Identification of a Gene Encoding a New Fungal Steroid 7-Hydroxylase and Its Functional Characterization in Yeast

Overview

Journal Int J Mol Sci

Publisher MDPI

Specialties Biochemistry
Chemistry
Molecular Biology

Date 2023 Dec 23

PMID 38139084

Authors

Vyacheslav Kollerov

Sergey Tarlachkov

Andrei Shutov

Alexey Kazantsev

Marina Donova

Affiliations

Soon will be listed here.

Abstract

The hydroxylation of steroids in the C7β position is one of the rare reactions that allow the production of value-added precursors in the synthesis of ursodeoxycholic acid and other pharmaceuticals. Recently, we discovered this activity in the ascomycete sp. VKM F-3040. In this study, the novel gene of 7-hydroxylase (P450) was identified as being heterologously expressed and functionally characterized in . Transcriptome data mining and differential expression analysis revealed that 12 putative genes in sp. mycelia significantly increased their expression in response to dehydroepiandrosterone (DHEA). The transcriptional level of the most up-regulated cytochrome P450 gene was increased more than 300-fold. A two-gene construct with a candidate P450 gene and the gene of its natural redox partner, NADPH-cytochrome P450 reductase (CPR), which is interconnected by a T2A element, was created. Using this construct, recombinant strains co-expressing fungal P450 and CPR genes were obtained. The functional activity of the recombinant P450 was studied in vivo during the bioconversion of androstane steroids. The fungal 7-monooxygenase predominantly catalyzed the 7β-hydroxylation of androstadienedione (ADD), DHEA, and androstenediol, whereas 1-dehydrotestosterone was hydroxylated by P450 mainly at the C7-Hα position. To our knowledge, this is the first report of a recombinant yeast capable of catalyzing the 7α/β-hydroxylation of ADD and DHEA.

References

Qian M, Zeng Y, Mao S, Jia L, Hua E, Lu F . Engineering of a fungal steroid 11α-hydroxylase and construction of recombinant yeast for improved production of 11α-hydroxyprogesterone. J Biotechnol. 2022; 353:1-8. DOI: 10.1016/j.jbiotec.2022.05.012. View

Kollerov V, Shutov A, Kazantsev A, Donova M . Steroid modification by filamentous fungus Drechslera sp.: Focus on 7-hydroxylase and 17β-hydroxysteroid dehydrogenase activities. Fungal Biol. 2021; 126(1):91-100. DOI: 10.1016/j.funbio.2021.11.002. View

Cresnar B, Petric S . Cytochrome P450 enzymes in the fungal kingdom. Biochim Biophys Acta. 2010; 1814(1):29-35. DOI: 10.1016/j.bbapap.2010.06.020. View

Lu W, Chen X, Feng J, Bao Y, Wang Y, Wu Q . A Fungal P450 Enzyme from Thanatephorus cucumeris with Steroid Hydroxylation Capabilities. Appl Environ Microbiol. 2018; 84(13). PMC: 6007115. DOI: 10.1128/AEM.00503-18. View

Chen J, Tang J, Xi Y, Dai Z, Bi C, Chen X . Production of 14α-hydroxysteroids by a recombinant Saccharomyces cerevisiae biocatalyst expressing of a fungal steroid 14α-hydroxylation system. Appl Microbiol Biotechnol. 2019; 103(20):8363-8374. DOI: 10.1007/s00253-019-10076-x. View

Durairaj P, Hur J, Yun H . Versatile biocatalysis of fungal cytochrome P450 monooxygenases. Microb Cell Fact. 2016; 15(1):125. PMC: 4950769. DOI: 10.1186/s12934-016-0523-6. View

Langmead B, Salzberg S . Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012; 9(4):357-9. PMC: 3322381. DOI: 10.1038/nmeth.1923. View

Kristan K, Rizner T . Steroid-transforming enzymes in fungi. J Steroid Biochem Mol Biol. 2011; 129(1-2):79-91. DOI: 10.1016/j.jsbmb.2011.08.012. View

Chen J, Fan F, Qu G, Tang J, Xi Y, Bi C . Identification of Absidia orchidis steroid 11β-hydroxylation system and its application in engineering Saccharomyces cerevisiae for one-step biotransformation to produce hydrocortisone. Metab Eng. 2019; 57:31-42. DOI: 10.1016/j.ymben.2019.10.006. View

10.

Liao Y, Smyth G, Shi W . featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2013; 30(7):923-30. DOI: 10.1093/bioinformatics/btt656. View

11.

Kollerov V, Tarlachkov S, Donova M . transcriptome assembly of sp. VKM F-3040, a promising steroid-modifying ascomycete. Microbiol Resour Announc. 2023; 12(11):e0066323. PMC: 10652950. DOI: 10.1128/MRA.00663-23. View

12.

Petric S, Hakki T, Bernhardt R, Zigon D, Cresnar B . Discovery of a steroid 11α-hydroxylase from Rhizopus oryzae and its biotechnological application. J Biotechnol. 2010; 150(3):428-37. DOI: 10.1016/j.jbiotec.2010.09.928. View

13.

Kolek T, Milecka N, Swizdor A, Panek A, Bialonska A . Hydroxylation of DHEA, androstenediol and epiandrosterone by Mortierella isabellina AM212. Evidence indicating that both constitutive and inducible hydroxylases catalyze 7α- as well as 7β-hydroxylations of 5-ene substrates. Org Biomol Chem. 2011; 9(15):5414-22. DOI: 10.1039/c1ob05350g. View

14.

Kozlowska E, Hoc N, Sycz J, Urbaniak M, Dymarska M, Grzeszczuk J . Biotransformation of steroids by entomopathogenic strains of Isaria farinosa. Microb Cell Fact. 2018; 17(1):71. PMC: 5948769. DOI: 10.1186/s12934-018-0920-0. View

15.

Li A, Acevedo-Rocha C, DAmore L, Chen J, Peng Y, Garcia-Borras M . Regio- and Stereoselective Steroid Hydroxylation at C7 by Cytochrome P450 Monooxygenase Mutants. Angew Chem Int Ed Engl. 2020; 59(30):12499-12505. PMC: 7384163. DOI: 10.1002/anie.202003139. View

16.

Jones E . The microbiological hydroxylation of steroids and related compounds. Pure Appl Chem. 1973; 33(1):39-52. DOI: 10.1351/pac197333010039. View

17.

Lathe R . Steroid and sterol 7-hydroxylation: ancient pathways. Steroids. 2002; 67(12):967-77. DOI: 10.1016/s0039-128x(02)00044-2. View

18.

Bolger A, Lohse M, Usadel B . Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15):2114-20. PMC: 4103590. DOI: 10.1093/bioinformatics/btu170. View

19.

Love M, Huber W, Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12):550. PMC: 4302049. DOI: 10.1186/s13059-014-0550-8. View

20.

Liu Z, Chen O, Wall J, Zheng M, Zhou Y, Wang L . Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci Rep. 2017; 7(1):2193. PMC: 5438344. DOI: 10.1038/s41598-017-02460-2. View