scholarly journals Family Shuffling of Expandase Genes To Enhance Substrate Specificity for Penicillin G

2004 ◽  
Vol 70 (10) ◽  
pp. 6257-6263 ◽  
Author(s):  
Jyh-Shing Hsu ◽  
Yunn-Bor Yang ◽  
Chan-Hui Deng ◽  
Chia-Li Wei ◽  
Shwu-Huey Liaw ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Kinetic Parameters ◽  
Substrate Inhibition ◽  
Fold Increase ◽  
Streptomyces Clavuligerus ◽  
Penicillin G ◽  
Dna Shuffling ◽  
Homologous Genes ◽  
Family Shuffling

ABSTRACT Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k cat/Km ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds.

Isolation and mapping of a mutant penicillin G acylase with altered substrate specificity from Escherichia coli

1995 ◽  
Vol 17 (1) ◽  
pp. 19-23 ◽  
Author(s):  
Helmut Niersbach ◽  
Walter Tischer ◽  
Marijke Weber ◽  
Frank Wedekind ◽  
Roland Plapp
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Penicillin G Acylase ◽  
Penicillin G

scholarly journals Engineering Streptomyces clavuligerus Deacetoxycephalosporin C Synthase for Optimal Ring Expansion Activity toward Penicillin G

2003 ◽  
Vol 69 (4) ◽  
pp. 2306-2312 ◽  
Author(s):  
Chia-Li Wei ◽  
Yunn-Bor Yang ◽  
Wen-Ching Wang ◽  
Wen-Chi Liu ◽  
Jyh-Shing Hsu ◽  
...  
Keyword(s):  
Random Mutagenesis ◽  
Fold Increase ◽  
Streptomyces Clavuligerus ◽  
Ring Expansion ◽  
Relative Activity ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Triple Mutant

ABSTRACT The deacetoxycephalosporin C synthase (DAOCS) from Streptomyces clavuligerus was engineered with the aim of enhancing the conversion of penicillin G into phenylacetyl-7-aminodeacetoxycephalosporanic acid, a precursor of 7-aminodeacetoxycephalosporanic acid, for industrial application. A single round of random mutagenesis followed by the screening of 5,500 clones identified three mutants, G79E, V275I, and C281Y, that showed a two- to sixfold increase in the k cat/Km ratio compared to the wild-type enzyme. Site-directed mutagenesis to modify residues surrounding the substrate resulted in three mutants, N304K, I305L, and I305M, with 6- to 14-fold-increased k cat/Km values. When mutants containing all possible combinations of these six sites were generated to optimize the ring expansion activity for penicillin G, the double mutant, YS67 (V275I, I305M), showed a significant 32-fold increase in the k cat/Km ratio and a 5-fold increase in relative activity for penicillin G, while the triple mutant, YS81 (V275I, C281Y, I305M), showed an even greater 13-fold increase in relative activity toward penicillin G. Our results demonstrate that this is a robust approach to the modification of DAOCS for an optimized DAOCS-penicillin G reaction.

scholarly journals Reconstitution of TCA cycle with DAOCS to engineer Escherichia coli into an efficient whole cell catalyst of penicillin G

2015 ◽  
Vol 112 (32) ◽  
pp. 9855-9859 ◽  
Author(s):  
Baixue Lin ◽  
Keqiang Fan ◽  
Jian Zhao ◽  
Junjie Ji ◽  
Linjun Wu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metabolic Flux ◽  
Fold Increase ◽  
Tca Cycle ◽  
Penicillin G ◽  
Whole Cell ◽  
Knock Out ◽  
Engineering Strategy ◽  
Acetate Accumulation ◽  
Biocatalytic Process

Many medically useful semisynthetic cephalosporins are derived from 7-aminodeacetoxycephalosporanic acid (7-ADCA), which has been traditionally made by the polluting chemical method. Here, a whole-cell biocatalytic process based on an engineered Escherichia coli strain expressing 2-oxoglutarate–dependent deacetoxycephalosporin C synthase (DAOCS) for converting penicillin G to G-7-ADCA is developed. The major engineering strategy is to reconstitute the tricarboxylic acid (TCA) cycle of E. coli to force the metabolic flux to go through DAOCS catalyzed reaction for 2-oxoglutarate to succinate conversion. Then the glyoxylate bypass was disrupted to eliminate metabolic flux that may circumvent the reconstituted TCA cycle. Additional engineering steps were taken to reduce the degradation of penicillin G and G-7-ADCA in the bioconversion process. These steps include engineering strategies to reduce acetate accumulation in the biocatalytic process and to knock out a host β-lactamase involved in the degradation of penicillin G and G-7-ADCA. By combining these manipulations in an engineered strain, the yield of G-7-ADCA was increased from 2.50 ± 0.79 mM (0.89 ± 0.28 g/L, 0.07 ± 0.02 g/gDCW) to 29.01 ± 1.27 mM (10.31 ± 0.46 g/L, 0.77 ± 0.03 g/gDCW) with a conversion rate of 29.01 mol%, representing an 11-fold increase compared with the starting strain (2.50 mol%).

