Expression of the autofluorescent protein, DsRed2, in the recombinants of the ectomycorrhizal basidiomycete, Suillus grevillei, generated by Agrobacterium-mediated transformation

Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5′ gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.     

Transformation of the white-rot basidiomycete Coriolus hirsutus using the ornithine carbamoyltransferase gene

An efficient transformation system for the basidiomycete Coriolus hirsutus was developed. A double-auxotrophic mutant of C. hirsutus, deficient both in ornithine carbamoyltransferase (OCTase) and 3-isopropylmalate dehydrogenase (3-IPM dehydrogenase), was transformed to Arg+ with each allelic type of the C. hirsutus genomic OCTase gene (arg1) newly cloned. The transformation frequency of 10(3)-10(4) transformants per mug DNA per 10(6)-10(7) oidial protoplasts was reached. Southern blots showed that the transforming DNA was integrated into chromosomal DNA with multi-copies. The Arg+ phenotype of the transformants was stably inherited through mitosis.     

Genetic introduction of foreign genes to <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> by restriction enzyme-mediated integration

Pleurotus eryngii was transformed via restriction enzyme-mediated integration. In order to construct the transformation plasmid, the enhanced cyan fluorescent protein (ECFP) gene was ligated next to the gpd promoter of the plasmid pAN7-1. Transformation was facilitated via the heat treatment of a transformation mixture containing 1 μg of the HindIII-digested plasmid DNA and 10⁶ mushroom protoplasts in 40% polyethyleneglycol solution, resulting in 10–40 hygromycin-resistant transformants. Successful transformation was evidenced by PCR, Southern blot, and confocal fluorescence microscopic analyses on the selected transformants. To date, this is the first report on the transformation of P. eryngii by REMI technique.     

Comparison of microbial hosts and expression systems for mammalian CYP1A1 catalysis

Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis. In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U g_CDW⁻¹, respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol g_CDW⁻¹). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical production, but are sufficient for preparative-scale drug metabolite synthesis.     

A highly conserved mycobacterial cholesterol catabolic pathway

Degradation of the cholesterol side‐chain in Mycobacterium tuberculosis is initiated by two cytochromes P450, CYP125A1 and CYP142A1, that sequentially oxidize C26 to the alcohol, aldehyde and acid metabolites. Here we report characterization of the homologous enzymes CYP125A3 and CYP142A2 from Mycobacterium smegmatis mc² 155. Heterologously expressed, purified CYP125A3 and CYP142A2 bound cholesterol, 4‐cholesten‐3‐one, and antifungal azole drugs. CYP125A3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4‐cholesten‐3‐one to the C‐26 alcohol and subsequently to the acid. The X‐ray structures of both substrate‐free CYP125A3 and CYP142A2 and of cholest‐4‐en‐3‐one‐bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M. tuberculosis proteins. Deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3. In contrast to M. tuberculosis, the M. smegmatis Δcyp125Δcyp142 double mutant retains its ability to grow on cholesterol albeit with a diminished capacity, indicating an additional level of redundancy within its genome.     

Interruption of glycerol pathway in industrial alcoholic yeasts to improve the ethanol production

The two homologous genes GPD1 and GPD2, encoding two isoenzymes of NAD⁺-dependent glycerol-3-phosphate dehydrogenase in industrial yeast Saccharomyces cerevisiae CICIMY0086, had been deleted. The obtained two kinds of mutants gpd1Δ and gpd2Δ were studied under alcoholic fermentation conditions. gpd1Δ mutants exhibited a 4.29% (relative to the amount of substrate consumed) decrease in glycerol production and 6.83% (relative to the amount of substrate consumed) increased ethanol yield while gpd2Δ mutants exhibited a 7.95% (relative to the amount of substrate consumed) decrease in glycerol production and 7.41% (relative to the amount of substrate consumed) increased ethanol yield compared with the parental strain. The growth rate of the two mutants were slightly lower than that of the wild type under the exponential phase whereas ANG1 (gpd1Δ) and the decrease in glycerol production was not accompanied by any decline in the protein content of the strain ANG1 (gpd1Δ) but a slight decrease in the strain ANG2 (gpd2Δ). Meanwhile, dramatic decrease of acetate acid formation was observed in strain ANG1 (gpd1Δ) and ANG2 (gpd2Δ) compared to the parental strain. Therefore, it is possible to improve the ethanol yield by interruption of glycerol pathway in industrial alcoholic yeast.     

