Pursuing the unlimited potential of microorganisms-progress and prospect of a fermentation company.

Production of pharmaceuticals and chemicals using microbial functions has bestowed numerous benefits onto society. The Nobel Prize awarded to Professor Ōmura, Distinguished Emeritus Professor of Kitasato University, showed the world the importance of the discovery and practical application of microorganisms. Now, increasing attention is turned toward the future path of this field.

Complete genome sequence of the motile actinomycete Actinoplanes missouriensis 431(T) (= NBRC 102363(T)).

Actinoplanes missouriensis Couch 1963 is a well-characterized member of the genus Actinoplanes, which is of morphological interest because its members typically produce sporangia containing motile spores. The sporangiospores are motile by means of flagella and exhibit chemotactic properties. It is of further interest that members of Actinoplanes are prolific sources of novel antibiotics, enzymes, and other bioactive compounds.

Insight into functional diversity of cytochrome P450 in the white-rot basidiomycete Phanerochaete chrysosporium: involvement of versatile monooxygenase.

To elucidate functional diversity of cytochrome P450 monooxygenases from the white-rot basidiomycete Phanerochaete chrysosporium (PcCYPs), we conducted a comprehensive functional screening using a wide variety of compounds. A functionomic survey resulted in characterization of novel PcCYP functions and discovery of versatile PcCYPs that exhibit broad substrate profiles. These results suggested that multifunctional properties of the versatile PcCYPs would play crucial roles in diversification of fungal metabolic systems involved in xenobiotic detoxification.

Construction of a novel expression vector in Pseudonocardia autotrophica and its application to efficient biotransformation of compactin to pravastatin, a specific HMG-CoA reductase inhibitor.

The novel plasmid vector (pTAOR4-Rev) suitable for gene expression in actinomycete strains of Pseudonocardia autotrophica was constructed from 2 P. autotrophica genetic elements, the novel replication origin and the acetone-inducible promoter. The replication origin was isolated from the endogenous plasmid of strain DSM 43082 and the acetone-inducible promoter was determined by analysis of the upstream region of an acetaldehyde dehydrogenase gene homologue in strain NBRC 12743.

Genome sequence of Kitasatospora setae NBRC 14216T: an evolutionary snapshot of the family Streptomycetaceae.

Kitasatospora setae NBRC 14216(T) (=KM-6054(T)) is known to produce setamycin (bafilomycin B1) possessing antitrichomonal activity. The genus Kitasatospora is morphologically similar to the genus Streptomyces, although they are distinguishable from each other on the basis of cell wall composition and the 16S rDNA sequence. We have determined the complete genome sequence of K.

Structural evidence for enhancement of sequential vitamin D3 hydroxylation activities by directed evolution of cytochrome P450 vitamin D3 hydroxylase.

Vitamin D(3) hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D(3) (VD(3)) to 1α,25-dihydroxyvitamin D(3) (1α,25(OH)(2)VD(3)) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD(3), i.

Metabolism of mono- and dichloro-dibenzo-p-dioxins by Phanerochaete chrysosporium cytochromes P450.

The white-rot fungus Phanerochaete chrysosporium possesses biodegradative capabilities of polychlorinated dibenzo-p-dioxins (PCDDs). One hundred twenty yeast clones expressing individual P450s of P. chrysosporum (PcCYPs), generated in our previous efforts, were screened for transformation of dioxin, and 40 positive clones were obtained.

Atypical kinetics of cytochromes P450 catalysing 3'-hydroxylation of flavone from the white-rot fungus Phanerochaete chrysosporium.

We cloned full-length cDNAs of 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium and successfully expressed 70 isoforms in Saccharomyces cerevisiae. To elucidate substrate specificity of P. chrysosporium P450s, we examined various substrates including steroid hormones, several drugs, flavonoids and polycyclic aromatic hydrocarbons using the recombinant S.

Directed evolution of the actinomycete cytochrome P450moxA (CYP105) for enhanced activity.

