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Pathway engineering in Corynebacterium glutamicum S9114 for 5-aminolevulinic acid production.

ABSTRACT: 5-Aminolevulinic acid (ALA) is a non-protein amino acid with a significant potential for cancer treatment and plant stress resistance. Microbial fermentation has gradually replaced the traditional chemical-based method for ALA production, thus increasing the need for high-ALA-producing strains. In this study, we engineered the glutamate producing strain, Corynebacterium glutamicum S9114, for ALA production. To efficiently convert l-glutamate to ALA, hemA and hemL from Salmonella typhimurium and Escherichia coli were tandemly overexpressed. In addition, ncgl1221 encoding a glutamate transporter was deleted to block glutamate secretion and thus improve ALA production. Furthermore, the intrinsic ribosome-binding site (RBS) of hemB was replaced by a relatively weak RBS to reduce the conversion of ALA to porphyrin. Transcriptional and fermentation data confirmed that inactivation of lysE and putP reduced the conversion of glutamate to arginine and proline, which also contribute to ALA production. The final SA14 strain produced 895 mg/L concentration of ALA after 72 h incubation in a shake flask. This amount was 58-fold higher than that obtained by the parent strain C. glutamicum S9114. The results demonstrate the potential of C. glutamicum S9114 for efficient ALA production and provide new targets for the development of ALA-producing strains.

SUBMITTER: Zhang B

PROVIDER: S-EPMC5940623 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Pathway engineering in Corynebacterium glutamicum S9114 for 5-aminolevulinic acid production.

Zhang Bin B Ye Bang-Ce BC

3 Biotech 20180508 5

5-Aminolevulinic acid (ALA) is a non-protein amino acid with a significant potential for cancer treatment and plant stress resistance. Microbial fermentation has gradually replaced the traditional chemical-based method for ALA production, thus increasing the need for high-ALA-producing strains. In this study, we engineered the glutamate producing strain, Corynebacterium glutamicum S9114, for ALA production. To efficiently convert l-glutamate to ALA, hemA and hemL from Sal ...[more]

PMID: 29744279

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Project description:BackgroundItaconic acid is a promising platform chemical for a bio-based polymer industry. Today, itaconic acid is biotechnologically produced with Aspergillus terreus at industrial scale from sugars. The production of fuels but also of chemicals from food substrates is a dilemma since future processes should rely on carbon sources which do not compete for food or feed. Therefore, the production of chemicals from alternative substrates such as acetate is desirable to develop novel value chains in the bioeconomy.ResultsIn this study, Corynebacterium glutamicum ATCC 13032 was engineered to efficiently produce itaconic acid from the non-food substrate acetate. Therefore, we rewired the central carbon and nitrogen metabolism by inactivating the transcriptional regulator RamB, reducing the activity of isocitrate dehydrogenase, deletion of the gdh gene encoding glutamate dehydrogenase and overexpression of cis-aconitate decarboxylase (CAD) from A. terreus optimized for expression in C. glutamicum. The final strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt) produced 3.43 ± 0.59 g itaconic acid L-1 with a product yield of 81 ± 9 mmol mol-1 during small-scale cultivations in nitrogen-limited minimal medium containing acetate as sole carbon and energy source. Lowering the cultivation temperature from 30 °C to 25 °C improved CAD activity and further increased the titer and product yield to 5.01 ± 0.67 g L-1 and 116 ± 15 mmol mol-1, respectively. The latter corresponds to 35% of the theoretical maximum and so far represents the highest product yield for acetate-based itaconic acid production. Further, the optimized strain C. glutamicum ΔramB Δgdh IDHR453C (pEKEx2-malEcadopt), produced 3.38 ± 0.28 g itaconic acid L-1 at 25 °C from an acetate-containing aqueous side-stream of fast pyrolysis.ConclusionAs shown in this study, acetate represents a suitable non-food carbon source for itaconic acid production with C. glutamicum. Tailoring the central carbon and nitrogen metabolism enabled the efficient production of itaconic acid from acetate and therefore this study offers useful design principles to genetically engineer C. glutamicum for other products from acetate.

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.

Project description:5-Aminovaleric acid (5AVA) is an important five-carbon platform chemical that can be used for the synthesis of polymers and other chemicals of industrial interest. Enzymatic conversion of L-lysine to 5AVA has been achieved by employing lysine 2-monooxygenase encoded by the davB gene and 5-aminovaleramidase encoded by the davA gene. Additionally, a recombinant Escherichia coli strain expressing the davB and davA genes has been developed for bioconversion of L-lysine to 5AVA. To use glucose and xylose derived from lignocellulosic biomass as substrates, rather than L-lysine as a substrate, we previously examined direct fermentative production of 5AVA from glucose by metabolically engineered E. coli strains. However, the yield and productivity of 5AVA achieved by recombinant E. coli strains remain very low. Thus, Corynebacterium glutamicum, a highly efficient L-lysine producing microorganism, should be useful in the development of direct fermentative production of 5AVA using L-lysine as a precursor for 5AVA. Here, we report the development of metabolically engineered C. glutamicum strains for enhanced fermentative production of 5AVA from glucose.Various expression vectors containing different promoters and origins of replication were examined for optimal expression of Pseudomonas putida davB and davA genes encoding lysine 2-monooxygenase and delta-aminovaleramidase, respectively. Among them, expression of the C. glutamicum codon-optimized davA gene fused with His6-Tag at its N-Terminal and the davB gene as an operon under a strong synthetic H36 promoter (plasmid p36davAB3) in C. glutamicum enabled the most efficient production of 5AVA. Flask culture and fed-batch culture of this strain produced 6.9 and 19.7 g/L (together with 11.9 g/L glutaric acid as major byproduct) of 5AVA, respectively. Homology modeling suggested that endogenous gamma-aminobutyrate aminotransferase encoded by the gabT gene might be responsible for the conversion of 5AVA to glutaric acid in recombinant C. glutamicum. Fed-batch culture of a C. glutamicum gabT mutant-harboring p36davAB3 produced 33.1 g/L 5AVA with much reduced (2.0 g/L) production of glutaric acid.Corynebacterium glutamicum was successfully engineered to produce 5AVA from glucose by optimizing the expression of two key enzymes, lysine 2-monooxygenase and delta-aminovaleramidase. In addition, production of glutaric acid, a major byproduct, was significantly reduced by employing C. glutamicum gabT mutant as a host strain. The metabolically engineered C. glutamicum strains developed in this study should be useful for enhanced fermentative production of the novel C5 platform chemical 5AVA from renewable resources.

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Pathway engineering in Corynebacterium glutamicum S9114 for 5-aminolevulinic acid production.

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Pathway engineering in <i>Corynebacterium glutamicum</i> S9114 for 5-aminolevulinic acid production.

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