Online estimation of changing metabolic capacities in continuous Corynebacterium glutamicum cultivations growing on a complex sugar mixture

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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CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

Biochemical Journal ◽

10.1042/bcj20190320 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3141-3159 ◽

Cited By ~ 2

Author(s):

Meiru Si ◽

Can Chen ◽

Zengfan Wei ◽

Zhijin Gong ◽

GuiZhi Li ◽

...

Keyword(s):

Stress Response ◽

Ligand Binding ◽

Corynebacterium Glutamicum ◽

Conformational Changes ◽

Cysteine Oxidation ◽

Transcriptional Regulatory ◽

Ligand Free ◽

Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

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Expression of the NADP+-Dependent Formate Dehydrogenase Gene from Pseudomonas Increases the Lysine Production in Corynebacterium glutamicum

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-21-29 ◽

2019 ◽

Vol 35 (6) ◽

pp. 21-29

Author(s):

T.E. Leonova ◽

T.E. Shustikova ◽

T.V. Gerasimova ◽

Т.А. Ivankova ◽

K.V. Sidorenko Sidorenko ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Formate Dehydrogenase ◽

Ministry Of Education ◽

Lysine Production ◽

Simultaneous Formation ◽

Resource Center ◽

Formate Oxidation ◽

Gene Coding ◽

Dehydrogenase Gene ◽

Lysine Synthesis

Thepsefdh_D221Q gene coding for a mutant formate dehydrogenase (PseFDG_D221Q) from Pseudomonas, which catalyzes the formate oxidation with the simultaneous formation of NADPH, has been expressed in the cells of lysine-producing Corynebacterium glutamicum strains. The psefdh_D221Q gene was introduced into С. glutamicum strains as part of an autonomous plasmid or was integrated into the chromosome with simultaneous inactivation of host formate dehydrogenase genes. It was shown that the С. glutamicum strains with NADP+ -dependent formate dehydrogenase have an increased level of L-lysine synthesis in the presence of formate, if their own formate dehydrogenase is inactivated. L-lysine, formate dehydrogenase, NADPH, Corynebacterium glutamicum The work was carried out using the equipment of the Multipurpose Scientific This work was carried out on the equipment of the Multipurpose Scientific Installation of «All-Russian Collection of Industrial Microorganisms», National Bio-Resource Center, NRC «Kurchatov Institute»- GosNIIgenetika. This work was financially supported by the Ministry of Education and Science of Russia (Unique Project Identifier - RFMEFI61017X0011).

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