生物合成己二酸的代谢工程研究进展

Metabolic engineering research progress of biosynthesis of adipic acid

己二酸是一种重要的大宗化学品,主要用于合成尼龙和聚氨酯泡沫塑料,市场需求巨大,其高效生物合成至今还未实现。本文概括了酿酒酵母中构建和优化新的己二酸合成途径研究进展。首先,通过体内及体外活性测试,对催化每一步反应的酶进行筛选,构建初步的代谢途径。利用组学分析诊断和定位生物合成途径的瓶颈。对于途径中的限速酶采用蛋白质工程手段进行改造。其次,利用合成生物学和代谢工程手段优化代谢途径。优化手段具体包括:通过模块化优化,平衡各个基因之间的表达;利用蛋白支架,构建酶反应的流水线,减少中间产物的扩散,提高反应效率;通过RNA干扰技术抑制竞争代谢途径的流量,提高目标代谢途径的通量。最后,利用CRISPR/Cas9及全局转录机器工程(gTME)等最新技术进行基因组编辑、重排转录网络,最终获得己二酸的高产菌株及适用于高效生产其他芳香族化合物的底盘酵母菌株。

Adipic acid is an important bulk chemical and is mainly used for the synthesis of nylon and polyurethane foam.It has a huge market,but the efficient total biosynthesis of this compound has not been achieved yet.This paper summarized the fabrication and optimization of novel artificial adipic acid biosynthetic pathway from Saccharomyces cerevisiae.First,the in vivo and in vitro assays were carried out to screen for efficient enzymes.The initial biosynthetic pathways for adipic acid production were constructed.The possible bottlenecks of the biosynthetic pathways were detected using transcriptomics,proteomics and metabolomics tools.The rate-limiting steps in the pathways were eliminated using protein engineering methods.Then,the metabolic pathways were optimized and adipic acid overproducing strains were obtained through strategies of synthetic biology and metabolic engineering.The strategies included：balancing gene expression using modular optimization;building enzyme pipelines using protein scaffolds,reducing the diffusion of intermediates,and improving the reaction efficiency;using anti-sense RNAs to turn down competing pathways and increase the carbon flux through target pathway.In the end,the most advanced technologies,such as genome editing and global transcription machinery engineering were used to obtain chassis yeast platform for the production of adipic acid and other aromatic compounds.