Flux Enforcement for Fermentative Production of 5-Aminovalerate and Glutarate by Corynebacterium glutamicum

Bio-based plastics represent an increasing percentage of the plastics economy. The fermentative production of bioplastic monomer 5-aminovalerate (5AVA), which can be converted to polyamide 5 (PA 5), has been established in Corynebacterium glutamicum via two metabolic pathways. l-lysine can be converted to 5AVA by either oxidative decarboxylation and subsequent oxidative deamination or by decarboxylation to cadaverine followed by transamination and oxidation. Here, a new three-step pathway was established by using the monooxygenase putrescine oxidase (Puo), which catalyzes the oxidative deamination of cadaverine, instead of cadaverine transaminase. When the conversion of 5AVA to glutarate was eliminated and oxygen supply improved, a 5AVA titer of 3.7 ± 0.4 g/L was reached in microcultivation that was lower than when cadaverine transaminase was used. The elongation of the new pathway by 5AVA transamination by GABA/5AVA aminotransferase (GabT) and oxidation by succinate/glutarate semialdehyde dehydrogenase (GabD) allowed for glutarate production. Flux enforcement by the disruption of the l-glutamic acid dehydrogenase-encoding gene gdh rendered a single transaminase (GabT) in glutarate production via the new pathway responsible for nitrogen assimilation, which increased the glutarate titer to 7.7 ± 0.7 g/L, i.e., 40% higher than with two transaminases operating in glutarate biosynthesis. Flux enforcement was more effective with one coupling site, thus highlighting requirements regarding the modularity and stoichiometry of pathway-specific flux enforcement for microbial production.

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Adaptation of Corynebacterium glutamicum to Ammonium Limitation: a Global Analysis Using Transcriptome and Proteome Techniques

Applied and Environmental Microbiology ◽

10.1128/aem.71.5.2391-2402.2005 ◽

2005 ◽

Vol 71 (5) ◽

pp. 2391-2402 ◽

Cited By ~ 50

Author(s):

Maike Silberbach ◽

Mathias Schäfer ◽

Andrea T. Hüser ◽

Jörn Kalinowski ◽

Alfred Pühler ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Metabolic Pathways ◽

Ribosomal Proteins ◽

Nitrogen Assimilation ◽

Global Analysis ◽

Cellular Transport ◽

Acid Biosynthesis ◽

Limited Growth ◽

Rate Dependent ◽

Genes Encoding

ABSTRACT Theresponse of Corynebacterium glutamicum to ammonium limitation was studied by transcriptional and proteome profiling of cells grown in a chemostat. Our results show that ammonium-limited growth of C. glutamicum results in a rearrangement of the cellular transport capacity, changes in metabolic pathways for nitrogen assimilation, amino acid biosynthesis, and carbon metabolism, as well as a decreased cell division. Since transcription at different growth rates was studied, it was possible to distinguish specific responses to ammonium limitation and more general, growth rate-dependent alterations in gene expression. The latter include a number of genes encoding ribosomal proteins and genes for FoF1-ATP synthase subunits.

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Utilization of a Wheat Sidestream for 5-Aminovalerate Production in Corynebacterium glutamicum

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.732271 ◽

2021 ◽

Vol 9 ◽

Author(s):

Arthur Burgardt ◽

Carina Prell ◽

Volker F. Wendisch

Keyword(s):

