scholarly journals Experimental evolution reveals an effective avenue to release catabolite repression via mutations in XylR

2017 ◽  
Vol 114 (28) ◽  
pp. 7349-7354 ◽  
Author(s):  
Christian Sievert ◽  
Lizbeth M. Nieves ◽  
Larry A. Panyon ◽  
Taylor Loeffler ◽  
Chandler Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lignocellulosic Biomass ◽  
Catabolite Repression ◽  
High Efficiency ◽  
Xylose Fermentation ◽  
Amino Acid Substitutions ◽  
Xylose Utilization ◽  
Microbial Conversion ◽  
E Coli ◽  
Product Titer

Microbial production of fuels and chemicals from lignocellulosic biomass provides promising biorenewable alternatives to the conventional petroleum-based products. However, heterogeneous sugar composition of lignocellulosic biomass hinders efficient microbial conversion due to carbon catabolite repression. The most abundant sugar monomers in lignocellulosic biomass materials are glucose and xylose. Although industrialEscherichia colistrains efficiently use glucose, their ability to use xylose is often repressed in the presence of glucose. Here we independently evolved threeE. colistrains from the same ancestor to achieve high efficiency for xylose fermentation. Each evolved strain has a point mutation in a transcriptional activator for xylose catabolic operons, either CRP or XylR, and these mutations are demonstrated to enhance xylose fermentation by allelic replacements. Identified XylR variants (R121C and P363S) have a higher affinity to their DNA binding sites, leading to a xylose catabolic activation independent of catabolite repression control. Upon introducing these amino acid substitutions into theE. coliD-lactate producer TG114, 94% of a glucose–xylose mixture (50 g⋅L−1each) was used in mineral salt media that led to a 50% increase in product titer after 96 h of fermentation. The two amino acid substitutions in XylR enhance xylose utilization and release glucose-induced repression in differentE. colihosts, including wild type, suggesting its potential wide application in industrialE. colibiocatalysts.

scholarly journals Experimental evolution reveals a novel avenue to release catabolite repression via mutations in XylR

2017 ◽  
Author(s):  
Christian Sievert ◽  
Lizbeth M. Nieves ◽  
Larry A. Panyon ◽  
Taylor Loeffler ◽  
Chandler Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Lignocellulosic Biomass ◽  
Catabolite Repression ◽  
High Efficiency ◽  
Xylose Fermentation ◽  
Amino Acid Substitutions ◽  
Xylose Utilization ◽  
Microbial Conversion ◽  
E Coli ◽  
Product Titer

AbstractMicrobial production of fuels and chemicals from lignocellulosic biomass provides promising bio-renewable alternatives to the conventional petroleum-based products. However, heterogeneous sugar composition of lignocellulosic biomass hinders efficient microbial conversion due to carbon catabolite repression. The most abundant sugar monomers in lignocel-lulosic biomass materials are glucose and xylose. While industrialEscherichia colistrains efficiently utilize glucose, their ability to utilize xylose is often repressed in the presence of glucose. Here we independently evolved threeE. colistrains from the same ancestor to achieve high efficiency for xylose fermentation. Each evolved strain has a point mutation in a transcriptional activator for xylose catabolic operons, either CRP or XylR, and these mutations are demonstrated to enhance xylose fermentation by allelic replacements. Identified XylR variants (R121C and P363S) have a higher affinity to their DNA binding sites, leading to a xylose catabolic activation independent of catabolite repression control. Upon introducing these amino acid substitutions into theE. coliD-lactate producer TG114, 94 % of a glucose-xylose mixture (50 g L-1each) was utilized in mineral salt media that led to a 50 % increase in product titer after 96 h of fermentation. The two amino acid substitutions in XylR enhance xylose utilization and release glucose-induced repression in differentE. colihosts, including wild-type, suggesting its potential wide application in industrialE. colibiocatalysts.

scholarly journals AmpC β-Lactamase in an Escherichia coli Clinical Isolate Confers Resistance to Expanded-Spectrum Cephalosporins