Evaluation of agrobacterium-mediated transformation of agricus bisporus using a range of promoters linked to hygromycin resistance

There is interest in establishing genetic modification technologies for the cultivated mushroom Agaricus bisporus, both for improved crop characteristics and for molecular pharming. For these methods to be successful, it is necessary to establish a set of transformation systems that include robust and reliable vectors for gene manipulation. In this article, we report the evaluation of a series of promoters for driving expression of the Escherichia coli hph gene encoding hygromycin phosphotransferase. This was achieved using the Aspergillus nidulans gpdA and the A. bisporus gpdII and trip2 promoters. The Coprinus cinereus β-tubulin promoter gave contrasting results depending on the size of promoter used, with a 393-bp region being effective, whereas the longer 453-bp fragment failed to yield any hygromycin-resistant transformants. The C. cinereus trp 1 and the A. bisporus lcc1 promoters both failed to yield transformants. We also show that transformation efficiency may be improved by careful selection of both appropriate Agrobacterium strains, with ALG-1 yielding more than LBA1126 and by the choice of the binary vectors used to mobilize the DNA, with pCAMBIA vectors appearing to be more efficient than either pBIN19- or pGREEN-based systems.     

Analysis of a genetically unstable region inStreptomyces lividans 66-TK64

Genetic and molecular analyses of an unstable region encompassing the gene loci cml arg and a 5.7 kb amplifiable unit of DNA were done. Spontaneous mutants from Cm1^R →Cml^S and the revertants from Cml^S →Cml^R were analysed for mutations at arg locus and amplification of amplifiable unit of DNA. Twenty-one revertants were analysed. Two of these had large-scale amplification and one of these was also Arg^-. Nine of the revertants which were Arg⁺ had low-level or intermediate-level amplification of the 5.7 kb DNA sequence but no deletions of the flanking sequences were detected. Five of the CmI^R’ revertants, which were also Arg⁺, had lost one of the two copies from the doublet of amplifiable unit of DNA. The remaining five revertants did not show any other change. The amplifiable unit of DNA, therefore, not only undergoes amplification but can also suffer specific deletion of one copy. Thus, this region as a whole is characterized by instability and the events appear to take place at more than one locus concomitantly with a high frequency.     

Muscle Contraction : (Outline Studies in Biology) by C.R. Bagshaw Chapman & Hall; London,New York, 1982 79 pages. £2.75

On incubation of B. subtilis RM125(arg15 leuA8 r_M^? m_M^?) with DNA from alkalophilic Bacillus, the transformants (Arg⁺Leu^? or Leu^?Arg⁺) appeared at pH 10. The transformants were able to grow even at pH 7. Alkalophilic Bacillus was resistant to bacteriophages π105D1C2·1012 grown on B. subtilis 1012(r^-m_M⁺) and π105D1C2·ISMR4 grown on B. subtilis ISMR4r_M⁺r_R⁺m_M⁺m_R⁺), but the recipient B. subtilis and the transformant(Arg⁺Leu^?) were susceptible to both the of the bacteriophages. The results indicate that the transformant is a B. subtilis derivative and that alkalophilicity of alkalophilic Bacillus was transferred to B. subtilis.     

Promotion of Monacolin K Production by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-Mediated Transformation in <Emphasis Type="Italic">Monascus albidus</Emphasis> 9901

The binary vector pCAMBIA3300-gpdA-hph-trpC with hygromycin B phosphotransferase (hph) was constructed and transformed into Monascus albidus 9901 by Agrobacterium tumefaciens-mediated transformation, with gene hph as the selective marker. In order to improve the efficiency of A. tumefaciens-mediated transformation in M. albidus 9901, we optimized various factors including concentration of M. albidus 9901 spores, cell density of A. tumefaciens, co-cultivation time, temperature, and acetosyringone concentration. Most transformants of M. albidus 9901 could grow stably on media containing 50 μg ml⁻¹ hygromycin B up to five generations. The presence of hph was identified by PCR. Two transformants H1 and H2 which produced more Monacolin K than M. albidus 9901 were screened, and the concentration of Monacolin K in the fermented millet by H1 and H2 increased by 42.15% and 40.34% respectively compared with that produced by M. albidus 9901.     

