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1.
Yuto Yamada Jianqiao Wang Hirokazu Kawagishi 《Bioscience, biotechnology, and biochemistry》2013,77(12):2128-2133
Glyoxal oxidase (GLOX) is a source of the extracellular H2O2 required for the oxidation reactions catalyzed by the ligninolytic peroxidases. In the present study, the GLOX-encoding gene (glx) of Phanerochaete chrysosporium was cloned, and bee2 promoter of P. sordida YK-624 was used to drive the expression of glx. The expression plasmid was transformed into a P. sordida YK-624 uracil auxotrophic mutant (strain UV-64), and 16 clones were obtained as GLOX-introducing transformants. These transformants showed higher GLOX activities than wild-type P. sordida YK-624 and control transformants harboring marker plasmid. RT-PCR analysis indicated that the increased GLOX activity was associated with elevated recombinant glx expression. Moreover, these transformants showed higher ligninolytic activity than control transformants. These results suggest that the ligninolytic properties of white-rot fungi can be improved by recombinant expression of glx. 相似文献
2.
Hirofumi Hirai Kenta Misumi Tomohiro Suzuki Hirokazu Kawagishi 《Current microbiology》2013,67(6):708-711
The manganese peroxidase (MnP) gene (mnp4) promoter of Phanerochaete sordida YK-624 was used to drive expression of 5-aminolevulinic acid synthase (als), which is a key heme biosynthesis enzyme. The expression plasmid pMnP4pro-als was transformed into P. sordida YK-624 uracil auxotrophic mutant UV-64, and 14 recombinant als expressing-transformants were generated. Average cumulative MnP activities in the transformants were 1.18-fold higher than that of control transformants. In particular, transformants A-14 and A-61 showed significantly higher MnP activity (approximately 2.8-fold) than wild type. RT-PCR analysis indicated that the increased MnP activity was caused by elevated recombinant als expression. These results suggest that the production of MnP is improved by high expression of als. 相似文献
3.
Enhancement of biodegradation of 2,7-dichlorodibenzo-p-dioxin by addition of fungal culture filtrate
Sato Akira Watanabe Tsuneo Watanabe Yoshio Kurane Ryuichiro 《World journal of microbiology & biotechnology》2003,19(4):439-441
The hydrophilicity of 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD), a model dioxin compound, increased when incubated with the culture filtrates of several strains of fungi. The possibility that the addition of these filtrates could enhance the biodegradation of 2,7-DCDD by the white-rot basidiomycetous fungus Phanerochaete sordida YK-624 was examined. The decrease of 2,7-DCDD after 3 weeks incubation in a YK-624 culture containing these filtrates was greater (30%) than that in the culture of YK-624 alone (15%). This is the first report describing the enhancement of dioxin decrease by the addition of a fungal filtrate. 相似文献
4.
The non-steroidal anti-inflammatory drugs diclofenac (DCF) and mefenamic acid (MFA) were treated with the white rot fungus
Phanerochaete sordida YK-624. DCF completely disappeared and MFA decreased by about 90% after 6 days of treatment. It was also confirmed that the
fungus almost completely removed the acute lethal toxicity of DCF and MFA towards the freshwater crustacean Thamnocephalus platyurus after 6 days of treatment. Mass spectrometric and 1H nuclear magnetic resonance analyses demonstrated that two mono-hydroxylated DCFs (4′-hydroxydiclofenac and 5-hydroxydiclofenac)
and one di-hydroxylated DCF (4′,5-dihydroxydiclofenac) were formed via fungal transformation. The four metabolites of MFA
were identified as 3′-hydroxymethylmefenamic acid (mono-hydroxylated MFA), 3′-hydroxymethyl-5-hydroxymefenamic acid (di-hydroxylated
MFA), 3′-hydroxymethyl-6′-hydroxymefenamic acid (di-hydroxylated MFA) and 3′-carboxymefenamic acid. These results suggest
that hydroxylation catalyzed by cytochrome P450 (CYP) in P. sordida YK-624 may be involved in the elimination and detoxification of DCF and MFA. This notion was further supported by the fact
that smaller decreases in DCF and MFA were observed in cultures of P. sordida YK-624 incubated with 1-aminobenzotriazole, a known inhibitor of CYP. 相似文献
5.