2004 ◽  
Vol 48 (10) ◽  
pp. 4050-4053 ◽  
Author(s):  
Hedi Mammeri ◽  
Hasan Nazic ◽  
Thierry Naas ◽  
Laurent Poirel ◽  
Sophie Léotard ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Clinical Isolate ◽  
Biochemical Analysis ◽  
Amino Acid Substitutions ◽  
E Coli ◽  
Extended Spectrum

ABSTRACT Cloning, sequencing, and biochemical analysis identified a novel AmpC-type β-lactamase conferring resistance to extended-spectrum cephalosporins in an Escherichia coli clinical isolate. This enzyme, exhibiting 14 amino acid substitutions compared to a reference AmpC cephalosporinase of E. coli, hydrolyzed ceftazidime and cefepime significantly.

scholarly journals Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Jung-Hoon Bae ◽  
Mi-Jin Kim ◽  
Bong Hyun Sung ◽  
Yong-Su Jin ◽  
Jung-Hoon Sohn
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Directed Evolution ◽  
Lignocellulosic Biomass ◽  
Xylose Fermentation ◽  
Xylose Isomerase ◽  
Carbon Substrate ◽  
Yeast Fermentation ◽  
Xylose Utilization ◽  
Secretory Expression ◽  
Xylose Consumption

Abstract Background Xylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways along with the introduction of a xylose transporter and amplification of the downstream pathway. However, the low utilization of xylose in the presence of glucose, due to the varying preference for cellular uptake, is a lingering challenge. Studies so far have mainly focused on xylose utilization inside the cells, but there have been little trials on the conversion of xylose to xylulose by cell before uptake. We hypothesized that the extracellular conversion of xylose to xylulose before uptake would facilitate better utilization of xylose even in the presence of glucose. To verify this, XI from Piromyces sp. was engineered and hyper-secreted in S. cerevisiae for the extracellular conversion of xylose to xylulose. Results The optimal pH of XI was lowered from 7.0 to 5.0 by directed evolution to ensure its high activity under the acidic conditions used for yeast fermentation, and hyper-secretion of an engineered XI-76 mutant (E56A and I252M) was accomplished by employing target protein-specific translational fusion partners. The purified XI-76 showed twofold higher activity than that of the wild type at pH 5. The secretory expression of XI-76 in the previously developed xylose utilizing yeast strain, SR8 increased xylose consumption and ethanol production by approximately 7–20% and 15–20% in xylose fermentation and glucose and xylose co-fermentation, respectively. Conclusions Isomerisation of xylose to xylulose before uptake using extracellular XI was found to be effective in xylose fermentation or glucose/xylose co-fermentation. This suggested that glucose competed less with xylulose than with xylose for uptake by the cell. Consequently, the engineered XI secretion system constructed in this study can pave the way for simultaneous utilization of C5/C6 sugars from the sustainable lignocellulosic biomass.

scholarly journals 698. Nacubactam Inhibits Class A β-lactamases

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S251-S252
Author(s):  
Melissa D Barnes ◽  
Caryn E Good ◽  
Saralee Bajaksouzian ◽  
Magdalena A Taracila ◽  
David Van Duin ◽  
...  
Keyword(s):  
Amino Acid ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
E Coli ◽  
Class A ◽  
Cell Extracts ◽  
Fixed Concentration ◽  
Genes Encoding ◽  
Inactivation Mechanism ◽  
Research Grant

