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1.
Enhancement and Selective Production of Oligomycin through Inactivation of Avermectin’s Starter Unit in Streptomyces avermitilis 总被引:2,自引:0,他引:2
Oligomycin and its analogues, produced by Streptomyces avermitilis and other actinomycetes, are of interest for their potent and selective biological activities. PCR-mediated gene replacement, targeting bkdF, one of avermectin’s starter unit encoding genes in S. avermitilis, was performed to yield an oligomycin producer, BIB0423. The engineered strain produced oligomycin A at 2.3 mg/ml compared to the wild type strain at 0.1 mg/ml. This resulting mutant was genetically stable and should be useful for the industrial production of oligomycin. 相似文献
2.
A polygalacturonase from the filamentous fungus Rhizopus oryzae strain sb (NRRL 29086), previously shown to be effective in the retting of flax fibers, was shown by the analysis of its
reaction products on polygalacturonic acid to be an endo-type. By zymogram analysis, the enzyme in the crude culture filtrate
appeared as two active species of 37 and 40 kD. The endopolygalacturonase-encoding gene was cloned in Escherichia coli and its translated 383-amino acid sequence found to be identical to that of a presumed exopolygalacturonase found in R. oryzae strain YM9901 and 96% identical to a hypothetical protein (RO3G_04731.1) in the sequenced genome of R. oryzae strain 99–880. Phylogenetic analysis revealed the presence of an unique cluster of Rhizopus polygalacturonase sequences that are separate from other fungal polygalacturonases. Conservation of 12 cysteines appears
to be a special feature of this family of Rhizopus polygalacturonase sequences.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
3.
Chlud Kaddor Karolin Biermann Rainer Kalscheuer Alexander Steinbüchel 《Applied microbiology and biotechnology》2009,84(1):143-155
The physiology of lipid production in Streptomyces avermitilis MA-4680 with regard to the fatty acid composition of the accumulated lipids and their cellular distribution was analyzed.
Cells were able to accumulate about ten to 30 lipid granules with diameters between 100 and 500 nm filling about 70–80% of
the cell cytoplasm. Gas chromatography/mass spectrometry analyses of total cellular lipids and from isolated triacylglycerols
(TAG) confirmed a similar fatty acid composition with a large portion of iso- and anteiso-methyl-branched fatty acids. De novo biosynthesis of wax esters (WE) appeared only during cocultivation on glucose and hexadecanol
as carbon source. Homology alignments with the wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT; AtfA)
from Acinetobacter baylyi strain ADP1 yielded one open reading frame in the genome databases of S. avermitilis MA-4680 referred to as SAV7256 with 25.3% homology. The highly conserved HHAxxDG active site motif found in AtfA, which is
present in SAV7256, as well as the similar hydrophobicity profiles of AtfA and SAV7256 indicate a similar structure and function
of both proteins. High acyl-CoA:diacylglycerol acyltransferase activity (DGAT; 143 pmol (mg min)−1) but low wax ester synthase activity (WS; 1.3 pmol (mg min)−1) were detected in crude extracts of S. avermitilis, which were consistent with the high TAG and negligible WE content of the cells. This indicates that TAG accumulation in
S. avermitilis MA-4680 is mediated by the classical acyl-CoA-dependent DGAT pathway. Heterologous expression experiments in recombinant
Escherichia coli BL21(DE3) demonstrated both WS and DGAT enzyme activity of SAV7256. Furthermore, substrate specificities of the acyltransferase
SAV7256 will be discussed.
Chlud Kaddor and Karolin Biermann contributed equally to this work. 相似文献
4.
Igor Loncaric Josua T. Oberlerchner Birgit Heissenberger Rudolf Moosbeckhofer 《Antonie van Leeuwenhoek》2009,95(2):165-178
Intra-specific diversity of 200 Aureobasidium pullulans strains isolated from different sources and their relatives Kabatiella lini CBS 125.21 T and Hormonema prunorum CBS 933.72 T were studied by assessment of macromorphological, and physiological tests, sodium dodecyl sulfate-polyacrylamide
gel electrophoresis technique (SDS–PAGE) of whole-cell proteins as well as enterobacterial repetitive intergenic consensus
(ERIC)-, repetitive extragenic palindromic (REP)- and BOX-PCR techniques (collectively known as rep-PCR). Rep-PCR is an efficient
procedure for discrimination of A. pullulans in terms of simplicity and rapidity. RFLP-PCR technique was applied for the identification of A. pullulans isolates and distinction from related species. This technique was insufficient for investigation of intra-specific diversity.
