一碳化合物利用新途径的设计和体外构建

摘要

一碳化合物作为糖类的廉价替代底物在工业生物技术中具有广泛的应用前景.传统途径中由一碳化合物或糖类生产乙酰辅酶A衍生产品时会有1/3的碳损失,导致产品得率较低,因此找到没有碳损失的新途径非常重要.基于MetaCyc数据库的代谢反应集和通量平衡分析的途径分析方法,计算得到一条由一碳化合物甲醛生成乙酰辅酶A衍生产品的新途径,该途径不仅无碳损失,并且无能量(ATP)和还原力消耗.为了验证途径的可行性,筛选途径相关酶,纯化后进行体外实验,通过乙酸的产量来间接表征途径的运行情况.结果发现:乙酸的生成浓度会随着体系初始甲醛加入量的增加而增加,在一定条件下,乙酸的总碳摩尔得率可以达到90％,超过了传统路径的最大理论得率67％.%C1 compounds can be widely used in industrial biotechnology as low-cost alternative substrates to sugars.In normal pathways for the synthesis of acetyl coenzyme A derived products from C1 compound, one third of the carbon atoms from the substrate is wasted as CO2 ,and thus the product yield is low. Therefore,it is important to find new carbon conserved synthesis pathways for acetyl-CoA derived products from C1 compounds.In this study,we built a large metabolic network based on reactions from MetaCyc database and used the flux balance analysis method to find a new pathway from formaldehyde to acetyl coenzyme A derived products.The new pathway is carbon conserved and ATP independent.We screened and purified the enzymes in the pathway and tested the pathway in vitro using acetate as the target product.The concentration of acetate produced from the system is enhanced with the increased addition of formaldehyde substrate.The carbon molar yield can achieve 90％ under certain conditions,exceeding the theoretical yield of 67％ from naturally existing pathways.