Cytochrome P450 125 (CYP125) catalyses C26‐hydroxylation to initiate sterol side‐chain degradation in Rhodococcus jostii RHA1

The cyp125 gene of Rhodococcus jostii RHA1 was previously found to be highly upregulated during growth on cholesterol and the orthologue in Mycobacterium tuberculosis (rv3545c) has been implicated in pathogenesis. Here we show that cyp125 is essential for R. jostii RHA1 to grow on 3‐hydroxysterols such as cholesterol, but not on 3‐oxo sterol derivatives, and that CYP125 performs an obligate first step in cholesterol degradation. The involvement of cyp125 in sterol side‐chain degradation was confirmed by disrupting the homologous gene in Rhodococcus rhodochrous RG32, a strain that selectively degrades the cholesterol side‐chain. The RG32Ωcyp125 mutant failed to transform the side‐chain of cholesterol, but degraded that of 5‐cholestene‐26‐oic acid‐3β‐ol, a cholesterol catabolite. Spectral analysis revealed that while purified ferric CYP125_RHA1 was < 10% in the low‐spin state, cholesterol (K_D^app = 0.20 ± 0.08 μM), 5α‐cholestanol (K_D^app = 0.15 ± 0.03 μM) and 4‐cholestene‐3‐one (K_D^app = 0.20 ± 0.03 μM) further reduced the low spin character of the haem iron consistent with substrate binding. Our data indicate that CYP125 is involved in steroid C26‐carboxylic acid formation, catalysing the oxidation of C26 either to the corresponding carboxylic acid or to an intermediate state.     

Molecular characterization of cytochrome P450 genes in the polycyclic aromatic hydrocarbon degrading Mycobacterium vanbaalenii PYR-1

Mycobacterium vanbaalenii PYR-1 has the ability to degrade low- and high-molecular-weight polycyclic aromatic hydrocarbons (PAHs). In addition to dioxygenases, cytochrome P450 monooxygenases have been implicated in PAH degradation. Three cytochrome P450 genes, cyp151 (pipA), cyp150, and cyp51, were detected and amplified by polymerase chain reaction from M. vanbaalenii PYR-1. The complete sequence of these genes was determined. The translated putative proteins were ≥80% identical to other GenBank-listed mycobacterial CYP151, CYP150, and CYP51. Genes pipA and cyp150 were cloned, and the proteins partially expressed in Escherchia coli as soluble heme-containing cytochrome P450s that exhibited a characteristic peak at 450 nm in reduced carbon monoxide difference spectra. Monooxygenation metabolites of pyrene, dibenzothiophene, and 7-methylbenz[α]anthracene were detected in whole cell biotransformations, with E. coli expressing pipA or cyp150 when analyzed by gas chromatography/mass spectrometry. The cytochrome P450 inhibitor metyrapone strongly inhibited the S-oxidation of dibenzothiophene. Thirteen other Mycobacterium strains were screened for the presence of pipA, cyp150, and cyp51 genes, as well as the initial PAH dioxygenase (nidA and nidB). The results indicated that many of the Mycobacterium spp. surveyed contain both monooxygenases and dioxygenases to degrade PAHs. Our results provide further evidence for the diverse enzymatic capability of Mycobacterium spp. to metabolize polycylic aromatic hydrocarbons.An erratum to this article can be found at     

A cytochrome P450 class I electron transfer system from Novosphingobium aromaticivorans

Cytochrome P450 (CYP) enzymes of the CYP101 and CYP111 families from Novosphingobium aromaticivorans are heme monooxygenases that catalyze the hydroxylation of a range of terpenoid compounds. CYP101D1 and CYP101D2 oxidized camphor to 5-exo-hydroxycamphor. CYP101B1 and CYP101C1 oxidized β-ionone to predominantly 3-R-hydroxy-β-ionone and 4-hydroxy-β-ionone, respectively. CYP111A2 oxidized linalool to 8-hydroxylinalool. Physiologically, these CYP enzymes could receive electrons from Arx, a [2Fe-2S] ferredoxin equivalent to putidaredoxin from the CYP101A1 system from Pseudomonas putida. A putative ferredoxin reductase (ArR) in the N. aromaticivorans genome, with high amino acid sequence homology to putidaredoxin reductase, has been over-produced in Escherichia coli and found to support substrate oxidation by these CYP enzymes via Arx with both high activity and coupling of product formation to NADH consumption. The ArR/Arx electron-transport chain has been co-expressed with the CYP enzymes in an E. coli host to provide in vivo whole-cell substrate oxidation systems that could produce up to 6.0 g L⁻¹ of 5-exo-hydroxycamphor at rates of up to 64 μM (gram of cell dry weight)⁻¹ min⁻¹. These efficient biocatalytic systems have potential uses in preparative scale whole-cell biotransformations.     

