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Nonsterile l-Lysine Fermentation Using Engineered Phosphite-Grown Corynebacterium glutamicum.

ABSTRACT: Fermentation using Corynebacterium glutamicum is an important method for the industrial production of amino acids. However, conventional fermentation processes using C. glutamicum are susceptible to microbial contamination and therefore require equipment sterilization or antibiotic dosing. To establish a more robust fermentation process, l-lysine-producing C. glutamicum was engineered to efficiently utilize xenobiotic phosphite (Pt) by optimizing the expression of Pt dehydrogenase in the exeR genome locus. This ability provided C. glutamicum with a competitive advantage over common contaminating microbes when grown on media containing Pt as a phosphorus source instead of phosphate. As a result, the engineered strain could produce 41.00 g/L l-lysine under nonsterile conditions during batch fermentation for 60 h, whereas the original strain required 72 h to produce 40.78 g/L l-lysine under sterile conditions. Therefore, the recombinant strain can efficiently produce l-lysine under nonsterilized conditions with unaffected production efficiency. Although this anticontamination strategy has been previously reported for other species, this is the first time it has been demonstrated in C. glutamicum; these findings should aid in the further development of cost-efficient amino acid fermentation processes.

SUBMITTER: Lei M

PROVIDER: S-EPMC8153679 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:N-alkylated amino acids occur widely in nature and can also be found in bioactive secondary metabolites such as the glycopeptide antibiotic vancomycin and the immunosuppressant cyclosporine A. To meet the demand for N-alkylated amino acids, they are currently produced chemically; however, these approaches often lack enantiopurity, show low product yields and require toxic reagents. Fermentative routes to N-alkylated amino acids like N-methyl-l-alanine or N-methylantranilate, a precursor of acridone alkaloids, have been established using engineered Corynebacterium glutamicum, which has been used for the industrial production of amino acids for decades. Here, we describe metabolic engineering of C. glutamicum for de novo production of N-methylphenylalanine based on reductive methylamination of phenylpyruvate. Pseudomonas putida Δ-1-piperideine-2-carboxylate reductase DpkA containing the amino acid exchanges P262A and M141L showed comparable catalytic efficiencies with phenylpyruvate and pyruvate, whereas the wild-type enzyme preferred the latter substrate over the former. Deletion of the anthranilate synthase genes trpEG and of the genes encoding branched-chain amino acid aminotransferase IlvE and phenylalanine aminotransferase AroT in a strain engineered to overproduce anthranilate abolished biosynthesis of l-tryptophan and l-phenylalanine to accumulate phenylpyruvate. Upon heterologous expression of DpkAP262A,M141L, N-methylphenylalanine production resulted upon addition of monomethylamine to the medium. In glucose-based minimal medium, an N-methylphenylalanine titer of 0.73 ± 0.05 g L-1, a volumetric productivity of 0.01 g L-1 h-1 and a yield of 0.052 g g-1 glucose were reached. When xylose isomerase gene xylA from Xanthomonas campestris and the endogenous xylulokinase gene xylB were expressed in addition, xylose as sole carbon source supported production of N-methylphenylalanine to a titer of 0.6 ± 0.04 g L-1 with a volumetric productivity of 0.008 g L-1 h-1 and a yield of 0.05 g g-1 xylose. Thus, a fermentative route to sustainable production of N-methylphenylalanine by recombinant C. glutamicum has been established.

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Nonsterile l-Lysine Fermentation Using Engineered Phosphite-Grown Corynebacterium glutamicum.

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