Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

Fujii, Yoshikazu; Kabumoto, Hiroki; Nishimura, Kenji; Fujii, Tadashi; Yanai, Satoshi; Takeda, Koji; Tamura, Noriko; Arisawa, Akira; Tamura, Tomohide

doi:10.1016/j.bbrc.2009.05.033

Cited by 65 publications

(53 citation statements)

References 15 publications

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“…CYP-sb13, previously named an S. benihana CYP506, was proposed to play a limited role in the CsA hydroxylation process, probably through indirect interactions with other CYP-FD-FDR systems (13). Eight other S. benihana CYPs (CYP-sb3-1, CYP-sb3-2, CYP-sb4, CYP-sb10, CYP-sb11, CYP-sb12, CYP-sb21, and CYP-sb24) belong to the bacterial CYP107 family (34)(35)(36). The remaining four S. benihana CYPs (CYP-sb8, CYP-sb9, CYP-sb15, and CYP-sb22) each belong to different bacterial CYP families.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Cyclosporine-Specific P450 Hydroxylase Gene through Targeted Cytochrome P450 Complement (CYPome) Disruption in Sebekia benihana

Lee

Kim

Yoon

et al. 2013

Appl Environ Microbiol

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c It was previously proposed that regio-specific hydroxylation of an immunosuppressive cyclosporine (CsA) at the 4th N-methyl leucine is mediated by cytochrome P450 hydroxylase (CYP) in the rare actinomycete Sebekia benihana. This modification is thought to be the reason for the hair growth-promoting side effect without the immunosuppressive activity of CsA. Through S. benihana genome sequencing and in silico analysis, we identified the complete cytochrome P450 complement (CYPome) of S. benihana, including 21 CYPs and their electron transfer partners, consisting of 7 ferredoxins (FDs) and 4 ferredoxin reductases (FDRs). Using Escherichia coli conjugation-based S. benihana CYPome-targeted disruption, all of the identified CYP, FD, and FDR genes in S. benihana were individually inactivated. Among the 32 S. benihana exconjugant mutants tested, only a single S. benihana CYP mutant, ⌬CYP-sb21, failed to exhibit CsA hydroxylation activity. The hydroxylation was restored by CYP-sb21 gene complementation. Since all S. benihana FD and FDR disruption mutants maintained CsA hydroxylation activity, it can be concluded that CYP-sb21, a new member of the bacterial CYP107 family, is the only essential component of the in vivo regio-specific CsA hydroxylation process in S. benihana. Moreover, expression of an extra copy of the CYP-sb21 gene increased CsA hydroxylation in wild-type S. benihana and an NADPH-enriched Streptomyces coelicolor mutant, by 2-fold and 1.5-fold, respectively. These results show for the first time that regio-specific hydroxylation of CsA is carried out by a specific P450 hydroxylase present in S. benihana, and they set the stage for the biotechnological application of regio-specific CsA hydroxylation through heterologous CYP-sb21 expression.

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Section: Resultsmentioning

confidence: 99%

Identification of a Cyclosporine-Specific P450 Hydroxylase Gene through Targeted Cytochrome P450 Complement (CYPome) Disruption in Sebekia benihana

Lee

Kim

Yoon

et al. 2013

Appl Environ Microbiol

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“…However, purification was difficult due to the occurrence of the phenomenon where the VD 3 hydroxylation activity became undetectable during the purification process. After much trial and error, we found that salt (NaCl and others) had to be present in the reaction solution for this enzyme to be active, and it was possible to follow the activity to the final step of purification [6] .…”

Section: Isolation Of the Enzyme And Identification Of The Genementioning

confidence: 99%

“…The quadruple mutant (Vdh-K1) that had the highest improvement showed about 12 times increase of 25-hydroxylase activity and about 25 times increase of 1 -hydroxylase activity compared to the wild type Vdh (Vdh-WT) [6] [8] . Interestingly, the four mutations were located far from the active site, and it is difficult to find such mutations by rational design.…”

Section: High Activation Of the Enzymementioning

confidence: 99%

Efficient production of active form of vitamin D3 by microbial conversion

Yasutake

Tamura

2012

Synthesiology English edition

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“…The enzymatic activity of Vdh was determined based on the bioconversion rate of the inactive vitamin D 3 into the hydroxylated active form of vitamin D 3 as previously described (10). Briefly, the cell pellet, twice washed with KPB, was resuspended in 1 ml of KPB containing 2% glucose, 0.5% methyl-␤-cyclodextrin (M␤CD; Junsei Chemical, Co., Ltd., Tokyo, Japan), 1 mM thiamine, and 0.5 mM vitamin D 3 (Sigma Chemical Co., St. Louis, MO) as a substrate.…”

Section: Colony Pcr a Rapid Preliminary Test For Characterization Ofmentioning

confidence: 99%

“…Confirmation of the stability of the integrated copies of the foreign DNA in the host cell genome. Southern hybridization analysis was conducted for three randomly selected mutants with multiple insertions to verify the stability of the integrated DNA after culturing for several generations (10,20,40, and 80) of serial growth in the absence of antibiotics. The integrated DNA seems stable in the genome throughout the culturing period.…”

Section: Vol 76 2010 Generating Multiple Integrations In Rhodococcumentioning

confidence: 99%

New Vector System for Random, Single-Step Integration of Multiple Copies of DNA into the Rhodococcus Genome

Sallam

Tamura

Imoto

et al. 2010

Appl Environ Microbiol

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We designed a new vector system for creating a random mutant library with multiple integrations of DNA fragments into the Rhodococcus genome in a single step. For this, we cotransformed two vectors into Rhodococcus by electroporation: pTip-istAB-sacB regulates the expression of the transposase (IstA) and its helper protein (IstB) under the influence of a thiostrepton-inducible promoter, and pRTSK-sacB provides the transposable-marker DNA. Both are multicopy vectors that are stable in the host cells; transposition of the transposable-marker DNA occurs only after the induction of IstA/IstB expression. With the addition of thiostrepton, all cultured cells harboring the two vectors, irrespective of the volume, can be mutated by random insertion of the transposable-marker DNA into their genome. Among the generated mutants examined, 30% showed multiple (two to five) insertion copies. The multiple integrated DNA copies were stable in the genome for more than 80 generations of serial growth without the addition of any selective antibiotics. This system can also be used for integrating various copy numbers of stably maintained protein expression cassettes in the host cell genome to modulate the expression level of biologically active recombinant proteins. We successfully applied this system to integrate multiple copies of expression cassettes for proline iminopeptidase and vitamin D 3 hydroxylase into the Rhodococcus genome and verified that the clones containing double or multiple copies of the integrated cassettes produced higher levels and showed higher enzymatic activities of the target protein than clones with only a single copy of integration.

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Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

Cited by 65 publications

References 15 publications

Identification of a Cyclosporine-Specific P450 Hydroxylase Gene through Targeted Cytochrome P450 Complement (CYPome) Disruption in Sebekia benihana

Identification of a Cyclosporine-Specific P450 Hydroxylase Gene through Targeted Cytochrome P450 Complement (CYPome) Disruption in Sebekia benihana

Efficient production of active form of vitamin D3 by microbial conversion

New Vector System for Random, Single-Step Integration of Multiple Copies of DNA into the Rhodococcus Genome

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