Department of Chemical and Biomolecular Engineering, The University of Tennessee, Knoxville, TN, USA; Center of Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA.
Center of Bioenergy Innovation, Oak Ridge National Laboratory, Oak Ridge, TN, USA; Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 37830, USA.
Metab Eng. 2022 Sep;73:38-49. doi: 10.1016/j.ymben.2022.05.003. Epub 2022 May 11.
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.
一碳递归酮酸延伸途径负责在活细胞中生成各种支链氨基酸、醛、醇、酮酸和醋酸酯。由于酶的混杂性、调控和代谢负担，控制这些目标分子在微生物中的高效选择性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的重新分配，以实现支链醋酸酯的选择性微生物生物合成。通过途径模块化，我们将支链酯途径分为四个子模块，包括将丙酮酸转化为酮异戊酸的酮异戊酸子模块、产生更长碳链酮酸的酮酸延伸子模块、将酮酸转化为醇的酮酸脱羧酶子模块，以及通过缩合醇和乙酰辅酶 A 生成支链醋酸酯的醇酰基转移酶子模块。通过系统地操纵途径基因复制和转录、这些子模块的第一关键步骤的酶特异性以及下游竞争途径，我们展示了异戊酸乙酯相对于异丁酸乙酯的选择性微生物生产。我们发现，优化的异戊酸乙酯途径全局重新分配了蛋白质组中的氨基酸分数，并需要高达 23-31%的蛋白质组重新分配，以牺牲其他细胞资源为代价，例如生成前体代谢物和能量以支持生长和氨基酸生物合成所需的资源。从葡萄糖补料分批发酵中，工程菌株生产的异戊酸乙酯最高达到 8.8 g/L（>0.25 g/L 毒性限制）的浓度、0.22 g/g 的产率（61%的最大理论值）和 86%的选择性，达到了迄今为止报道的异戊酸乙酯的最高浓度、产率和选择性。
