scholarly journals Heterologous redox partners supporting the efficient catalysis of epothilone B biosynthesis by EpoK in Schlegelella brevitalea

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Junheng Liang ◽  
Huimin Wang ◽  
Xiaoying Bian ◽  
Youming Zhang ◽  
Guoping Zhao ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Electron Transport ◽  
Cytochrome P450 Enzyme ◽  
Whole Cell ◽  
Epothilone B ◽  
Redox Partners ◽  
Conversion Rates ◽  
Epothilone A ◽  
P450 Enzyme ◽  
Whole Cell Biotransformation

Abstract Background Epothilone B is a natural product that stabilizes microtubules, similar to paclitaxel (Taxol); therefore, epothilone B and several derivatives have shown obvious antitumour activities. Some of these products are in clinical trials, and one (ixabepilone, BMS) is already on the market, having been approved by the FDA in 2007. The terminal step in epothilone B biosynthesis is catalysed by the cytochrome P450 enzyme EpoK (CYP167A1), which catalyses the epoxidation of the C12–C13 double bond (in epothilone C and D) to form epothilone A and B, respectively. Although redox partners from different sources support the catalytic activity of EpoK in vitro, the conversion rates are low, and these redox partners are not applied to produce epothilone B in heterologous hosts. Results Schlegelella brevitalea DSM 7029 contains electron transport partners that efficiently support the catalytic activity of EpoK. We screened and identified one ferredoxin, Fdx_0135, by overexpressing putative ferredoxin genes in vivo and identified two ferredoxin reductases, FdR_0130 and FdR_7100, by whole-cell biotransformation of epothilone C to effectively support the catalytic activity of EpoK. In addition, we obtained strain H7029-3, with a high epothilone B yield and found that the proportion of epothilone A + B produced by this strain was 90.93%. Moreover, the whole-cell bioconversion strain 7029-10 was obtained; this strain exhibited an epothilone C conversion rate of 100% in 12 h. Further RT-qPCR experiments were performed to analyse the overexpression levels of the target genes. Gene knock-out experiments showed that the selected ferredoxin (Fdx_0135) and its reductases (FdR_0130 and FdR_7100) might participate in critical physiological processes in DSM 7029. Conclusion Gene overexpression and whole-cell biotransformation were effective methods for identifying the electron transport partners of the P450 enzyme EpoK. In addition, we obtained an epothilone B high-yield strain and developed a robust whole-cell biotransformation system. This strain and system hold promise for the industrial production of epothilone B and its derivatives.

Human CYP4Z1 catalyzes the in-chain hydroxylation of lauric acid and myristic acid

2009 ◽  
Vol 390 (4) ◽  
Author(s):  
Andy Zöllner ◽  
Calin-Aurel Dragan ◽  
Dominik Pistorius ◽  
Rolf Müller ◽  
Helge B. Bode ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Fission Yeast ◽  
Lauric Acid ◽  
Myristic Acid ◽  
Poor Prognosis ◽  
Catalytic Properties ◽  
Human Cancer ◽  
Cytochrome P450 Enzyme ◽  
P450 Enzyme ◽  
Whole Cell Biotransformation

Abstract Overexpression of human CYP4Z1, a cytochrome P450 enzyme, has been correlated with poor prognosis in human cancer. However, its catalytic properties are not yet known. We expressed this P450 in Schizosaccharomyces pombe and demonstrate by whole-cell biotransformation assays CYP4Z1-dependent in-chain hydroxylation of lauric and myristic acid, which in both cases leads to the formation of four different monohydroxylated products at positions ω-2, ω-3, ω-4, and ω-5, respectively. The CYP4Z1-expressing fission yeast should be a new valuable tool for testing cancer drugs or for the development of new prodrug strategies.

scholarly journals Reconstitution of theIn VitroActivity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System

2015 ◽  
Vol 81 (18) ◽  
pp. 6268-6275 ◽  
Author(s):  
Li Ma ◽  
Lei Du ◽  
Hui Chen ◽  
Yue Sun ◽  
Shan Huang ◽  
...  
Keyword(s):  
Redox System ◽  
Cytochrome P450 Enzyme ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
Site Specific ◽  
Whole Cell ◽  
Content Type ◽  
P450 Enzyme

ABSTRACTThe cytochrome P450 enzyme CYP-sb21 fromSebekia benihanais capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylationin vivo, thein vitroactivity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted thein vitroactivity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed ofseFdx andseFdR fromSynechococcus elongatusPCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst usingEscherichia coliwas also constructed, and the permeability problem was solved by usingN-cetyl-N,N,N-trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application.

