Advances in metabolic engineering of <i>Corynebacterium glutamicum</i> to produce high-value active ingredients for food, feed, human health, and well-being

Wolf, Sabrina; Becker, Jörg; Tsuge, Yota; Kawaguchi, Hideo; Kondo, Akihiko; Marienhagen, Jan; Bott, Michael; Wendisch, Volker F.; Wittmann, Christoph

doi:10.1042/ebc20200134

Cited by 85 publications

(48 citation statements)

References 147 publications

(225 reference statements)

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“…An interesting candidate for biotechnological production of bacteriocins is C. glutamicum , one of the most extensively used platform organisms for biotechnological production of more than 70 compounds including high value active ingredients and therapeutic proteins ( Becker et al, 2018 ; Eggeling and Bott, 2015 ; Wolf et al, 2021 ; Yim et al, 2014 , 2016 ). This platform organism bears the advantage that well-defined, low-cost, minimal media as well as appropriate industry-scale production processes are available.…”

Section: Introductionmentioning

confidence: 99%

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum

Goldbeck

Desef

Ovchinnikov

et al. 2021

Metabolic Engineering

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Bacteriocins are antimicrobial peptides produced by bacteria to inhibit competitors in their natural environments. Some of these peptides have emerged as commercial food preservatives and, due to the rapid increase in antibiotic resistant bacteria, are also discussed as interesting alternatives to antibiotics for therapeutic purposes. Currently, commercial bacteriocins are produced exclusively with natural producer organisms on complex substrates and are sold as semi-purified preparations or crude fermentates. To allow clinical application, efficacy of production and purity of the product need to be improved. This can be achieved by shifting production to recombinant microorganisms. Here, we identify Corynebacterium glutamicum as a suitable production host for the bacteriocin pediocin PA-1. C. glutamicum CR099 shows resistance to high concentrations of pediocin PA-1 and the bacteriocin was not inactivated when spiked into growing cultures of this bacterium. Recombinant C. glutamicum expressing a synthetic pedACD Cgl operon releases a compound that has potent antimicrobial activity against Listeria monocytogenes and Listeria innocua and matches size and mass:charge ratio of commercial pediocin PA-1. Fermentations in shake flasks and bioreactors suggest that low levels of dissolved oxygen are favorable for production of pediocin. Under these conditions, however, reduced activity of the TCA cycle resulted in decreased availability of the important pediocin precursor l -asparagine suggesting options for further improvement. Overall, we demonstrate that C. glutamicum is a suitable host for recombinant production of bacteriocins of the pediocin family.

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Section: Introductionmentioning

confidence: 99%

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum

Goldbeck

Desef

Ovchinnikov

et al. 2021

Metabolic Engineering

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“…), it is widely used in fermentation processes today ( Becker and Wittmann, 2012 ). Besides production of amino acids and other bulk chemicals ( Wendisch, 2020 , 2016 ), it is also applied for production of high-value compounds ( Wolf et al, 2021 ). While many C. glutamicum strains have been isolated, to our knowledge just two are widely used in basic research and biotechnology: strain ATCC 13 032 and strain R. This might in part be due to the presence of the paracrystalline protein surface (S) layer in many other C. glutamicum strains ( Hansmeier et al, 2004 ) that was shown to interfere with DNA transfer by electroporation in Caulobatcer species ( Gilchrist and Smit, 1991 ) and in part due to the early availability of complete genome sequences for the two strains ( Ikeda and Nakagawa, 2003 ; Kalinowski et al, 2003 ; Yukawa et al, 2007 ).…”

Section: Introductionmentioning

confidence: 99%

Construction of an IS-Free Corynebacterium glutamicum ATCC 13 032 Chassis Strain and Random Mutagenesis Using the Endogenous ISCg1 Transposase

Linder

Haak

Botes

et al. 2021

Front. Bioeng. Biotechnol.