Characterization of bacterial β-carotene 3,3′-hydroxylases, CrtZ, and P450 in astaxanthin biosynthetic pathway and adonirubin production by gene combination in Escherichia coli

β-Carotene hydroxylase (CrtZ) is one of rate-limiting enzymes for astaxanthin production. A complementation analysis was conducted using Escherichia coli transformants to compare the catalytic efficiency of bacterial CrtZ from Brevundimonas sp. SD212, Paracoccus sp. PC1 (formerly known as Alcaligenes sp. PC-1), Paracoccus sp. N81106 (Agrobacterium aurantiacum), Pantoea ananatis (Erwinia uredovora 20D3), marine bacterium P99-3, and P450 monooxygenase (CYP175A1) from Thermus thermophilus HB27. Each crtZ or CYP175A1 gene was expressed in E. coli transformants synthesizing canthaxanthin and β-carotene due to the respective presence of plasmids pAC-Cantha and pACCAR16ΔcrtX. The carotenoids that accumulated in the resulting recombinant E. coli cells were examined by chromatographic and spectroscopic analyses. E. coli carrying Brevundimonas sp. SD212 crtZ showed the highest astaxanthin production efficiency among the transformants examined, while there was no significant difference in the catalytic efficiency for conversion from β-carotene to zeaxanthin. Recombinant E. coli expressing the CYP175A1 gene, in addition to the genes for canthaxanthin synthesis, surprisingly accumulated adonirubin (phoenicoxanthin) as the main product, although the other recombinant E. coli did not accumulate any adonirubin. The present results suggest that the Brevundimonas sp. SD212 crtZ and T. thermophilus HB27 CYP175A1 genes could, respectively, be used for the efficient production of astaxanthin and adonirubin in heterologous hosts.     

The spliceophilin CYP18-2 is mainly involved in the splicing of retained introns under heat stress in Arabidopsis

Peptidyl-prolyl isomerase-like 1(PPIL1) is associated with the human spliceosome complex. However, its function in pre-mRNA splicing remains unclear. In this study, we show that Arabidopsis thaliana CYCLOPHILIN 18-2(AtCYP18-2), a PPIL1 homolog,plays an essential role in heat tolerance by regulating pre-mRNA splicing. Under heat stress conditions,AtCYP18-2 expression was upregulated in mature plants and GFP-tagged AtCYP18-2 redistributed to nuclear and cytoplasmic puncta. We determined that AtCYP...     

Biotransformation of dichloro-, trichloro-, and tetrachlorodibenzo-p-dioxin by the white-rot fungus Phlebia lindtneri

The model polychlorinated dibenzo-p-dioxins (PCDDs) 2,7-dichloro-, 2,3,7-trichloro, 1,2,6,7-, 1,2,8,9-, and 1,3,6,8-tetrachlorodibenzo-p-dioxin were used as substrates for a degradation experiment with the white-rot fungus Phlebia lindtneri. 2,7-Dichlorodibenzo-p-dioxin (2,7-diCDD) was biotransformed to hydroxylated diCDD and methoxylated diCDD. With the exception of 1,3,6,8-tetrachlorodibenzo-p-dioxin, the tri- and tetrachlorodibenzo-p-dioxins were biotransformed to hydroxyl and methoxyl compounds by P. lindtneri. The degradation rate of 1,2,6,7-tetrachlorodibenzo-p-dioxin was higher than that of 2,3,7-trichlorodibenzo-p-dioxin and no degradation of 1,3,6,8-tetrachlorodibenzo-p-dioxin was observed. These results indicate that the degradation of these PCDDs depends on the chlorination patterns of the substrates. This is the first report of the hydroxylation and methoxylation of tri- to tetra-CDDs by a fungal strain.