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

• Published in: Microbial cell factories Vol. 11; no. 1; p. 95
• Main Authors: Makino, Takuya, Otomatsu, Toshihiko, Shindo, Kazutoshi, Kitamura, Emi, Sandmann, Gerhard, Harada, Hisashi, Misawa, Norihiko
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 18.07.2012; BioMed Central; BMC
• Subjects: Aromatic compounds; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biocatalysis; Cyanobacterium; CYP110; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Cytochrome P450; Design of experiments; E coli; Enzymes; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fatty acids; Fixation; Flavone synthase; Flavones - biosynthesis; Flavones - chemistry; Functional foods; Gene Expression; Genes; Genetic aspects; Genetic Engineering; Hydroxides; Instrument industry; Isoflavones; Life sciences; Microbial genetic engineering; Microbiology; Molecular Structure; Nitrogen; NMR; Nostoc - enzymology; Nostoc - genetics; Nostoc sp. strain PCC 7120; Nuclear magnetic resonance; Phylogenetics; Phylogeny; Physiological aspects; Plasmids; Production processes; Proteins; Self sufficiency; Sesquiterpenes; Zerumbone; United States; Japan; Germany
• ISSN: 1475-2859; 1475-2859
• DOI: 10.1186/1475-2859-11-95

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. 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. 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.