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Mobile genetic elements (MGEs) contribute to instability of the host genome and plasmids. Previously, removal of the prophages in the industrial amino acid producer Corynebacterium glutamicum ATCC 13 032 resulted in strain MB001 which showed better survival under stress conditions and increased transformability. Still, eight families of Insertion Sequence (IS) elements with 27 potentially active members remain in MB001, two of which were demonstrated to be detrimental in biotechnological processes. In this study, systematical deletion of all complete IS elements in MB001 resulted in the MGE-free strain CR101. CR101 shows growth characteristics identical to the wildtype and the increased transformability of MB001. Due to its improved genome stability, we consider this strain to be an optimal host for basic research and biotechnology. As a “zero-background” host, it is also an ideal basis to study C. glutamicum IS elements. Re-sequencing of CR101 revealed that only five spontaneous point mutations had occurred during the construction process, highlighting the low mutation rate of C. glutamicum on the nucleotide level. In a second step, we developed an easily applicable ISCg1-based transposon mutagenesis system to randomly transpose a selectable marker. For optimal plasmid stability during cloning in Escherichia coli, the system utilizes a genetic switch based on the phage integrase Bxb1. Use of this integrase revealed the presence of a functional attB site in the C. glutamicum genome. To avoid cross-talk with our system and increase ease-of-use, we removed the attB site and also inserted the Bxb1 encoding gene into the chromosome of CR101. Successful insertion of single markers was verified by sequencing randomly selected mutants. Sequencing pooled mutant libraries revealed only a weak target site specificity, seemingly random distribution of insertion sites and no general strand bias. The resulting strain, ML103, together with plasmid pML10 provides a easily customizable system for random mutagenesis in an otherwise genomically stable C. glutamicum. Taken together, the MGE-free C. glutamicum strain CR101, the derivative ML103, and the plasmid pML10 provide a useful set of tools to study C. glutamicum in the future.

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“…Since then, Corynebacterium glutamicum has been used successfully to produce specialty glutamine and specialty amino acids to meet global demand. The advent of accessible whole-genome sequencing and mutagenesis methods have enabled a clearer understanding of how specific isolates can overproduce these desired molecules, as well as how they maintain productivity across volumetrically-larger scales ( Becker et al, 2018 ; Pérez-García and Wendisch, 2018 ; Wolf et al, 2021 ). Using C. glutamicum to produce non-native metabolites as next-generation biofuels is an attractive large-volume market with the potential to reduce global carbon emissions.…”

Section: Introductionmentioning

confidence: 99%

Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation Under Ionic Liquid Stress

Banerjee

Eng

Sasaki

et al. 2021

Front. Bioeng. Biotechnol.

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Corynebacterium glutamicum is an ideal microbial chassis for production of valuable bioproducts including amino acids and next generation biofuels. Here we resequence engineered isopentenol (IP) producing C. glutamicum BRC-JBEI 1.1.2 strain and assess differential transcriptional profiles using RNA sequencing under industrially relevant conditions including scale transition and compare the presence vs absence of an ionic liquid, cholinium lysinate ([Ch][Lys]). Analysis of the scale transition from shake flask to bioreactor with transcriptomics identified a distinct pattern of metabolic and regulatory responses needed for growth in this industrial format. These differential changes in gene expression corroborate altered accumulation of organic acids and bioproducts, including succinate, acetate, and acetoin that occur when cells are grown in the presence of 50 mM [Ch][Lys] in the stirred-tank reactor. This new genome assembly and differential expression analysis of cells grown in a stirred tank bioreactor clarify the cell response of an C. glutamicum strain engineered to produce IP.

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Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being

Cited by 85 publications

References 147 publications

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum

Establishing recombinant production of pediocin PA-1 in Corynebacterium glutamicum

Construction of an IS-Free Corynebacterium glutamicum ATCC 13 032 Chassis Strain and Random Mutagenesis Using the Endogenous ISCg1 Transposase

Genomics Characterization of an Engineered Corynebacterium glutamicum in Bioreactor Cultivation Under Ionic Liquid Stress

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