Engineering of a Glycerol Utilization Pathway for Amino Acid Production by Corynebacterium glutamicum

ABSTRACT The amino acid-producing organism Corynebacterium glutamicum cannot utilize glycerol, a stoichiometric by-product of biodiesel production. By heterologous expression of Escherichia coli glycerol utilization genes, C. glutamicum was engineered to grow on glycerol. While expression of the E. coli genes for glycerol kinase (glpK) and glycerol 3-phosphate dehydrogenase (glpD) was sufficient for growth on glycerol as the sole carbon and energy source, additional expression of the aquaglyceroporin gene glpF from E. coli increased growth rate and biomass formation. Glutamate production from glycerol was enabled by plasmid-borne expression of E. coli glpF, glpK, and glpD in C. glutamicum wild type. In addition, a lysine-producing C. glutamicum strain expressing E. coli glpF, glpK, and glpD was able to produce lysine from glycerol as the sole carbon substrate as well as from glycerol-glucose mixtures.

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Molecular Characterization of Genes Coding for Wild-Type and Sulfonylurea-Resistant Acetolactate Synthase in the Cyanobacterium Synechococcus PC C7942

Zeitschrift für Naturforschung C ◽

10.1515/znc-1990-0541 ◽

1990 ◽

Vol 45 (5) ◽

pp. 538-543 ◽

Cited By ~ 6

Author(s):

D. Friedberg ◽

J. Seijffers

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Molecular Characterization ◽

Amino Acid Level ◽

Acetolactate Synthase ◽

Mutant Gene ◽

Wild Type ◽

E Coli

We present here the isolation and molecular characterization of acetolactate synthase (ALS) genes from the cyanobacterium Synechococcus PCC7942 which specify a sulfonylurea-sensitive enzyme and from the sulfonylurea-resistant mutant SM3/20, which specify resistance to sulfonylurea herbicides. The ALS gene was cloned and mapped by complementation of an Escherichia coli ilv auxotroph that requires branched-chain amino acids for growth and lacks ALS activity. The cyanobacterial gene is efficiently expressed in this heterologous host. The ALS gene codes for 612 amino acids and shows high sequence homology (46%) at the amino acid level with ALS III of E. coli and with the tobacco ALS. The resistant phenotype is a consequence of proline to serine substitution in residue 115 of the deduced amino acid sequence. Functional expression of the mutant gene in wild-type Synechococcus and in E. coli confirmed that this amino-acid substitution is responsible for the resistance. Yet the deduced amino-acid sequence as compared with othjer ALS proteins supports the notion that the amino-acid context of the substitution is important for the resistance.

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PBP5, PBP6 and DacD play different roles in intrinsic β-lactam resistance of Escherichia coli

Microbiology ◽

10.1099/mic.0.046227-0 ◽

2011 ◽

Vol 157 (9) ◽

pp. 2702-2707 ◽

Cited By ~ 25

Author(s):

Sujoy Kumar Sarkar ◽

Mouparna Dutta ◽

Chiranjit Chowdhury ◽

Akash Kumar ◽

Anindya S. Ghosh

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Parent Strain ◽

Amino Acid Identity ◽

Enzymic Activity ◽

Exponential Phase ◽

Wild Type ◽

Acid Identity ◽

Penicillin Binding Proteins ◽

E Coli

Escherichia coli PBP5, PBP6 and DacD, encoded by dacA, dacC and dacD, respectively, share substantial amino acid identity and together constitute ~50 % of the total penicillin-binding proteins of E. coli. PBP5 helps maintain intrinsic β-lactam resistance within the cell. To test if PBP6 and DacD play simlar roles, we deleted dacC and dacD individually, and dacC in combination with dacA, from E. coli 2443 and compared β-lactam sensitivity of the mutants and the parent strain. β-Lactam resistance was complemented by wild-type, but not dd-carboxypeptidase-deficient PBP5, confirming that enzymic activity of PBP5 is essential for β-lactam resistance. Deletion of dacC and expression of PBP6 during exponential or stationary phase did not alter β-lactam resistance of a dacA mutant. Expression of DacD during mid-exponential phase partially restored β-lactam resistance of the dacA mutant. Therefore, PBP5 dd-carboxypeptidase activity is essential for intrinsic β-lactam resistance of E. coli and DacD can partially compensate for PBP5 in this capacity, whereas PBP6 cannot.

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Isolation and Characterization of vicH, Encoding a New Pleiotropic Regulator in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.182.7.2026-2032.2000 ◽

2000 ◽

Vol 182 (7) ◽

pp. 2026-2032 ◽

Cited By ~ 29

Author(s):

Christian Tendeng ◽

Cyril Badaut ◽

Evelyne Krin ◽

Pierre Gounon ◽

Saravuth Ngo ◽

...

