Comparative functional characterization of a novel benzoate hydroxylase cytochrome P450 of Fusarium oxysporum

Durairaj, Pradeepraj; Jung, E.; Park, Hyun Ho; Kim, Byung‐Gee; Yun, Hyungdon

doi:10.1016/j.enzmictec.2014.12.013

Cited by 18 publications

(23 citation statements)

References 29 publications

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“…To overcome this dilemma, one way is to create CYP‐CPR fusion protein (Renault, Bassard, Hamberger, & Werck Reichhart, ). Another promising alternative is to select a suitable CPR with high electron transfer efficiency towards CYP, where a CPR with the same origin as CYP is often preferred (Durairaj, Jung, Park, Kim, & Yun, ). Our previous work indicated a predicted CPR ( gl20687 ) was unlikely to support the function of CYP5150L8 in S. cerevisiae (Wang et al, ).…”

Section: Introductionmentioning

confidence: 99%

Efficient biosynthesis of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase

Lan

Wei

Wang

et al. 2019

Biotech & Bioengineering

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Ganoderic acid 3-hydroxy-lanosta-8,24-dien-26-oic acid (GA-HLDOA), an antitumor triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, is considered as a key precursor for biosynthesizing other ganoderic acids (GAs) with superior antitumor activities. Our previous study identified CYP5150L8 from G. lucidum as a lanosterol oxidase, and achieved heterologous biosynthesis of GA-HLDOA in Saccharomyces cerevisiae. However, low production of GA-HLDOA in either G. lucidum or heterologous host hindered its further investigation and application. In this study, we constructed a dual tunable system for balancing the expression of CYP5150L8 and a Ganoderma P450 reductase iGLCPR, and performed a comprehensive optimization of CYP5150L8 expression, iGLCPR expression, and glycerol usage. Then, we investigated the fermentation behavior of the best strain in optimized condition in flask and achieved 154.45 mg/L GA-HLDOA production, which was 10.7-fold higher compared with previous report. This study may facilitate the wide-spread application of GA-HLDOA and the discovery of unknown cytochrome P450s in downstream GAs biosynthesis.

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Section: Introductionmentioning

confidence: 99%

Efficient biosynthesis of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase

Lan

Wei

Wang

et al. 2019

Biotech & Bioengineering

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“…Historically, studies involving heterologous P450 expression have generally compensated for insufficient electron supply from natural CPR systems by either overexpressing the native CPR of the host (Nthangeni et al ., ; Schiffler et al ., ) or coexpressing the natural CPR partner from the P450 source organism (Gudiminchi et al ., ; Syed et al ., ). Both of these approaches were recently applied in studies on the expression of benzoate hydroxylases and fatty acid ω‐hydroxylases from Fusarium oxysporum in Saccharomyces cerevisiae (Durairaj et al ., ,b). Regardless of the F. oxysporum P450 expressed in S. cerevisiae , the cooverexpressed F. oxysporum CPR (source) enhanced activity better than the overexpressed S. cerevisiae CPR (host).…”

Section: Discussionmentioning

confidence: 99%

“…Regardless of the F. oxysporum P450 expressed in S. cerevisiae , the cooverexpressed F. oxysporum CPR (source) enhanced activity better than the overexpressed S. cerevisiae CPR (host). Additionally, when the unrelated CPR from Candida albicans was also overexpressed with CYP53A19 in S. cerevisiae , poorer results were obtained than in both of the other cases (Durairaj et al ., 2015b). Interestingly, CPR assays performed in that study on CPRs in isolation indicated relatively similar activities among all of the CPRs tested, suggesting that differences in P450 activities were due to specific CPR‐P450 interactions.…”

Section: Discussionmentioning

confidence: 99%

“…As such, they have been identified as potential targets for antifungal drugs (Jawallapersand et al, 2014;Koro sec et al, 2014). BA and the CYP53B1 hydroxylation product, phydroxybenzoic acid (pHBA), are industrially versatile chemicals, with applications as intermediates in drug synthesis, in food preservatives and polyester fibres (Durairaj et al, 2015b). In addition, we previously established reasonably rapid and convenient assays for detection and quantification of BA and pHBA (Shiningavamwe et al, 2006;Theron, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Heterologous coexpression of the benzoate‐para‐hydroxylase CYP53B1 with different cytochrome P450 reductases in various yeasts

