scholarly journals Biocatalytic Conversion of Avermectin to 4″-Oxo-Avermectin: Improvement of Cytochrome P450 Monooxygenase Specificity by Directed Evolution

2007 ◽  
Vol 73 (13) ◽  
pp. 4317-4325 ◽  
Author(s):  
Axel Trefzer ◽  
Volker Jungmann ◽  
István Molnár ◽  
Ajit Botejue ◽  
Dagmar Buckel ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
High Throughput Screening ◽  
Random Mutagenesis ◽  
Reaction System ◽  
Industrial Process ◽  
Product Formation ◽  
Site Directed Mutagenesis ◽  
Process Conditions ◽  
P450 Monooxygenase ◽  
Biocatalytic Reaction

ABSTRACT Discovery of the CYP107Z subfamily of cytochrome P450 oxidases (CYPs) led to an alternative biocatalytic synthesis of 4″-oxo-avermectin, a key intermediate for the commercial production of the semisynthetic insecticide emamectin. However, under industrial process conditions, these wild-type CYPs showed lower yields due to side product formation. Molecular evolution employing GeneReassembly was used to improve the regiospecificity of these enzymes by a combination of random mutagenesis, protein structure-guided site-directed mutagenesis, and recombination of multiple natural and synthetic CYP107Z gene fragments. To assess the specificity of CYP mutants, a miniaturized, whole-cell biocatalytic reaction system that allowed high-throughput screening of large numbers of variants was developed. In an iterative process consisting of four successive rounds of GeneReassembly evolution, enzyme variants with significantly improved specificity for the production of 4″-oxo-avermectin were identified; these variants could be employed for a more economical industrial biocatalytic process to manufacture emamectin.

scholarly journals Main Structural Targets for Engineering Lipase Substrate Specificity

Catalysts ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 747
Author(s):  
Samah Hashim Albayati ◽  
Malihe Masomian ◽  
Siti Nor Hasmah Ishak ◽  
Mohd Shukuri bin Mohamad Ali ◽  
Adam Leow Thean ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Experimental Studies ◽  
Random Mutagenesis ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Process Conditions

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.

scholarly journals Directed Evolution of a Thermostable Phosphite Dehydrogenase for NAD(P)H Regeneration

2005 ◽  
Vol 71 (10) ◽  
pp. 5728-5734 ◽  
Author(s):  
Tyler W. Johannes ◽  
Ryan D. Woodyer ◽  
Huimin Zhao
Keyword(s):  
Directed Evolution ◽  
Half Life ◽  
High Throughput Screening ◽  
Thermal Inactivation ◽  
Random Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Candida Boidinii ◽  
Enzymatic System ◽  
Nadh Regeneration ◽  
Phosphite Dehydrogenase

ABSTRACT NAD(P)H-dependent oxidoreductases are valuable tools for synthesis of chiral compounds. The expense of the cofactors, however, requires in situ cofactor regeneration for preparative applications. We have attempted to develop an enzymatic system based on phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri to regenerate the reduced nicotinamide cofactors NADH and NADPH. Here we report the use of directed evolution to address one of the main limitations with the wild-type PTDH enzyme, its low stability. After three rounds of random mutagenesis and high-throughput screening, 12 thermostabilizing amino acid substitutions were identified. These 12 mutations were combined by site-directed mutagenesis, resulting in a mutant whose T 50 is 20°C higher and half-life of thermal inactivation at 45°C is >7,000-fold greater than that of the parent PTDH. The engineered PTDH has a half-life at 50°C that is 2.4-fold greater than the Candida boidinii formate dehydrogenase, an enzyme widely used for NADH regeneration. In addition, its catalytic efficiency is slightly higher than that of the parent PTDH. Various mechanisms of thermostabilization were identified using molecular modeling. The improved stability and effectiveness of the final mutant were shown using the industrially important bioconversion of trimethylpyruvate to l-tert-leucine. The engineered PTDH will be useful in NAD(P)H regeneration for industrial biocatalysis.

scholarly journals The efficient and selective catalytic oxidation of para-substituted cinnamic acid derivatives by the cytochrome P450 monooxygenase, CYP199A4

RSC Advances ◽  
2016 ◽  
Vol 6 (60) ◽  
pp. 55286-55297 ◽  
Author(s):  
Rebecca R. Chao ◽  
James J. De Voss ◽  
Stephen G. Bell
Keyword(s):  
Cytochrome P450 ◽  
Catalytic Oxidation ◽  
Cinnamic Acid ◽  
Product Formation ◽  
Cytochrome P450 Enzyme ◽  
Cinnamic Acids ◽  
P450 Monooxygenase ◽  
Cinnamic Acid Derivatives ◽  
Selective Catalytic Oxidation ◽  
P450 Enzyme

The cytochrome P450 enzyme, CYP199A4 oxidised para substituted alkyloxy- and alkyl-cinnamic acids, with high product formation activity.