Keyword(s):

Amino Acid ◽

Vibrio Cholerae ◽

Genomic Library ◽

Single Gene ◽

Wild Type ◽

Semisolid Medium ◽

E Coli ◽

Isolation And Characterization ◽

Serovar Typhimurium

ABSTRACT During the last decade, the hns gene and its product, the H-NS protein, have been extensively studied in Escherichia coli. H-NS-like proteins seem to be widespread in gram-negative bacteria. However, unlike in E. coli and inSalmonella enterica serovar Typhimurium, little is known about their role in the physiology of those organisms. In this report, we describe the isolation of vicH, an hns-like gene in Vibrio cholerae, the etiological agent of cholera. This gene was isolated from a V. cholerae genomic library by complementation of different phenotypes associated with anhns mutation in E. coli. It encodes a 135-amino-acid protein showing approximately 50% identity with both H-NS and StpA in E. coli. Despite a low amino acid conservation in the N-terminal part, VicH is able to cross-react with anti-H-NS antibodies and to form oligomers in vitro. ThevicH gene is expressed as a single gene from two promoters in tandem and is induced by cold shock. A V. choleraewild-type strain expressing a vicHΔ92 gene lacking its 3′ end shows pleiotropic alterations with regard to mucoidy and salicin metabolism. Moreover, this strain is unable to swarm on semisolid medium. Similarly, overexpression of the vicH wild-type gene results in an alteration of swarming behavior. This suggests that VicH could be involved in the virulence process in V. cholerae, in particular by affecting flagellum biosynthesis.

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Active-Site Residues of Escherichia coli DNA Gyrase Required in Coupling ATP Hydrolysis to DNA Supercoiling and Amino Acid Substitutions Leading to Novobiocin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.3.1037-1046.2003 ◽

2003 ◽

Vol 47 (3) ◽

pp. 1037-1046 ◽

Cited By ~ 47

Author(s):

Christian H. Gross ◽

Jonathan D. Parsons ◽

Trudy H. Grossman ◽

Paul S. Charifson ◽

Steven Bellon ◽

...

Keyword(s):

Amino Acid ◽

Atp Hydrolysis ◽

Dna Gyrase ◽

Dna Supercoiling ◽

Wild Type ◽

Wild Type Enzyme ◽

Mutant Proteins ◽

E Coli ◽

Novobiocin Resistance

ABSTRACT DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP · PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A2B2 gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

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Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

Biochemical Journal ◽

10.1042/bj3140063 ◽

1996 ◽

Vol 314 (1) ◽

pp. 63-71 ◽

Cited By ~ 10

Author(s):

Johanneke L. H. BUSCH ◽

Jacques L. J. BRETON ◽

Barry M. BARTLETT ◽

Richard JAMES ◽

E. Claude HATCHIKIAN ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Magnetic Circular Dichroism ◽

Wild Type ◽

E Coli ◽

Excess Iron ◽

Expression In Escherichia Coli ◽

Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

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Carbon source-dependent transcriptional regulation of the mitochondrial glycerol-3-phosphate dehydrogenase gene, GUT2, from Saccharomyces cerevisiae

Canadian Journal of Microbiology ◽

10.1139/w00-105 ◽

2000 ◽

Vol 46 (12) ◽

pp. 1096-1100 ◽

Cited By ~ 24

Author(s):

Morten Grauslund ◽

Birgitte Rønnow

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Regulation ◽

Glucose Repression ◽

Carbon Sources ◽

Negative Regulator ◽

Transcriptional Analysis ◽

Dna Binding Site ◽

Glycerol Utilization ◽

Glycerol 3 Phosphate Dehydrogenase ◽

Utilization Pathway

Cytosolic glycerol kinase (Gut1p) and mitochondrial glycerol-3-phosphate dehydrogenase (Gut2p) constitute the glycerol utilization pathway in Saccharomyces cerevisiae. Transcriptional analysis of the GUT2 gene showed that it was repressed by glucose and derepressed on the non-fermentable carbon sources, glycerol, lactate and ethanol. Derepression of GUT2 requires the protein kinase Snf1p as well as the heteromeric protein complex, Hap2/3/4/5, and its putative DNA-binding site (UASHAP) located in the promoter region. Furthermore, glucose repression of GUT2 requires the negative regulator, Opi1p.Key words: GUT2, mitochondrial glycerol-3-phosphate dehydrogenase, transcriptional regulation, Saccharomyces cerevisiae.

