Genetic engineering approaches for the fermentative production of phenylglycines

Abstract

L-phenylglycine (L-Phg) is a rare non-proteinogenic amino acid, which only occurs in some natural compounds, such as the streptogramin antibiotics pristinamycin I and virginiamycin S or the bicyclic peptide antibiotic dityromycin. Industrially, more interesting than L-Phg is the enantiomeric D-Phg as it plays an important role in the fine chemical industry, where it is used as a precursor for the production of semisynthetic β-lactam antibiotics. Based on the natural L-Phg operon from Streptomyces pristinaespiralis and the stereo-inverting aminotransferase gene hpgAT from Pseudomonas putida, an artificial D-Phg operon was constructed. The natural L-Phg operon, as well as the artificial D-Phg operon, was heterologously expressed in different actinomycetal host strains, which led to the successful production of Phg. By rational genetic engineering of the optimal producer strains S. pristinaespiralis and Streptomyces lividans, Phg production could be improved significantly. Here, we report on the development of a synthetic biology-derived D-Phg pathway and the optimization of fermentative Phg production in actinomycetes by genetic engineering approaches. Our data illustrate a promising alternative for the production of Phgs.

Overview on Multienzymatic Cascades for the Production of Non-canonical α-Amino Acids

“…The same strategy was used to produce L-PheGly, but using L-4-hydroxyphenylglycine transaminases from A. orientalis and S. coelicolor (Liu et al, 2014). Another synthetic biology-derived D-Phg operon has been recently developed on the basis of the natural lpg operon from S. pristinaespiralis; substitution of the natural PglE TA by the stereoinverting HpgAT allowed the creation of different plasmids which were utilized for the synthesis of D-PheGly in different engineered actinomycetal expression strains ( Figure 11A, Moosmann et al, 2020).…”

“…values ranging from 94 to >99% (Figure 11F, Parmeggiani et al, 2019b). One (Moosmann et al, 2020). (B) Conversion of styrenes to L-PheGly derivatives with E. coli multimodular systems.…”

“…PglE, L-PheGly aminotransferase. Inclusion of the stereoinverting D-(4-hydroxy)-PheGly aminotransferase from Pseudomonas putida (HpgAT) allowed the production of D -PheGly ( Moosmann et al, 2020 ). (B) Conversion of styrenes to L -PheGly derivatives with E. coli multimodular systems.…”

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

“…Most of them are non-proteinogenic chiral amino acid, which are becoming increasingly important to modern drug discovery. These amino acids are used as antimetabolites of common amino acids and serve as effective inhibitors for a series of metabolic targets [ 2 ]. It is estimated that optically active amine including amino acids make up approximately 40% of all chiral intermediates involved in the production of active pharmaceutical ingredients [ 3 ].…”

“…A fermentative process to produce chiral amino acids is preferable because of its high enantioselectivity, sustainable raw material, and relatively reduced environmental detriments. Previous studies have reported overproduction of L-phg based on glucose, l -phenylalanine (L-phe), and other substrates [ 2 , [8] , [9] , [10] ].…”