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Enhanced production of neoagarobiose from agar with Corynebacterium glutamicum producing exo-type and endo-type β-agarases.

ABSTRACT: Neoagarobiose (NA2) derived from agar marine biomass is a rare reagent that acts as an anti-melanogenesis reagent and moisturizer. Here, for the economical manufacturing of NA2, we developed the co-secretory production system of endo-type β-agarases (DagA) and exo-type β-agarases (EXB3) in Corynebacterium glutamicum. For this purpose, we first developed a secretory system of DagA via Tat pathway. To improve the secretion efficiency, we coexpressed two Tat pathway components (TatA and TatC), and to improve the purity of secreted DagA in the culture supernatant, two endogenous protein genes (Cg2052 and Cg1514) were removed. Using the engineered strain (C. glutamicum SP002), we confirmed that DagA as high as 1.53 g l^-1 was successfully produced in the culture media with high purity (72.7% in the supernatant protein fraction). Next, we constructed the expression system (pHCP-CgR-DagA-EXB3) for the simultaneous secretion of EXB3 via Sec-pathway together with DagA, and it was clearly confirmed that DagA and EXB3 were successfully secreted as high as 54% and 24.5%, respectively. Finally, using culture medium containing DagA and EXB3, we successfully demonstrated the conversion of high-concentration agar (40 g l^-1 ) into NA2 via a two-stage hydrolysis process.

SUBMITTER: Jeon EJ

PROVIDER: S-EPMC8449658 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Enhanced production of neoagarobiose from agar with Corynebacterium glutamicum producing exo-type and endo-type β-agarases.

Jeon Eun Jung EJ Choi Jae Woong JW Cho Min Soo MS Jeong Ki Jun KJ

Microbial biotechnology 20210726 5

Neoagarobiose (NA2) derived from agar marine biomass is a rare reagent that acts as an anti-melanogenesis reagent and moisturizer. Here, for the economical manufacturing of NA2, we developed the co-secretory production system of endo-type β-agarases (DagA) and exo-type β-agarases (EXB3) in Corynebacterium glutamicum. For this purpose, we first developed a secretory system of DagA via Tat pathway. To improve the secretion efficiency, we coexpressed two Tat pathway components (TatA and TatC), and ...[more]

PMID: 34310855

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Project description:Background5-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.ResultsVarious 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.ConclusionsCorynebacterium 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.

Project description:To date, no information has been made available on the genetic traits that lead to increased carbon flow into the fatty acid biosynthetic pathway of Corynebacterium glutamicum. To develop basic technologies for engineering, we employed an approach that begins by isolating a fatty acid-secreting mutant without depending on mutagenic treatment. This was followed by genome analysis to characterize its genetic background. The selection of spontaneous mutants resistant to the palmitic acid ester surfactant Tween 40 resulted in the isolation of a desired mutant that produced oleic acid, suggesting that a single mutation would cause increased carbon flow down the pathway and subsequent excretion of the oversupplied fatty acid into the medium. Two additional rounds of selection of spontaneous cerulenin-resistant mutants led to increased production of the fatty acid in a stepwise manner. Whole-genome sequencing of the resulting best strain identified three specific mutations (fasR20, fasA63(up), and fasA2623). Allele-specific PCR analysis showed that the mutations arose in that order. Reconstitution experiments with these mutations revealed that only fasR20 gave rise to oleic acid production in the wild-type strain. The other two mutations contributed to an increase in oleic acid production. Deletion of fasR from the wild-type strain led to oleic acid production as well. Reverse transcription-quantitative PCR analysis revealed that the fasR20 mutation brought about upregulation of the fasA and fasB genes encoding fatty acid synthases IA and IB, respectively, by 1.31-fold ± 0.11-fold and 1.29-fold ± 0.12-fold, respectively, and of the accD1 gene encoding the β-subunit of acetyl-CoA carboxylase by 3.56-fold ± 0.97-fold. On the other hand, the fasA63(up) mutation upregulated the fasA gene by 2.67-fold ± 0.16-fold. In flask cultivation with 1% glucose, the fasR20 fasA63(up) fasA2623 triple mutant produced approximately 280 mg of fatty acids/liter, which consisted mainly of oleic acid (208 mg/liter) and palmitic acid (47 mg/liter).