Corynebacterium Glutamicum ◽

Xanthomonas Campestris ◽

Raw Materials ◽

Amino Acid Production ◽

Biotechnological Production ◽

Oxidase Gene ◽

E Coli ◽

Putrescine Oxidase ◽

Encoding Gene ◽

Hydrolysis Of

Production of plastics from petroleum-based raw materials extensively contributes to global pollution and CO2 emissions. Biotechnological production of functionalized monomers can reduce the environmental impact, in particular when using industrial sidestreams as feedstocks. Corynebacterium glutamicum, which is used in the million-ton-scale amino acid production, has been engineered for sustainable production of polyamide monomers. In this study, wheat sidestream concentrate (WSC) from industrial starch production was utilized for production of l-lysine–derived bifunctional monomers using metabolically engineered C. glutamicum strains. Growth of C. glutamicum on WSC was observed and could be improved by hydrolysis of WSC. By heterologous expression of the genes xylAXcBCg (xylA from Xanthomonas campestris) and araBADEc from E. coli, xylose, and arabinose in WSC hydrolysate (WSCH), in addition to glucose, could be consumed, and production of l-lysine could be increased. WSCH-based production of cadaverine and 5-aminovalerate (5AVA) was enabled. To this end, the lysine decarboxylase gene ldcCEc from E. coli was expressed alone or for conversion to 5AVA cascaded either with putrescine transaminase and dehydrogenase genes patDAEc from E. coli or with putrescine oxidase gene puoRq from Rhodococcus qingshengii and patDEc. Deletion of the l-glutamate dehydrogenase–encoding gene gdh reduced formation of l-glutamate as a side product for strains with either of the cascades. Since the former cascade (ldcCEc-patDAEc) yields l-glutamate, 5AVA production is coupled to growth by flux enforcement resulting in the highest 5AVA titer obtained with WSCH-based media.

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Carbon-flux distribution in the central metabolic pathways of Corynebacterium glutamicum during growth on fructose

European Journal of Biochemistry ◽

10.1046/j.1432-1327.1998.2540096.x ◽

1998 ◽

Vol 254 (1) ◽

pp. 96-102 ◽

Cited By ~ 126

Author(s):

Helene Dominguez ◽

Catherine Rollin ◽

Armel Guyonvarch ◽

Jean-Luc Guerquin-Kern ◽

Muriel Cocaign-Bousquet ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Metabolic Pathways ◽

Carbon Flux ◽

Flux Distribution ◽

Carbon Flux Distribution

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Overexpression of the phosphofructokinase encoding gene is crucial for achieving high production of D-lactate in Corynebacterium glutamicum under oxygen deprivation

Applied Microbiology and Biotechnology ◽

10.1007/s00253-015-6546-9 ◽

2015 ◽

Vol 99 (11) ◽

pp. 4679-4689 ◽

Cited By ~ 33

Author(s):

Yota Tsuge ◽

Shogo Yamamoto ◽

Naoto Kato ◽

Masako Suda ◽

Alain A. Vertès ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Oxygen Deprivation ◽

High Production ◽

Encoding Gene

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Mutagenesis of the C1 Oxidation Pathway in Methanosarcina barkeri: New Insights into the Mtr/Mer Bypass Pathway

Journal of Bacteriology ◽

10.1128/jb.01424-07 ◽

2008 ◽

Vol 190 (6) ◽

pp. 1928-1936 ◽

Cited By ~ 33

Author(s):

Paula V. Welander ◽

William W. Metcalf

Keyword(s):

Metabolic Pathways ◽

Methanosarcina Barkeri ◽

Growth Substrate ◽

Oxidation Of Methanol ◽

Cell Extracts ◽

Oxidation Reduction ◽

Absolute Requirement ◽

Genes Encoding ◽

Encoding Gene

ABSTRACT A series of Methanosarcina barkeri mutants lacking the genes encoding the enzymes involved in the C1 oxidation/reduction pathway were constructed. Mutants lacking the methyl-tetrahydromethanopterin (H4MPT):coenzyme M (CoM) methyltransferase-encoding operon (Δmtr), the methylene-H4MPT reductase-encoding gene (Δmer), the methylene-H4MPT dehydrogenase-encoding gene (Δmtd), and the formyl-methanofuran:H4MPT formyl-transferase-encoding gene (Δftr) all failed to grow using either methanol or H2/CO2 as a growth substrate, indicating that there is an absolute requirement for the C1 oxidation/reduction pathway for hydrogenotrophic and methylotrophic methanogenesis. The mutants also failed to grow on acetate, and we suggest that this was due to an inability to generate the reducing equivalents needed for biosynthetic reactions. Despite their lack of growth on methanol, the Δmtr and Δmer mutants were capable of producing methane from this substrate, whereas the Δmtd and Δftr mutants were not. Thus, there is an Mtr/Mer bypass pathway that allows oxidation of methanol to the level of methylene-H4MPT in M. barkeri. The data further suggested that formaldehyde may be an intermediate in this bypass; however, no methanol dehydrogenase activity was found in Δmtr cell extracts, nor was there an obligate role for the formaldehyde-activating enzyme (Fae), which has been shown to catalyze the condensation of formaldehyde and H4MPT in vitro. Both the Δmer and Δmtr mutants were able to grow on a combination of methanol plus acetate, but they did so by metabolic pathways that are clearly distinct from each other and from previously characterized methanogenic pathways.

