基于橡胶聚合酶及自裂解的的一步法合成异戊橡胶基因工程菌构建的研究

Rubber is one of the most important resources; however, native rubber is very limited due to the strong depandence of plant growing on climate. Synthetic rubber is produced from petroleum, while the fast growing of economy,sustainable development is the current research focus and difficult problem.Biomass is very abundant on the earth,and the key resource is the unlimited solar energy. Thus, transformation of biomass into rubber maybe one of the suitable route to solve this problem. It is reported that the precusor of rubber isoprene could be produced in E. coli. However, isoprene is gas form under room temperature, shows a little cytotoxicity and difficult to be recovered. We propose a novel method that we produce rubber directly in E. coli. In the nature, plant produces rubber rather than isoprene by rubber cis-polyprenylcistransferase which catalyze polymerization of isopentenyl diphosphate and farnesyl diphosphate to accumulate native rubber. In our preliminary work, we constructed a genetically engineered E. coli to produce isoprene. Though this work, we understood the relevant metabolic pathway and prepared the experimental basis. Thus, by introducing the rubber cis-polyprenylcistransferase into E. coli we will realize microbial production of rubber. In this study, we will screen and investigate several rubber cis-polyprenylcistransferases from different plant sources. After that, the metabolic pathway will be optimized to facilitate the production of rubber in E. coli. To release rubber into fermentation medium, an autolysis system will be also investigated. This study will pave the way for biological production of rubber.

橡胶是极为重要的战略物资，天然橡胶依赖地理环境产量有限，而合成橡胶严重依赖石油，随着经济快速增长，如何可持续发展是目前的热点和难题。生物质资源丰富，且能源来自于取之不尽的太阳能，通过转化生物质为橡胶将是解决该问题的有效途径之一。虽然有报道用大肠杆菌产橡胶的前体分子异戊二烯，但由于其常温下为气态，又一定的细胞毒性并且回收困难，因此本项目另辟蹊径，提出在大肠杆菌中直接产异戊橡胶的新思路。植物通过橡胶合成酶将异戊二烯焦磷酸（亦为异戊二烯前体）聚合成异戊橡胶，并通过可控释放，积累天然橡胶。本项目预试验中，已成功地构建了产异戊二烯的大肠杆菌基因工程菌，取得了较好结果，并深入理解的相关代谢途径和夯实了所需的实验基础。因此，通过在大肠杆菌中导入橡胶合成酶，将可实现异戊橡胶的微生物合成。项目将筛选、研究不同来源橡胶合成酶的特性，及异戊橡胶积累响应型自裂解机理，为一步法产异戊橡胶奠定理论依据和实践基础。

本项目首先以大肠杆菌为宿主菌，以前体物异戊二烯为目标产物，探究了改造MEP与MVA途径对工程菌产异戊二烯的影响。通过对MEP代谢途径中的关键酶和限速酶基因进行过表达，强化了MEP途径，同时利用RNA干扰技术对副产物分流途径的基因进行弱化，相较与野生菌异戊二烯的产量提高了7倍，达到16 mg/L；在构建MVA途径过程中，通过表达外源基因，实现了该途径中异戊二烯的积累，其产量从调控MEP代谢途径的16 mg/L提高到55.4 mg/L，进一步结合启动子优化，基因表达盒子优化，发酵条件优化以及原位萃取分离技术等手段增强了异戊二烯的产量，最终可达到约587 mg/L，达到了较高水平。为分析高产工程菌的代谢网络，本项目采用荧光定量PCR对MEP及MVA代谢途径关键基因的转录情况进行了探究，结合代谢中间产物检测解析了异戊二烯的积累途径。在此基础上，本项目通过表达异戊二烯低聚体产物epi-isozizaene， pentalenene、α-isocomene、紫穗槐二烯、角鲨烯的合成酶打通了异戊二烯低聚体产物的合成途径，结合启动子及表达盒子的优化，代谢途径的强化、辅因子的供给、前体物的积累等手段，最终异戊二烯低聚体产物浓度分别达到epi-isozzaene 2.5mg/L，pentalenene 0.45mg/L，α-isocomene 0.013 mg/L，紫穗槐二烯24.4 mg/L，角鲨烯355 mg/L，为进一步合成异戊二烯高聚体奠定了基础。本项目分别表达了来源于橡胶树与拟南芥的橡胶合成酶基因，但由于该类酶的表达机制及机理不清晰，目前文献中也没有相关聚合酶的报道，因此本项目对该酶做了大量的改造及探索性工作，最终仍没有检测到聚异戊二烯高聚体的产出，对该聚合酶的表达以及调控机制还有待进一步研究。