Cloning and heterologous expression of P450Lent4B11, a novel bacterial P450 gene, for hydroxylation of an antifungal agent sordaricin.

Microbial transformation is known to be one of promising options to add functional groups such as a hydroxyl moiety to active base compounds to generate their derivatives. Sordaricin, a diterpene aglycone of the natural product sordarin, is an antifungal agent to selectively inhibit fungal protein synthesis by stabilizing the ribosome/EF-2 (elongation factor 2) complex. We screened actinomycetes to catalyze hydroxylation of sordaricin on the basis that the hydroxyl moiety would make it easier to generate derivatives of sordaricin. As a result of the screening, 6-hydroxylation of sordaricin was found to be catalyzed by Lentzea sp. 7887. We found that the cytochrome P450 inhibitor metyrapone inhibited this reaction, suggesting that a cytochrome P450 may be responsible for the biotransformation. As a next step, we cloned multiple cytochrome P450 genes, one of which were named P450Lent4B11, using degenerate PCR primers. The expressed cytochrome P450 derived from the P450Lent4B11 gene provided a different absorbance spectrum pattern from original one when it was incubated with sordaricin. Moreover, in cell-free conditions, the corresponding cytochrome P450 displayed the 6-hydroxylation activity toward sordaricin. Taken together, these results indicate that P450Lent4B11, derived from Lentzea sp. 7887, should be responsible for catalyzing 6-hydroxylation of sordaricin.

Enhanced oxidative activities of cytochrome P450Rhf from Rhodococcus sp. expressed using the hyper-inducible expression system.

Bacterial cytochrome P450 enzymes catalyze the oxidative biotransformation of various types of compounds. Although the functional expression of Actinomycetes P450 in a closely related heterologous host can serve as a useful biocatalyst in whole-cell biotransformation assays, the co-expression of an electron transfer partner is required. To overcome this limitation, P450Rhf from Rhodococcus sp. NCIMB 9784 is an ideal candidate, because it is fused to a reductase domain at the C terminus and does not require an electron transfer partner. Here, we cloned P450Rhf into the hyper-inducible expression vector pSH19 in Streptomyces lividans TK24 for developing an efficient whole-cell biotransformation system with bacterial P450. The recombinant strain displayed high conversion activity (79.1%) of 7-ethoxycoumarin to 7-hydroxycoumarin after 48 h, and the observed activity was markedly higher than those for 7-methoxycoumarin and 7-propoxycoumarin used as substrates. We next screened several commercially available substrates possessing an ethyl phenyl ether moiety, which is also present in 7-ethoxycoumarin, and found that 4'-ethoxy-2'-hydroxyacetphenone was almost completely dealkylated (95.0%), and that 7-ethoxy-4-methylcoumarin was converted to two products, 7-hydroxy-4-methylcoumarin and 7-ethoxy-4-hydroxymethyl-coumarin. Our research suggests that enhancement of heterologous P450 expression using the pSH19 system in whole-cell biotransformation assays is valuable for identifying novel substrates of P450, as well as for obtaining high yields of conversion products.

Cloning and heterologous expression of P450Um-1, a novel bacterial P450 gene, for hydroxylation of immunosuppressive agent AS1387392.

Biotransformation technology involving enzymatic modification of original substrates by organisms such as microbes is a valuable tool in improving pharmacokinetics or physicochemical properties of the base compounds. The fungal metabolite AS1387392 is a histone deacetylase inhibitor with potential as a therapeutic immunosuppressant. However, its paucity of functional groups, essential to synthesizing derivatives, is a drawback. Amycolatopsis azurea JCM-3275 catalyzed hydroxylation of AS1387392 to AS1429716, which may facilitate the synthesis of more derivatives by the additional hydroxyl moiety present in AS1429716. This reaction was inhibited by cytochrome P450 inhibitor metyrapone, indicating that cytochrome P450 may be responsible for the transformation. Degenerate PCR primers were subsequently constructed and used to clone genes encoding cytochrome P450 from the genomic DNA of A. azurea JCM-3275. We cloned an entire novel P450 gene (1209 bp) and named it P450Um-1. Its deduced amino acid sequence was homologous with that of the CYP105 subfamily. Further cloning of the upstream region, which may contain the native promoter site, was followed by insertion of the open reading frame with the upstream area into Streptomycetes high copy vector pIJ702, giving the expression plasmid pNUm-1. P450Um-1 was specifically expressed in Streptomyces lividans TK24, and this recombinant strain converted AS1387392 to AS1429716 without any redox partners. These results show that P450Um-1, a novel bacterial P450, catalyzed hydroxylation of AS1387392 to AS1429716. This resultant recombinant strain is expected to be an efficient biocatalyst with application to more suitable redox systems than those tested here.