The tested strains of A. pullulans could be divided into two groups based on their macromorphological, protein patterns obtained after SDS-PAGE as well as rep-PCR
patterns. The first group of strains shared similar characteristics and was very different from the second one, designated
as “complex group”, consisting of strains with very little similarities within the group. Phenetic analysis of ERIC banding
patterns failed to group the isolates on the basis of their substrate or geographical origin. Using 18S rDNA gene sequence
analysis of selected isolates, three strains: HoHe3 km, A. pullulans DSM 62074 and H. prunorum CBS 933.72 T were distinguished from all other analysed members of genera Aureobasidium and Kabatiella.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
5.
Xiuping Zou Demou Li Xiaoying Luo Keming Luo Yan Pei 《In vitro cellular & developmental biology. Plant》2008,44(3):169-177
Highly efficient Agrobacterium-mediated transformation of trifoliate orange (Poncirus trifoliata (L.) Raf.) was achieved via indirect shoot organogenesis. Stable transformants were obtained from epicotyl segments infected
with Agrobacterium strain EHA 105 harboring the binary vector pBI121, which contained the neomycin phosphotransferase gene (NPTII) as a selectable
marker and the β-glucuronidase (GUS) gene as a reporter. The effects of regeneration and selection conditions on the transformation
efficiency of P. trifoliata (L.) Raf. have been investigated. A 7-d cocultivation on a medium with 8.86 μM 6-benzylaminopurine (BA)+1.43 μM indole-3-acetic
acid (IAA) was used to improve callus formation from epicotyl segments after transformation. A two-step selection strategy
was developed to select kanamycin-resistant calluses and to improve rooting of transgenic shoots. Transgenic shoots were multiplied
on shoot induction medium with 1.11 μM BA + 5.71 μM IAA. Using the optimized transformation procedure, transformation efficiency
and rooting frequency reached 417% and 96%, respectively. Furthermore, the number of regenerated escape shoots was dramatically
reduced. Stable integration of the transgenes into the genome of transgenic citrus plants was confirmed by GUS histochemical
assay, PCR, and Southern blot analysis. 相似文献
6.
The role of the extracytoplasmic function (ECF) σ factor Sig6 (SAV663) in avermectin production by Streptomyces avermitilis was investigated by gene-deletion, complementation and over-expression experiments. Inactivation of Sig6 had no major effect
on growth, stress responses, or morphology. Avermectin yield was increased 2- to 2.7-fold (~680 μg/ml) relative to the wild-type
strain by deletion of the sig6 gene, and was restored to the wild-type level by introduction of a single copy of sig6. Introduction of extra multi-copy or integrative sig6 vectors into the wild-type decreased avermectin yield by 56–63%. Taken together, these findings indicate that Sig6 plays
a negative regulatory role in avermectin production in S. avermitilis. RT-PCR analysis demonstrated that this role of Sig6 is mediated by the pathway-specific activator gene aveR. 相似文献
7.
To understand the metabolic characteristics of Clostridium acetobutylicum and to examine the potential for enhanced butanol production, we reconstructed the genome-scale metabolic network from its
annotated genomic sequence and analyzed strategies to improve its butanol production. The generated reconstructed network
consists of 502 reactions and 479 metabolites and was used as the basis for an in silico model that could compute metabolic and growth performance for comparison with fermentation data. The in silico model successfully predicted metabolic fluxes during the acidogenic phase using classical flux balance analysis. Nonlinear
programming was used to predict metabolic fluxes during the solventogenic phase. In addition, essential genes were predicted
via single gene deletion studies. This genome-scale in silico metabolic model of C. acetobutylicum should be useful for genome-wide metabolic analysis as well as strain development for improving production of biochemicals,
including butanol.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
J. L. and H. Y. equally contributed to this work. 相似文献
8.
Partial gyrB sequences (>1 kb) were obtained from 34 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships
within the genus. The use of gyrB sequence analysis as an alternative to DNA–DNA hybridization was also assessed for distinguishing closely related species.
The gyrB based phylogeny mostly confirmed the conventional 16S rRNA gene-based phylogeny and thus provides additional support for
certain of these 16S rRNA gene-based phylogenetic groupings. Although pairwise gyrB sequence similarity cannot be used to predict the DNA relatedness between type strains, the gyrB genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the
genus Amycolatopsis. In particular a genetic distance of >0.02 between two Amycolatopsis strains (based on a 315 bp variable region of the gyrB gene) is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization
of the unknown strain is worth undertaking).