Biotransformation of acetamiprid by the white-rot fungus Phanerochaete sordida YK-624 总被引:1,自引:0,他引:1
Acetamiprid (ACE) belongs to the neonicotinoid class of systemic broad-spectrum insecticides, which are the most highly effective
and largest-selling insecticides worldwide for crop protection. As neonicotinoid insecticides persist in crops, biotransformation
of these insecticides represents a promising approach for improving the safety of foods. Here, the elimination of ACE from
a liquid medium by the white-rot fungus Phanerochaete sordida YK-624 was examined. Under ligninolytic and non-ligninolytic conditions, 45% and 30% of ACE were eliminated, respectively,
after 15 days of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses
of a metabolite identified in the culture supernatant suggested that ACE was N-demethylated to (E)-N
1-[(6-chloro-3-pyridyl)-methyl]-N
2-cyano-acetamidine, which has a much lower toxicity than ACE. In addition, we investigated the effect of the cytochrome P450
inhibitor piperonyl butoxide (PB) on the elimination of ACE. The elimination rate of ACE by P. sordida YK-624 was markedly reduced by the addition of either 0.01 or 0.1 mM PB to the culture medium. These results suggest that
cytochrome P450 plays an important role in the N-demethylation of ACE by P. sordida YK-624. 相似文献
6.
Abida Yasmeen Bushra Mirza Samia Inayatullah Naila Safdar Maryam Jamil Shawkat Ali M. Fayyaz Choudhry 《Plant Molecular Biology Reporter》2009,27(1):20-28
In this study, in planta transformation of tomato (Solanum lycopersicum L.), using fruit injection and floral dip, is reported. Agrobacterium tumefaciens strain EHA 105 containing one of three constructs, i.e., pROKIIAP1GUSint (carrying the Apetala 1 [AP1] gene), pROKIILFYGUSint (carrying the LEAFY [LFY] gene), or p35SGUSint (carrying the β-glucuronidase [GUS] gene), was used for plant transformation. For fruit injection transformation, no significant effects (p > 0.05) of the construct used were observed. The highest frequency of transformation was obtained following 48-h incubation
of tomato fruit with bacterial cells harboring either one of the three constructs; transformation frequencies of 17%, 19%,
and 21% for AP1, LFY, and GUS gene constructs, respectively, were obtained. When fruit maturity was evaluated in fruit injection experiments, mature red
fruit resulted in higher frequency of transformants than immature green fruit with 40%, 35%, and 42% for AP1, LFY, and GUS gene constructs, respectively. For floral dip transformation, a higher number of transformants was obtained when the GUS gene construct was used instead of either the AP1 or LFY gene construct, thus suggesting a possible inhibitory effect of the flowering genes used. When flowers were transformed prior
to rather than following pollination, they yielded a higher transformation frequency, 12% for the LFY construct and 23% for the GUS construct (p < 0.05), although no transformant was obtained with the AP1 gene construct. All putative GUS-positive transformants were analyzed using polymerase chain reaction and confirmed for the
presence of the transgene. Compared to control plants, transgenic plants carrying either the AP1 or LFY transgene flowered earlier and showed several different morphological characters. 相似文献
7.
Hiroyuki Uehara Bok Du Choi Enoch Y. Park Mitsuyasu Okabe 《Biotechnology and Bioprocess Engineering》2000,5(1):7-12
The expression of the mouse α-amylase gene in the methylotrophic yeast,P. pastoris was investigated. The mouse α-amylase gene was inserted into the multi-cloning site of a Pichia expression vector, pPIC9,
yielding a new expression vector pME624. The plasmid pME624 was digested withSalI orBglII, and was introduced intoP. pastoris strain GS115 by the PEG1000 method. Fifty-three transformants were obtained by the transplacement of pME624 digested withSalI orBglII into theHIS
4 locus (38 of Mut+ clone) or into theAOX1 locus (45 of Muts clone). Southern blot was carried out in 11 transformants, which showed that the mouse α-amylase gene was integrated into
thePichia chromosome. When the second screening was performed in shaker culture, transformant G2 showed the highest α-amylase activity,
290 units/ml after 3-day culture, among 53 transformants. When this expression level of the mouse α-amylase gene is compared
with that in recombinantSaccharomyces cerevisiae harboring a plasmid encoding the same mouse α-amylase gene, the specific enzyme activity is eight fold higher than that of
the recombinantS. cerevisiae. 相似文献
8.
