Journal Article

Production of p-amino-l-phenylalanine (l-PAPA) from glycerol by metabolic grafting of Escherichia coli

Sprenger, Georg A., Youn, Jung-Won, Mohammadi Nargesi, Behrouz, Trachtmann, Natalie

2018

Overview

Background The non-proteinogenic aromatic amino acid, p -amino- l -phenylalanine ( l -PAPA) is a high-value product with a broad field of applications. In nature, l -PAPA occurs as an intermediate of the chloramphenicol biosynthesis pathway in Streptomyces venezuelae . Here we demonstrate that the model organism Escherichia coli can be transformed with metabolic grafting approaches to result in an improved l -PAPA producing strain. Results Escherichia coli K-12 cells were genetically engineered for the production of l -PAPA from glycerol as main carbon source. To do so, genes for a 4-amino-4-deoxychorismate synthase ( pabAB from Corynebacterium glutamicum ), and genes encoding a 4-amino-4-deoxychorismate mutase and a 4-amino-4-deoxyprephenate dehydrogenase ( papB and papC , both from Streptomyces venezuelae ) were cloned and expressed in E. coli W3110 (lab strain LJ110). In shake flask cultures with minimal medium this led to the formation of ca. 43 ± 2 mg l −1 of l -PAPA from 5 g l −1 glycerol. By expression of additional chromosomal copies of the tktA and glpX genes, and of plasmid-borne aroFBL genes in a tyrR deletion strain, an improved l -PAPA producer was obtained which gave a titer of 5.47 ± 0.4 g l −1 l -PAPA from 33.3 g l −1 glycerol (0.16 g l -PAPA/g of glycerol) in fed-batch cultivation (shake flasks). Finally, in a fed-batch fermenter cultivation, a titer of 16.7 g l −1 l -PAPA was obtained which is the highest so far reported value for this non-proteinogenic amino acid. Conclusion Here we show that E. coli is a suitable chassis strain for l -PAPA production. Modifying the flux to the product and improved supply of precursor, by additional gene copies of glpX , tkt and aroFBL together with the deletion of the tyrR gene, increased the yield and titer.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Amino acids / Antibiotics / Applied Microbiology / Bacteria / Batch culture / Biosynthesis / Biotechnology / Carbon sources / Cell culture / Chassis / Chemistry / Chemistry and Materials Science / Chloramphenicol / Chloromycetin / Clonal deletion / Cloning / Coliforms / Corynebacterium glutamicum / Cultivation / Dehydrogenases / E coli / Engineering / Enzymology / Escherichia coli / Flasks / Gene deletion / Gene expression / Genes / Genetic aspects / Genetic Engineering / Genetic research / Genetic transformation / Glycerol / Grafting / Laboratories / Metabolic grafting / Metabolism / Metabolites / Microbial Genetics and Genomics / Microbiology / Microorganisms / Non-proteinogenic aromatic amino acids / p-Amino-l-phenylalanine / Phenylalanine / Properties / Streptomyces venezuelae / TyrR gene