scholarly journals Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

2016 ◽  
Vol 113 (26) ◽  
pp. 7136-7141 ◽  
Author(s):  
Immacolata Venditto ◽  
Ana S. Luis ◽  
Maja Rydahl ◽  
Julia Schückel ◽  
Vânia O. Fernandes ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Plant Cell Wall ◽  
Industrial Process ◽  
Site Directed Mutagenesis ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Ruminococcus Flavefaciens ◽  
Pectic Polysaccharide ◽  
Degrading Bacterium ◽  
Carbohydrate Binding Modules

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

Molecular evolution of a cytochrome P450 for the synthesis of potential antidepressant (2R,6R)-hydroxynorketamine

2021 ◽  
Author(s):  
Ansgar Bokel ◽  
Michael C. Hutter ◽  
Vlada B. Urlacher
Keyword(s):  
Cytochrome P450 ◽  
Molecular Evolution ◽  
Cytochrome P450 Monooxygenase ◽  
P450 Monooxygenase ◽  
Effective Synthesis

Engineered cytochrome P450 monooxygenase CYP154E1 enables the effective synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine via N-demethylation and regio- and stereoselective hydroxylation of (R)-ketamine.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

scholarly journals Cytochrome P450 Monooxygenase-Mediated Metabolic Utilization of Benzo[a]Pyrene by Aspergillus Species

mBio ◽  
2019 ◽  
Vol 10 (3) ◽  
Author(s):  
Erin M. Ostrem Loss ◽  
Mi-Kyung Lee ◽  
Ming-Yueh Wu ◽  
Julia Martien ◽  
Wanping Chen ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Environmental Pollutants ◽  
Energy Generation ◽  
Fungal Species ◽  
Metabolic Changes ◽  
Cytochrome P450 Monooxygenase ◽  
Cellular Growth ◽  
P450 Monooxygenase ◽  
Content Type ◽  
Fundamental Knowledge

ABSTRACT Soil-dwelling fungal species possess the versatile metabolic capability to degrade complex organic compounds that are toxic to humans, yet the mechanisms they employ remain largely unknown. Benzo[a]pyrene (BaP) is a pervasive carcinogenic contaminant, posing a significant concern for human health. Here, we report that several Aspergillus species are capable of degrading BaP. Exposing Aspergillus nidulans cells to BaP results in transcriptomic and metabolic changes associated with cellular growth and energy generation, implying that the fungus utilizes BaP as a growth substrate. Importantly, we identify and characterize the conserved bapA gene encoding a cytochrome P450 monooxygenase that is necessary for the metabolic utilization of BaP in Aspergillus. We further demonstrate that the fungal NF-κB-type velvet regulators VeA and VelB are required for proper expression of bapA in response to nutrient limitation and BaP degradation in A. nidulans. Our study illuminates fundamental knowledge of fungal BaP metabolism and provides novel insights into enhancing bioremediation potential. IMPORTANCE We are increasingly exposed to environmental pollutants, including the carcinogen benzo[a]pyrene (BaP), which has prompted extensive research into human metabolism of toxicants. However, little is known about metabolic mechanisms employed by fungi that are able to use some toxic pollutants as the substrates for growth, leaving innocuous by-products. This study systemically demonstrates that a common soil-dwelling fungus is able to use benzo[a]pyrene as food, which results in expression and metabolic changes associated with growth and energy generation. Importantly, this study reveals key components of the metabolic utilization of BaP, notably a cytochrome P450 monooxygenase and the fungal NF-κB-type transcriptional regulators. Our study advances fundamental knowledge of fungal BaP metabolism and provides novel insight into designing and implementing enhanced bioremediation strategies.

scholarly journals Evolutionary design of optimal surface topographies for biomaterials

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Aliaksei Vasilevich ◽  
Aurélie Carlier ◽  
David A. Winkler ◽  
Shantanu Singh ◽  
Jan de Boer
Keyword(s):  
High Throughput Screening ◽  
Random Mutagenesis ◽  
Material Design ◽  
Fitness Assessment ◽  
Surface Topographies ◽  
Optimal Surface ◽  
Design Production ◽  
Brute Force Approach ◽  
Survival Of The Fittest ◽  
Selection Of

AbstractNatural evolution tackles optimization by producing many genetic variants and exposing these variants to selective pressure, resulting in the survival of the fittest. We use high throughput screening of large libraries of materials with differing surface topographies to probe the interactions of implantable device coatings with cells and tissues. However, the vast size of possible parameter design space precludes a brute force approach to screening all topographical possibilities. Here, we took inspiration from Nature to optimize materials surface topographies using evolutionary algorithms. We show that successive cycles of material design, production, fitness assessment, selection, and mutation results in optimization of biomaterials designs. Starting from a small selection of topographically designed surfaces that upregulate expression of an osteogenic marker, we used genetic crossover and random mutagenesis to generate new generations of topographies.

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