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Background: The disposal of plastic waste is a major environmental challenge. With recent advances in microbial genetic and metabolic engineering technologies, microbial polyhydroxyalkanoates (PHAs) are being used as next-generation biomaterials to replace petroleum-based synthetic plastics in a sustainable future. However, the relatively high production cost of bioprocesses hinders the production and application of microbial PHAs on an industrial scale., Results: Here, we describe a rapid strategy to rewire metabolic networks in an industrial microorganism, Corynebacterium glutamicum, for the enhanced production of poly(3-hydroxybutyrate) (PHB). A three-gene PHB biosynthetic pathway in Rasltonia eutropha was refactored for high-level gene expression. A fluorescence-based quantification assay for cellular PHB content using BODIPY was devised for the rapid fluorescence-activated cell sorting (FACS)-based screening of a large combinatorial metabolic network library constructed in C. glutamicum. Rewiring metabolic networks across the central carbon metabolism enabled highly efficient production of PHB up to 29% of dry cell weight with the highest cellular PHB productivity ever reported in C. glutamicum using a sole carbon source., Conclusions: We successfully constructed a heterologous PHB biosynthetic pathway and rapidly optimized metabolic networks across central metabolism in C. glutamicum for enhanced production of PHB using glucose or fructose as a sole carbon source in minimal media. We expect that this FACS-based metabolic rewiring framework will accelerate strain engineering processes for the production of diverse biochemicals and biopolymers., (© 2023. The Author(s).)

Ectoine is formed in various bacteria as cell protectant against all kinds of stress. Its preservative and protective effects have enabled various applications in medicine, cosmetics, and biotechnology, and ectoine therefore has high commercial value. Industrially, ectoine is produced in a complex high-salt process, which imposes constraints on the costs, design, and durability of the fermentation system. Here, Corynebacterium glutamicum is upgraded for the heterologous production of ectoine from sugar and molasses. To overcome previous limitations, the ectoine pathway taken from Pseudomonas stutzeri is engineered using transcriptional balancing. An expression library with 185,193 variants is created, randomly combining 19 synthetic promoters and three linker elements. Strain screening discovers several high-titer mutants with an improvement of almost fivefold over the initial strain. High production thereby particularly relies on a specifically balanced ectoine pathway. In an optimized fermentation process, the new top producer C. glutamicum ectABC opt achieves an ectoine titer of 65 g L -1 and a specific productivity of 120 mg g -1  h -1 . This process is the first reported example of a simple fermentation process under low-salt conditions using well-established feedstocks to produce ectoine with industrial efficiency. There is a compelling case for more intensive implementation of transcriptional balancing in future metabolic engineering of C. glutamicum., (© 2019 The Authors. Biotechnology Journal Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the cell surface display, the choice of host cell and anchoring motif are the most crucial for the efficient display of passenger proteins. Corynebacterium glutamicum has mycolic acid layer in outer membrane and the use of protein in the mycolic acid layer as an anchoring motif can provide a potential platform for surface display in C. glutamicum. All 19 mycolic acid layer proteins of C. glutamicum are analyzed, and two proteins, NCgl0535 and NCgl1337, which have a signal peptide and predicted O-mycoloylation site, are selected as anchoring motifs candidates. Among them, NCgl1337, which shows better expression with higher display efficiency, is chosen as a potential anchoring motif. Two forms of the NCgl1337 anchoring motif, a full-length (1-324 amino acids) and a short-length (1-50 amino acids) containing only signal peptide and O-mycoloylation site, are constructed and their abilities for surface display are examined using two protein models, endoxylanase from Streptomyces coelicolor and α-amylase from Streptococcus bovis. For both model proteins, the short-length NCgl1337 anchoring motif exhibits higher yield of protein display on the surface of C. glutamicum than the full-length NCgl1337. Finally, with C. glutamicum displaying α-amylase, a batch fermentation is performed for the production of l-lysine from starch degradation, and a production of l-lysine as high as 10.8 ± 0.92 g L -1 was achieved after 18 h of culture., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Beyond its traditional role as an L-amino acid producer, Corynebacterium glutamicum has recently received significant attention regarding its use in the production of various biochemicals and recombinant proteins. However, despite these attributes, limitations in genetic tools are still hampering the engineering of C. glutamicum for use in more potential hosts. Here, we engineered a C. glutamicum via adaptive laboratory evolution to enhance the production of recombinant proteins. During the continuous cultivation, C. glutamicum producing enhanced green fluorescent proteins was screened using high-speed flow cytometer, and in the end, we successfully isolated an evolved strain with a fluorescence intensity 4.5-fold higher than that of the original strain. Extensive analysis of the evolved strain confirmed that the plasmid prepared from the evolved strain contains the nonsense mutation in the parB locus, which mutation contributed to increasing the copy number of plasmid by approximately 10-fold compared to that of the wild type. To validate the usefulness of the high-copy-number plasmid, we examined the secretory production of endoxylanase and the bioconversion of xylose to xylonate using xylonate dehydrogenase. In the fed-batch cultivation, the use of the high-copy-number plasmid led to 1.4-fold increase in the production of endoxylanase (~ 1.54 g/L in culture medium) without cell growth retardation comparing cultivation with cells harboring original plasmid. The expression of xylonate dehydrogenase in the high-copy-number plasmid also improved the bioconversion into xylonic acid by approximately 1.5-fold compared to the original plasmid.