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Microbial metabolism of the pyridine ring. Metabolic pathways of pyridine biodegradation by soil bacteria

Biochemical Journal ◽

10.1042/bj1460157 ◽

1975 ◽

Vol 146 (1) ◽

pp. 157-172 ◽

Cited By ~ 72

Author(s):

G K Watson ◽

R B Cain

Keyword(s):

Metabolic Pathways ◽

Carbonyl Compounds ◽

Soil Bacteria ◽

Pyridine Ring ◽

Formate Dehydrogenase ◽

Isocitrate Lyase ◽

Rapid Loss ◽

Semialdehyde Dehydrogenase ◽

Nocardia Sp ◽

Succinate Semialdehyde

1. Two bacteria, a Bacillus sp. and a Nocardia sp. (strain Z1) were isolated from soil by enrichment with 0.1 percent (v/v) pyridine and grew rapidly on this compound as sole C, N and energy source. The monohydroxypyridines, tetrahydropyridine, piperidine and some other analogues were not utilized for growth or oxidized by washed suspensions of either bacterium. 2. Cell-free extracts were unable to metabolize pyridine even after supplementation with a variety of cofactors or protecting agents. Treatment of cells with toluene led to rapid loss of the ability to oxidize pyridine. 3. In the presence of 10mM-semicarbazide at pH 6.0, Nocardia Z1 accumulated a semialdehyde idenditied as its 2,4-dinitrophenylhydrazone by chromatography, mixed melting point, mass spectrometry and isotope trapping from [2,6(-14)C]pyridine as glutarate semialdehyde. 4. Extracts of this bacterium prepared from cells grown with pyridine or exposed to the gratuitous inducer 2-picoline, contained high activities of a specific glutarate semialdehyde dehydrogenase. 5. Cells grown with pyridine or glutarate also contained a glutaric dialdehyde dehydrogenase, an acyl-CoA synthetase and elevated amounts of isocitrate lyase but no glutaryl-CoA dehydrogenase. 6. Bacillus 4 accumulated in the presence of 10mM-semicarbazide several acidic carbonyl compounds from pyridine among which was succinate semialdehyde. Extracts of this bacillus after growth of the cells with pyridine contained an inducible succinate semialdehyde dehydrogenase in amounts at least 50-fold over those found in succinate-grown cells. 7. Two mutants of this bacillus, selected for their inability to grow on pyridine were deficient in succinate semialdehyde dehydrogenase. 8. In the presence of 0.2mM-KCN, washed suspensions of Bacillus 4 accumulated formate and possibly formamide from pyridine. The use of [14C]pyridine showed that formate was derived from C-2 of the pyridine ring. 9. The organism had a specific formamide amidohydrolase cleaving formamide quantitatively to formate and NH3. 10. Formate was further oxidized by the particle fraction. There was no soluble formate dehydrogenase in extracts.