Project description:BackgroundCorynebacterium glutamicum is a traditional food-grade industrial microorganism, in which an efficient endotoxin-free recombinant protein expression factory is under developing in recent years. However, the intrinsic disadvantage of low recombinant protein expression level is still difficult to be solved. Here, according to the bacteria-specific polycistronic feature that multiple proteins can be translated in one mRNA, efforts have been made to insert a leading peptide gene upstream of target genes as an expression enhancer, and it is found that this can remarkably improve the expression level of proteins under the control of inducible tac promoter in C. glutamicum.ResultsIn this research, the Escherichia coli (E. coli) tac promoter combined with 24 different fore-cistron sequences were constructed in a bicistronic manner in C. glutamicum. Three strong bicistronic expression vectors were isolated and exhibited high efficiency under different culture conditions. The compatibility of these bicistronic vectors was further validated using six model proteins- aldehyde dehydrogenase (ALDH), alcohol dehydrogenase (ADH), RamA (regulator of acetate metabolism), Bovine interferon-α (BoIFN-α), glycoprotein D protein (gD) of infectious bovine rhinotracheitis virus (IBRV) and procollagen type Ι N-terminal peptide (PΙNP). All examined proteins were highly expressed compared with the original vector with tac promoter. Large-scale production of PΙNP was also performed in fed-batch cultivation, and the highest PΙNP production level was 1.2 g/L.ConclusionIn this study, the strength of the inducible tac promoter for C. glutamicum was improved by screening and inserting fore-cistron sequences in front of the target genes. Those vectors with bicistronic expression patterns have strong compatibility for expressing various heterogeneous proteins in high yield. This new strategy could be used to further improve the performance of inducible promoters, achieving double competence of inducible control and high yield.

Project description:BackgroundIn most bacteria, various jumping genetic elements including insertion sequences elements (IS elements) cause a variety of genetic rearrangements resulting in harmful effects such as genome and recombinant plasmid instability. The genetic stability of a plasmid in a host is critical for high-level production of recombinant proteins, and in this regard, the development of an IS element-free strain could be a useful strategy for the enhanced production of recombinant proteins. Corynebacterium glutamicum, which is a workhorse in the industrial-scale production of various biomolecules including recombinant proteins, also has several IS elements, and it is necessary to identify the critical IS elements and to develop IS element deleted strain.ResultsFrom the cultivation of C. glutamicum harboring a plasmid for green fluorescent protein (GFP) gene expression, non-fluorescent clones were isolated by FACS (fluorescent activated cell sorting). All the isolated clones had insertions of IS elements in the GFP coding region, and two major IS elements (ISCg1 and ISCg2 families) were identified. By co-cultivating cells harboring either the isolated IS element-inserted plasmid or intact plasmid, it was clearly confirmed that cells harboring the IS element-inserted plasmids became dominant during the cultivation due to their growth advantage over cells containing intact plasmids, which can cause a significant reduction in recombinant protein production during cultivation. To minimize the harmful effects of IS elements on the expression of heterologous genes in C. glutamicum, two IS element free C. glutamicum strains were developed in which each major IS element was deleted, and enhanced productivity in the engineered C. glutamicum strain was successfully demonstrated with three models: GFP, poly(3-hydroxybutyrate) [P(3HB)] and γ-aminobutyrate (GABA).ConclusionsOur findings clearly indicate that the hopping of IS elements could be detrimental to the production of recombinant proteins in C. glutamicum, emphasizing the importance of developing IS element free host strains.

Dataset Information

Enhanced production of neoagarobiose from agar with Corynebacterium glutamicum producing exo-type and endo-type β-agarases.

Publications

Enhanced production of neoagarobiose from agar with Corynebacterium glutamicum producing exo-type and endo-type β-agarases.

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