Construction And Optimization Of 1,2,4-butanetriol Synthetic Pathway In Engineered Escherichia Coli

1,2,4-Butanetriol(BT) is an important intermediate chemical, which has a number of industrial applications. In medical applications, BT is a known precursor of cationic lipids for drug delivery; it also can be a potential precursor for the synthesis of several chiral chemcals in pharmaceuticals. One of its most important applications is to synthesize 1,2,4-butanetriol trinitrate(BTTN), which is a good propellant and an energetic plasticizer, that has the potential to replace nitroglycerin. BT can also be used as raw materials to synthesize polymeric materials, cigarette additive and colour developer, etc.Chemical synthesis is currently used for commercial production of BT, which requires extreme reaction conditions and has results in many unwanted byproducts. Recently biological transformation strategy has emerged as an attractive field due to its low cost, mild condition and environmental friendly nature. Microbial conversion of D-xylose into BT has been reported using recombinant E. coli strains in a few literatures. However, the low transformation efficiency of BT still hindered the commercial scale BT production. To further improve BT transformation efficiency, the BT synthetic pathway has to be optimized, the limited steps identified and expression level of each enzymes precisely modulated.In this work,competing pathways for xylose and 2-keto-3-deoxy-D-xylonate in E. coli BW25113 were blocked by disrupting xyl AB, yag E/yjh H and yia E/ycd W. The xdh(encoding D-xylose dehydrogenase, the first step) and mdl C(benzylformate decarboxylase, the third step) gene were heterologously introduced into E. coli to assemble the BT synthetic pathway from xylose. The strain BW-011 which could produce 0.3 g/L of BT was successfully constructed.In order to improve BT production, the potential limiting step was analyzed and new 2-keto decarboxylase from different sources were expressed and screened for the third catalytic step. Shake flask transformation results showed that the strain BW-025 which harbored kiv D from Lactococcus lactis produced nearly two times more BT than BW-011 which carried mdl C gene. Rational design and modification of Kiv D with of aim of improving its performance was attempted. Expression levels of other enzymes were also tuned in search of new bottleneck for BT production. Finally, a new strain named BW-074 with overexpressed xdh gene was identified, which produced 48.62% more BT than BW-025.In addition, we also explored the effect of conversion conditions on BT production. After systematical optimization, a final BT titer of 2.38 g/L was achieved for BW-025 after 72 h of conversion.As the result of the process above, BT production of the engineered E. coli was improved significantly. And strategies of BT microbial synthesis improvement were provided in this study.