Dataset Information

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 | 2021 Apr

REPOSITORIES: biostudies-literature

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

Lei Ming M Peng Xiwei X Sun Wenjun W Zhang Di D Wang Zhenyu Z Yang Zhengjiao Z Zhang Chong C Yu Bin B Niu Huanqing H Ying Hanjie H Ouyang Pingkai P Liu Dong D Chen Yong Y

ACS omega 20210407 15

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 dehydro ...[more]

PMID: 34056170

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Project description:BackgroundExtremolytes enable microbes to withstand even the most extreme conditions in nature. Due to their unique protective properties, the small organic molecules, more and more, become high-value active ingredients for the cosmetics and the pharmaceutical industries. While ectoine, the industrial extremolyte flagship, has been successfully commercialized before, an economically viable route to its highly interesting derivative 5-hydroxyectoine (hydroxyectoine) is not existing.ResultsHere, we demonstrate high-level hydroxyectoine production, using metabolically engineered strains of C. glutamicum that express a codon-optimized, heterologous ectD gene, encoding for ectoine hydroxylase, to convert supplemented ectoine in the presence of sucrose as growth substrate into the desired derivative. Fourteen out of sixteen codon-optimized ectD variants from phylogenetically diverse bacterial and archaeal donors enabled hydroxyectoine production, showing the strategy to work almost regardless of the origin of the gene. The genes from Pseudomonas stutzeri (PST) and Mycobacterium smegmatis (MSM) worked best and enabled hydroxyectoine production up to 97% yield. Metabolic analyses revealed high enrichment of the ectoines inside the cells, which, inter alia, reduced the synthesis of other compatible solutes, including proline and trehalose. After further optimization, C. glutamicum Ptuf ectDPST achieved a titre of 74 g L-1 hydroxyectoine at 70% selectivity within 12 h, using a simple batch process. In a two-step procedure, hydroxyectoine production from ectoine, previously synthesized fermentatively with C. glutamicum ectABCopt, was successfully achieved without intermediate purification.ConclusionsC. glutamicum is a well-known and industrially proven host, allowing the synthesis of commercial products with granted GRAS status, a great benefit for a safe production of hydroxyectoine as active ingredient for cosmetic and pharmaceutical applications. Because ectoine is already available at commercial scale, its use as precursor appears straightforward. In the future, two-step processes might provide hydroxyectoine de novo from sugar.

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.

Dataset Information

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

Publications

Nonsterile l-Lysine Fermentation Using Engineered Phosphite-Grown <i>Corynebacterium glutamicum</i>.

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