Actinomycete cytochrome P450 from Nonomuraea recticatena NBRC 14525 (P450moxA) catalyzes the hydroxylation of a broad range of substrates, including fatty acids, steroids, and various aromatic compounds. Hence, the enzyme is potentially useful in medicinal applications, but the activity is insufficient for practical use. Here we applied directed evolution to enhance the activity.

Comparison of two vectors for functional expression of a bacterial cytochrome P450 gene in Escherichia coli using CYP153 genes.

Two vectors, pT7NScamAB and pRED, have been used for the functional expression of bacterial class I cytochrome P450 (P450) genes in Escherichia coli, which utilize putidaredoxin reductase (CamA) and putidaredoxin (CamB), and the reductase domain of a self-sufficient P450RhF respectively, for electron transfer from NAD(P)H to a P450 protein. We here compared the efficiency of bioconversion with the two vectors towards n-octane, cyclohexane, n-butylbenzene, and 2-n-butylbenzofuran using two well-characterized CYP153A genes, aciA and CYP153A13a (P450balk). As for n-octane bioconversion, aciA and pT7camAB was the best combination for the production of 1-octanol and 1,8-octanediol.

Regioselective biooxidation of (+)-valencene by recombinant E. coli expressing CYP109B1 from Bacillus subtilis in a two-liquid-phase system.

Background: (+)-Nootkatone (4) is a high added-value compound found in grapefruit juice. Allylic oxidation of the sesquiterpene (+)-valencene (1) provides an attractive route to this sought-after flavoring. So far, chemical methods to produce (+)-nootkatone (4) from (+)-valencene (1) involve unsafe toxic compounds, whereas several biotechnological approaches applied yield large amounts of undesirable byproducts.

Enzymatic properties of cytochrome P450 catalyzing 3'-hydroxylation of naringenin from the white-rot fungus Phanerochaete chrysosporium.

We cloned full-length cDNAs of more than 130 cytochrome P450s (P450s) derived from Phanerochaete chrysosporium, and successfully expressed 70 isoforms using a co-expression system of P. chrysosporium P450 and yeast NADPH-P450 reductase in Saccharomyces cerevisiae. Of these P450s, a microsomal P450 designated as PcCYP65a2 consists of 626 amino acid residues with a molecular mass of 68.

Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica.

Vitamin D(3) (VD(3)) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD(3) into its physiologically active forms, namely, 25(OH)VD(3) or 1alpha,25(OH)(2)VD(3). In this study, we isolated and characterized Vdh (vitamin D(3) hydroxylase), which hydroxylates VD(3) from P.

Crystallization and preliminary X-ray diffraction studies of vitamin D3 hydroxylase, a novel cytochrome P450 isolated from Pseudonocardia autotrophica.

Vitamin D(3) hydroxylase (Vdh) is a novel cytochrome P450 monooxygenase isolated from the actinomycete Pseudonocardia autotrophica and consisting of 403 amino-acid residues. Vdh catalyzes the activation of vitamin D(3) via sequential hydroxylation reactions: these reactions involve the conversion of vitamin D(3) (cholecalciferol or VD3) to 25-hydroxyvitamin D(3) [25(OH)VD3] and the subsequent conversion of 25(OH)VD3 to 1alpha,25-dihydroxyvitamin D(3) [calciferol or 1alpha,25(OH)(2)VD3]. Overexpression of recombinant Vdh was carried out using a Rhodococcus erythropolis expression system and the protein was subsequently purified and crystallized.

Organization of the biosynthetic gene cluster for the polyketide antitumor macrolide, pladienolide, in Streptomyces platensis Mer-11107.

Pladienolides are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107. They show strong antitumor activity and are a potential lead in the search for novel antitumor agents. We sequenced the 65-kb region covering the biosynthetic gene cluster, and found four polyketide synthase genes (pldAI-pldAIV) composed of 11 modules, three genes involved in post-modifications (pldB-D), and a luxR-family regulatory gene (pldR).