P. Lu B. P. Davis J. Hendrick T. W. Jeffries 《Applied microbiology and biotechnology》1998,49(2):141-146
Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (PsURA3) to disrupt P. stipitis LEU2 in a P. stipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX26) was selected for resistance to 5′-fluoroorotic acid to obtain P. stipitis FPL-UC7 (ura3-3). A URA3:lacZ“pop-out” cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu− Ura+ phenotype. The ura3 marker was recovered from this strain by plating cells onto 5′-fluoroorotate and screening for spontaneous URA3 deletion mutants. Excision of the flanked PsURA3 gene resulted in the Leu−Ura− phenotype. The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replication-sequence-based plasmids.
Received: 17 June 1997 / Received revision: 10 September 1997 / Accepted: 14 October 1997 相似文献
9.
Expression of genes from the marine bacteriumAlteromonas haloplanktis 214 inEscherichia coli K-12 总被引:1,自引:0,他引:1
MacLeod Robert A. Hadley R. G. Szalay A. A. Vink Bernadette MacLeod Patricia R. 《Archives of microbiology》1985,142(3):248-252
DNA from the marine bacteriumAlteromonas haloplanktis 214 was partially digested withSau 3A and inserted into theBam HI site of the cloning vector pBR322. The ligation mixture was used to transformEscherichia coli HB101. The gene bank plasmid preparation obtained was used to transformEscherichia coli K-12 strain EO2717, an organism auxotrophic for histidine, arginine, adenine, uracil and thiamin. Prototrophic transformants for each of the five metabolites were isolated using appropriate minimal media for selection. Plasmids isolated from each of the transformants were shown by hybridization to containA. haloplanktis DNA and to be capable of complementing the appropriate mutation inE. coli EO2717. Restriction maps showed that each of the plasmids was different. 相似文献
10.
Cong-Ping Tan Fang-Qing Zhao Zhong-Liang Su Cheng-Wei Liang Song Qin 《Journal of applied phycology》2007,19(4):347-355
A carotenoid gene (crtR-B) from the green alga Haematococcus pluvialis, encoding β-carotene hydroxylase that was able to catalyze the conversion of β-carotene to zeaxanthin and canthaxanthin to
astaxanthin, was cloned into Chlamydomonas reinhardtii chloroplast expression vector p64D to yield plasmid p64DcrtR-B. The vector p64DcrtR-B was transferred to the chloroplast
genome of C. reinhardtii using micro-particle bombardment. PCR and Southern blot analyses indicated that crtR-B was integrated into the chloroplast genome of the transformants. RT-PCR assays showed that the H. pluvialis
crt R-B gene was expressed in C. reinhardtii transformants. The transformants rapidly synthesized carotenoids in larger quantities than the wild-type upon being transferred
from moderate to high-intensity white light. This research provides a foundation for further study to elucidate the possible
mechanism of photo-protection by xanthophylls and other carotenoids in high light conditions or through exposure to UV radiation. 相似文献
11.
A new lipase gene designated as SlLipA was isolated from Serratia liquefaciens S33 DB-1 by the genomic-walking method. The cloned gene contained an open reading frame (ORF) of 1,845 bp encoding 615 amino
acids with a conserved GXSXG motif. Genome sequence analysis showed that an aldo/keto reductase gene closed to the SlLipA gene. The lipase gene was cloned into the expression vector pPICZαA and successfully integrated into the heterologous host,
methylotrophic yeast Pichia pastoris GS115. Five transformants could be expressed as secreted recombinant proteins with the high activity on Triglyceride–Agarose
plate and as candidates to produce the recombinant enzyme. A C-terminal His tag was used for its purification. The lipase
activity of different transformants against substrate para-nitrophenyl laurate (p-NPL) varied from 14 to 16 U ml−1. For the substrates para-nitrophenyl caprate (p-NPC), p-NPL, para-nitrophenyl myristate (p-NPM), para-nitrophenyl palmitate (p-NPP), and para-nitrophenyl stearate (p-NPS), the specific activity was shown to be preferred to long acyl chain length of p-NPS. 相似文献
12.