Xylonic acid is a promising platform chemical with various applications in the fields of food, pharmaceuticals, and agriculture. However, in the current process, xylonic acid is mainly produced by the conversion of xylose, whose preparation requires substantial cost and time. Here, Corynebacterium glutamicum is engineered for the consolidated bioconversion of hemicellulosic biomass (xylan) into xylonic acid in a single cultivation. First, for the efficient conversion of xylose to xylonic acid, xylose dehydrogenase (Xdh) and xylonolactonase (XylC) from Caulobacter crescentus are evaluated together with a previously optimized xylose transporter module (XylE of Escherichia coli), and cells expressing xdh and xylE genes with an optimized expression system can produce xylonic acid from xylose with 100% conversion yield. Next, to directly process xylan as a substrate, an engineered xylan-degrading module is introduced, in which two xylan-degrading enzymes (endoxylanase and xylosidase) are secreted into the culture medium. The engineered C. glutamicum successfully produce 6.23 g L -1 of xylonic acid from 20 g L -1 of xylan. This is the first report on xylonic acid production in C. glutamicum and this robust system will contribute to development of an industrially relevant platform for production of xylonic acid from raw biomass., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Corynebacterium glutamicum is a non-pathogenic, non-sporulating Gram-positive soil bacterium that has been used for the industrial production of various proteins and chemicals. To achieve enhanced and economical production of target molecules, the development of strong auto-inducible promoters is desired, which can be activated without expensive inducers and has significant advantages for industrial-scale use. Here, we developed a stationary-phase gene expression system by engineering a sigma factor B (SigB)-dependent promoter that can be activated during the transition phase between exponential and stationary growth phases in C. glutamicum. First, the inducibilities of three well-known SigB-dependent promoters were examined using super-folder green fluorescent protein as a reporter protein, and we found that promoter of cg3141 (P cg3141 ) exhibited the highest inducibility. Next, a synthetic promoter library was constructed by randomizing the flanking and space regions of P cg3141 , and the stationary-phase promoters exhibiting high strengths were isolated via FACS-based high-throughput screening. The isolated synthetic promoter (P4-N14) showed a 3.5-fold inducibility and up to 20-fold higher strength compared to those of the original cg3141 promoter. Finally, the use of the isolated P4-N14 for fed-batch cultivation was verified with the production of glutathione S-transferase as a model protein in a lab-scale (5-L) bioreactor.

