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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, mainly used in the synthesis of nylon and polyurethane foam, the huge market demand, the efficient biosynthesis has not yet been achieved. This review summarizes the recent advances in the construction and optimization of new adipate biosynthetic pathways in Saccharomyces cerevisiae. First of all, through the in vivo and in vitro activity tests, the enzymes that catalyze each reaction are screened to construct a preliminary metabolic pathway. Use of omics to diagnose and locate bottlenecks in biosynthetic pathways. The rate-limiting enzyme in the pathway was engineered using protein engineering. Second, the use of synthetic biology and metabolic engineering means to optimize metabolic pathways. Optimization means specifically include: modulating the expression of each gene through modular optimization; using a protein scaffold to build a pipeline of enzyme reactions to reduce the diffusion of intermediates and improve the reaction efficiency; inhibiting the flow of competing metabolic pathways by RNA interference technology and improving The flux of the target metabolic pathway. Finally, genomic editing and rearrangement of the transcribed network using the latest technologies such as CRISPR / Cas9 and gTME have led to the production of high-yielding strains of adipic acid and to bottom-plate yeast strains for the efficient production of other aromatic compounds.