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.

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.

Analysis of reading frame and expressional regulation of randomly selected promoter-proximal genes in Escherichia coli.

The expression of seventy-seven randomly cloned genes of Escherichia coli was examined following a variety of treatments including heat shock, glucose starvation, phosphate starvation, ammonium starvation or osmotic shock, with the aid of lacZ reporter gene protein fusions on multicopy plasmids. Two of 77 genes (amr and yigL) had not previously been identified as protein encoding open-reading frames (ORFs) in annotations of the E. coli genome database. Thirteen genes exhibited significant changes in expression in response to at least one of the treatments, and six of them appeared to be controlled by more than one sigma (sigma) factor of RNA polymerase. This study thus allows us not only to identify the reading frame of the genomic genes but also to support the hypothesis earlier proposed that a significant proportion of genes in E. coli are involved in adaptations to various stresses to which the organism is likely to be exposed in the environment.

PCR detection of DNAs of animal origin in feed by primers based on sequences of short and long interspersed repetitive elements.

PCR primers for the detection of materials derived from ruminants, pigs, and chickens were newly designed on the basis of sequences of the Art2 short interspersed repetitive element (SINE), PRE-1 SINE, and CR1 long interspersed repetitive element (LINE), respectively. These primers amplified the SINE or LINE from total DNA extracted from the target animals and from test feed containing commercial meat and bone meal (MBM). With the primers, detection of Art2, PRE-1, or CR1 in test feed at concentrations of 0.01% MBM or less was possible. This method was suitable for the detection of microcontamination of feed by animal materials.