整合型1,2,4-丁三醇重组菌的构建及共底物发酵

Construction of a 1,2,4-butanetriol-integrated strain and fermentation of its co-substrate

1,2,4-丁三醇(BT)是重要的非天然化学品.为构建整合型BT合成菌株,实现木糖、葡萄糖共底物发酵,通过Red系统将基因kivD、xdh整合至Escherichia coli基因组的xylAB、ptsHI、ptsG、crr位点,并尝试利用廉价的乳糖替代IPTG诱导外源基因表达.结果表明,外源基因整合至xylAB后,生物量降低28%,重组菌Escherichia coli W021能够代谢木糖合成BT(0.7 g/L).添加葡萄糖为共底物后生物量提高36%,但碳分解代谢抑制作用限制了木糖的代谢,BT产量降低14%.进一步整合代谢基因至不同的磷酸转移酶系统(PTS)位点,其中整合至ptsHI基因后BT产量最高,达到2.8 g/L.优化葡萄糖、木糖浓度后,BT产量提高到3.6 g/L,进一步优化乳糖替代IPTG后BT产量为1.9 g/L.最后经发酵罐优化,BT产量提高到3.9 g/L,转化率为0.3 mol/mol.本研究构建整合型菌株在廉价乳糖诱导下共底物发酵合成BT,为后续放大研究提供了借鉴.

1,2,4-Butanetriol (BT) is an important non-natural chemical. The exogenous genes kivD and xdh were integrated into the xylAB site of the Escherichia coli genome by the Red system to construct a BT-producing strain E. coli W021. To realize glucose/xylose co-fermentation, kivD and xdh were respectively integrated into ptsHI, ptsG, and crr sites in E. coli W021 to obtain recombinant strains E. coli W031, E. coli W032, and E. coli W033. Non-toxic and cheap lactose was used to replace IPTG to induce the expression of exogenous genes. The results showed the recombinant strain E. coli W021 produced 0.7 g/L BT from xylose, with a 28% decrease in biomass. After adding glucose as a co-substrate, the biomass increased by 36%. However, xylose metabolism was inhibited due to carbon catabolite repression (CCR), resulting in a BT production decrease of 14%. Upon modifying the phosphotransferase system (PTS) to alleviate the CCR effect, E.coli W031 produced BT at the highest titer of 2.8 g/L. BT production was further increased to 3.6 g/L by optimizing glucose and xylose concentrations. Additionally, using lactose as the inducer instead of IPTG yielded 1.9 g/L of BT. Finally, the BT yield reached 3.9 g/L and the conversion rate was 0.3 mol/mol during batch fermentation. In this study, an integrated strain was used to synthesize BT induced by lactose under co-substrate fermentation, thus providing a reference for subsequent amplification studies.