Discovery of anti-varicella zoster virus activity of polyether antibiotic CP-44161.

In search for new anti-varicella zoster virus (VZV) compounds with new mechanism of action, we applied a DNA hybridization assay (dot blot method) for screening. Using this method, we screened microbial products and found the polyether compound CP-44161 from the culture broth of an actinomycete strain. CP-44161 was previously reported as an anticoccidal agent, but there has been no claim of its antiviral activities. CP-44161 showed strong anti-VZV activity against pOka strain by plaque reduction assay. Moreover, CP-44161 showed lower cytotoxicity than other antiviral polyethers, such as monensin and nigericin. Its better safety margin and strong anti-VZV properties make it a good candidate for a new anti-VZV agent.

Anti-herpes virus activity of polyether antibiotic CP-44161 in vivo.

In the previous study, we discovered a polyether antibiotic CP-44161, which was reported earlier as an anticoccidal agent, as an anti-varicella zoster virus compound. In this study, we demonstrated that CP-44161 had a very strong and broad anti-herpes virus activities against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro. To determine the antiviral activity of CP-44161 in vivo, we examined its effect on the cutaneous HSV-2 infection model in Balb/c mice. CP-44161 showed inhibitory effect on lesion development as well as acyclovir (ACV) when the treatment was started from day 3. Meanwhile, in case the start of treatment was delayed until day 4, when ACV was no longer effective, the effectiveness of CP-44161 still remained. In this model, CP-44161 also showed inhibitory effect on the proliferation of HSV-2 DNA in dorsal root ganglia. This is the first article to report that polyether antibiotics can be effective on viral infection in vivo.

Oxidative activities of heterologously expressed CYP107B1 and CYP105D1 in whole-cell biotransformation using Streptomyces lividans TK24.

Cytochrome P450 enzymes are a major class of biocatalysts related to the oxidative metabolism of many drugs, assisted by electron transfer partners. The functional expression of the P450 gene in a heterologous host will lead to efficient biotransformation and biodegradation, which are useful in pharmaceutical improvement or environmental cleanup. The soluble cytochrome P450 monooxygenase systems CYP105D1 and CYP107B1 involved in the biotransformation of some xenobiotics, such as secondary metabolites or environmental pollutants, were expressed in Streptomyces lividans TK24 with the Streptomyces expression vector pIJ6021. In whole-cell biotransformation assay using these recombinant strains, the oxidative dealkylation of 7-ethoxycoumarin was detected without any foreign redox partners in the case of CYP107B1, while the activity of CYP105D1 was not monitored until this gene was coexpressed with the ferredoxin gene located downstream of the CYP105D1 gene, and the ferredoxin reductase gene SCF 15.02 from Streptomyces coelicolor A3(II). This result suggests that CYP107B1 is capable of utilizing an endogenous electron transfer partner from the host but not CYP105D1, and that CYP105D1 is complemented by some redox partner imported from closely related strains.

Suppressive effect of antibiotic siomycin on antibody production.

The antibiotic thiazole compound siomycin, which we have found from the culture broth of Actinomycetes (strain No.806097) in search of antibody production inhibitor, showed the in vitro immunosuppressive property against B-cells stimulated with T-cell independent antigen DNP-LPS (dinitrophenyl-lipopolysaccharide) while it also showed inhibitory effect against T-cell proliferation. Its inhibitory mechanism was considered to be different from that of FK506, the representative of T-cell immunosuppressant. Moreover, siomycin showed inhibitory effect in both T-cell dependent and independent murine antibody production models and decreased the severity in murine collagen arthritis model. Therefore, siomycin is a unique immunosuppressant which has potential for the treatment of some antibody-mediated diseases.