Molecular cloning, expression of CPR gene from Rhizopus oryzae into Rhizopus nigericans and its application in the 11α-hydroxylation of 16α, 17-epoxy-progesterone

Chen, Xiaolong; X, Luo; Cao, Fuyang; Zhu, Tingheng; Yang, Fan; Xing, Jia; Shen, Yin‐Chu

doi:10.1016/j.enzmictec.2014.08.002

Cited by 8 publications

(8 citation statements)

References 17 publications

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“…The lower conversion rate of the biohydroxylation is the bottleneck of 11α-hydroxylation of EP by R. nigricans which is used in steroid industry [9]. Cytochrome P450 monooxygenase was suggested to play a crucial role in steroid hydroxylation conducted by microorganisms [19,20].…”

Section: Discussionmentioning

confidence: 99%

“…The sequence of CYP509C12 (RO3G-05077.1) of R. oryzae were downloaded from the Broad Institute Database (http://www.broad.mit.edu/annotation/genome/rhizopus_oryzae/MultiHome.html, 7.9.2009) and was used to design primers for amplification of RoCYP . The cDNA was synthesized from the total RNA isolated from R. oryzae treated with 300 µM EP as described previously [9] using the RT-PCR kit (Takara, Dalian, China). The coding sequence of putative RoCYP was amplified by PCR using the primers listed in Table 1, and cloned into the pMD19-T vector (Takara, Dalian, China) to generate pMD19T-CYP for sequencing.…”

Section: Methodsmentioning

confidence: 99%

“…The two main classes of CYPs are the mitochondrial/bacterial type and the microsomal type [7]. Microsomal CYPs are membrane bound and accept electrons from a microsomal CPR, which is an essential redox partner of CYPs, and whose function is to accept two electrons from NADPH and transfer them sequentially to CYPs [9,10]. The activities of CYP and CPR are crucial for various oxidation reactions performed by microsomal enzymes [11].…”

Section: Introductionmentioning

confidence: 99%

“…The 11α-hydroxylation of 16α, 17-epoxyprogesterone (EP) with R. nigricans (but not with R. oryzae ) is an essential and important step in the process to produce 11α-hydroxy-16α, 17-epoxyprogesterone (HEP), and for decades, this has been a crucial step in the production of glucocorticoids in China [9]. Improving the conversion rate of 11α-hydroxylation is of great importance for the pharmaceutical industry.…”

Section: Introductionmentioning

confidence: 99%

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Heterologous Expression ofRhizopus Oryzae CYP509C12Gene inRhizopus NigricansEnhances Reactive Oxygen Species Production and 11α-Hydroxylation Rate of 16α, 17-Epoxyprogesterone

Shen

Gao

et al. 2019

Mycobiology

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The 11α-hydroxylation of 16α, 17-epoxyprogesterone (EP) catalyzed by Rhizopus nigricans is crucial for the steroid industry. However, lower conversion rate of the biohydroxylation restricts its potential industrial application. The 11α-steroid hydroxylase CYP509C12 from R. oryzae were reported to play a crucial role in the 11α-hydroxylation in recombinant fission yeast. In the present study, the CYP509C12 of R. oryzae (RoCYP) was introduced into R. nigricans using the liposome-mediated mycelial transformation. Heterologous expression of RoCYP resulted in increased fungal growth and improved intracellular reactive oxygen species content in R. nigricans. The H2O2 levels in RoCYP transformants were approximately 2-folder that of the R. nigricans wild type (RnWT) strain, with the superoxide dismutase activities increased approximately 45% and catalase activities decreased approximately 68%. Furthermore, the 11α-hydroxylation rates of EP in RoCYP transformants (C4, C6 and C9) were 39.7%, 38.3% and 38.7%, which were 12.1%, 8.2% and 9.4% higher than the rate of the RnWT strain, respectively. This paper investigated the effect of heterologous expression of RoCYP in R. nigricans, providing an effective genetic method to construct the engineered strains for steroid industry.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Heterologous Expression ofRhizopus Oryzae CYP509C12Gene inRhizopus NigricansEnhances Reactive Oxygen Species Production and 11α-Hydroxylation Rate of 16α, 17-Epoxyprogesterone

Shen

Gao

et al. 2019

Mycobiology

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“…In many types of yeast, XR activity has been enhanced or its coenzyme specificity has been changed through site‐direct mutagenesis technique, and the main active sites and binding sites of these XRs have been clarified . Many genes in R. oryzae have been identified and characterized since its genomic sequences were published . However, the XR gene in R. oryzae remained unidentified.…”

Section: Introductionmentioning

confidence: 99%

Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae

Zhang

Jiang

Zheng

et al. 2015

J. Basic Microbiol.

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Rhizopus oryzae is valuable as a producer of organic acids via lignocellulose catalysis. R. oryzae metabolizes xylose, which is one component of lignocellulose hydrolysate. In this study, a novel NADPH-dependent xylose reductase gene from R. oryzae AS 3.819 (Roxr) was cloned and expressed in Pichia pastoris GS115. Homology alignment suggested that the 320-residue protein contained domains and active sites belonging to the aldo/keto reductase family. SDS-PAGE demonstrated that the recombinant xylose reductase has a molecular weight of approximately 37 kDa. The optimal catalytic pH and temperature of the purified recombinant protein were 5.8 and 50 °C, respectively. The recombinant protein was stable from pH 4.4 to 6.5 and at temperatures below 42 °C. The recombinant enzyme has bias for D-xylose and L-arabinose as substrates and NADPH as its coenzyme. Real-time quantitative reverse transcription PCR tests suggested that native Roxr expression is regulated by a carbon catabolite repression mechanism. Site-directed mutagenesis at two possible key sites involved in coenzyme binding, Thr(226) → Glu(226) and Val(274) → Asn(274), were performed, respectively. The coenzyme specificity constants of the resulted RoXR(T226E) and RoXR(V274N) for NADH increased 18.2-fold and 2.4-fold, which suggested possibility to improve the NADH preference of this enzyme through genetic modification.

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Molecular cloning, expression of CPR gene from Rhizopus oryzae into Rhizopus nigericans and its application in the 11α-hydroxylation of 16α, 17-epoxy-progesterone

Cited by 8 publications

References 17 publications

Heterologous Expression ofRhizopus Oryzae CYP509C12Gene inRhizopus NigricansEnhances Reactive Oxygen Species Production and 11α-Hydroxylation Rate of 16α, 17-Epoxyprogesterone

Heterologous Expression ofRhizopus Oryzae CYP509C12Gene inRhizopus NigricansEnhances Reactive Oxygen Species Production and 11α-Hydroxylation Rate of 16α, 17-Epoxyprogesterone

Cloning, expression, and characterization of a novel xylose reductase from Rhizopus oryzae

Nitroreduction of flutamide by Cunninghamella elegans NADPH: Cytochrome P450 reductase

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