Hemicellulose, which is the second most abundant polysaccharide in nature after cellulose, has the potential to become a major feedstock for microbial fermentation to produce various biofuels and chemicals. To utilize hemicellulose economically, it is necessary to develop a consolidated bioprocess (CBP), in which all processes from biomass degradation to the production of target products occur in a single bioreactor. Here, we report a modularly engineered Corynebacterium glutamicum strain suitable for CBP using hemicellulosic biomass (xylan) as a feedstock. The hemicellulose-utilizing pathway was divided into three distinct modules, and each module was separately optimized. In the module for xylose utilization, the expression level of the xylose isomerase (xylA) and xylulokinase (xylB) genes was optimized with synthetic promoters of different strengths. Then, the module for xylose transport was engineered with combinatorial sets of synthetic promoters and heterologous transporters to achieve the fastest cell growth rate on xylose (0.372 h(-1)). Next, the module for the enzymatic degradation of xylan to xylose was also engineered with different combinations of promoters and signal peptides to efficiently secrete both endoxylanase and xylosidase into the extracellular medium. Finally, each optimized module was integrated into a single plasmid to construct a highly efficient xylan-utilizing pathway. Subsequently, the direct production of lysine from xylan was successfully demonstrated with the engineered pathway. To the best of our knowledge, this is the first report of the development of a consolidated bioprocessing C. glutamicum strain for hemicellulosic biomass.

Corynebacterium glutamicum, which has been for long an industrial producer of various L-amino acids, nucleic acids, and vitamins, is now also regarded as a potential host for the secretory production of recombinant proteins. To harness its potential as an industrial platform for recombinant protein production, the development of an efficient secretion system is necessary. Particularly, regarding protein production in large-scale bioreactors, it would be appropriate to develop a secretory expression system that is specialized for high cell density cultivation conditions. Here we isolated a new signal peptide that mediates the efficient secretion of recombinant proteins under high cell density cultivation conditions. The secretome of C. glutamicum ATCC 13032 under high cell density cultivation conditions was initially investigated, and one major protein was identified as a hypothetical protein encoded by cg1514. Novel secretory production systems were then developed using the Cg1514 signal peptide and its own promoter. Efficient protein secretion was demonstrated using three protein models: endoxylanase, α-amylase, and camelid antibody fragment (VHH). For large-scale production, fed-batch cultivations were also conducted and high yields were successfully achieved--as high as 1.07 g/L (endoxylanase), 782.6 mg/L (α-amylase), and 1.57 g/L (VHH)--in the extracellular medium. From the culture media, all model proteins could be simply purified by one-step column chromatography with high purities and recovery yields. To the best of our knowledge, this is the first report of the development of an efficient secretory expression system by secretome analysis under high cell density cultivation conditions in C. glutamicum., (© 2015 Wiley Periodicals, Inc.)

Background: In 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., Results: From 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)., Conclusions: Our 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.

Background: Gamma-aminobutylate (GABA) is an important chemical in pharmacetucal field and chemical industry. GABA has mostly been produced in lactic acid bacteria by adding L-glutamate to the culture medium since L-glutamate can be converted into GABA by inherent L-glutamate decarboxylase. Recently, GABA has gained much attention for the application as a major building block for the synthesis of 2-pyrrolidone and biodegradable polyamide nylon 4, which opens its application area in the industrial biotechnology. Therefore, Corynebacterium glutamicum, the major L-glutamate producing microorganism, has been engineered to achieve direct fermentative production of GABA from glucose, but their productivity was rather low., Results: Recombinant C. glutamicum strains were developed for enhanced production of GABA from glucose by expressing Escherichia coli glutamate decarboxylase (GAD) mutant, which is active in expanded pH range. Synthetic PH36, PI16, and PL26 promoters, which have different promoter strengths in C. glutamicum, were examined for the expression of E. coli GAD mutant. C. glutamicum expressing E. coli GAD mutant under the strong PH36 promoter could produce GABA to the concentration of 5.89±0.35 g/L in GP1 medium at pH 7.0, which is 17-fold higher than that obtained by C. glutamicum expressing wild-type E. coli GAD in the same condition (0.34±0.26 g/L). Fed-bath culture of C. glutamicum expressing E. coli GAD mutant in GP1 medium containing 50 μg/L of biotin at pH 6, culture condition of which was optimized in flask cultures, resulted in the highest GABA concentration of 38.6±0.85 g/L with the productivity of 0.536 g/L/h., Conclusion: Recombinant C. glutamicum strains developed in this study should be useful for the direct fermentative production of GABA from glucose, which allows us to achieve enhanced production of GABA suitable for its application area in the industrial biotechnology.

