scholarly journals Laboratory Evolution of Toluene Dioxygenase To Accept 4-Picoline as a Substrate

2001 ◽  
Vol 67 (9) ◽  
pp. 3882-3887 ◽  
Author(s):  
Takeshi Sakamoto ◽  
John M. Joern ◽  
Akira Arisawa ◽  
Frances H. Arnold
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Stop Codon ◽  
Random Mutagenesis ◽  
Product Formation ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Laboratory Evolution ◽  
Toluene Dioxygenase ◽  
Naturally Occurring

ABSTRACT We are using directed evolution to extend the range of dioxygenase-catalyzed biotransformations to include substrates that are either poorly accepted or not accepted at all by the naturally occurring enzymes. Here we report on the oxidation of a heterocyclic substrate, 4-picoline, by toluene dioxygenase (TDO) and improvement of the enzyme's activity by laboratory evolution. The biotransformation of 4-picoline proceeds at only ∼4.5% of the rate of the natural reaction on toluene. Random mutagenesis, saturation mutagenesis, and screening directly for product formation using a modified Gibbs assay generated mutant TDO 3-B38, in which the wild-type stop codon was replaced with a codon encoding threonine. Escherichia coli-expressed TDO 3-B38 exhibited 5.6 times higher activity toward 4-picoline and ∼20% more activity towards toluene than wild-type TDO. The product of the biotransformation of 4-picoline is 3-hydroxy-4-picoline; no cis-diols of 4-picoline were observed.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals Correction: Directed evolution of mevalonate kinase in Escherichia coli by random mutagenesis for improved lycopene

RSC Advances ◽  
2019 ◽  
Vol 9 (16) ◽  
pp. 8934-8934
Author(s):  
Hailin Chen ◽  
Changqing Liu ◽  
Meijie Li ◽  
Haibo Zhang ◽  
Mo Xian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Random Mutagenesis ◽  
Mevalonate Kinase

Correction for ‘Directed evolution of mevalonate kinase in Escherichia coli by random mutagenesis for improved lycopene’ by Hailin Chen et al., RSC Adv., 2018, 8, 15021–15028.

scholarly journals Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

2002 ◽  
Vol 184 (3) ◽  
pp. 695-705 ◽  
Author(s):  
Joseph C. Chen ◽  
Michael Minev ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Random Mutagenesis ◽  
Dominant Negative ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Negative Effects ◽  
Mutant Proteins ◽  
Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

scholarly journals Directed evolution of mevalonate kinase in Escherichia coli by random mutagenesis for improved lycopene

RSC Advances ◽  
2018 ◽  
Vol 8 (27) ◽  
pp. 15021-15028 ◽  
Author(s):  
Hailin Chen ◽  
Changqing Liu ◽  
Meijie Li ◽  
Haibo Zhang ◽  
Mo Xian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Random Mutagenesis ◽  
Mevalonate Kinase

Lycopene is a terpenoid pigment that has diverse applications in the fields of food and medicine.

scholarly journals Natural Genetic Transformation of Clinical Isolates of Escherichia coli in Urine and Water

2002 ◽  
Vol 68 (1) ◽  
pp. 440-443 ◽  
Author(s):  
Markus Woegerbauer ◽  
Bernard Jenni ◽  
Florian Thalhammer ◽  
Wolfgang Graninger ◽  
Heinz Burgmann
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Current Knowledge ◽  
Antibiotic Resistance Genes ◽  
Aquatic Environments ◽  
Wild Type ◽  
E Coli ◽  
Naturally Occurring ◽  
Natural Genetic Transformation ◽  
Type Strains

ABSTRACT Transfer of plasmid-borne antibiotic resistance genes in Escherichia coli wild-type strains is possible by transformation under naturally occurring conditions in oligotrophic, aquatic environments containing physiologic concentrations of calcium. In contrast, transformation is suppressed in nitrogen-rich body fluids like urine, a common habitat of uropathogenic strains. Current knowledge indicates that transformation of these E. coli wild-type strains is of no relevance for the acquisition of resistance in this clinically important environment.

scholarly journals Conversion of Escherichia coli pyruvate oxidase to an ‘α-ketobutyrate oxidase’

2000 ◽  
Vol 352 (3) ◽  
pp. 717-724 ◽  
Author(s):  
Ying-Ying CHANG ◽  
John E. CRONAN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Random Mutagenesis ◽  
Fold Increase ◽  
Natural Substrate ◽  
Wild Type ◽  
Pyruvate Oxidase ◽  
Mutant Proteins ◽  
Essentially Normal

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate (‘α-ketobutyrate’), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in Km for pyruvate and a 10-fold lower Vmax with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

scholarly journals REGULATION OF NEWLY EVOLVED ENZYMES. IV. DIRECTED EVOLUTION OF THE EBG REPRESSOR

Genetics ◽  
1978 ◽  
Vol 90 (4) ◽  
pp. 673-681
Author(s):  
Barry G Hall
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Enzyme Synthesis ◽  
Regulatory Function ◽  
Wild Type ◽  
Selection For ◽  
Selection Of