Effects of ginseng components on c-DNA-expressed cytochrome P450 enzyme catalytic activity

Life Sciences ◽  
1999 ◽  
Vol 65 (15) ◽  
pp. PL209-PL214 ◽  
Author(s):  
G.L. Henderson ◽  
M.R. Harkey ◽  
M.E. Gershwin ◽  
R.M. Hackman ◽  
J.S. Stern ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Catalytic Activity ◽  
Cytochrome P450 Enzyme ◽  
P450 Enzyme

Biocatalytic conversion of avermectin into 4″-oxo-avermectin: discovery, characterization, heterologous expression and specificity improvement of the cytochrome P450 enzyme

2006 ◽  
Vol 34 (6) ◽  
pp. 1236-1240 ◽  
Author(s):  
I. Molnár ◽  
V. Jungmann ◽  
J. Stege ◽  
A. Trefzer ◽  
J.P. Pachlatko
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Electron Transport ◽  
Natural Product ◽  
High Throughput Screening ◽  
Streptomyces Coelicolor ◽  
Cytochrome P450 Enzyme ◽  
Expression Systems ◽  
P450 Enzyme ◽  
Solvent Tolerant

4″-Oxo-avermectin is a key intermediate in the manufacture of the insecticide emamectin benzoate from the natural product avermectin. Seventeen Streptomyces strains with the ability to oxidize avermectin to 4″-oxo-avermectin in a regioselective manner have been discovered, and the enzymes responsible for this reaction were found to be CYPs (cytochrome P450 mono-oxygenases). The genes for these enzymes have been cloned, sequenced and compared to reveal a new subfamily of CYPs. The biocatalytic enzymes have been overexpressed in Escherichia coli, Streptomyces lividans and solvent-tolerant Pseudomonas putida strains using different promoters and vectors. FDs (ferredoxins) and FREs (ferredoxin:NADP+ reductases) were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains, and tested in co-expression systems to optimize the electron transport. Subsequent studies showed that increasing the biocatalytic conversion levels to commercial relevance results in the production of several side products in significant amounts. Chimaeric Ema CYPs were created by sequential rounds of GeneReassembly™, a proprietary directed evolution method, and selected for improved substrate specificity by high-throughput screening.

Polymorphisms in alcohol metabolizing genes ADH2 and ADH3 and susceptibility to pancreatitis in alcoholics

2017 ◽  
Vol 6 (03) ◽  
pp. 5297
Author(s):  
Vedangi Aaren* ◽  
Godi Sudhakar ◽  
Girinadh L.R.S.
Keyword(s):  
Frequency Distribution ◽  
Ethanol Metabolism ◽  
Alcoholic Pancreatitis ◽  
Cytochrome P450 Enzyme ◽  
Increased Risk ◽  
Significant Difference ◽  
Pcr Rflp ◽  
P450 Enzyme ◽  
Acute Alcoholic Pancreatitis ◽  
Acute And Chronic Pancreatitis

In both developed and developing countries, overuse of alcohol is a considered as the major cause of acute and chronic pancreatitis. Prolonged overconsumption of alcohol for 5–10 years typically precedes the initial attack of acute alcoholic pancreatitis. It is observed that only a minority (around 5%) of alcoholics develop pancreatitis. It is now established that the pancreas has the capacity to metabolize ethanol. Previous studies have shown that there are two major pathways of ethanol metabolism, oxidative and non-oxidative. Oxidative ethanol metabolism involves the conversion of ethanol to acetaldehyde, a reaction that is catalysed by aldehyde dehydrogenase (ADH) with contributions from cytochrome P450 enzyme (CYP2E1) and possibly also catalase. Genetic factors regulating alcohol metabolism could predispose in developing alcoholic pancreatitis (AP). We investigated the association of polymorphisms in ADH enzymes with the alcoholic pancreatitis in North coastal Andhra Pradesh. Patients with alcoholic pancreatitis (AP; n = 100), alcoholic controls (AC; n = 100), and healthy controls (HC; n = 100) were included in the study. Blood samples were collected from the subjects in EDTA coated vials. DNA was extracted and genotyping for ADH2 and ADH3 was done by PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism). The products were analysed by gel electrophoresis. The frequency distribution of ADH3*1/*1 genotype was significantly higher in AP group (54%) compared with AC (35%), and HC (42%), and was found to be associated with increased risk of alcoholic pancreatitis. There was no statistically significant difference between the frequency distribution of ADH3*1/*1, ADH3*1/*2, and ADH3*2/*2 genotypes between AC and HC. There was no statistically significant difference between the frequency distribution of ADH2*1/*1, ADH2*1/*2, and ADH2*2/*2 genotypes in AP compared with AC and HC. This study shows that carriers of ADH3*1/*1 individuals consuming alcohol are at higher risk for alcoholic pancreatitis than those with other genotypes such as ADH3*1/*2 and ADH3*2/*2. 

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