We describe the development of a new secretory production system for the enhanced production of a single-chain variable fragment (scFv) against the anthrax toxin in Corynebacterium glutamicum. For efficient secretory production of the antibody fragment, the following components were examined: (1) signal peptides, (2) codon usage of antibody fragment, (3) promoters, (4) 5' untranslated region (5' UTR) sequence, and (5) transcriptional terminator. Among all the systems examined, the use of a codon-optimized gene sequence, a Sec-dependent PorB signal peptide, and a fully synthetic H36 promoter, allowed the highest production of antibody fragments in a culture medium. For large-scale production, fed-batch cultivations were also conducted in a 5-L lab-scale bioreactor. When cells were cultivated in semi-defined media, cells could grow up to an OD600 of 179 for 32 h and an antibody fragment concentration as high as 68 mg/L could be obtained in a culture medium with high purity. From the culture medium, the secreted antibody was successfully purified using a simple purification procedure, with correct binding activity confirmed by enzyme-linked immunosorbent assay. To the best of our knowledge, this is the first report of a fed-batch cultivation for antibody fragment production in C. glutamicum.

Corynebacterium glutamicum is an important industrial organism that is widely used in the production of amino acids, nucleotides and vitamins. To extend its product spectrum and improve productivity, C. glutamicum needs to undergo further engineering, including the development of applicable promoter system. Here, we isolated new promoters from the fully synthetic promoter library consisting of 70-bp random sequences in C. glutamicum. Using green fluorescent protein (GFP) as a reporter, highly fluorescent cells were screened from the library by fluorescent activated cell sorting (FACS). Twenty potential promoters of various strengths were isolated and characterized through extensive analysis of DNA sequences and mRNA transcripts. Among 20 promoters, 6 promoters which have different strengths were selected and their activities were successfully demonstrated using two model proteins (antibody fragment and endoxylanase). Finally, the strongest promoter (P(H36)) was employed for the secretory production of endoxylanase in fed-batch cultivation, achieving production levels of 746 mg/L in culture supernatant. This is the first report of synthetic promoters constructed in C. glutamicum, and our screening strategy together with the use of synthetic promoters of various strengths will contribute to the future engineering of C. glutamicum., (© 2013 Wiley Periodicals, Inc.)

Corynebacterium glutamicum is one of the useful hosts for the secretory production of heterologous proteins because of intrinsic attributes such as the presence of few endogenous proteins and proteases in culture medium. Here, we report the development of a new secretory system for the production of heterologous proteins by using the porin B (PorB) signal peptide in C. glutamicum. We examined two different endoxylanases and an antibody fragment (scFv) as model proteins for secretory production. In the flask cultivations, all the examined proteins were successfully produced as active forms into the culture medium with high efficiency. For the high-level production of endoxylanase, fed-batch cultivation was also performed in a lab-scale (5L) bioreactor, and the endoxylanases were efficiently secreted in the culture medium at levels as high as 615mg/L. From the culture supernatant, the secreted endoxylanases could be purified with high purity via one-step affinity column chromatography., (Copyright © 2013 Elsevier Inc. All rights reserved.)