Abstract Background Nacubactam, formerly RG6080 and OP0595 (Figure 1A), is a bridged diazabicyclooctane (DBO) that inactivates class A and class C β-lactamases. Unlike avibactam, the DBO that is approved for use in combination with ceftazidime, nacubactam also inhibits penicillin binding proteins (i.e., PBP2) in Enterobacteriaceae. We set out to determine the effectiveness of meropenem-nacubactam against Klebsiella pneumoniae clinical strains and to elucidate the structure–function relationships. Methods Minimal inhibitory concentration (MIC) measurements using broth microdilution according to Clinical and Laboratory Standards Institute for meropenem (MERO) ± nacubactam (fixed concentration of 4 mg/L or fixed 1:1 ratio) was performed on 50 clinical K. pneumoniae strains (6 having OXA-48-like β-lactamases and 44 harboring KPC-2 or KPC-3) and 47 isogenic Escherichia coli strains harboring bla genes encoding K. pneumoniae carbapenemase (KPC) variants with single amino acid substitutions in residues that are involved in catalysis. IC50s for selected KPC-2 variants were determined on periplasmic extracts with varying concentrations of nacubactam using nitrocefin as a reporter substrate. Results The MERO combinations with either 4 mg/L or a 1:1 ratio of nacubactam effectively lowered the MERO MICs of K. pneumoniae strains (Figure 1B). Similarly, all E. coli strains expressing blaKPC-2 variants were susceptible to the MERO-nacubactam combinations based on the breakpoint of MERO. The strains harboring K73R, S130G, and K234R had slightly elevated MERO-nacubactam MICs relative to wild type but did not have corresponding increases in MERO MICs. Strains with pBC SK-KPC2, K73R or S130G had 0.015 mg/L MERO MICs. The pBR322-K234R strain had a twofold lower MERO MIC than pBR322-KPC-2 (Figure 1C). The IC50 of cell extracts containing the K234R variant is 781 µM, which is 12-fold higher than that for KPC-2 (66 µM) (Figure 1C). Extracts containing the S130G variant were not inhibited by nacubactam (IC50 > 2.6 mM). Conclusion Meropenem-nacubactam is an effective β-lactam β-lactamase inhibitor combination for Enterobacteriaceae with KPC or OXA-48 β-lactamases. The single amino acid substitutions K73R, S130G, and K234R in KPC-2 affect the inactivation mechanism. Disclosures M. R. Jacobs, F. Hoffmann-La Roche Ltd.: Grant Investigator, Research grant. K. M. Papp-Wallace, F. Hoffmann-La Roche Ltd.: Grant Investigator, Research grant. R. A. Bonomo, F. Hoffmann-La Roche Ltd.: Grant Investigator, Research grant.

scholarly journals Host Mutations (miaA and rpsL) Reduce Tetracycline Resistance Mediated by Tet(O) and Tet(M)

1998 ◽  
Vol 42 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Diane E. Taylor ◽  
Catharine A. Trieber ◽  
Gudrun Trescher ◽  
Michelle Bekkering
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Tetracycline Resistance ◽  
Acceptor Site ◽  
Amino Acid Substitutions ◽  
Trna Modification ◽  
E Coli ◽  
Translational Accuracy ◽  
Host Genes ◽  
Ribosomal Protection

ABSTRACT The effects of mutations in host genes on tetracycline resistance mediated by the Tet(O) and Tet(M) ribosomal protection proteins, which originated in Campylobacter spp. andStreptococcus spp., respectively, were investigated by using mutants of Salmonella typhimuriumand Escherichia coli. The miaA,miaB, and miaAB double mutants of S. typhimurium specify enzymes for tRNA modification at the adenosine at position 37, adjacent to the anticodon in tRNA. InS. typhimurium, this involves biosynthesis ofN 6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A). The miaA mutation reduced the level of tetracycline resistance mediated by both Tet(O) and Tet(M), but the latter showed a greater effect, which was ascribed to the isopentenyl (i6) group or to a combination of the methylthioadenosine (ms2) and i6 groups but not to the ms2 group alone (specified by miaB). In addition, mutations in E. coli rpsL genes, generating both streptomycin-resistant and streptomycin-dependent strains, were also shown to reduce the level of tetracycline resistance mediated by Tet(O) and Tet(M). The single-site amino acid substitutions present in the rpsL mutations were pleiotropic in their effects on tetracycline MICs. These mutants affect translational accuracy and kinetics and suggest that Tet(O) and Tet(M) binding to the ribosome may be reduced or slowed in theE. coli rpsL mutants in which the S12 protein is altered. Data from both the miaA and rpsL mutant studies indicate a possible link between stability of the aminoacyl-tRNA in the ribosomal acceptor site and tetracycline resistance mediated by the ribosomal protection proteins.

scholarly journals FunctionalDomains of the RhlR Transcriptional Regulator ofPseudomonasaeruginosa

2003 ◽  
Vol 185 (24) ◽  
pp. 7129-7139 ◽  
Author(s):  
Janet R. Lamb ◽  
Hetal Patel ◽  
Timothy Montminy ◽  
Victoria E. Wagner ◽  
Barbara H. Iglewski
Keyword(s):  
Amino Acid ◽  
Transcriptional Activation ◽  
Gene Activation ◽  
Transcriptional Regulator ◽  
Homoserine Lactone ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Mutant Form ◽  
E Coli ◽  
Terminal Domain