Jianqiao Wang Yotaro Yamamoto Hirofumi Hirai Hirokazu Kawagishi 《Current microbiology》2013,66(6):544-547
Bisphenol A (BPA) was treated with hyper lignin-degrading fungus Phanerochaete sordida YK-624 under ligninolytic condition. After preculturing P. sordida YK-624 for 4 days, BPA (final concentration, 1 and 0.1 mM) was added to cultures. Both 1- and 0.1-mM BPA were effectively decreased within a 24-h treatment and two metabolites were detected. Two metabolites (5,5′-bis-[1-(4-hydroxy-phenyl)1-methyl-ethyl]-biphenyl-2,2′-diol and 4-(2-(4-hydroxy-phenyl) propan-2-yl)-2-(4-(2-(4-hydroxyphenyl) propan-2-yl) phenoxy)phenol) were identified by ESI–MS and NMR analysis. These results indicated that BPA was oxidized to BPA phenoxy radicals by ligninolytic enzymes and then dimerized at extracellular region. 相似文献
13.
Cira LA González GA Torres JC Pelayo C Gutiérrez M Ramírez J 《Antonie van Leeuwenhoek》2008,93(3):259-266
This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to α-tomatine. Two
transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 μM
α-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 μM. Tomatinase activity was detected
by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase
activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity
was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave
tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions. 相似文献
14.
Ruirong Yi Takashi Tachikawa Hiroyuki Mukaiyama Yusuke Mochida Mariko Ishikawa Tadanori Aimi 《Mycoscience》2009,50(2):123-129
We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker
and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was
about 88.8 transformants/μg pMBsip2 DNA using 5 × 106 protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the efficiency of hygromycin B resistance transformation
was about 122.4 transformants/μg pMBhph1 DNA using 5 × 106 protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced
sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more. 相似文献
15.
James A. Sweigard Forrest G. Chumley Barbara Valent 《Molecular & general genetics : MGG》1992,232(2):183-190
Summary Using a one-step strategy to disrupt CUT1, a gene for cutinase, cut1
– mutants were generated in two strains of Magnaporthe grisea. One strain, pathogenic on weeping lovegrass and barley and containing the arg3–12 mutation, was transformed with a disruption vector in which the Aspergillus nidulans ArgB
+ gene was inserted into CUT1. Prototrophic transformants were screened by Southern hybridization, and 3 of 53 tested contained a disrupted CUT1 gene (cut1 : : ArgB
+). A second strain, pathogenic on rice, was transformed with a disruption vector in which a gene for hyg B resistance was inserted into CUT1. Two of the 57 transformants screened by Southern hybridization contained a disrupted CUT1 gene (cut1:. Hyg). CUT1 mRNA was not detectable in transformants that contained a disrupted gene. Transformants with a disrupted CUT1 gene failed to produce a cutin-inducible esterase that is normally detected by activity staining on non-denaturing polyacrylamide gels. Enzyme activity, measured either with tritiated cutin or with p-nitrophenyl butyrate as a substrate, was reduced but not eliminated in strains with a disrupted CUT1 gene. The infection efficiency of the cut1
– disruption transformants was equal to that of the parent strains on all three host plants. Lesions produced by these mutants had an appearance and a sporulation rate similar to those produced by the parent strains. We conclude that the M. grisea CUT1 gene is not required for pathogenicity. 相似文献
16.
Symptoms of fairy rings caused by Lepista sordida have been reported on Zoysiagrass (Zoysia spp.) turf maintained at fairway height (2 cm), but not on bentgrass (Agrostis spp.) maintained at putting green height (0.5 cm). The mycelia of this fungus inhabit primarily the upper 0–2 cm layer of
the soil extending into the thatch. To compare conditions for the mycelial growth in Z. matrella turf to those in A. palustris turf, we examined the effects of nutrients, temperature, water potential, and pH in the field as well as in the laboratory.
Greater growth of the mycelia was observed in medium that included hot water extracts from soil of the 0–1 cm zone in Z. matrella turf compared to that from A. palustris. The upper soil layer in Z. matrella turf contained more organic matter from clippings than that in A. palustris. The temperature and water potential of the 0–2 cm soil zone in Z. matrella turf were also more favorable for the mycelial growth. The soil pH values of this zone in Z. matrella turf were less favorable compared to A. palustris but within the range for accelerating mycelial growth.
