副产物途径的缺失对大肠杆菌合成D-1,2,4-丁三醇的影响

Influence of the deficiency of by-product pathways on biosynthesis of D-1,2,4-butanetriol in Escherichia coli

【目的】敲除副产物途径,提高重组大肠杆菌D-1,2,4-丁三醇(D-1,2,4-Butanetriol,BT)产量。【方法】利用Red重组技术敲除木糖途径xyl AB基因及2-酮-3-脱氧木糖酸代谢途径的yag E及yjh H基因,考察其对重组菌生长、BT生产及副产物积累的影响。【结果】敲除xyl AB基因后,重组菌生物量降低57%,BT产量降低20%,单位菌体产量提高84%,木糖酸积累量提高52%。yag E或yjh H基因单独缺失重组菌生物量分别提高10%和5%,BT产量提高36%和14%。基因共同缺失后重组菌生物量降低了21%,BT产量提高184%,达到2.44 g/L,单位菌体产量提高258%。而共同敲除两途径,生物量降低了72%,虽然单位菌体产量提高了约4倍,但BT产量仅提高43%。p H调控下,重组菌木糖酸积累量下降,BT产量进一步提高,最高达3.11 g/L。【结论】xyl AB基因缺失后,虽有利于提高BT途径的效率,但由于木糖无法进入PPP途径及木糖酸积累,造成生物量降低,不利于BT合成。单独敲除yag E或yjh H后BT产量略有提高,而共同敲除这两基因更为有效地调整碳流向BT合成偏转。两途径共同敲除利于BT的合成,但由于菌体量的减少,无法大量获得BT。

[Objective] In order to improve the D-1,2,4-butanetriol（BT） titer in recombinant Escherichia coli, two by-product pathways were blocked. [Methods] The xyl AB, yag E and yjh H were knocked out by Red system and the cell growth, BT production and accumulation of byproduct of the resultant strains were detected. [Results] The biomass and BT titer of xyl AB-deficient strain were repressed by 57% and 20%, with the BT yield per cell increased by 84%. In contrast, the biomass of yag E-deficient and yjh H-deficient strains was increased by 10% and 5%, respectively. The BT titers of these two strains showed increase of 36% and 14%, respectively. Co-knocking out these two genes led to the decrease of biomass by 21%, but the BT titer was improved by 184%, up to 2.44 g/L, and BT yield per cell was increased by 258%. Meanwhile, blocking these two by-product pathways simultaneously resulted in the decrease of biomass by 72% and raised BT titer per cell by 4 times, and the final BT titer was showed 43% improvement. The xylonate titers of recombinant strains were decreased, and the BT titer was further improved by p H-controlled fermentation, up to 3.11 g/L. [Conclusion] Although knocking out xyl AB is beneficial to BT produce, the repressed xylose utilization ability through the PPP pathway and accumulated xylonate lead to the reduced biomass and BT titer. Knocking out the yag E or yjh H showed slightly positive effect on BT production. Further co-deficiency of these two genes shifts more 2-keto-3-deoxy-xylonate（KDX） into BT pathway, leading to significantly improved BT titer. Finally, blocking these two by-product pathways shows negative effect on cell growth but slightly improvement on BT production.