短链酯作为香料具有广泛的用途,香水,溶剂,和生物燃料。控制酯微生物生物合成的选择性一直是一个突出的代谢工程问题。这里,我们提出了一个可推广的框架,以实现丁酰辅酶A衍生的设计酯的从头发酵微生物生物合成(例如,乙酸丁酯,丁酸乙酯,丁酸丁酯)具有可控的选择性。采用模块化设计原则,我们产生了丁酰辅酶A衍生的酯途径,作为可交换的生产模块,与工程化的底盘细胞兼容,用于厌氧生产设计酯。我们设计了这些源自酰基CoA子模块的模块(例如,乙酰辅酶A,丁酰辅酶A),酒精子模块(例如,乙醇,丁醇),辅因子再生子模块(例如,NADH),和醇乙酰转移酶(AAT)子模块(例如,ATF1、SAAT)通过操纵复制(例如,质粒拷贝数),转录(例如,promotors),翻译(例如,密码子优化),途径酶,和途径诱导条件。为了进一步提高高选择性设计酯的产量,我们使用蛋白质融合标签和分子伴侣系统地筛选了各种蛋白质增溶策略,以提高多个途径酶的可溶性表达。最后,我们的工程产酯菌株可以使乙酸丁酯产量增加19倍(0.64g/L,96%的选择性),丁酸乙酯产量增加6倍(0.41g/L,86%选择性),丁酸丁酯产量增加13倍(0.45克/升,54%的选择性)与初始菌株相比。总的来说,这项研究提出了一个可推广的框架来设计模块化微生物平台,用于从可再生原料厌氧生产丁酰辅酶A衍生的设计酯。
Short-chain esters have broad utility as flavors, fragrances, solvents, and biofuels. Controlling selectivity of ester microbial biosynthesis has been an outstanding metabolic engineering problem. Here, we present a generalizable framework to enable the de novo fermentative microbial biosynthesis of butyryl-CoA-derived designer esters (e.g., butyl acetate, ethyl butyrate, butyl butyrate) with controllable selectivity. Using the modular design principles, we generated the butyryl-CoA-derived ester pathways as exchangeable production modules compatible with an engineered chassis cell for anaerobic production of designer esters. We designed these modules derived from an acyl-CoA submodule (e.g., acetyl-CoA, butyryl-CoA), an alcohol submodule (e.g., ethanol, butanol), a cofactor regeneration submodule (e.g., NADH), and an alcohol acetyltransferase (AAT) submodule (e.g., ATF1, SAAT) for rapid module construction and optimization by manipulating replication (e.g., plasmid copy number), transcription (e.g., promoters), translation (e.g., codon optimization), pathway enzymes, and pathway induction conditions. To further enhance production of designer esters with high selectivity, we systematically screened various strategies of protein solubilization using protein fusion tags and chaperones to improve the soluble expression of multiple pathway enzymes. Finally, our engineered ester-producing strains could achieve 19-fold increase in butyl acetate production (0.64 g/L, 96% selectivity), 6-fold increase in ethyl butyrate production (0.41 g/L, 86% selectivity), and 13-fold increase in butyl butyrate production (0.45 g/L, 54% selectivity) as compared to the initial strains. Overall, this study presented a generalizable framework to engineer modular microbial platforms for anaerobic production of butyryl-CoA-derived designer esters from renewable feedstocks.