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Characterization of Genes Involved in γ-Aminobutyric Acid Metabolic Pathways Response to Metabolites Accumulation in Embryos during Barley Germination

International Journal of Agriculture and Biology ◽

10.17957/ijab/15.1730 ◽

2021 ◽

Vol 25 (04) ◽

pp. 786-794

Author(s):

Mengyuan Jin

Keyword(s):

Gene Expression ◽

Metabolic Pathways ◽

Aminobutyric Acid ◽

Acid Decarboxylase ◽

Succinic Semialdehyde ◽

Succinic Semialdehyde Dehydrogenase ◽

Semialdehyde Dehydrogenase ◽

Key Enzyme ◽

Gaba Content ◽

Gaba Accumulation

To reveal the key enzyme genes involved in γ-aminobutyric acid (GABA) metabolic pathways response to elevated metabolite storage in embryos during barley germination, this study investigated the GABA content, cloned GABA metabolic pathway genes and analyzed their expression levels, respectively. In barley embryos, GABA content continued to rise during the soaking process and then decreased after the germination. Three genes including glutamic acid decarboxylase (GAD), GABA transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) involved in the GABA pathway were cloned and characterized from the barley embryos, respectively. Before the germination, the expression of GAD gene was up-regulated, while GABA-T gene expression was down-regulated. After the germination, GAD gene expression was lowered, but GABA-T gene expression was rapidly increased. The SSADH gene expression remained stable after soaking of 4 h, and then down-regulated. There is evidence that the high GABA content in germinating barley seeds is parallel with the upregulation of the GAD gene, and down-regulation of GABA-T gene. These results indicate that the expression level of the genes involved in GABA pathway is a crucial factor in GABA accumulation during soaking and germination. This study is beneficial for the development of GABA-rich barley products by germination. © 2021 Friends Science Publishers

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Fermentative Production of N-Alkylated Glycine Derivatives by Recombinant Corynebacterium glutamicum Using a Mutant of Imine Reductase DpkA From Pseudomonas putida

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2019.00232 ◽

2019 ◽

Vol 7 ◽

Cited By ~ 6

Author(s):

Melanie Mindt ◽

Silvin Hannibal ◽

Maria Heuser ◽

Joe Max Risse ◽

Keerthi Sasikumar ◽

...

Keyword(s):

Pseudomonas Putida ◽

Corynebacterium Glutamicum ◽

Fermentative Production ◽

Glycine Derivatives

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Highlighting Human Enzymes Active in Different Metabolic Pathways and Diseases: The Case Study of EC 1.2.3.1 and EC 2.3.1.9

Biomedicines ◽

10.3390/biomedicines8080250 ◽

2020 ◽

Vol 8 (8) ◽

pp. 250

Author(s):

Giulia Babbi ◽

Davide Baldazzi ◽

Castrense Savojardo ◽

Martelli Pier Luigi ◽

Rita Casadio

Keyword(s):

Metabolic Pathways ◽

Molecular Mechanisms ◽

Mitochondrial Gene ◽

Enzyme Commission Number ◽

Multiple Roles ◽

Acetyl Coa ◽

Functional Activities ◽

Interaction Sites ◽

Semialdehyde Dehydrogenase

Enzymes are key proteins performing the basic functional activities in cells. In humans, enzymes can be also responsible for diseases, and the molecular mechanisms underlying the genotype to phenotype relationship are under investigation for diagnosis and medical care. Here, we focus on highlighting enzymes that are active in different metabolic pathways and become relevant hubs in protein interaction networks. We perform a statistics to derive our present knowledge on human metabolic pathways (the Kyoto Encyclopaedia of Genes and Genomes (KEGG)), and we found that activity aldehyde dehydrogenase (NAD(+)), described by Enzyme Commission number EC 1.2.1.3, and activity acetyl-CoA C-acetyltransferase (EC 2.3.1.9) are the ones most frequently involved. By associating functional activities (EC numbers) to enzyme proteins, we found the proteins most frequently involved in metabolic pathways. With our analysis, we found that these proteins are endowed with the highest numbers of interaction partners when compared to all the enzymes in the pathways and with the highest numbers of predicted interaction sites. As specific enzyme protein test cases, we focus on Alpha-Aminoadipic Semialdehyde Dehydrogenase (ALDH7A1, EC 2.3.1.9) and Acetyl-CoA acetyltransferase, cytosolic and mitochondrial (gene products of ACAT2 and ACAT1, respectively; EC 2.3.1.9). With computational approaches we show that it is possible, by starting from the enzyme structure, to highlight clues of their multiple roles in different pathways and of putative mechanisms promoting the association of genes to disease.

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