Significance of the Cgl1427 gene encoding cytidylate kinase in microaerobic growth of Corynebacterium glutamicum

Bacterial metabolism shifts from aerobic respiration to fermentation at the transition from exponential to stationary growth phases in response to limited oxygen availability. Corynebacterium glutamicum, a Gram-positive, facultative aerobic bacterium used for industrial amino acid production, excretes L-lactate, acetate, and succinate as fermentation products. The ldhA gene encoding L-lactate dehydrogenase is solely responsible for L-lactate production. Its expression is repressed at the exponential phase and prominently induced at the transition phase. ldhA is transcriptionally repressed by the sugar-phosphate-responsive regulator SugR and L-lactate-responsive regulator LldR. Although ldhA expression is derepressed even at the exponential phase in the sugR and lldR double deletion mutant, a further increase in its expression is still observed at the stationary phase, implicating the action of additional transcription regulators. In this study, involvement of the cAMP receptor protein-type global regulator GlxR in the regulation of ldhA expression was investigated. The GlxR-binding site found in the ldhA promoter was modified to inhibit or enhance binding of GlxR. The ldhA promoter activity and expression of ldhA were altered in proportion to the binding affinity of GlxR. Similarly, L-lactate production was also affected by the binding site modification. Thus, GlxR was demonstrated to act as a transcriptional activator of ldhA.

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Platform Engineering of Corynebacterium glutamicum with Reduced Pyruvate Dehydrogenase Complex Activity for Improved Production of l-Lysine, l-Valine, and 2-Ketoisovalerate

Applied and Environmental Microbiology ◽

10.1128/aem.01741-13 ◽

2013 ◽

Vol 79 (18) ◽

pp. 5566-5575 ◽

Cited By ~ 64

Author(s):

Jens Buchholz ◽

Andreas Schwentner ◽

Britta Brunnenkan ◽

Christina Gabris ◽

Simon Grimm ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Pyruvate Dehydrogenase ◽

Pyruvate Dehydrogenase Complex ◽

Fed Batch ◽

Complex Activity ◽

Gene Encoding ◽

Content Type ◽

Genes Encoding ◽

Cell Densities ◽

Dehydrogenase Complex

ABSTRACTExchange of the nativeCorynebacterium glutamicumpromoter of theaceEgene, encoding the E1p subunit of the pyruvate dehydrogenase complex (PDHC), with mutateddapApromoter variants led to a series ofC. glutamicumstrains with gradually reduced growth rates and PDHC activities. Upon overexpression of thel-valine biosynthetic genesilvBNCE, all strains producedl-valine. Among these strains,C. glutamicum aceEA16 (pJC4ilvBNCE) showed the highest biomass and product yields, and thus it was further improved by additional deletion of thepqoandppcgenes, encoding pyruvate:quinone oxidoreductase and phosphoenolpyruvate carboxylase, respectively. In fed-batch fermentations at high cell densities,C. glutamicum aceEA16 Δpqo Δppc(pJC4ilvBNCE) produced up to 738 mM (i.e., 86.5 g/liter)l-valine with an overall yield (YP/S) of 0.36 mol per mol of glucose and a volumetric productivity (QP) of 13.6 mM per h [1.6 g/(liter × h)]. Additional inactivation of the transaminase B gene (ilvE) and overexpression ofilvBNCDinstead ofilvBNCEtransformed thel-valine-producing strain into a 2-ketoisovalerate producer, excreting up to 303 mM (35 g/liter) 2-ketoisovalerate with aYP/Sof 0.24 mol per mol of glucose and aQPof 6.9 mM per h [0.8 g/(liter × h)]. The replacement of theaceEpromoter by thedapA-A16 promoter in the twoC. glutamicuml-lysine producers DM1800 and DM1933 improved the production by 100% and 44%, respectively. These results demonstrate thatC. glutamicumstrains with reduced PDHC activity are an excellent platform for the production of pyruvate-derived products.

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The surface (S)-layer gene cspB of Corynebacterium glutamicum is transcriptionally activated by a LuxR-type regulator and located on a 6 kb genomic island absent from the type strain ATCC 13032

Microbiology ◽

10.1099/mic.0.28673-0 ◽

2006 ◽

Vol 152 (4) ◽

pp. 923-935 ◽

Cited By ~ 37

Author(s):

Nicole Hansmeier ◽

Andreas Albersmeier ◽

Andreas Tauch ◽

Thomas Damberg ◽

Robert Ros ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Affinity Purification ◽

