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 Omura, 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. As people involved in the microorganism industry, we will review the industrial activities thus far and consider the possible future developments in this field and its potential contribution to society.

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. Here, we describe the features of A. missouriensis 431(T), together with the complete genome sequence and annotation. The 8,773,466 bp genome contains 8,125 protein-coding and 79 RNA genes.

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. To our knowledge, this is the first report describing multifunctional properties of versatile P450s from the fungal kingdom. An increased compilation of PcCYP functions will facilitate a thorough understanding of metabolic diversity in basidiomycetes and provide new insights that could also expedite practical applications in the biotechnology sector.

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. P. autotrophica strains transformed with pTAOR4-P450, carrying a gene for cytochrome P450 monooxygenase, expressed P450 from the acetone-inducible promoter, as verified by SDS-PAGE and spectral analysis. The biotransformation test of acetone-induced resting cells prepared from a strain of P. autotrophica carrying pTAOR4 that harbors a compactin (CP)-hydroxylating P450 gene revealed 3.3-fold increased production of pravastatin (PV), a drug for hypercholesterolemia. Biotransformation of CP by the same strain in batch culture yielded PV accumulation of 14.3 g/l after 100 h. The expression vector pTAOR4-Rev and its function-enhancing derivatives provide a versatile approach to industrial biotransformation by Pseudonocardia strains, which can be good hosts for P450 monooxygenase expression.

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. setae NBRC 14216(T) as the first Streptomycetaceae genome other than Streptomyces. The genome is a single linear chromosome of 8,783,278 bp with terminal inverted repeats of 127,148 bp, predicted to encode 7569 protein-coding genes, 9 rRNA operons, 1 tmRNA and 74 tRNA genes. Although these features resemble those of Streptomyces, genome-wide comparison of orthologous genes between K. setae and Streptomyces revealed smaller extent of synteny. Multilocus phylogenetic analysis based on amino acid sequences unequivocally placed K. setae outside the Streptomyces genus. Although many of the genes related to morphological differentiation identified in Streptomyces were highly conserved in K. setae, there were some differences such as the apparent absence of the AmfS (SapB) class of surfactant protein and differences in the copy number and variation of paralogous components involved in cell wall synthesis.

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.e. the conversion of VD(3) to 25-hydroxyvitamin D(3) (25(OH)VD(3)) and then of 25(OH)VD(3) to 1α,25(OH)(2)VD(3), a hormonal form of VD(3). Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD(3)-bound, and 25(OH)VD(3)-bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substrate-bound structure of Vdh-K1 accommodates both VD(3) and 25(OH)VD(3) but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD(3) hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD(3) 25-hydroxylase and 25(OH)VD(3) 1α-hydroxylase activities.

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. Of these clones, six clones showed metabolism of 2-chloro-dibenzo-p-dioxin, and a microsomal PcCYP designated as PcCYP11a3 showed much higher activity than any other PcCYPs. The turnover numbers of hydroxylation activities of PcCYP11a3 toward 1-MCDD (58 min(-1)) and 2-MCDD (13 min(-1)) are more than 200 times higher than those of previously reported PcCYP65a2. In addition, PcCYP11a3 catalyzes hydroxylation of 2,3-dichlorodibenzo-p-dioxin. To our best knowledge, PcCYP11a3 has the highest activity toward PCDDs among the known CYPs derived from microorganisms. Although PcCYP11a3 showed no detectable activity toward 2,7-dichloro-dibenzop-dioxin and 2,3,7-trichloro-dibenzo-p-dioxin, PcCYP11a3 is promising as a template whose activity would be enhanced by site-directed mutagenesis.

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. cerevisiae cells. Of these P450s, two CYPs designated as PcCYP50c and PcCYP142c with 14% identity in their amino acid sequences catalyse 3'-hydroxylation of flavone and O-deethylation of 7-ethoxycoumarin. Kinetic data of both enzymes on both reactions fitted not to the Michaelis-Menten equation but to Hill's equation with a coefficient of 2, suggesting that two substrates bind to the active site. Molecular modelling of PcCYP50c and a docking study of flavone to its active site supported this hypothesis. The enzymatic properties of PcCYP50c and PcCYP142c resemble mammalian drug-metabolizing P450s, suggesting that their physiological roles are metabolism of xenobiotics. It is noted that these unique P. chrysosporium P450s have a potential for the production of useful flavonoids.

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. A random mutagenesis library was screened using 7-ethoxycoumarin as a substrate to retrieve 17 variants showing >2-fold activities. Twenty-five amino acid substitutions were found in the variants, of which five mutations were identified to have the largest effects (Q87W, T115A, H132L, R191W, and G294D). These mutations additively increased the activity; the quintet mutant had 20-times the activity of the wildtype. These five single mutations also increased in activity toward structurally distinct substrates (diclofenac and naringenin). Based on the three-dimensional structure of the enzyme, we discerned that mutations in the substrate recognition site improved the activity, which was substrate dependent; mutations apart from the active site improved the activity as well as the substrates did.

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. As for the bioconversion of cyclohexane, n-butylbenzene and 2-n-butylbenzofuran, CYP153A13a with pRED achieved the most efficient bioconversion, as compared by conversion ratio per active CYP153A protein content. It was also found that 2-n-butylbenzofuran is biotransformed into 4-benzofuran-2-yl-butyric acid via 4-benzofuran-2-yl-butan-1-ol with E. coli cells expressing CYP153A.

