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 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

Molecular Characterization and Substrate Preference of a Polycyclic Aromatic Hydrocarbon Dioxygenase from Cycloclasticus sp. Strain A5

Cycloclasticus sp. strain A5 is able to grow with petroleum polycyclic aromatic hydrocarbons (PAHs), including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. A set of genes responsible for the degradation of petroleum PAHs was isolated by using the ability of the organism to oxidize indole to indigo. This 10.5-kb DNA fragment was sequenced and found to contain 10 open reading frames (ORFs). Seven ORFs showed homology to previously characterized genes for PAH degradation and were designated phn genes, although the sequence and order of these phn genes were significantly different from the sequence and order of the known PAH-degrading genes. The phnA1, phnA2, phnA3, and phnA4 genes, which encode the α and β subunits of an iron-sulfur protein, a ferredoxin, and a ferredoxin reductase, respectively, were identified as the genes coding for PAH dioxygenase. The phnA4A3 gene cluster was located 3.7 kb downstream of the phnA2 gene. PhnA1 and PhnA2 exhibited moderate (less than 62%) sequence identity to the α and β subunits of other aromatic ring-hydroxylating dioxygenases, but motifs such as the Fe(II)-binding site and the [2Fe-2S] cluster ligands were conserved. Escherichia coli cells possessing the phnA1A2A3A4 genes were able to convert phenanthrene, naphthalene, and methylnaphthalene in addition to the tricyclic heterocycles dibenzofuran and dibenzothiophene to their hydroxylated forms. Significantly, the E. coli cells also transformed biphenyl and diphenylmethane, which are ordinarily the substrates of biphenyl dioxygenases

New and Rare Carotenoids Isolated from Marine Bacteria and Their Antioxidant Activities

Marine bacteria have not been examined as extensively as land bacteria. We screened carotenoids from orange or red pigments-producing marine bacteria belonging to rare or novel species. The new acyclic carotenoids with a C30 aglycone, diapolycopenedioc acid xylosylesters A–C and methyl 5-glucosyl-5,6-dihydro-apo-4,4′-lycopenoate, were isolated from the novel Gram-negative bacterium Rubritalea squalenifaciens, which belongs to phylum Verrucomicrobia, as well as the low-GC Gram-positive bacterium Planococcus maritimus strain iso-3 belonging to the class Bacilli, phylum Firmicutes, respectively. The rare monocyclic C40 carotenoids, (3R)-saproxanthin and (3R,2′S)-myxol, were isolated from novel species of Gram-negative bacteria belonging to the family Flavobacteriaceae, phylum Bacteroidetes. In this review, we report the structures and antioxidant activities of these carotenoids, and consider relationships between bacterial phyla and carotenoid structures

Isolation and Characterization of o-Xylene Oxygenase Genes from Rhodococcus opacus TKN14

o-Xylene is one of the most difficult-to-degrade environmental pollutants. We report here Rhodococcus genes mediating oxygenation in the first step of o-xylene degradation. Rhodococcus opacus TKN14, isolated from soil contaminated with o-xylene, was able to utilize o-xylene as the sole carbon source and to metabolize it to o-methylbenzoic acid. A cosmid library from the genome of this strain was constructed in Escherichia coli. A bioconversion analysis revealed that a cosmid clone incorporating a 15-kb NotI fragment had the ability to convert o-xylene into o-methylbenzyl alcohol. The sequence analysis of this 15-kb region indicated the presence of a gene cluster significantly homologous to the naphthalene-inducible dioxygenase gene clusters (nidABCD) that had been isolated from Rhodococcus sp. strain I24. Complementation studies, using E. coli expressing various combinations of individual open reading frames, revealed that a gene (named nidE) for rubredoxin (Rd) and a novel gene (named nidF) encoding an auxiliary protein, which had no overall homology with any other proteins, were indispensable for the methyl oxidation reaction of o-xylene, in addition to the dioxygenase iron-sulfur protein genes (nidAB). Regardless of the presence of NidF, the enzyme composed of NidABE was found to function as a typical naphthalene dioxygenase for converting naphthalene and various (di)methylnaphthalenes into their corresponding cis-dihydrodiols. All the nidABEF genes were transcriptionally induced in R. opacus TKN14 by the addition of o-xylene to a mineral salt medium. It is very likely that these genes are involved in the degradation pathways of a wide range of aromatic hydrocarbons by Rhodococcus species as the first key enzyme

Structural Elucidation of Humulone Autoxidation Products and Analysis of Their Occurrence in Stored Hops

The transformation of α-acids [in hops (<i>Humulus lupulus</i> L.)] to iso-α-acids (in beer) during the brewing process is well known, but the occurrence and structure of the oxidized α-acids during hop storage are not well documented. Because an understanding of these oxidized compounds is essential to optimize the effects of oxidized hops on the quality of beer, we investigated the autoxidation products of humulone (a representative congener of α-acids) using a simplified autoxidation model. Among the oxidation products, tricyclooxyisohumulones A (<b>1</b>) and B (<b>2</b>), tricycloperoxyisohumulone A (<b>3</b>), deisopropyltricycloisohumulone (<b>4</b>), and the hemiacetal <b>5</b> of tricycloperoxyhumulone A (<b>5</b>′) were isolated, and their structures were elucidated for the first time. The occurrence of compounds <b>1</b>–<b>4</b> in stored hops was verified using LC/MS/MS analysis. We also monitored the levels of compounds <b>1</b>–<b>4</b> during hop storage using LC/MS/MS analysis