Theron

Labuschagne

Albertyn

et al. 2018

Microbial Biotechnology

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Summary Cytochrome P450 monooxygenases (P450) are enzymes with high potential as biocatalysts for industrial applications. Their large‐scale applications are, however, limited by instability and requirement for coproteins and/or expensive cofactors. These problems are largely overcome when whole cells are used as biocatalysts. We previously screened various yeast species heterologously expressing self‐sufficient P450s for their potential as whole‐cell biocatalysts. Most P450s are, however, not self‐sufficient and consist of two or three protein component systems. Therefore, in the present study, we screened different yeast species for coexpression of P450 and P450‐reductase (CPR) partners, using CYP53B1 from Rhodotorula minuta as an exemplary P450. The abilities of three different coexpressed CPR partners to support P450 activity were investigated, two from basidiomycetous origin and one from an ascomycete. The various P450‐CPR combinations were cloned into strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, Hansenula polymorpha, Yarrowia lipolytica and Arxula adeninivorans, using a broad‐range yeast expression vector. The results obtained supported the previous finding that recombinant A. adeninivorans strains perform excellently as whole‐cell biocatalysts. This study also demonstrated for the first time the P450 reductase activity of the CPRs from R. minuta and U. maydis. A very interesting observation was the variation in the supportive activity provided by the different reductase partners tested and demonstrated better P450 activity enhancement by a heterologous CPR compared to its natural partner CPR. This study highlights reductase selection as a critical variable for consideration in the pursuit of optimal P450‐based catalytic systems. The usefulness of A. adeninivorans as both a host for recombinant P450s and whole‐cell biocatalyst was emphasized, supporting earlier findings.

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“…The hydroxylation on specific carbon atoms of aliphatic compound is important in industrial chemical synthesis. In fact, omega fatty acids terminally oxidized are useful for the synthesis of industrial compounds with pharmaceutical and commercial interests such as potential anticancer drugs, cosmetic intermediates, adhesives and bioplastics (Durairaj et al 2015). Enzymatic hydroxylation of specific C-H bonds is an interesting solution since a specific carbon atoms are hydroxylated.…”

Section: )mentioning

confidence: 99%

Catalytically self-sufficient cytochromes P450 for green production of fine chemicals

Ciaramella

Minerdi

Gilardi

2016

Rend. Fis. Acc. Lincei

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This review covers our current knowledge on catalytically self-sufficient cytochromes P450. These enzymes present all the advantages of the regio-and stereo-selectivity of typical P450s combined with the fact that, due to the natural fusion with their reductases, their application in biocatalysis does not need the addition of other proteins: only NAD(P)H and substrate are needed to trigger catalysis. To this date only few catalytically self-sufficient P450s have been isolated from bacteria, fungi and plants. Their low number does not detract from their importance, as the reactions they catalyse and the turnover numbers of which they are capable (in some cases higher than 15,000 s-1) makes them highly relevant to the 1 This contribution is the written, peer-reviewed version of a paper presented by a participant to the Conference ""Concepts in catalysis : from heterogeneous to homogeneous and enzymatic catalysis" held at Accademia Nazionale dei Lincei in Rome on February 25-26 , 2016. 3 chemical and pharmaceutical industry. These enzymes will be covered in this review, together with their specific reactions relevant to the field of biocatalysis.

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Comparative functional characterization of a novel benzoate hydroxylase cytochrome P450 of Fusarium oxysporum

Cited by 18 publications

References 29 publications

Efficient biosynthesis of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase

Efficient biosynthesis of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase

Heterologous coexpression of the benzoate‐para‐hydroxylase CYP53B1 with different cytochrome P450 reductases in various yeasts

Catalytically self-sufficient cytochromes P450 for green production of fine chemicals

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