Improved l-phenylglycine synthesis by introducing an engineered cofactor self-sufficient system

Abstract

l -phenylglycine (L-phg) is a valuable non-proteinogenic amino acid used as a precursor to β-lactam antibiotics, antitumor agent taxol and many other pharmaceuticals. L-phg synthesis through microbial bioconversion allows for high enantioselectivity and sustainable production, which will be of great commercial and environmental value compared with organic synthesis methods. In this work, an L-phg synthesis pathway was built in Escherichia coli resulting in 0.23 mM L-phg production from 10 mM l -phenylalanine. Then, new hydroxymandelate synthases and hydroxymandelate oxidases were applied in the L-phg synthesis leading to a 5-fold increase in L-phg production. To address 2-oxoglutarate, NH 4 + , and NADH shortage, a cofactor self-sufficient system was introduced, which converted by-product l -glutamate and NAD + to these three cofactors simultaneously. In this way, L-phg increased 2.5-fold to 2.82 mM. Additionally, in order to reduce the loss of these three cofactors, a protein scaffold between synthesis pathway and cofactor regeneration modular was built, which further improved the L-phg production to 3.72 mM with a yield of 0.34 g/g L-phe. This work illustrated a strategy applying for whole-cell biocatalyst converting amino acid to its value-added chiral amine in a cofactor self-sufficient manner.

Design of a cofactor self‐sufficient whole‐cell biocatalyst for enzymatic asymmetric reduction via engineered metabolic pathways and multi‐enzyme cascade

“…Many strategies focused on the supply and regeneration of NAD(P)H cofactor have been developed in biocatalysis and biotransformation by researchers. [ 5–10 ] For example, cheaper artificial analogs of nicotinamide cofactors have emerged as alternatives to more expensive natural nicotinamide cofactors because of their similar chemical structure [ 11 ] ; however, using artificial cofactors resulted in the enzymatic efficiencies being substantially decreased compared with the same amount of stoichiometric use of natural cofactors. Besides, NAD(P)H was recycled to regenerate under a sacrificial substrate as the electron donor by introducing a heterologous NAD(P)+ dependent enzyme coupled with NAD(P)H dependent enzyme.…”

Engineering of Hydroxymandelate Oxidase and Cascade Reactions for High-Yielding Conversion of Racemic Mandelic Acids to Phenylglyoxylic Acids and (R)- and (S)-Phenylglycines

“…The amination of racemic alcohols to enantiopure amines could be achieved by one-pot cascade reactions consisting of full oxidation of racemic alcohols and enantioselective amination of ketones. The enzyme cascades could combine ADH or AOx, with transaminase (TA), amine dehydrogenase, or amino acid dehydrogenase (AADH), ,,,− ,− but their practical application depends on the enzymes for the full oxidation of racemic alcohols and enantioselective amination, as well as the system for the recycling of the necessary cofactor. We are interested in engineering AOx-MR-TA cascade reactions to convert racemic α-hydroxy acids to enantiopure α-amino acids (Scheme b).…”