ABSTRACT In Escherichia coli, the wild-type repressor of ebg (evolved β-galactosidase) enzyme synthesis, specified by the ebgR  + gene, responds very weakly to lactulose (fructose-β-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 x 10-10. ebgR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.

scholarly journals Improvement of an Unusual Twin-Arginine Transporter Leader Peptide by a Codon-Based Randomization Approach

2006 ◽  
Vol 72 (5) ◽  
pp. 3797-3801 ◽  
Author(s):  
Olga Monroy-Lagos ◽  
Xavier Soberon ◽  
Paul Gaytan ◽  
Joel Osuna
Keyword(s):  
Escherichia Coli ◽  
Protein Secretion ◽  
Signal Peptide ◽  
Random Mutagenesis ◽  
Penicillin Acylase ◽  
Gene Region ◽  
Leader Peptide ◽  
Wild Type ◽  
Region Coding

ABSTRACT Secretion of Escherichia coli penicillin acylase was improved by codon-based random mutagenesis of its signal peptide. The mutagenesis technology was applied to the gene region coding for positions Lys2 to Thr13 (N half) and Ala14 to Leu25 (C half) of the signal peptide. Protein secretion was higher in several signal peptide variants (up to fourfold with respect to the wild-type value).

scholarly journals Improved Secretory Production of Recombinant Proteins by Random Mutagenesis of hlyB, an Alpha-Hemolysin Transporter from Escherichia coli

2005 ◽  
Vol 71 (2) ◽  
pp. 656-662 ◽  
Author(s):  
Yasuhiro Sugamata ◽  
Toshikazu Shiba
Keyword(s):  
Escherichia Coli ◽  
Low Temperature ◽  
Fusion Protein ◽  
Signal Sequence ◽  
Random Mutagenesis ◽  
Single Amino Acid ◽  
Wild Type ◽  
Single Chain ◽  
Alpha Hemolysin ◽  
Subtilisin E

ABSTRACT Fusion proteins with an alpha-hemolysin (HlyA) C-terminal signal sequence are known to be secreted by the HlyB-HlyD-TolC translocator in Escherichia coli. We aimed to establish an efficient Hly secretory expression system by random mutagenesis of hlyB and hlyD. The fusion protein of subtilisin E and the HlyA signal sequence (HlyA218) was used as a marker protein for evaluating secretion efficiency. Through screening of more than 1.5 × 104 E. coli JM109 transformants, whose hlyB and hlyD genes had been mutagenized by error-prone PCR, we succeeded in isolating two mutants that had 27- and 15-fold-higher levels of subtilisin E secretion activity than the wild type did at 23°C. These mutants also exhibited increased activity levels for secretion of a single-chain antibody-HlyA218 fusion protein at 23 and 30°C but unexpectedly not at 37°C, suggesting that this improvement seems to be dependent on low temperature. One mutant (AE104) was found to have seven point mutations in both HlyB and HlyD, and an L448F substitution in HlyB was responsible for the improved secretion activity. Another mutant (AE129) underwent a single amino acid substitution (G654S) in HlyB. Secretion of c-Myc-HlyA218 was detected only in the L448F mutant (AE104F) at 23°C, whereas no secretion was observed in the wild type at any temperature. Furthermore, for the PTEN-HlyA218 fusion protein, AE104F showed a 10-fold-higher level of secretion activity than the wild type did at 37°C. This result indicates that the improved secretion activity of AE104F is not always dependent on low temperature.

scholarly journals Phylogenetic Evidence for Horizontal Transfer ofmutS Alleles among Naturally Occurring Escherichia coli Strains

2001 ◽  
Vol 183 (5) ◽  
pp. 1631-1644 ◽  
Author(s):  
Eric W. Brown ◽  
J. Eugene LeClerc ◽  
Baoguang Li ◽  
William L. Payne ◽  
Thomas A. Cebula
Keyword(s):  
Escherichia Coli ◽  
Horizontal Transfer ◽  
Hot Spot ◽  
Enzyme Electrophoresis ◽  
Wild Type ◽  
Polymorphic Region ◽  
E Coli ◽  
Naturally Occurring ◽  
Phylogenetic Discordance ◽  
Homeologous Recombination

ABSTRACT mutS mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the mutS phenotype, the evolutionary structure of the mutS + gene inEscherichia coli remains unclear. The physical proximity ofmutS to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify E. coli strains in whichmutS alleles have recombined. Comparison ofmutS phylogeny against predicted E. coli“whole-chromosome” phylogenies (derived from multilocus enzyme electrophoresis and mdh sequences) revealed striking levels of phylogenetic discordance among mutS alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among mutS alleles. Examination of additional sites surrounding mutS also revealed incongruous distributions compared to E. coli strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-minmutS-rpoS region is a recombinational hot spot within theE. coli chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted bymutS mutators through rescue of defective mutSalleles with wild-type sequences.

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