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. In the present work 125 cytochrome P450 enzymes from bacteria were tested for regioselective oxidation of (+)-valencene (1) at allylic C2-position to produce (+)-nootkatone (4) via cis- (2) or trans-nootkatol (3). The P450 activity was supported by the co-expression of putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida in Escherichia coli. RESULTS: Addressing the whole-cell system, the cytochrome CYP109B1 from Bacillus subtilis was found to catalyze the oxidation of (+)-valencene (1) yielding nootkatol (2 and 3) and (+)-nootkatone (4). However, when the in vivo biooxidation of (+)-valencene (1) with CYP109B1 was carried out in an aqueous milieu, a number of undesired multi-oxygenated products has also been observed accounting for approximately 35% of the total product. The formation of these byproducts was significantly reduced when aqueous-organic two-liquid-phase systems with four water immiscible organic solvents - isooctane, n-octane, dodecane or hexadecane - were set up, resulting in accumulation of nootkatol (2 and 3) and (+)-nootkatone (4) of up to 97% of the total product. The best productivity of 120 mg l-1 of desired products was achieved within 8 h in the system comprising 10% dodecane. CONCLUSION: This study demonstrates that the identification of new P450s capable of producing valuable compounds can basically be achieved by screening of recombinant P450 libraries. The biphasic reaction system described in this work presents an attractive way for the production of (+)-nootkatone (4), as it is safe and can easily be controlled and scaled up.

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 68.3kDa. Sequence alignment of PcCYP65a2 and human CYP1A2 revealed a unique structure of PcCYP65a2. Functional analysis of PcCYP65a2 using the recombinant S. cerevisiae cells demonstrated that this P450 catalyzes 3'-hydroxylation of naringenin to yield eriodictyol, which has various biological and pharmacological properties. In addition, the recombinant S. cerevisiae cells expressing PcCYP65a2 metabolized such polyaromatic compounds as dibenzo-p-dioxin (DD), 2-monochloroDD, biphenyl, and naphthalene. These results suggest that PcCYP65a2 is practically useful for both bioconversion and bioremediation.

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. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V(max) values for VD(3) 25-hydroxylation and 25(OH)VD(3) 1alpha-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD(3). We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD(3) 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD(3) into 25(OH)VD(3) was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD(3) by a single cytochrome P450.

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. Two different crystal forms were obtained by the hanging-drop vapour-diffusion method at 293 K using polyethylene glycol as a precipitant. The form I crystal belonged to the trigonal space group P3(1), with unit-cell parameters a = b = 61.7, c = 98.8 A. There is one Vdh molecule in the asymmetric unit, with a solvent content of 47.6%. The form II crystal was grown in the presence of 25(OH)VD3 and belonged to the orthorhombic system P2(1)2(1)2(1), with unit-cell parameters a = 63.4, b = 65.6 c = 102.2 A. There is one Vdh molecule in the asymmetric unit, with a solvent content of 46.7%. Native data sets were collected to resolutions of 1.75 and 3.05 A for form I and form II crystals, respectively, using synchrotron radiation. The structure solution was obtained by the molecular-replacement method and model refinement is in progress for the form I crystal.

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). The thioesterase domain of pldAIV was more dissimilar to that of polyketide synthase systems synthesizing 12/14-membered macrolide polyketides than to that of systems synthesizing other cyclic polyketides. The pldB gene was identified as a 6-hydroxylase belonging to a cytochrome P450 of the CYP107 family. This was clarified by a disruption experiment on pldB, in which the disruptant produced 6-dehydroxy pladienolide B. Two genes located downstream of pldB, designated pldC and pldD, are thought to be a probable genes for 7-O-acetylase and 18, 19-epoxydase respectively.

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. Unexpectedly, the biotransformed compounds showed weak antifungal activities in vitro compared with those of benanomicin A.

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. We identified psmA from S. bungoensis A-1544, which encoded a pladienolide B 16-hydroxylase PsmA belonging to the CYP105 family of cytochrome P450. To increase the efficiency of pladienolide D production, we constructed recombinant S. bungoensis A-1544 overexpressing psmA and performed biotransformation of pladienolide B to pladienolide D. This biotransformation achieved a production level 15-fold higher than that using the control strain S. bungoensis A-1544/pIJ702.

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. Structural comparison with various bacterial and human microsomal P450s reveals that the P450moxA structure is most closely related to that of the fungal nitric oxide reductase P450nor (CYP55A1). Final refined model of P450moxA comprises almost all the residues, including the "BC-loop" and "FG-loop" regions pivotal for substrate recognition, and the current structure thus defines a well-ordered substrate-binding pocket. Clear electron density map reveals that the MES molecule is bound to the substrate-binding site, and the sixth coordination position of the heme iron is not occupied by a water molecule, probably due to the presence of MES molecule in the vicinity of the heme. The unexpected binding of the MES molecule might reflect the ability of P450moxA to accommodate a broad range of structurally diverse compounds.

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. In an alternative approach with the cell-free reaction system, the conversion ratio increased up to 17%.

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. In addition, the initial production rate of 1-octanol was maintained throughout the entire incubation period. These results indicate that we have achieved the functional and stable expression of a CYP in E. coli for the first time. Further, our biotransformation system showed alpha,omega-diterminal oxidation activity of n-alkanes, and a large amount of 1,8-octanediol (722 mg per liter) was produced from 1-octanol after 24 h of incubation. This is the first report on the bioproduction of alpha,omega-alkanediols from n-alkanes or 1-alkanols.