Dataset Information

Reverse Engineering Targets for Recombinant Protein Production in Corynebacterium glutamicum Inspired by a Fast-Growing Evolved Descendant.

ABSTRACT: We previously reported a Corynebacterium glutamicum JH41 strain with a 58% faster growth rate through application of adaptive laboratory evolution. To verify that the fast-reproducing strain was useful as a host for recombinant protein expression, we introduced a plasmid responsible for the secretory production of a recombinant protein. The JH41 strain harboring the plasmid indeed produced the secretory recombinant protein at a 2.7-fold greater rate than its ancestral strain. To provide the reverse engineering targets responsible for boosting recombinant protein production and cell reproduction, we compared the genome sequence of the JH41 strain with its ancestral strain. Among the 15 genomic variations, a point mutation was confirmed in the 14 bases upstream of NCgl1959 (encoding a presumed siderophore-binding protein). This mutation allowed derepression of NCgl1959, thereby increasing iron consumption and ATP generation. A point mutation in the structural gene ramA (A239G), a LuxR-type global transcription regulator involved in central metabolism, allowed an increase in glucose consumption. Therefore, mutations to increase the iron and carbon consumption were concluded as being responsible for the enhanced production of recombinant protein and cell reproduction in the evolved host.

SUBMITTER: Lee MJ

PROVIDER: S-EPMC7755716 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Reverse Engineering Targets for Recombinant Protein Production in <i>Corynebacterium glutamicum</i> Inspired by a Fast-Growing Evolved Descendant.

Lee Min Ju MJ Park Jihoon J Park Kyunghoon K Kim Jihyun F JF Kim Pil P

Frontiers in bioengineering and biotechnology 20201209

We previously reported a <i>Corynebacterium glutamicum</i> JH41 strain with a 58% faster growth rate through application of adaptive laboratory evolution. To verify that the fast-reproducing strain was useful as a host for recombinant protein expression, we introduced a plasmid responsible for the secretory production of a recombinant protein. The JH41 strain harboring the plasmid indeed produced the secretory recombinant protein at a 2.7-fold greater rate than its ancestral strain. To provide t ...[more]

PMID: 33363126

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Project description:The Gram-positive soil bacterium Corynebacterium glutamicum is widely used in industrial fermentative processes for the production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose is taken up into the C. glutamicum cell by the phosphotransferase system PTS which can be replaced and/or enhanced by a permease and a glucokinase. Heterologous expression of the gene for the high-affinity glucose permease from Streptomyces coelicolor and of the Bacillus subitilis glucokinase gene fully compensated for the absence of the PTS in ï??hpr strains and strains grew as fast with glucose as C. glutamicum wild type. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and the glucokinase from Bacillus subtilis were overproduced using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid, a 40% higher L-lysine titer and a 30% higher volumetric productivity as compared to GRLys1(pEKEx3) resulted. The non-proteinogenic amino acid pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the L-lysine producing strain, the L-Lysine dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were expressed as synthetic operon. This enabled C. glutamicum to L-PA with a yield of 0.49 Â± 0.03 gg-1 and a volumetric productivity of 0.04 Â± 0.00 gL-1h-1.To the best of our knowledge, this is the first fermentative process for the production of L-PA. Two conditions tested, 200 mM NaCl Vs 200 mM pipecolic supplemented in the culture medium, control experiments done with the addition of 200mM of NaCl. Four technical replicates.

Dataset Information

Reverse Engineering Targets for Recombinant Protein Production in Corynebacterium glutamicum Inspired by a Fast-Growing Evolved Descendant.

Publications

Reverse Engineering Targets for Recombinant Protein Production in <i>Corynebacterium glutamicum</i> Inspired by a Fast-Growing Evolved Descendant.

Similar Datasets

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