scholarly journals Differential Substrate Specificity and Kinetic Behavior of Escherichia coli YfdW and Oxalobacter formigenes Formyl Coenzyme A Transferase

2008 ◽  
Vol 190 (7) ◽  
pp. 2556-2564 ◽  
Author(s):  
Cory G. Toyota ◽  
Catrine L. Berthold ◽  
Arnaud Gruez ◽  
Stefán Jónsson ◽  
Ylva Lindqvist ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Coenzyme A ◽  
Substrate Inhibition ◽  
Kinetic Behavior ◽  
Oxalobacter Formigenes ◽  
Structural Genomic ◽  
E Coli ◽  
Coa Transferase ◽  
Acidic Conditions

ABSTRACT The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions.

scholarly journals Duck liver ‘malic’ enzyme. Expression in Escherichia coli and characterization of the wild-type enzyme and site-directed mutants

1992 ◽  
Vol 284 (3) ◽  
pp. 869-876 ◽  
Author(s):  
R Y Hsu ◽  
M J Glynias ◽  
J Satterlee ◽  
R Feeney ◽  
A R Clarke ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Kinetic Parameters ◽  
Malic Enzyme ◽  
Dissociation Constants ◽  
Substrate Inhibition ◽  
Oxidative Decarboxylation ◽  
Lacz Gene ◽  
Wild Type ◽  
Recombinant Enzymes ◽  
Wild Type Enzyme

A cDNA for duck liver ‘malic’ enzyme (EC 1.1.1.40) was subcloned into pUC-8, and the active enzyme was expressed in Escherichia coli TG-2 cells as a fusion protein including a 15-residue N-terminal leader from beta-galactosidase coded by the lacZ′ gene. C99S and R70Q mutants of the enzyme were generated by the M13 mismatch technique. The recombinant enzymes were purified to near homogeneity by a simple two-step procedure and characterized relative to the enzyme isolated from duck liver. The natural duck enzyme has a subunit molecular mass of approx. 65 kDa, and the following kinetic parameters for oxidative decarboxylation of L-malate at pH 7.0: Km NADP+ (4.6 microM); Km L-malate (73 microM); kcat (160 s-1); Ka (2.4 microM) and Ka′ (270 microM), dissociation constants of Mn2+ at ‘tight’ (activating) and ‘weak’ metal sites; and substrate inhibition (51% of kcat. at 8 mM-L-malate). Properties of the E. coli-derived recombinant wild-type enzyme are indistinguishable from those of the natural duck enzyme. Kinetic parameters of the R70Q mutant are relatively unaltered, indicating that Arg-70 is not required for the reaction. The C99S mutant has unchanged Km for NADP+ and parameters for the ‘weak’ sites (i.e. inhibition by L-malate, Ka′); however, kcat. decreased 3-fold and Km for L-malate and Ka each increased 4-fold, resulting in a catalytic efficiency [kcat./(Km NADP+ x Km L-malate x Ka)] equal to 3.7% of the natural duck enzyme. These results suggest that the positioning of Cys-99 in the sequence is important for proper binding of L-malate and bivalent metal ions.

scholarly journals Specific phenotypic, genomic, and fitness evolutionary trajectories toward streptomycin resistance induced by pesticide co-stressors in Escherichia coli

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Yue Xing ◽  
Xiaoxi Kang ◽  
Siwei Zhang ◽  
Yujie Men
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Selective Pressure ◽  
Fold Increase ◽  
Streptomycin Resistance ◽  
Evolutionary Stage ◽  
Fitness Costs ◽  
Evolutionary Trajectories ◽  
K 12 ◽  
Late Stages

AbstractTo explore how co-occurring non-antibiotic environmental stressors affect evolutionary trajectories toward antibiotic resistance, we exposed susceptible Escherichia coli K-12 populations to environmentally relevant levels of pesticides and streptomycin for 500 generations. The coexposure substantially changed the phenotypic, genotypic, and fitness evolutionary trajectories, resulting in much stronger streptomycin resistance (>15-fold increase) of the populations. Antibiotic target modification mutations in rpsL and rsmG, which emerged and dominated at late stages of evolution, conferred the strong resistance even with less than 1% abundance, while the off-target mutations in nuoG, nuoL, glnE, and yaiW dominated at early stages only led to mild resistance (2.5–6-fold increase). Moreover, the strongly resistant mutants exhibited lower fitness costs even without the selective pressure and had lower minimal selection concentrations than the mildly resistant ones. Removal of the selective pressure did not reverse the strong resistance of coexposed populations at a later evolutionary stage. The findings suggest higher risks of the selection and propagation of strong antibiotic resistance in environments potentially impacted by antibiotics and pesticides.

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