短链硫酯酶缺失对大肠杆菌合成聚羟基丁酸乳酸酯的影响

Effect of short-chain thioesterase deficiency on P(3HB-co-LA) biosynthesis in Escherichia coli

摘要:

聚羟基脂肪酸酯 (Polyhydroxyalkanoates，PHAs) 是一种具有优质生物相容性的可降解生物基材料，其理化性质优越，具备替代石油基塑料的潜力。P(3HB-co-LA) 是PHAs的一种，融合了聚乳酸 (Polylactic acid，PLA) 和聚3-羟基丁酸 (poly(3-hydroxybutyrate)，P(3HB)) 共同的优点，具有更好的韧性和透明度。文中首先在大肠杆菌MG1655中通过质粒表达外源基因phaA、phaB、phaCm和pctth，发酵生产出乳酸含量为23.8 mol%的P(3HB-co-LA)，在此基础上缺失dld基因得到菌株WXJ01-03，其合成的聚合物中乳酸组分含量提升至37.2 mol%。当硫酯酶基因ydiI和yciA基因继续被敲除后，生产的共聚物中乳酸组分进一步提升至42.3 mol%和41.1 mol%。最后将3个基因dld、yciA和ydiI同时缺失得到重组菌株WXJ03-03，并通过该重组菌株获得了乳酸组分含量为46.1 mol%的共聚物。通过比较不同碳源的发酵结果得知，木糖有利于提高共聚物中乳酸组分含量。上述实验结果表明，在木糖发酵中短链硫酯酶基因缺失阻碍了大肠杆菌胞内的LA-CoA被降解，可有效提高聚合物中乳酸组分的摩尔百分比。

Abstract:

Polyhydroxyalkanoates (PHAs) have obtained much attention in biomaterial fields due to their similar physicochemical properties to those of the petroleum-derived plastics. Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] is one member of the PHAs family, and has better toughness and transparency compared to existing polylactic acid (PLA) and poly[(R)-3-hydroxybutyrate] [P(3HB)]. First, we confirmed the one-step biosynthesis of P(LA-co-3HB) with the lactate fraction of 23.8 mol% by introducing P(3HB-co-LA) production module into Escherichia coli MG1655. Then, the lactate fraction was increased to 37.2 mol% in the dld deficient strain WXJ01-03. The genes encoding the thioesterases, ydiI and yciA, were further knocked out, and the lactate fraction in the P(3HB-co-LA) was improved to 42.3 mol% and 41.1 mol% respectively. Strain WXJ03-03 with dld, ydiI and yciA deficient was used for the production of the LA-enriched polymer, and the lactate fraction was improved to 46.1 mol%. Notably, the lactate fraction in P(3HB-co-LA) from xylose was remarkably higher than from glucose, indicating xylose as a potent carbon source for P(3HB-co-LA) production. Therefore, the deficiency of thioesterase may be considered as an effective strategy to improve the lactate fraction in P(3HB-co-LA) in xylose fermentation.