Kinetic compartmentalization by unnatural reaction for itaconate production

Abstract

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.

Recent Progress in Metabolic Engineering of Escherichia coli for the Production of Various C4 and C5-Dicarboxylic Acids

“…Consequently, it may underline the need for an integrative, system-wide approach . With the aid of computational tools and genetically encoded biosensors, in silico metabolic analysis and adaptive laboratory evolution capable of genome-scale engineering have been applied in metabolic engineering. , These in silico tools and biosensors are also anticipated to reveal previously unknown enzymes with superior catalytic activity and enable more effective enzyme engineering than known enzymes. , They are expected to be also applied in DCAs production in E. coli . ,, …”

“…Rather than using enzymes directly derived from nature, enzyme engineering to have modified catalytic activity, stability, and ligand specificity can effectively enhance production performance. ,− These modifications use information related to sequence or structure. , Various strategies for enzyme engineering have been developed, such as rational design supported by in silico analysis or a mix-and-match approach that utilizes biosensor-guided high throughput screening. , …”

“…123,127−129 These modifications use information related to sequence or structure. 127,130 Various strategies for enzyme engineering have been developed, such as rational design supported by in silico analysis or a mix-and-match approach that utilizes biosensor-guided high throughput screening. 127,130 This method can be especially useful to boost the efficiency of itaconate production.…”

“…127,130 Various strategies for enzyme engineering have been developed, such as rational design supported by in silico analysis or a mix-and-match approach that utilizes biosensor-guided high throughput screening. 127,130 This method can be especially useful to boost the efficiency of itaconate production. The enzyme aconitase typically converts citrate to isocitrate with cis-aconitate as an intermediate.…”

“…This was accomplished by modifying 2-methylcitrate dehydratase (PrpD), an enzyme that can react with citrate, which shares structural similarities with 2methylcitrate, the natural substrate of PrpD. 127 The modified version of PrpD exhibited increased selectivity for citrate compared to that of the unaltered enzyme, verified by a biosensor capable of detecting itaconate. By balancing metabolic flux at the isocitrate junction, the final bacterial strain produced 5.06 g/L of itaconate with a yield of 0.33 g/g acetate in 80 h. This newly engineered enzyme was also used in glucose-based fermentation, causing a more than 3-fold increase in itaconate production compared to the traditional strain without the icd gene.…”