Regulatory Protein ◽

Direct Repeat ◽

Peptide Mass Fingerprinting ◽

Gene Encoding ◽

Force Microscopy ◽

Flight Mass Spectrometry ◽

Peptide Mass ◽

Rapid Amplification

The surface (S)-layer gene region of the Gram-positive bacterium Corynebacterium glutamicum ATCC 14067 was identified on fosmid clones, sequenced and compared with the genome sequence of C. glutamicum ATCC 13032, whose cell surface is devoid of an ordered S-layer lattice. A 5·97 kb DNA region that is absent from the C. glutamicum ATCC 13032 chromosome was identified. This region includes cspB, the structural gene encoding the S-layer protomer PS2, and six additional coding sequences. PCR experiments demonstrated that the respective DNA region is conserved in different C. glutamicum wild-type strains capable of S-layer formation. The DNA region is flanked by a 7 bp direct repeat, suggesting that illegitimate recombination might be responsible for gene loss in C. glutamicum ATCC 13032. Transfer of the cloned cspB gene restored the PS2− phenotype of C. glutamicum ATCC 13032, as confirmed by visualization of the PS2 proteins by SDS-PAGE and imaging of ordered hexagonal S-layer lattices on living C. glutamicum cells by atomic force microscopy. Furthermore, the promoter of the cspB gene was mapped by 5′ rapid amplification of cDNA ends PCR and the corresponding DNA fragment was used in DNA affinity purification assays. A 30 kDa protein specifically binding to the promoter region of the cspB gene was purified. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and peptide mass fingerprinting of the purified protein led to the identification of the putative transcriptional regulator Cg2831, belonging to the LuxR regulatory protein family. Disruption of the cg2831 gene in C. glutamicum resulted in an almost complete loss of PS2 synthesis. These results suggested that Cg2831 is a transcriptional activator of cspB gene expression in C. glutamicum.

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Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence

FEMS Microbiology Letters ◽

10.1016/0378-1097(94)90405-7 ◽

1994 ◽

Vol 119 (1-2) ◽

pp. 137-145

Author(s):

Jung-Kee Lee

Keyword(s):

Nucleotide Sequence ◽

Corynebacterium Glutamicum ◽

Protein Sequence ◽

Gene Encoding

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Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3418-3428.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3418-3428 ◽

Cited By ~ 163

Author(s):

Hideo Kawaguchi ◽

Alain A. Vertï¿½s ◽

Shohei Okino ◽

Masayuki Inui ◽

Hideaki Yukawa

Keyword(s):

Carbon Source ◽

Corynebacterium Glutamicum ◽

Xylose Isomerase ◽

Mineral Medium ◽

Gene Encoding ◽

Xylose Consumption ◽

E Coli ◽

High Copy Number Plasmid ◽

Sugar Mixture ◽

Pentose Sugar

ABSTRACT The aerobic microorganism Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar xylose, which is commonly found in agricultural residues and other lignocellulosic biomass. We demonstrated the functionality of the corynebacterial xylB gene encoding xylulokinase and constructed two recombinant C. glutamicum strains capable of utilizing xylose by cloning the Escherichia coli gene xylA encoding xylose isomerase, either alone (strain CRX1) or in combination with the E. coli gene xylB (strain CRX2). These genes were provided on a high-copy-number plasmid and were under the control of the constitutive promoter trc derived from plasmid pTrc99A. Both recombinant strains were able to grow in mineral medium containing xylose as the sole carbon source, but strain CRX2 grew faster on xylose than strain CRX1. We previously reported the use of oxygen deprivation conditions to arrest cell replication in C. glutamicum and divert carbon source utilization towards product production rather than towards vegetative functions (M. Inui, S. Murakami, S. Okino, H. Kawaguchi, A. A. Vertï¿½s, and H. Yukawa, J. Mol. Microbiol. Biotechnol. 7:182-196, 2004). Under these conditions, strain CRX2 efficiently consumed xylose and produced predominantly lactic and succinic acids without growth. Moreover, in mineral medium containing a sugar mixture of 5% glucose and 2.5% xylose, oxygen-deprived strain CRX2 cells simultaneously consumed both sugars, demonstrating the absence of diauxic phenomena relative to the new xylA-xylB construct, albeit glucose-mediated regulation still exerted a measurable influence on xylose consumption kinetics.

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Growth promotion in Corynebacterium glutamicum by overexpression of the NCgl2986 gene encoding a protein homologous to peptidoglycan amidases

The Journal of General and Applied Microbiology ◽

10.2323/jgam.2019.03.002 ◽

2020 ◽

Vol 66 (1) ◽

pp. 1-7

Author(s):

Mia Fitria Utami ◽

Yoshihiko Matsuda ◽

Ayako Takada ◽

Noritaka Iwai ◽

Takashi Hirasawa ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Growth Promotion ◽

Gene Encoding

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Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.00792-15 ◽

2015 ◽

Vol 81 (12) ◽

pp. 4173-4183 ◽

Cited By ~ 10

Author(s):

Akira Watanabe ◽

Kazumi Hiraga ◽

Masako Suda ◽

Hideaki Yukawa ◽

Masayuki Inui

Keyword(s):

Heterologous Expression ◽

Corynebacterium Glutamicum ◽

Abc Transporter ◽

Binding Protein ◽

Functional Characterization ◽

Product Inhibition ◽

Gene Encoding ◽

Content Type ◽

Industrial Strains ◽

Efficient Fermentation

ABSTRACTTheCorynebacterium alkanolyticumxylEFGDgene cluster comprises thexylDgene that encodes an intracellular β-xylosidase next to thexylEFGoperon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover forp-nitrophenyl-β-d-xylopyranoside of 111 ± 4 s−1), weak α-l-arabinofuranosidase activity (turnover forp-nitrophenyl-α-l-arabinofuranoside of 5 ± 1 s−1), and high tolerance to product inhibition (Kifor xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutivetacpromoter in theCorynebacterium glutamicumxylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended oncgR_2369, a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguousxylDgene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability ofC. glutamicumto take up xylooligosaccharides, an ability that is enhanced by in the presence of a functionalxylEFG-encoded xyloside ABC transporter. The finding thatxylEFGimparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.

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