Biocatalytic synthesis of flavones and hydroxyl-small molecules by recombinant Escherichia coli cells expressing the cyanobacterial CYP110E1 gene

Background: Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria inclu
Background: Cyanobacteria possess several cytochrome P450s, but very little is known about their catalytic functions. CYP110 genes unique to cyanaobacteria are widely distributed in heterocyst-forming cyanobacteria including nitrogen-fixing genera Nostoc and Anabaena. We screened the biocatalytic functions of all P450s from three cyanobacterial strains of genus Nostoc or Anabaena using a series of small molecules that contain flavonoids, sesquiterpenes, low-molecular-weight drugs, and other aromatic compounds.

Results: Escherichia coli cells carrying each P450 gene that was inserted into the pRED vector, containing the RhFRed reductase domain sequence from Rhodococcus sp. NCIMB 9784 P450RhF (CYP116B2), were co-cultured with substrates and products were identified when bioconversion reactions proceeded. Consequently, CYP110E1 of Nostoc sp. strain PCC 7120, located in close proximity to the first branch point in the phylogenetic tree of the CYP110 family, was found to be promiscuous for the substrate range mediating the biotransformation of various small molecules. Naringenin and (hydroxyl) flavanones were respectively converted to apigenin and (hydroxyl) flavones, by functioning as a flavone synthase. Such an activity is reported for the first time in prokaryotic P450s. Additionally, CYP110E1 biotransformed the notable sesquiterpene zerumbone, anti-inflammatory drugs ibuprofen and flurbiprofen (methylester forms), and some aryl compounds such as 1-methoxy and 1-ethoxy naphthalene to produce hydroxylated compounds that are difficult to synthesize chemically, including novel compounds.

Conclusion: We elucidated that the CYP110E1 gene, C-terminally fused to the P450RhF RhFRed reductase domain sequence, is functionally expressed in E. coli to synthesize a robust monooxygenase, which shows promiscuous substrate specificity (affinity) for various small molecules, allowing the biosynthesis of not only flavones (from flavanones) but also a variety of hydroxyl-small molecules that may span pharmaceutical and nutraceutical industries.
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  • vector for the functional expression of class I P450 genes inE. coli.Figure S3: List of screened substrates (47 samples). Figure S4: CO difference spectral analysis of CYP110E1 C-terminally fused to RhFRed. Cell extracts from E. coli BL21 (DE3) carrying plasmid pCYP110E1-Red (three samples, S1, S2, and S3) were measured for CO difference spectra.

  • Additional file 2: Spectroscopic data of the individual converted products.

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Author:Takuya Makino, Toshihiko Otomatsu, Kazutoshi Shindo, Emi Kitamura, Gerhard Sandmann, Hisashi Harada, Norihiko Misawa
URN:urn:nbn:de:hebis:30:3-256542
DOI:http://dx.doi.org/10.1186/1475-2859-11-95
ISSN:1475-2859
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=22809492
Parent Title (English):Microbial cell factories
Publisher:BioMed Central
Place of publication:London
Document Type:Article
Language:English
Date of Publication (online):2012/07/18
Date of first Publication:2012/07/18
Publishing Institution:Univ.-Bibliothek Frankfurt am Main
Release Date:2012/08/15
Tag:CYP110; Cyanobacterium; Cytochrome P450; Flavone synthase; Nostoc sp. strain PCC 7120; Zerumbone
Volume:11
Issue:11:95
Pagenumber:13
First Page:1
Last Page:13
Institutes:Biowissenschaften
Dewey Decimal Classification:570 Biowissenschaften; Biologie
Sammlungen:Universitätspublikationen
Sondersammelgebiets-Volltexte
Licence (German):License LogoCreative Commons - Namensnennung 3.0

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