Metabolic Engineering ( IF 8.4 ) Pub Date : 2022-05-11 , DOI: 10.1016/j.ymben.2022.05.003 Hyeongmin Seo 1 , Richard J Giannone 2 , Yung-Hun Yang 3 , Cong T Trinh 1
The one-carbon recursive ketoacid elongation pathway is responsible for making various branched-chain amino acids, aldehydes, alcohols, ketoacids, and acetate esters in living cells. Controlling selective microbial biosynthesis of these target molecules at high efficiency is challenging due to enzyme promiscuity, regulation, and metabolic burden. In this study, we present a systematic modular design approach to control proteome reallocation for selective microbial biosynthesis of branched-chain acetate esters. Through pathway modularization, we partitioned the branched-chain ester pathways into four submodules including ketoisovalerate submodule for converting pyruvate to ketoisovalerate, ketoacid elongation submodule for producing longer carbon-chain ketoacids, ketoacid decarboxylase submodule for converting ketoacids to alcohols, and alcohol acyltransferase submodule for producing branched-chain acetate esters by condensing alcohols and acetyl-CoA. By systematic manipulation of pathway gene replication and transcription, enzyme specificity of the first committed steps of these submodules, and downstream competing pathways, we demonstrated selective microbial production of isoamyl acetate over isobutyl acetate. We found that the optimized isoamyl acetate pathway globally redistributed the amino acid fractions in the proteomes and required up to 23–31% proteome reallocation at the expense of other cellular resources, such as those required to generate precursor metabolites and energy for growth and amino acid biosynthesis. From glucose fed-batch fermentation, the engineered strains produced isoamyl acetate up to a titer of 8.8 g/L (>0.25 g/L toxicity limit), a yield of 0.22 g/g (61% of maximal theoretical value), and 86% selectivity, achieving the highest titers, yields and selectivity of isoamyl acetate reported to date.
中文翻译:
蛋白质组重新分配使非线性、支链乙酸酯的选择性从头生物合成成为可能
一碳递归酮酸延伸途径负责在活细胞中制造各种支链氨基酸、醛、醇、酮酸和乙酸酯。由于酶混杂、调节和代谢负担,高效控制这些靶分子的选择性微生物生物合成具有挑战性。在这项研究中,我们提出了一种系统的模块化设计方法来控制蛋白质组重新分配,以用于支链乙酸酯的选择性微生物生物合成。通过途径模块化,我们将支链酯途径划分为四个子模块,包括用于将丙酮酸转化为酮异戊酸的酮异戊酸子模块、用于产生更长碳链酮酸的酮酸延长子模块、用于将酮酸转化为醇的酮酸脱羧酶子模块、和醇酰基转移酶子模块,用于通过缩合醇和乙酰辅酶A生产支链乙酸酯。通过系统操作途径基因复制和转录、这些子模块的第一个承诺步骤的酶特异性以及下游竞争途径,我们证明了乙酸异戊酯的选择性微生物生产优于乙酸异丁酯。我们发现优化的乙酸异戊酯途径在全球范围内重新分配了蛋白质组中的氨基酸部分,并且需要以牺牲其他细胞资源为代价进行高达 23-31% 的蛋白质组重新分配,例如产生前体代谢物和生长所需能量以及氨基酸所需的资源生物合成。通过葡萄糖补料分批发酵,工程菌株产生的乙酸异戊酯滴度高达 8.8 g/L(>0.25 g/L 毒性极限),产量为 0。