异戊二烯新甲羟戊酸(MVA)生物合成途径构建及其动态感应调控系统研究

Isoprene is an important platform chemical, 95% of which was exploited for the production of synthetic rubber for tires. Furthermore, isoprene can also be widely used in the fields of isoprenoid medicines, fragrances, and aviation fuel. The usage of traditional chemical was largely limited by material resources. Microbial synthesis of isoprene by fermentation has become a promising and attractive route for environmental production, renewable resources and sustainable development. However, the microbial process is facing a severe problem of low yield as a result of lengthy steps of pathway and imbalanced metabolism. In this study, to construct a novel mevalonate(MVA)-mediated pathway, the different origins of enzymes including decarboxylase and dehydroxylase will be selected and evaluated. In comparison with traditional MVA pathway, the novel pathway is shortened by 3 steps. Meanwhile, the multiple-site saturation mutagenesis, fluorescence-activated cell sorting (FACS) and whole-genome transcript arrays will be used to establish a dynamic sensor-regulator system in the cell. The dynamic transcription level of genes of novel MVA-mediated pathway and the isoprene content in E. coli under the different regulation system will be analyzed by Real-Time PCR and chromatography analysis. This study is expected to lay the foundation for exploration on the relation between function and structure of two key enzymes of the novel MVA pathway. It will also provide a novel method for the regulation of metabolic pathway for the production of bio-isoprene as well as other bio-based chemicals.

异戊二烯是一种重要平台化合物，95%用于合成橡胶单体，亦广泛应用于医药、香料和航空燃料等领域。传统的化学工艺受原料瓶颈限制，生物法合成异戊二烯已成为国际研究热点。针对生物转化过程中由于野生代谢途径步骤冗长及代谢调控失衡而导致效率低下等问题，拟开展不同来源的脱羧酶和脱羟基酶的功能评价及改造研究，将天然MVA途径缩短3步反应，创制一条新的高效甲羟戊酸（MVA）代谢途径；在新途径构建基础上，为避免因中间代谢产物积累导致代谢调控失衡，拟采用饱和定点突变、荧光激活细胞分选技术及全基因组转录分析技术，在细胞体内开展途径动态感应调控系统的研究，通过实时定量PCR及色谱技术，考察动态调控系统对新途径基因转录及产物合成的影响规律，解析动态精确调控过程。本研究有望阐明新途径的两种关键酶结构与功能的关系，对改造生物合成代谢途径提供新的例证，为生物合成异戊二烯及其他生物基产品的代谢途径调控研究提供新的科学方法。

异戊二烯是一种重要平台化合物，95%用于合成橡胶单体，亦广泛应用于医药、香料和航空燃料等领域。传统的化学工艺受原料瓶颈限制，生物法合成异戊二烯已成为国际研究热点。针对天然生物合成途径步骤冗长及辅因子不平衡而导致效率低下的问题，本项目首先研究了不同来源的脱羧酶和脱水酶的功能性质，将天然MVA途径缩短3步反应，创制了一条新的高效甲羟戊酸（MVA）代谢途径；其次，本研究利用异戊二烯和1,3-丙二醇联产的新策略实现了辅因子代谢平衡调控。异戊二烯合成途径还原力不平衡，每生成1分子异戊二烯同时净生成4分子的还原力NAD(P)H，也是导致异戊二烯产率降低的主要原因。我们成功构建了联产菌株，并通过对诱导条件、补料策略和通气氧浓度等的优化，使得异戊二烯和1,3-PDO产率分别达到9.1%和62%。与优化前相比，异戊二烯和1,3-PDO的产率分别提高2.5倍和3.3倍。另外还通过构建非磷酸葡萄糖转移酶系统（简称PTS系统）依赖型葡萄糖转运途径，增加了NADPH在1,3-PDO合成中的有效供应，联产菌株合成1,3-PDO所消耗的NADPH占异戊二烯合成途径净生成NADPH总量的比例提高到85.2%，比PTS野生型联产菌株提高了2.6倍。第三，本研究探索了以花生壳和木质素水解液为原料，生物转化合成异戊二烯，为生物基异戊二烯的可持续工业化发展提供了新原料技术路线。综上所述，本研究完成了甲羟戊酸途径的改造，简化了天然代谢途径，实现了还原力的平衡，探索了新原料合成异戊二烯的路线，为天然生物合成途径的改造提供了新的例证，为生物合成异戊二烯及其他生物基产品的代谢途径理性设计和改造提供新的科学方法。