scholarly journals Exploring the Potential of Corynebacterium glutamicum to Produce the Compatible Solute Mannosylglycerate

2021 ◽  
Vol 9 ◽  
Author(s):  
Andreas Schwentner ◽  
Heiko Neugebauer ◽  
Serin Weinmann ◽  
Helena Santos ◽  
Bernhard J. Eikmanns
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Freeze Drying ◽  
Cold Water ◽  
Dry Weight ◽  
Compatible Solute ◽  
Protein Stabilization ◽  
Gene Encoding ◽  
Dehalococcoides Mccartyi ◽  
Growth Production

The compatible solute mannosylglycerate (MG) has exceptional properties in terms of protein stabilization and protection under salt, heat, and freeze-drying stresses as well as against protein aggregation. Due to these characteristics, MG possesses large potential for clinical and biotechnological applications. To achieve efficient MG production, Corynebacterium glutamicum was equipped with a bifunctional MG synthase (encoded by mgsD and catalyzing the condensation of 3-phosphoglycerate and GDP-mannose to MG) from Dehalococcoides mccartyi. The resulting strain C. glutamicum (pEKEx3 mgsD) intracellularly accumulated about 111 mM MG (60 ± 9 mg gCDW−1) with 2% glucose as a carbon source. To enable efficient mannose metabolization, the native manA gene, encoding mannose 6-phosphate isomerase, was overexpressed. Combined overexpression of manA and mgsD from two plasmids in C. glutamicum resulted in intracellular MG accumulation of up to ca. 329 mM [corresponding to 177 mg g cell dry weight (CDW)−1] with glucose, 314 mM (168 mg gCDW−1) with glucose plus mannose, and 328 mM (176 mg gCDW−1) with mannose as carbon source(s), respectively. The product was successfully extracted from cells by using a cold water shock, resulting in up to 5.5 mM MG (1.48 g L−1) in supernatants. The two-plasmid system was improved by integrating the mgsD gene into the manA-bearing plasmid and the resulting strain showed comparable production but faster growth. Repeated cycles of growth/production and extraction of MG in a bacterial milking-like experiment showed that cells could be recycled, which led to a cumulative MG production of 19.9 mM (5.34 g L−1). The results show that the newly constructed C. glutamicum strain produces MG from glucose and mannose and that a cold water shock enables extraction of MG from the cytosol into the medium.

scholarly journals Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

2010 ◽  
Vol 76 (9) ◽  
pp. 2884-2894 ◽  
Author(s):  
Efraín Manilla-Pérez ◽  
Alvin Brian Lange ◽  
Stephan Hetzler ◽  
Marc Wältermann ◽  
Rainer Kalscheuer ◽  
...  
Keyword(s):  
Carbon Source ◽  
Sole Carbon Source ◽  
Marine Bacterium ◽  
Neutral Lipids ◽  
Nile Red ◽  
Dry Weight ◽  
Wax Ester ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Key Enzyme

ABSTRACT In many microorganisms, the key enzyme responsible for catalyzing the last step in triacylglycerol (TAG) and wax ester (WE) biosynthesis is an unspecific acyltransferase which is also referred to as wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT; AtfA). The importance and function of two AtfA homologues (AtfA1 and AtfA2) in the biosynthesis of TAGs and WEs in the hydrocarbon-degrading marine bacterium Alcanivorax borkumensis SK2 have been described recently. However, after the disruption of both the AtfA1 and AtfA2 genes, reduced but substantial accumulation of TAGs was still observed, indicating the existence of an alternative TAG biosynthesis pathway. In this study, transposon-induced mutagenesis was applied to an atfA1 atfA2 double mutant to screen for A. borkumensis mutants totally defective in biosynthesis of neutral lipids in order to identify additional enzymes involved in the biosynthesis of these lipids. At the same time, we have searched for a totally TAG-negative mutant in order to study the function of TAGs in A. borkumensis. Thirteen fluorescence-negative mutants were identified on Nile red ONR7a agar plates and analyzed for their abilities to synthesize lipids. Among these, mutant 2 M131 was no longer able to synthesize and accumulate TAGs if pyruvate was used as the sole carbon source. The transposon insertion was localized in a gene encoding a putative cytochrome c family protein (ABO_1185). Growth and TAG accumulation experiments showed that the disruption of this gene resulted in the absence of TAGs in 2 M131 but that growth was not affected. In cells of A. borkumensis SK2 grown on pyruvate as the sole carbon source, TAGs represented about 11% of the dry weight of the cells, while in the mutant 2 M131, TAGs were not detected by thin-layer and gas chromatography analyses. Starvation and lipid mobilization experiments revealed that the lipids play an important role in the survival of the cells. The function of neutral lipids in A. borkumensis SK2 is discussed.

scholarly journals Engineering of a Xylose Metabolic Pathway in Corynebacterium glutamicum

