Model assisted multi step pathway design and construction for synthesis of B alanine in engineered E COLI

Abstract

Pathway engineering using multi-step enzymatic processes presents a newlinerobust alternative to chemical catalysis for the production of pharmaceuticals, newlinefine chemicals, and bulk chemicals, including biofuels and bio-polymers. newlineOvercoming major bottlenecks, such as the challenge of overexpressing newlinemultiple enzymes in soluble and functional form, enzyme inefficiency, and newlineregulatory obstacles, is crucial for achieving commercially viable product newlinetiters. This research work summarizes and demonstrates strategies for newlinedeveloping multi-enzymatic processes, aiming to understand regulatory newlinehurdles and optimize pathway design using and#946;-alanine as a model product. newlineExploration of and#946;-alanine production via a multi-cascade polyamine newlinepathway using 1,3-diaminopropane as a substrate achieved a titer of 1 g/L newlinewith Apaldh, NoxE, catalase, and DAO enzymes. LC-MS analysis confirmed newlineand#946;-alanine presence, with DAO identified as a major rate-limiting enzyme, newlinesuggesting efficient DAO scouting could increase titers. newlineAn efficient synthetic acrylate pathway was pursued as an alternative newlinepathway for and#946;-alanine production. Utilizing the acrylate pathway chassis for newlinepropionic acid production with enzymes including Pct, Acr from E. coli, and newlineLcd from Megasphaera elsdenii, based on previous experimental insights, newlineresulted in improved kinetic parameters and reduced size. Functional newlineexpression strategies, including sorbitol-induced folding and low newlinetemperatures, yielded significantly higher soluble protein yields for Pct, Acr, newlineand Lcd. newline