弱化呼吸链水平对代谢工程大肠杆菌聚羟基丁酸乳酸酯合成的影响

Effect of reducing the activity of respiratory chain on biosynthesis of poly(3-hydroxybutyrate-co-lactate) in Escherichia coli

摘要:

3-羟基丁酸乳酸酯[Poly(3-hydroxybutyrate-co-lactate)，P(3HB-co-LA)]，属于聚羟基脂肪酸酯(Polyhydroxyalkanoates，PHA) 家族的一员，是一种具有良好生物相容性和可降解性的天然高分子生物材料。文中通过在大肠杆菌中引入来源于富养罗尔斯通氏菌Ralstonia eutropha的β-酮硫解酶、乙酰乙酰CoA还原酶、来源于丙酸梭菌Clostridium propionicum的丙酰CoA转移酶突变体以及荧光假单胞菌Pseudomonas fluorescens strain 2P24来源的PHA合成酶突变体等异源酶，成功实现了一步法利用葡萄糖合成P(3HB-co-LA)，其中乳酸组分的摩尔百分比达到1.6%，聚合物含量为83.9 wt%。在此基础上，通过敲除辅酶Q8合成所需的黄素异戊烯基转移酶基因 (ubiX) 来弱化呼吸链水平，从而增强乳酸积累，并进一步缺失乳酸脱氢酶基因 (dld) 以减少乳酸在发酵后期转化成丙酮酸，最终将P(3HB-co-LA) 中乳酸组分的摩尔百分比提高至14.1%，而聚合物含量为81.7 wt%。上述实验结果表明，采用弱化呼吸链水平策略可有效提高聚合物中乳酸组分的摩尔百分比，从而提供了一种改变生物合成聚合物中单体组分含量的新思路。

Abstract:

Poly(3-hydroxybutyrate-co-lactate) [P(3HB-co-LA)] belongs to the polyhydroxyalkanoates (PHA) family and possesses promising properties including biocompatibility and biodegradability. In this study, we directly synthesized P(3HB-co-LA) with glucose by introducing the β-ketothiolase and acetoacetyl-CoA reductase from Ralstonia eutropha, the engineered propionate CoA transferase from Clostridium propionicum and the engineered polyhydroxyalkanoate synthase from Pseudomonas fluorescens strain 2P24 into Escherichia coli. The polymer content was 83.9% (W/W), and the molar percentage of lactate reached 1.6%. On this basis, in order to accumulate lactate, we reduced the activity of respiratory chain by deleting the ubiX gene, which is involved in the synthesis of coenzyme Q8. Moreover, we removed the dld gene to avoid the conversion of lactate to pyruvate during the fermentation. With these manipulations, the molar percentage of lactate in the polymer was improved to 14.1%, with an 81.7% (W/W) of polymer content. The test results indicated that the strategy of reducing the activity of respiratory chain effectively increased the lactate units in the polymer, and it contributed a new approach to change the content of monomer components in the polymer.