Generation of new benanomicin analogues by biotransformation using Escherichia coli expressing actinomycete cytochrome P450.

Benanomicins were found as antifungal antibiotics from the culture of an actinomycete with potent antifungal activities in vitro and in vivo. We aimed to generate derivatives superior to benanomicin A by biotransformation using Escherichia coli constructed with bacterial P450 expression system. We found transformation of benanomicin A into two derivatives, 10-hydroxybenanomicin A and 11-O-demethylbenanomicin A by one of the P450-expressed strains which harbored a plasmid carrying a CYP105C1-homologous gene.

Increase in pladienolide D production rate using a Streptomyces strain overexpressing a cytochrome P450 gene.

Pladienolide B and its 16-hydroxylated derivative (pladienolide D) are novel 12-membered macrolides produced by Streptomyces platensis Mer-11107 showing strong in vitro and in vivo antitumor activity. While pladienolide B is mainly produced by this strain, pladienolide D is produced to a lesser extent. To facilitate the production of pladienolide D by biotransformation, we found that Streptomyces bungoensis A-1544 was able to hydroxylate pladienolide B at 16-position.

Crystal structure of cytochrome P450 MoxA from Nonomuraea recticatena (CYP105).

Cytochrome P450 MoxA (P450moxA) from a rare actinomycete Nonomuraea recticatena belongs to the CYP105 family and exhibits remarkably broad substrate specificity. Here, we demonstrate that P450moxA acts on several luciferin derivatives, which were originally identified as substrates of the human microsomal P450s. We also describe the crystal structure of P450moxA in substrate-free form.

Hydroxylation of oleanolic acid to queretaroic acid by cytochrome P450 from Nonomuraea recticatena.

A gene for cytochrome P450 (moxA) from Nonomuraea recticatena, coexpressed with camAB for pseudomonad redox partners in Escherichia coli, hydroxylated oleanolic acid to produce queretaroic acid. When we used the P450-induced whole-cell as a catalyst, only a small amount of queretaroic acid was produced, probably due to poor permeability of oleanolic acid into the E. coli cell.

Production of alpha, omega-alkanediols using Escherichia coli expressing a cytochrome P450 from Acinetobacter sp. OC4.

Our biotransformation using Escherichia coli expressing a cytochrome P450 (CYP) belonging to the CYP153A family from Acinetobacter sp. OC4 produced a great amount of 1-octanol (2,250 mg per liter) from n-octane after 24 h of incubation. This level of production is equivalent to the maximum level previously achieved in biotransformation experiments of alkanes.

Hydroxylation of testosterone by bacterial cytochromes P450 using the Escherichia coli expression system.

Two hundred thirteen cytochrome P450 (P450) genes were collected from bacteria and expressed based on an Escherichia coli expression system to test their hydroxylation ability to testosterone. Twenty-four P450s stereoselectively monohydroxylated testosterone at the 2alpha-, 2beta-, 6beta-, 7beta-, 11beta-, 12beta-, 15beta-, 16alpha-, and 17-positions (17-hydroxylation yields 17-ketoproduct). The hydroxylation site usage of the P450s is not the same as that of human P450s, while the 2alpha-, 2beta-, 6beta-, 11beta-, 15beta-, 16alpha-, and 17-hydroxylation are reactions common to both human and bacterial P450s.

The rpoZ gene, encoding the RNA polymerase omega subunit, is required for antibiotic production and morphological differentiation in Streptomyces kasugaensis.

The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.

Streptomyces serine protease (DHP-A) as a new biocatalyst capable of forming chiral intermediates of 1,4-dihydropyridine calcium antagonists.

Streptomyces viridosporus A-914 was screened as a producer of an enzyme to effectively form chiral intermediates of 1,4-dihydropyridine calcium antagonists. The supernatant liquid of the growing culture of this strain exhibited high activity for enantioselective hydrolysis of prochiral 1,4-dihydropyridine diesters to the corresponding (4R) half esters. The responsible enzyme (termed DHP-A) was purified to apparent homogeneity and characterized.