Reconstruction of the Diaminopimelic Acid Pathway to Promote L-lysine Production in Corynebacterium glutamicum

The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and potential to increase the production of L-lysine. The effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in a high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3 ± 4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening the tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189 ± 8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35 ± 0.05 g/(g·h) in a 5-L jar fermenter. The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.

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Reconstruction of the Diaminopimelic Acid Pathway to Promote L-lysine Production in Corynebacterium Glutamicum

10.21203/rs.3.rs-539166/v1 ◽

2021 ◽

Author(s):

Ning Liu ◽

Ting-Ting Zhang ◽

Zhi-Ming Rao ◽

Wei-Guo Zhang ◽

Jian-Zhong Xu

Keyword(s):

Corynebacterium Glutamicum ◽

Regulatory Protein ◽

Lysine Biosynthesis ◽

Diaminopimelic Acid ◽

Specific Rate ◽

Original Strain ◽

Lysine Production ◽

Final Strain ◽

Acid Pathway ◽

Maximum Specific Rate

Abstract Background: The dehydrogenase pathway and the succinylase pathway are involved in the synthesis of L-lysine in Corynebacterium glutamicum. Despite the low contribution rate to L-lysine production, the dehydrogenase pathway is favorable for its simple steps and great potentials to increase the production of L-lysine. Results: The aim of this work is to enhance the carbon flux in dehydrogenase pathway to promote L-lysine production. Firstly, the effect of ammonium (NH4+) concentration on L-lysine biosynthesis was investigated, and the results indicated that the biosynthesis of L-lysine can be promoted in high NH4+ environment. In order to reduce the requirement of NH4+, the nitrogen source regulatory protein AmtR was knocked out, resulting in an 8.5% increase in L-lysine production (i.e., 52.3±4.31 g/L). Subsequently, the dehydrogenase pathway was upregulated by blocking or weakening tetrahydrodipicolinate succinylase (DapD)-coding gene dapD and overexpressing the ddh gene to further enhance L-lysine biosynthesis. The final strain XQ-5-W4 could produce 189±8.7 g/L L-lysine with the maximum specific rate (qLys,max.) of 0.35±0.05 g/(g·h) in a 5-L jar fermenter. Conclusions: The L-lysine titer and qLys,max achieved in this study is about 25.2% and 59.1% higher than that of the original strain without enhancement of dehydrogenase pathway, respectively. The results indicated that the dehydrogenase pathway could serve as a breakthrough point to reconstruct the diaminopimelic acid (DAP) pathway and promote L-lysine production.

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Lysine biosynthesis inMethanobacterium thermoautotrophicum is by the diaminopimelic acid pathway

Current Microbiology ◽

10.1007/bf01627254 ◽

1984 ◽

Vol 10 (4) ◽

pp. 195-198 ◽

Cited By ~ 23

Author(s):

Nouna Bakhiet ◽

Fred W. Forney ◽

Donald P. Stahly ◽

Lacy Daniels

Keyword(s):

Lysine Biosynthesis ◽

Diaminopimelic Acid ◽

Acid Pathway

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In-Depth Profiling of Lysine-Producing Corynebacterium glutamicum by Combined Analysis of the Transcriptome, Metabolome, and Fluxome

Journal of Bacteriology ◽

10.1128/jb.186.6.1769-1784.2004 ◽

2004 ◽

Vol 186 (6) ◽

pp. 1769-1784 ◽

Cited By ~ 147

Author(s):

Jens Olaf Krömer ◽

Oliver Sorgenfrei ◽

Kai Klopprogge ◽

Elmar Heinzle ◽

Christoph Wittmann

Keyword(s):

Gene Expression ◽

Glucose Uptake ◽

Corynebacterium Glutamicum ◽

Tca Cycle ◽

Lysine Biosynthesis ◽

Intracellular Metabolite ◽

Lysine Production ◽

The Tca Cycle ◽

Depth Analysis

ABSTRACT An in-depth analysis of the intracellular metabolite concentrations, metabolic fluxes, and gene expression (metabolome, fluxome, and transcriptome, respectively) of lysine-producing Corynebacterium glutamicum ATCC 13287 was performed at different stages of batch culture and revealed distinct phases of growth and lysine production. For this purpose, 13C flux analysis with gas chromatography-mass spectrometry-labeling measurement of free intracellular amino acids, metabolite balancing, and isotopomer modeling were combined with expression profiling via DNA microarrays and with intracellular metabolite quantification. The phase shift from growth to lysine production was accompanied by a decrease in glucose uptake flux, the redirection of flux from the tricarboxylic acid (TCA) cycle towards anaplerotic carboxylation and lysine biosynthesis, transient dynamics of intracellular metabolite pools, such as an increase of lysine up to 40 mM prior to its excretion, and complex changes in the expression of genes for central metabolism. The integrated approach was valuable for the identification of correlations between gene expression and in vivo activity for numerous enzymes. The glucose uptake flux closely corresponded to the expression of glucose phosphotransferase genes. A correlation between flux and expression was also observed for glucose-6-phosphate dehydrogenase, transaldolase, and transketolase and for most TCA cycle genes. In contrast, cytoplasmic malate dehydrogenase expression increased despite a reduction of the TCA cycle flux, probably related to its contribution to NADH regeneration under conditions of reduced growth. Most genes for lysine biosynthesis showed a constant expression level, despite a marked change of the metabolic flux, indicating that they are strongly regulated at the metabolic level. Glyoxylate cycle genes were continuously expressed, but the pathway exhibited in vivo activity only in the later stage. The most pronounced changes in gene expression during cultivation were found for enzymes at entry points into glycolysis, the pentose phosphate pathway, the TCA cycle, and lysine biosynthesis, indicating that these might be of special importance for transcriptional control in C. glutamicum.

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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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Saccharopine, an Intermediate of the Aminoadipic Acid Pathway of Lysine Biosynthesis

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)97357-6 ◽

1965 ◽

Vol 240 (6) ◽

pp. 2524-2530 ◽

Cited By ~ 2

Author(s):

J.S. Trupin ◽

Harry P. Broquist

Keyword(s):

Lysine Biosynthesis ◽

Acid Pathway

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Structural basis for redox sensitivity in Corynebacterium glutamicum diaminopimelate epimerase: an enzyme involved in l-lysine biosynthesis

Scientific Reports ◽

10.1038/srep42318 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 10

Author(s):

Hye-Young Sagong ◽

Kyung-Jin Kim

Keyword(s):

Corynebacterium Glutamicum ◽

Lysine Biosynthesis ◽

Structural Basis ◽

Redox Sensitivity

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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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