基于葡萄糖和乙酸共利用的聚乳酸-乙醇酸共聚物的可控生物合成研究

Poly(lactic-co-glycolic acid) (PLGA) is a kind of polymers with favorable biocompatible and biodegradable properties. Currently, PLGA is mainly produced through chemical synthesis. In this project, we propose to construct Escherichia coli strains which could coutilize glucose and acetate to produce PLGA with controllable monomer compositions and structures. Firstly, the central metabolic pathway of E. coli will be modified to obtain strains which can consume glucose and acetate simultaneously, and then the metabolic mechanism of coutilization will be studied to improve the consumption rate of acetate. Subsequently, the PLGA synthetic network, which comprises three modules including lactate synthesis from glucose, glycolate synthesis from acetate, and polymerization of lactate and glycolate, will be constructed. Next, the expression level of each functional module will be regulated through a modular optimization approach, with the aim to study the effects of different adaptation conditions on PLGA synthesis and monomer composition. Finally, a gene toggle switch will be employed to regulate the monomer synthesis of lactate and glycolate, achieving the dynamic supply of polymerization precursors, which would help to find out the expression strategy of gene circuit to produce PLGA block copolymers. Our project will construct a novel synthetic system, using glucose and acetate as substrates to synthesize PLGA with various compositions and structures, which could provide the research basis for fermentation production of PLGA. The relevant research ideas and technologies of this project can also provide references for other biosynthetic processes using acetate as the carbon source, thus indicating high value of both theoretical and practical significances.

聚乳酸-乙醇酸共聚物（PLGA）是一种具有良好生物相容性和生物可降解性的聚合物，目前主要通过化学法合成。本项目拟构建共利用葡萄糖和乙酸生产PLGA的大肠杆菌，并实现PLGA单体比例和结构的可控合成。首先，改造大肠杆菌中心代谢途径，获得共利用葡萄糖和乙酸的菌株，研究其代谢机制，提高乙酸利用速度。其次，构建包含葡萄糖合成乳酸、乙酸合成乙醇酸、乳酸乙醇酸聚合三个模块的PLGA合成网络。再次，利用模块化优化技术调节各功能模块的表达，研究不同适配条件影响PLGA合成及其单体比例的规律。最后，利用拨动开关控制乳酸和乙醇酸的单体合成，实现聚合前体的动态供给，寻找能够合成嵌段共聚物的基因线路表达策略。本项目将构建出新型人工合成体系，以葡萄糖和乙酸为底物获得组成和结构多样的PLGA，为其发酵法生产提供研究基础。项目相关研究思路和技术也能为其它以乙酸为碳源的生物合成过程提供参考，具有重要的理论和现实意义。

聚羟基脂肪酸酯是具有良好生物相容性和生物可降解性的高分子材料，目前主要由微生物利用葡萄糖等碳源发酵获得。本项目的主要研究内容是构建能够共利用葡萄糖和乙酸的新型重组大肠杆菌，利用动态调控技术控制代谢途径中关键酶的表达，获得一系列单体组成和结构不同的新型聚羟基脂肪酸酯。项目取得的重要结果和关键数据有：.（1）对大肠杆菌的中心代谢途径进行理性设计和改造，通过阻断丙酮酸向乙酰辅酶A的转化，成功获得了能够共利用葡萄糖和乙酸的工程菌株，利用10.27g/L葡萄糖和2g/L乙酸获得了9.61g/L丙酮酸，葡萄糖转化率达到理论得率的95%。.（2）对大肠杆菌的乙酸利用机制进行研究，通过过表达磷酸转乙酰酶和乙酸激酶，提高了乙酸的利用速度，以乙酸为碳源成功合成出乙醇酸单体，得率达到0.58g/g。.（3）利用葡萄糖和乙酸为混合碳源，合成出乳酸和3-羟基丁酸共聚酯，乳酸单体含量能够在一定范围内进行调控，磷酸转乙酰酶和乙酸激酶的过表达对乙酸代谢有明显促进作用。.（4）在大肠杆菌中引入密码子扩展技术，对影响乳酸单体聚合的辅酶A转移酶的表达进行动态调控，成功获得了组成和结构多样的乳酸和3-羟基丁酸共聚酯。.（5）项目还筛选到能够高效利用乙酸等挥发性脂肪酸高产聚-3-羟基丁酸酯的盐弧菌等新型菌株，乙酸耐受浓度超过100g/L，与自然界中耐受性最强的醋酸杆菌属相当，分批补料发酵的聚-3-羟基丁酸酯产量超过50g/L。.项目成功构建了能够共利用葡萄糖和乙酸生产聚羟基脂肪酸酯的工程菌株，并且筛选出高效利用挥发性脂肪酸产聚羟基脂肪酸酯的新菌，项目相关研究思路和技术也能为其它以乙酸等挥发性脂肪酸为碳源的生物合成过程提供参考，具有重要的理论和现实意义。