Metabolic engineering of Escherichia coli for quinolinic acid production by assembling L-aspartate oxidase and quinolinate synthase as an enzyme complex.

Quinolinic acid (QA) is a key intermediate of nicotinic acid (Niacin) which is an essential human nutrient and widely used in food and pharmaceutical industries. In this study, a quinolinic acid producer was constructed by employing comprehensive engineering strategies. Firstly, the quinolinic acid production was improved by deactivation of NadC (to block the consumption pathway), NadR (to eliminate the repression of L-aspartate oxidase and quinolinate synthase), and PtsG (to slow the glucose utilization rate and achieve a more balanced metabolism, and also to increase the availability of the precursor phosphoenolpyruvate). Further modifications to enhance quinolinic acid production were investigated by increasing the oxaloacetate pool through overproduction of phosphoenolpyruvate carboxylase and deactivation of acetate-producing pathway enzymes. Moreover, quinolinic acid production was accelerated by assembling NadB and NadA as an enzyme complex with the help of peptide-peptide interaction peptides RIAD and RIDD, which resulted in up to 3.7 g/L quinolinic acid being produced from 40 g/L glucose in shake-flask cultures. A quinolinic acid producer was constructed in this study, and these results lay a foundation for further engineering of microbial cell factories to efficiently produce quinolinic acid and subsequently convert this product to nicotinic acid for industrial applications. The authors engineered E. coli MG1655 to produce quinolinic acid, a key precursor of nicotinic acid, by employing comprehensive engineering strategies, including 1) enabling quinolinate production by deactivation of nadC (blocking consumption), nadR (eliminating transcriptional repression), and ptsG (increasing PEP availability for PEPC); 2) improving quinolinate production by overexpression of gene encoding PEPC (raising the OAA pool for aspartate synthesis), and deactivation of by-product acetate producing pathways; 3) accelerating quinolinate production by assembling NadA and NadB as an enzyme complex at a ratio of 2:1 with the help of peptide-peptide interaction peptides RIDD and RIAD. Up to 3.7 g/L quinolinate was produced from 40 g/L glucose in shake-flask cultures. [Display omitted] • Quinolinate producer was constructed by employing comprehensive engineering strategies. • Quinolinate production was improved by assembling NadA and NadB as an enzyme complex at a ratio of 2:1. • Up to 3.7 g/L quinolinate was produced in E. coli strain FZ763 with NadA-RIDD and NadB-RIAD. [ABSTRACT FROM AUTHOR]