA,xylA2,phenylglyoxal dehydrogenase gene feaB,aldehyde dehydrogenase gene aldB,2-ketoacid reductase genes ycdW,ycdW2,EIICBGlc protein encoding gene ptsG in phosphoglucose transferase system,phosphoglucose isomerase gene pgi,respectively.The fermentation results showed that the knockout of xylA,xylA2,feaB,aldB and pgi alone could promote BT synthesis,and the BT yield of recombinant bacteria reached 4.5g·L-1,4.4 g·L-1,3.9 g·L-1,3.6 g·L-1,3.9 g·L-1,respectively,which was 36%,33%,18%,9%,18%higher than K.pneumoniae ZG25-BT,respectively.Meanwhile,the fermentation results showed that the absence of ycdW,ycdW2 and ptsG were detrimental to the co-production of1,3-PDO and BT.In order to enhance 1,3-PDO and BT co-production,multi-strategic modifications were used in K.pneumoniae ZG25.By combined knockout of xylA,aldB,feaB,pgi,the yields of1,3-PDO and BT were 18.9 g·L-1 and 5.8 g·L-1,respectively,at the shaking bottle level.The culture conditions were preliminarily optimized on the 5 L fermentation tank,the fermentation results showed that the yields of 1,3-PDO and BT were 39.9 g·L-1 and 14.8 g·L-1,respectively.The results showed that the co-production of 1,3-PDO and BT could be improved by using multiple strategies such as constructing a BT synthesis pathway in K.pneumoniae,bioengineering BT synthesis pathway and reducing carbon catabolite repression,thus laying a foundation for the co-production of high value-added products with multiple cheap substrates. |
PDF Full Text Request