2006 ◽  
Vol 72 (5) ◽  
pp. 3418-3428 ◽  
Author(s):  
Hideo Kawaguchi ◽  
Alain A. Vert�s ◽  
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. Vert�s, 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.

scholarly journals The transcriptional regulators RamA and RamB are involved in the regulation of glycogen synthesis in Corynebacterium glutamicum

Microbiology ◽  
2010 ◽  
Vol 156 (4) ◽  
pp. 1256-1263 ◽  
Author(s):  
Gerd M. Seibold ◽  
Christian T. Hagmann ◽  
Melanie Schietzel ◽  
Denise Emer ◽  
Marc Auchter ◽  
...  
Keyword(s):  
Carbon Source ◽  
Corynebacterium Glutamicum ◽  
Transcriptional Control ◽  
Glycogen Content ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Dry Weight ◽  
Transcriptional Regulators ◽  
Negative Regulator

When grown in glucose-, fructose- or sucrose-containing medium, the amino acid producer Corynebacterium glutamicum transiently accumulates large amounts of glycogen (up to 10 % of its dry weight), whereas only a marginal amount of glycogen is formed during growth with acetate. This carbon-source-dependent regulation is at least partially due to transcriptional control of glgC, encoding ADP-glucose pyrophosphorylase, the first enzyme of glycogen synthesis from glucose-1-phosphate. Here, we have analysed a possible regulatory role for the transcriptional regulators RamA and RamB on glycogen content of the cells and on control of expression of glgC and of glgA, which encodes the second enzyme of glycogen synthesis, glycogen synthase. Determination of the glycogen content of RamA- and RamB-deficient C. glutamicum indicated that RamA and RamB influence glycogen synthesis positively and negatively, respectively. In accordance with the identification of putative RamA and RamB binding sites upstream of glgC and glgA, both regulators were found to bind specifically to the glgC–glgA intergenic promoter region. Promoter activity assays in wild-type and RamA- and RamB-deficient strains of C. glutamicum revealed that (i) RamA is a positive regulator of glgC and glgA, (ii) RamB is a negative regulator of glgA and (iii) neither RamA nor RamB alone is responsible for the carbon-source-dependent regulation of glycogen synthesis in C. glutamicum.

Accumulation of Neutral Lipids and β-Carotene by the Yarrowia lipolytica Yeast on a Medium with Sucrose as a Carbon Source

2021 ◽  
Vol 37 (3) ◽  
pp. 29-41
Author(s):  
Yu.M. Kosikhina ◽  
E.B. Vinogradova ◽  
D.A. Dementev ◽  
V.S. Korobov ◽  
V.A. Zolottsev ◽  
...  
Keyword(s):  
Carbon Source ◽  
Yarrowia Lipolytica ◽  
Neutral Lipids ◽  
Sucrose Concentration ◽  
Dry Weight ◽  
Rich Medium ◽  
Kurchatov Institute ◽  
Gene Encoding ◽  
Dry Biomass ◽  
Β Carotene

Recombinant Yarrowia lipolytica yeast has been used as a host strain for the expression of the Saccharomyces cerevisiae ScSUC2 gene and Y. lipolytica YlHXK1 gene encoding invertase and hexakinase, respectively. The expression was carried out on the background of the enhanced pathway of the synthesis of neutral lipids. This allowed the yeast to efficiently utilize glucose as a single carbon source. The engineered strain accumulated neutral lipids in amount of 50.7% of biomass dry weight, when cultured in tubes in minimal medium with nitrogen limitation and high sucrose content. The next metabolic engineering step was the use of the CRISPR-Cas9 editing system to introduce a heterologous β-carotene synthesis pathway by the expression of the Mucor circinelloides genes CarRP and CarB, encoding the bifunctional enzyme phytoene synthase/licopene-β-cyclase (carRP) and phytoene dehydrogenase (carB), as well as an increase in the expression level of the Y. lipolytica YlGGS1 gene, encoding geranyl diphosphate synthase enzyme. The β-carotene production on a sucrose-containing medium was shown for the first time; it amounted for 24.0 mg/g dry biomass, or 406.9 mg/L, on the 5th day of cultivation in tubes in a rich medium with a sucrose portioned supply (50 g/L). The corresponding values for a rich medium with a higher sucrose concentration (90 g/L) were 21.9 mg/g dry biomass and 625.8 mg/L. Yarrowia lipolytica, β-carotene, sucrose, CRISPR-Cas9 The plasmids pdKu70Yl-URA3, pLTet-SP-CAT, pARS-Cre-reverse, pMW-att-Cm and pGPD1Yl were kindly provided by Ph.D. I.A. Laptev ("Kurchatov Institute"-GOSNIIGENETIKA NRC). The work was carried out using the equipment of the Multipurpose Scientific Facility of the "Russian State Collection of Industrial Microorganisms" National Bio-Resource Center, NRC «Kurchatov Institute»-GOSNIIGENETIKA. The work was supported by state assignment no. 14 of 25.07.2012 AAAA-A20-120093090016-9. The quantitative analysis of lipids and β-carotene was carried out by V.A. Zolottsev within the framework of the "Long-Term Program of Fundamental Scientific Research in the Russian Federation (2021-2030)".

