“代谢晶体管”模式介导大肠杆菌可控合成聚3-羟基丁酸乳酸酯的研究

Poly(lactate-co-3-hydroxybutyrate) [P(LA-co-3HB)], a new member of the bacterial bio-polyesters, has the great biomaterial properties including thermal plasticity, biocompatibility, and biodegradability, and will be a great candidate of bio-plastics for industrial application. The monomer ratio of the polymer (3-hydroxybutyryl CoA and 2-hydroxypropionyl CoA) can be adjusted to synthesize different P(3HB-co-LA) with different properties. The change of the monomer ratio are mainly affected by the change of added lactic acid or dissolved oxygen, but local differences of the concentration of the added lactic acid and oxygen in the large fermentation process will directly affect the composition of polymer, and it will cause the polymer’s heterogeneity. In pervious study, a novel "metabolic transistor" strategy, which was built on the manipulation of Q8 biosynthesis in E. coli, was established to finely regulate the aerobic carbon metabolism of E. coli and control the biosynthesis of acetyl-CoA (precursor of 3-hydroxybutyryl CoA) and lactate (precursor of 2-hydroxypropionyl CoA). Our objective in this project is to construct metabolically engineered E. coli strains which can produce P(3HB-co-LA) from glucose though multivariate modular metabolic engineering. The "metabolic transistor" strategy will be applied for fine regulation of synthesizing the precursor ratio of P(3HB-co-LA) combining with the modular metabolic engineering. The project aims to establish the novel strategy for green-polymer biosynthesis with controllable ratio of components in the engineered E. coli strains.

聚3-羟基丁酸乳酸酯，P(3HB-co-LA)，是一种新型胞内天然高分子生物材料，具备良好的可塑性、生物相容性和可降解性，有广泛的应用前景。聚合物单体（3-羟基丁酸和乳酸）的比例决定P(3HB-co-LA) 的理化性能。目前调节单体比例主要通过乳酸的外源添加或供氧的变化，然而大规模发酵过程中反应器内添加乳酸和供氧浓度的局部差异会直接影响聚合物的组成变化，造成聚合物的不均一性。前期研究表明，基于呼吸链改造的“代谢晶体管”调控模式，对乙酰CoA（3-羟基丁酸CoA前体）和乳酸（2-羟基丙酰CoA前体）比例进行可控性调节。本研究拟结合模块化代谢工程和“代谢晶体管”调控模式，系统研究胞内3-羟基丁酰CoA和2-羟基丙酰CoA的可控性合成机制，实现对P(3HB-co-LA)的组分的精细调控，并建立组分可控的聚酯生物材料合成策略。

聚羟基丁酸乳酸酯[P(3HB-co-LA)]，是一种微生物合成的天然高分子生物材料。它作为聚羟基脂肪酸酯(PHA)的一员，除具有PHA大类共有的生物相容性良好和可降解性外，还拥有出色的弹性和透明度，可以作为化学塑料的替代品，缓解目前危害较大的由塑料堆积引发的白色污染等问题。本研究以大肠杆菌E.coli MG1655作为背景菌株，通过表达聚羟基脂肪酸酯合成所需的β-酮硫解酶(phaA，来源于Ralstonia eutropha)和乙酰乙酰辅酶A还原酶(phaB，来源于Ralstonia eutropha)，突变后的PHA合成酶(phaCm，来源于Pseudomonas fluorescens strain 2P24)，用于共聚物前体LA-CoA合成所需的丙酸辅酶A转移酶突变体(pctth，来源于Clostridium propionicum)，优化途径关键酶的表达强度，实现了从葡萄糖和木糖直接合成聚羟基丁酸乳酸酯。采用“代谢晶体管”策略，实现了通过诱导剂浓度调控工程菌合成聚合物中的乳酸组分。工程菌株中得到了乳酸组分含量不同的聚合物（10.6-27.7 mol% LA）。为了进一步提高调控组分的范围，进一步缺失了细胞色素氧化酶基因。在细胞色素氧化酶基因cyoABCD和cbdAB缺失菌株中结合“代谢晶体管”策略得到了乳酸组分含量不同的聚合物(29.3-41.8 mol% LA)。此外，通过实验筛选出硫酯酶对LA-CoA具有降解作用，敲除两个硫酯酶基因ydiI和yciA后获得重组菌株木糖发酵中可获得乳酸组分含量为46.1 mol%的P(3HB-co-LA)。最后通过发酵优化实现了葡萄糖和木糖的混合糖发酵生产P(3HB-co-LA)，实现了菌株利用玉米秸秆水解液中葡萄糖和木糖合成P(3HB-co-LA)，为以后利用廉价易得的木质素水解液发酵生产生物基聚合物打下基础。