Part of this study was presented orally at the 46th meeting of the Mycological Society of Japan in 2002 相似文献
17.
Summary A transformation system has been developed for Mucor circinelloides, by direct cloning of a wild-type methionine gene that complements the auxotrophic mutation. The marker gene isolated was associated with an autonomous replication sequence (ARS) functional in this zygomycete. Southern hybridisation analyses of transformants showed sequence homology both with vector DNA and with Mucor wild-type DNA. The transformation frequency (up to 6000 per g DNA) and the mitotic instability of the transformed cells were studied. The hybridisation pattern of undigested DNA from the transformants suggests that the inserts contain a novel autonomous replication element for this filamentous fungus. 相似文献
18.
Interleukin-18 gene polymorphism,but not interleukin-2 gene polymorphism,is associated with rheumatoid arthritis 总被引:1,自引:0,他引:1
To investigate whether polymorphisms of IL-2 and IL-18 genes are associated with rheumatoid arthritis (RA), polymorphisms of IL-2 and IL-18 genes were detected by polymerase-chain-reaction-based restriction analysis in the patients with RA and normal controls.
The results for the IL-18 gene revealed a significant difference between the patients and the normal controls (p = 0.000003), but there was no significant difference for the IL-2 gene (p = 0.876). The IL-18 gene 105A allele was associated with RA in Chinese patients. Individuals possessing the 105A allele had a higher incidence
of RA. A lack of association of IL-2 gene polymorphism between RA patients and healthy individuals was noted. The results of this study provide genetic evidence
that IL-18-105A/C polymorphism may play an effective role in RA. 相似文献
19.
Frederico Ozanan Barros Monteiro Leandro Nassar Coutinho Eliane do Socorro de Souza Pompeu Paulo Henrique Gomes de Castro Celsemy Eleutério Maia Washington Luis Assunção Pereira Wilter Ricardo Russiano Vicente 《International journal of primatology》2009,30(2):327-336
We evaluated the uterus and ovaries of owl monkeys (Aotus azarai infulatus) via gynecological ultrasound examination. We evaluated the subjects in 2 different time periods. The first period (P1) was
characterized by the absence of mating, with daily examinations, during 4 mo (n = 10). At the end of P1, we paired the subjects for 30 d, but without ultrasonographic evaluation. The second period (P2)
was characterized by the presence of mating, with examinations once a week, during 7 consecutive months (n = 9). We evaluated the uterus and ovaries in sagittal and transverse scans, using a 5–12 MHz linear array probe. The uterine
volume (UV) was directly proportional to the number of previous parturitions. The right ovary volume (RtOV) is greater than
the left (LtOV) in P1 and P2. There is a positive correlation (p < 0.05) between the females’ mass, RtOV (r = 0.28) and LtOV (r = 0.16). 相似文献
20.
The present study aimed to obtain analgesic-antitumor peptide (AGAP) gene expression in plants. The analgesic-antitumor peptide (AGAP) gene was from the venom of Buthus martensii Karsch. Previous studies showed that AGAP has both analgesic and antitumor activities, suggesting that AGAP would be useful in clinical situations as an antitumor drug. Given that using a plant as an expression vector has more advantages
than prokaryotic expression, we tried to obtain transgenic plants containing AGAP. In the present study, the AGAP gene was cloned into the plasmid pBI121 to obtain the plant expression vector pBI-AGAP. By tri-parental mating and freeze–thaw transformation, pBI-AGAP was transformed into Agrobacterium tumefaciens LBA4404. Tobacco (Nicotiana tabacum) and tomato (Lycopersicom esculentum) were transformed by the method of Agrobacterium-mediated leaf disc transformation. The transformants were then screened to grow and root on media containing kanamycin. Finally,
transformations were confirmed by analysis of PCR, RT-PCR and western blotting. The results showed that the AGAP gene was integrated into the genomic DNA of tobacco and tomato and was successfully expressed. Therefore, the present study
suggests a potential industrial application of AGAP expressed in plants. 相似文献