Effects of an Autoregulatory Senescence-inhibitor Gene Construct on Nicotiana alata Link and Otto.

HortScience ◽  
1998 ◽  
Vol 33 (3) ◽  
pp. 519d-519 ◽  
Author(s):  
Kenneth R. Schroeder ◽  
Dennis P. Stimart
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Plant Height ◽  
Dry Weight ◽  
Nicotiana Alata ◽  
Wild Type ◽  
Gene Encoding ◽  
Delayed Senescence ◽  
Gene Construct ◽  
Shoot Dry Weight ◽  
Inhibitor System

Nicotiana alata Link and Otto. was transformed via Agrobacterium tumefaciens encoding a senescence-specific promoter SAG12 cloned from Arabidopsis thaliana fused to a Agrobacterium tumefaciens gene encoding isopentenyl transferase (IPT) that catalyzes cytokinin synthesis. This was considered an autoregulatory senescence-inhibitor system. In 1996, we reported delayed senescence of intact flowers by 2 to 6 d and delayed leaf senescence of transgenic vs. wild-type N. alata. Further evaluations in 1997 revealed several other interesting effects of the SAG12-IPT gene construct. Measurement of chlorophyll content of mature leaves showed higher levels of both chlorophyll a and b in transgenic material under normal fertilization and truncated fertilization regimes. At 4 to 5 months of age transgenic plants expressed differences in plant height, branching, and dry weight. Plant height was reduced by 3 to 13 cm; branch counts increased 2 to 3 fold; and shoot dry weight increased up to 11 g over wild-type N. alata. These observations indicate the system is not tightly autoregulated and may prove useful to the floriculture industry for producing compact and more floriferous plants.

scholarly journals The ldhA Gene Encoding Fermentative L-Lactate Dehydrogenase in Corynebacterium glutamicum Is Positively Regulated by the Global Regulator GlxR

2021 ◽  
Vol 9 (3) ◽  
pp. 550
Author(s):  
Koichi Toyoda ◽  
Masayuki Inui
Keyword(s):  
Lactate Dehydrogenase ◽  
Binding Site ◽  
Corynebacterium Glutamicum ◽  
Lactate Production ◽  
Exponential Phase ◽  
Aerobic Bacterium ◽  
Global Regulator ◽  
Gene Encoding ◽  
Fermentation Products ◽  
Ldha Gene

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.

scholarly journals Mutation in the ntrR Gene, a Member of the vap Gene Family, Increases the Symbiotic Efficiency of Sinorhizobium meliloti

2001 ◽  
Vol 14 (7) ◽  
pp. 887-894 ◽  
Author(s):  
Boglárka Oláh ◽  
Erno Kiss ◽  
Zoltán Györgypál ◽  
Judit Borzi ◽  
Gyöngyi Cinege ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Pathogenic Bacteria ◽  
Sinorhizobium Meliloti ◽  
Root Nodules ◽  
Nifh Gene ◽  
Dry Weight ◽  
Nodule Occupancy ◽  
Gene Encoding ◽  
Symbiotic Efficiency ◽  
Host Interactions

In specific plant organs, namely the root nodules of alfalfa, fixed nitrogen (ammonia) produced by the symbiotic partner Sinorhizobium meliloti supports the growth of the host plant in nitrogen-depleted environment. Here, we report that a derivative of S. meliloti carrying a mutation in the chromosomal ntrR gene induced nodules with enhanced nitrogen fixation capacity, resulting in an increased dry weight and nitrogen content of alfalfa. The efficient nitrogen fixation is a result of the higher expression level of the nifH gene, encoding one of the subunits of the nitrogenase enzyme, and nifA, the transcriptional regulator of the nif operon. The ntrR gene, controlled negatively by its own product and positively by the symbiotic regulator syrM, is expressed in the same zone of nodules as the nif genes. As a result of the nitrogen-tolerant phenotype of the strain, the beneficial effect of the mutation on efficiency is not abolished in the presence of the exogenous nitrogen source. The ntrR mutant is highly competitive in nodule occupancy compared with the wild-type strain. Sequence analysis of the mutant region revealed a new cluster of genes, termed the “ntrPR operon,” which is highly homologous to a group of vap-related genes of various pathogenic bacteria that are presumably implicated in bacterium-host interactions. On the basis of its favorable properties, the strain is a good candidate for future agricultural utilization.

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 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 ◽  
2006 ◽  
Vol 152 (4) ◽  
pp. 923-935 ◽  
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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