Enhanced production of d-lactate from mixed sugars in Corynebacterium glutamicum by overexpression of glycolytic genes encoding phosphofructokinase and triosephosphate isomerase

2019 ◽  
Vol 127 (3) ◽  
pp. 288-293 ◽  
Author(s):  
Yota Tsuge ◽  
Naoto Kato ◽  
Shogo Yamamoto ◽  
Masako Suda ◽  
Masayuki Inui
Keyword(s):  
Corynebacterium Glutamicum ◽  
Triosephosphate Isomerase ◽  
Mixed Sugars ◽  
Enhanced Production ◽  
Genes Encoding

Disruption of genes for the enhanced biosynthesis of α-ketoglutarate in Corynebacterium glutamicum

2012 ◽  
Vol 58 (3) ◽  
pp. 278-286 ◽  
Author(s):  
Jae-Hyung Jo ◽  
Hye-Young Seol ◽  
Yun-Bom Lee ◽  
Min-Hong Kim ◽  
Hyung-Hwan Hyun ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Biosynthetic Pathway ◽  
Isocitrate Lyase ◽  
Glutamate Synthase ◽  
Flask Culture ◽  
Control Strain ◽  
Enhanced Production ◽  
Key Enzyme ◽  
Microbial Strains ◽  
Glyoxylate Pathway

The development of microbial strains for the enhanced production of α-ketoglutarate (α-KG) was investigated using a strain of Corynebacterium glutamicum that overproduces of l-glutamate, by disrupting three genes involved in the α-KG biosynthetic pathway. The pathways competing with the biosynthesis of α-KG were blocked by knocking out aceA (encoding isocitrate lyase, ICL), gdh (encoding glutamate dehydrogenase, l-gluDH), and gltB (encoding glutamate synthase or glutamate-2-oxoglutarate aminotransferase, GOGAT). The strain with aceA, gltB, and gdh disrupted showed reduced ICL activity and no GOGAT and l-gluDH activities, resulting in up to 16-fold more α-KG production than the control strain in flask culture. These results suggest that l-gluDH is the key enzyme in the conversion of α-KG to l-glutamate; therefore, prevention of this step could promote α-KG accumulation. The inactivation of ICL leads the carbon flow to α-KG by blocking the glyoxylate pathway. However, the disruption of gltB did not affect the biosynthesis of α-KG. Our results can be applied in the industrial production of α-KG by using C. glutamicum as producer.

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

2013 ◽  
Vol 79 (18) ◽  
pp. 5566-5575 ◽  
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.

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

2005 ◽  
Vol 71 (5) ◽  
pp. 2391-2402 ◽  
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.

scholarly journals Functional Caracterization of CapBCA in Controlling Poly-γ-Glutamic Acid Synthesis in Corynebacterium Glutamicum

2021 ◽  
Author(s):  
Guoqiang Xu ◽  
Jiyue Wang ◽  
Luning Gu ◽  
Yaxin Zhu ◽  
Jian Zha ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Glutamic Acid ◽  
Environmental Protection ◽  
Corynebacterium Glutamicum ◽  
Bioinformatics Analysis ◽  
Acid Synthesis ◽  
Transcription Level ◽  
Enhanced Production ◽  
Medium Level ◽  
Shed Light

Abstract Background Poly-γ-glutamic acid (γ-PGA) is a natural anionic biopolymer widely used in various fields, including medicine, food, cosmetics, and environmental protection. The γ-PGA synthase complex, CapBCA, is the only polyprotein complex responsible for γ-PGA synthesis. However, systematic and in-depth research on the function of each component involved in γ-PGA synthesis is scarce, which limits enhanced production of γ-PGA. Results To address this limitation, γ-PGA synthase components were localized, and their functions associated with γ-PGA synthesis were investigated in Corynebacterium glutamicum. Bioinformatics analysis and confocal microscopic observations of CapB-sfGFP, CapC-sfGFP, and CapA-sfGFP proteins revealed that γ-PGA synthase components CapB, CapC, and CapA were all localized on the cell membrane. More importantly, γ-PGA was detected only when CapB, CapC, and CapA were expressed in combination in C. glutamicum. Furthermore, enhancement of CapB or CapC transcription levels (from low to high) and maintaining medium-level CapA transcription led to 35.44% and 76.53% increase in γ-PGA yield (γ-PGA yield-to-biomass), respectively. However, maintaining medium-level CapB and CapC transcription, and moderate enhancement of CapA transcription level (from low to medium) led to 35.01% increase in γ-PGA yield, whereas a further increase in CapA expression (from medium to high) led to 10.36% decrease in γ-PGA yield. Notably, CapC had the greatest influence (accounting for 68.24%) on γ-PGA synthesis. Conclusions The present study determined the membrane localization of γ-PGA synthase components, CapB, CapC, and CapA, in C. glutamicum and confirmed the significance of these components in γ-PGA production. Furthermore, CapC was found to have the greatest influence on controlling γ-PGA synthesis. These findings shed light into the effect of γ-PGA synthase component expression on γ-PGA synthesis, and provide insights for further improvement in γ-PGA production.

scholarly journals Metabolic Engineering of Corynebacterium glutamicum for 2-Ketoisovalerate Production

2010 ◽  
Vol 76 (24) ◽  
pp. 8053-8061 ◽  
Author(s):  
Felix S. Krause ◽  
Bastian Blombach ◽  
Bernhard J. Eikmanns
Keyword(s):  
Corynebacterium Glutamicum ◽  
Competitive Inhibition ◽  
Pyruvate Dehydrogenase Complex ◽  
Product Yield ◽  
Acetohydroxyacid Synthase ◽  
Gene Encoding ◽  
Keto Acids ◽  
Genes Encoding ◽  
Cell Densities ◽  
Branched Chain

ABSTRACT 2-Ketoisovalerate is used as a therapeutic agent, and a 2-ketoisovalerate-producing organism may serve as a platform for products deriving from this 2-keto acid. We engineered the wild type of Corynebacterium glutamicum for the growth-decoupled production of 2-ketoisovalerate from glucose by deletion of the aceE gene encoding the E1p subunit of the pyruvate dehydrogenase complex, deletion of the transaminase B gene ilvE, and additional overexpression of the ilvBNCD genes, encoding the l-valine biosynthetic enzymes acetohydroxyacid synthase (AHAS), acetohydroxyacid isomeroreductase, and dihydroxyacid dehydratase. 2-Ketoisovalerate production was further improved by deletion of the pyruvate:quinone oxidoreductase gene pqo. In fed-batch fermentations at high cell densities, the newly constructed strains produced up to 188 ± 28 mM (21.8 ± 3.2 g liter−1) 2-ketoisovalerate and showed a product yield of about 0.47 ± 0.05 mol per mol (0.3 ± 0.03 g per g) of glucose and a volumetric productivity of about 4.6 ± 0.6 mM (0.53 ± 0.07 g liter−1) 2-ketoisovalerate per h in the overall production phase. In studying the influence of the three branched-chain 2-keto acids 2-ketoisovalerate, 2-ketoisocaproate, and 2-keto-3-methylvalerate on the AHAS activity, we observed a competitive inhibition of the AHAS enzyme by 2-ketoisovalerate.

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