ABSTRACT The RhlR transcriptional regulator of Pseudomonas aeruginosa, along with its cognate autoinducer, N-butyryl homoserine lactone (C4-HSL), regulates gene expression in response to cell density. With an Escherichia coli LexA-based protein interaction system, we demonstrated that RhlR multimerized and that the degree of multimerization was dependent on the C4-HSL concentration. Studies with an E. coli lasB::lacZ lysogen demonstrated that RhlR multimerization was necessary for it to function as a transcriptional activator. Deletion analysis of RhlR indicated that the N-terminal domain of the protein is necessary for C4-HSL binding. Single amino acid substitutions in the C-terminal domain of RhlR generated mutant RhlR proteins that had the ability to bind C4-HSL and multimerize but were unable to activate lasB expression, demonstrating that the C-terminal domain is important for target gene activation. Single amino acid substitutions in both the N-terminal and C-terminal domains of RhlR demonstrated that both domains possess residues involved in multimerization. RhlR with a C-terminal deletion and an RhlR site-specific mutant form that possessed multimerization but not transcriptional activation capabilities were able to inhibit the ability of wild-type RhlR to activate rhlA expression in P. aeruginosa. We conclude that C4-HSL binding is necessary for RhlR multimerization and that RhlR functions as a multimer in P. aeruginosa.

scholarly journals Epidemiological Survey of Amoxicillin-Clavulanate Resistance and Corresponding Molecular Mechanisms in Escherichia coliIsolates in France: New Genetic Features ofblaTEM Genes

2000 ◽  
Vol 44 (10) ◽  
pp. 2709-2714 ◽  
Author(s):  
V. Leflon-Guibout ◽  
V. Speldooren ◽  
B. Heym ◽  
M.-H. Nicolas-Chanoine
Keyword(s):  
Amino Acid ◽  
Molecular Mechanisms ◽  
Consensus Sequence ◽  
Epidemiological Survey ◽  
Amino Acid Substitutions ◽  
Single Strand Conformational Polymorphism ◽  
E Coli ◽  
Genetic Features ◽  
Amoxicillin Clavulanate ◽  
Medical Wards

ABSTRACT Amoxicillin-clavulanate resistance (MIC >16 μg/ml) and the corresponding molecular mechanisms were prospectively studied inEscherichia coli over a 3-year period (1996 to 1998) in 14 French hospitals. The overall frequency of resistant E. coli isolates remained stable at about 5% over this period. The highest frequency of resistant isolates (10 to 15%) was observed, independently of the year, among E. coli isolated from lower respiratory tract samples, and the isolation rate of resistant strains was significantly higher in surgical wards than in medical wards in 1998 (7.8 versus 2.8%). The two most frequent mechanisms of resistance for the 3 years were the hyperproduction of the chromosomal class C β-lactamase (48, 38.4, and 39.7%) and the production of inhibitor-resistant TEM (IRT) enzymes (30.4, 37.2, and 41.2%). By using the single-strand conformational polymorphism–PCR technique and sequencing methods, we determined that 59 IRT enzymes corresponded to previously described IRT enzymes whereas 8 were new. Three of these new enzymes derived from TEM-1 by only one amino acid substitution (Ser130Gly, Arg244Gly, and Asn276Asp), whereas three others derived by two amino acid substitutions (Met69Leu and Arg244Ser, Met69Leu and Ile127Val, and Met69Val and Arg275Gln). The two remaining new IRTs showed three amino acid substitutions (Met69Val, Trp165Arg, and Asn276Asp and Met69Ile, Trp165Cys, and Arg275Gln). New genetic features were also found inbla TEM genes, namely,bla TEM-1B with either the promotersPa and Pb, P4, or a promoter displaying a C→G transversion at position 3 of the −35 consensus sequence and new bla TEM genes, notably one encoding TEM-1 but possessing the silent mutations originally described in bla TEM-2 and then in somebla TEM-encoding IRT enzymes.

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