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.

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.

Background Owing to increasing concerns about climate change and the depletion of fossil fuels, the development of efficient microbial processes for biochemical production from lignocellulosic biomass has been a key issue. Because process efficiency is greatly affected by the inherent metabolic activities of host microorganisms, it is essential to utilize a microorganism that can rapidly convert biomass-derived sugars. Here, we report a novel Vibrio-based microbial platform that can rapidly and simultaneously consume three major lignocellulosic sugars (i.e., glucose, xylose, and arabinose) faster than any previously reported microorganisms. Results The xylose isomerase pathway was constructed in Vibrio sp. dhg, which naturally displays high metabolic activities on glucose and arabinose but lacks xylose catabolism. Subsequent adaptive laboratory evolution significantly improved xylose catabolism of initial strain and led to unprecedently high growth and sugar uptake rate (0.67 h−1 and 2.15 g gdry cell weight−1 h−1, respectively). Furthermore, we achieved co-consumption of the three sugars by deletion of PtsG and introduction of GalP. We validated its superior performance and applicability by demonstrating efficient lactate production with high productivity (1.15 g/L/h) and titer (83 g/L). Conclusions In this study, we developed a Vibrio-based microbial platform with rapid and simultaneous utilization of the three major sugars from lignocellulosic biomass by applying an integrated approach of rational and evolutionary engineering. We believe that the developed strain can be broadly utilized to accelerate the production of diverse biochemicals from lignocellulosic biomass.

No abstract

No abstract