Physical compartmentalization of metabolism using membranous organelles in eukaryotes is helpful for chemical biosynthesis to ensure the availability of substrates from competitive metabolic reactions. Bacterial hosts lack such a membranous system, which is one of the major limitations for efficient metabolic engineering. Here, we employ kinetic compartmentalization with the introduction of an unnatural enzymatic reaction by an engineered enzyme as an alternative strategy to enable substrate availability from competitive reactions through kinetic isolation of metabolic pathways. As a proof of concept, we kinetically isolate the itaconate synthetic pathway from the tricarboxylic acid cycle in Escherichia coli, which is natively separated by mitochondrial membranes in Aspergillus terreus. Specifically, 2-methylcitrate dehydratase is engineered to alternatively catalyze citrate and kinetically secure cis-aconitate for efficient production using a high-throughput screening system. Itaconate production can be significantly improved with kinetic compartmentalization and its strategy has the potential to be widely applicable.

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As an alternative to petrochemical synthesis, well-established industrial microbes, such as Escherichia coli, are employed to produce a wide range of chemicals, including dicarboxylic acids (DCAs), which have significant potential in diverse areas including biodegradable polymers. The demand for biodegradable polymers has been steadily rising, prompting the development of efficient production pathways on four-(C4) and five-carbon (C5) DCAs derived from central carbon metabolism to meet the increased demand via the biosynthesis. In this context, E. coli is utilized to produce these DCAs through various metabolic engineering strategies, including the design or selection of metabolic pathways, pathway optimization, and enhancement of catalytic activity. This review aims to highlight the recent advancements in metabolic engineering techniques for the production of C4 and C5 DCAs in E. coli.