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GlpD and PlsB Participate in Persister Cell Formation in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00369-06 ◽

2006 ◽

Vol 188 (14) ◽

pp. 5136-5144 ◽

Cited By ~ 127

Author(s):

Amy L. Spoering ◽

Marin Vulić ◽

Kim Lewis

Keyword(s):

Escherichia Coli ◽

Cell Formation ◽

Wild Type ◽

Expression Library ◽

Genetic Studies ◽

Bacterial Populations ◽

E Coli ◽

Overexpression Strain ◽

Multidrug Tolerance ◽

Glycerol 3 Phosphate Dehydrogenase

ABSTRACT Bacterial populations produce dormant persister cells that are resistant to killing by all antibiotics currently in use, a phenomenon known as multidrug tolerance (MDT). Persisters are phenotypic variants of the wild type and are largely responsible for MDT of biofilms and stationary populations. We recently showed that a hipBA toxin/antitoxin locus is part of the MDT mechanism in Escherichia coli. In an effort to find additional MDT genes, an E. coli expression library was selected for increased survival to ampicillin. A clone with increased persister production was isolated and was found to overexpress the gene for the conserved aerobic sn-glycerol-3-phosphate dehydrogenase GlpD. The GlpD overexpression strain showed increased tolerance to ampicillin and ofloxacin, while a strain with glpD deleted had a decreased level of persisters in the stationary state. This suggests that GlpD is a component of the MDT mechanism. Further genetic studies of mutants affected in pathways involved in sn-glycerol-3-phosphate metabolism have led to the identification of two additional multidrug tolerance loci, glpABC, the anaerobic sn-glycerol-3-phosphate dehydrogenase, and plsB, an sn-glycerol-3-phosphate acyltransferase.

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The porin OmpC ofSalmonella typhimuriummediates adherence to macrophages

Canadian Journal of Microbiology ◽

10.1139/w99-053 ◽

1999 ◽

Vol 45 (8) ◽

pp. 658-669 ◽

Cited By ~ 25

Author(s):

Robert S Negm ◽

Thomas G Pistole

Keyword(s):

Amino Acid ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Virulent Strain ◽

Salmonella Typhi ◽

Amino Acid Sequences ◽

Wild Type ◽

E Coli ◽

Host Cell Recognition

Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequences of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.Key words: Salmonella, porin, macrophage, outer membrane protein, DNA sequencing.

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Molecular characterization ofGluconobacter oxydans recAgene and its inhibitory effect on the function of the host wild-typerecAgene

Canadian Journal of Microbiology ◽

10.1139/w97-140 ◽

1998 ◽

Vol 44 (2) ◽

pp. 149-156 ◽

Cited By ~ 3

Author(s):

Yu-Tien Liu ◽

Der-Chiang Chao ◽

Fan Lee ◽

Chia-Geun Chen ◽

Dar-Der Ji

Keyword(s):

Pseudomonas Aeruginosa ◽

Amino Acid ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

Reca Protein ◽

Gluconobacter Oxydans ◽

Inhibitory Effect ◽

Reca Gene ◽

Wild Type ◽

E Coli

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-typerecA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.Key words: Gluconobacter oxydans, recA gene, recombination, SOS function, interference.

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Cloning, sequencing and overexpression in Escherichia coli of the alginatelyase-encoding aly gene of Pseudomonas alginovora: identification of three classes of alginate lyases

Biochemical Journal ◽

10.1042/bj3190575 ◽

1996 ◽

Vol 319 (2) ◽

pp. 575-583 ◽

Cited By ~ 27

Author(s):

Frederic CHAVAGNAT ◽

Colette DUEZ ◽

Micheline GUINAND ◽

Philippe POTIN ◽

Tristan BARBEYRON ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Alginate Lyase ◽

Wild Type ◽

E Coli ◽

Step Procedure ◽

Amino Acid Precursor ◽

One Step ◽

Alginate Lyases ◽

Pcr Techniques

A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery of PCR techniques and sequenced. It encodes a 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, in the form of a 233-amino-acid precursor. P. alginovora Aly has been overproduced in E. coli with a His-tag sequence fused at the C-terminal end under conditions in which the formation of inclusion bodies is avoided. His-tagged P. alginovora Aly has the same enzymic properties as the wild-type enzyme and has the specificity of a mannuronate lyase. It can be purified in a one-step procedure by affinity chromatography on Ni2+-nitriloacetate resin. The yield is of 5 mg of enzyme per litre of culture. The amplification factor is 12.5 compared with the level of production by wild-type P. alginovora. The six alginate lyases of known primary structure fall into three distinct classes, one of which comprises the pair P. alginovora Aly and Klebsiella pneumoniae Aly.

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