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
The GenBank accession numbers for the gyrB gene sequences obtained in this study are shown in Table 1. 相似文献
9.
Yu YJ Wu SC Chan HH Chen YC Chen ZY Yang MT 《Applied microbiology and biotechnology》2008,81(3):523-532
A novel microbial transglutaminase (TGase) from the cultural filtrate of Streptomyces netropsis BCRC 12429 (Sn) was purified. The specific activity of the purified TGase was 18.2 U/mg protein with an estimated molecular
mass of 38 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. The TGase gene of S. netropsis was cloned and an open reading frame of 1,242 bp encoding a protein of 413 amino acids was identified. The Sn TGase was synthesized
as a precursor protein with a preproregion of 82 amino acid residues. The deduced amino acid sequence of the mature S. netropsis TGase shares 78.9–89.6% identities with TGases from Streptomyces spp. A high level of soluble Sn TGase with its N-terminal propeptide fused with thioredoxin was expressed in E. coli. A simple and efficient process was applied to convert the purified recombinant protein into an active enzyme and showed
activity equivalent to the authentic mature TGase.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
10.
A new bacterial strain producing succinic acid was enriched from bovine rumen content. It is facultatively anaerobic, belongs
to the family Pasteurellaceae and has similarity to the genus Mannheimia. In batch cultivations with D-glucose or sucrose the strain produced up to 5.8 g succinic acid l−1 with a productivity and a yield of up to 1.5 g l−1 h−1 and 0.6 g g−1, respectively. With crude glycerol up to 8.4 g l−1, 0.9 g l−1 h−1 and 1.2 g g−1 were obtained.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
11.
Khan SA Hamayun M Kim HY Yoon HJ Seo JC Choo YS Lee IJ Kim SD Rhee IK Kim JG 《Biotechnology letters》2009,31(2):283-287
Plant growth-promoting endophytic fungi with gibberellin-producing ability were isolated from the roots of Carex kobomugi Ohwi, a common sand-dune plant, and bioassayed for plant growth-promotion. A new strain, Arthrinium phaeospermum KACC43901, promoted growth of waito-c rice and Atriplex gemelinii. Analysis of its culture filtrate showed the presence of bioactive GA1 (0.5 ng/ml), GA3 (8.8 ng/ml), GA4 (4.7 ng/ml) and GA7 (2.2 ng/ml) along with physiologically inactive GA5 (0.4 ng/ml), GA9 (0.6 ng/ml), GA12 (0.4 ng/ml), GA15 (0.4 ng/ml), GA19 (0.9 ng/ml) and GA24 (1.8 ng/ml). The fungal isolate was identified through sequence homology and phylogenetic analysis of 18S rDNA (internal
transcribed region).
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
12.
13.
Sumita Kumari Prabhjeet Singh Sneh L. Singla-Pareek Ashwani Pareek 《Molecular biotechnology》2009,42(2):195-204
14.
15.
We have characterized cytochromes P450, CYP710A13, and CYP710A14, as the sterol C22-desaturase in the moss Physcomitrella patens. GC–MS analyses demonstrated that P. patens accumulated stigmasterol as the major sterol (56–60% of total sterol) and sitosterol to a lesser extent (8–12%); this sterol
profile contrasts with those in higher plants accumulating stigmasterol as a minor component. Recombinant CYP710A13 and CYP710A14
proteins prepared using a baculovirus/insect cell system exhibited the C22-desaturase activity with β-sitosterol to produce
stigmasterol, while campesterol and 24-epi-campesterol were not accepted as the substrates. The K
m values for β-sitosterol of CYP710A13 (1.0 ± 0.043 μM) and CYP710A14 (2.1 ± 0.17 μM) were at comparable levels of those reported
with higher plant CYP710A proteins. In Arabidopsis T87 cells over-expressing CYP710A14, stigmasterol contents reached a level 20- to 72-fold higher than those in the basal
level of T87 cells, confirming the C22-desaturase activity of this P450 enzyme. The occurrence of the end-products together
with the enzymes involved in the last step of the pathway substantiated the presence of an entire sterol biosynthetic pathway
in P. patens, providing evidence for the conservation of the sterol biosynthetic pathway through the evolutionary process of land plants.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
16.
Yemendzhiev H Gerginova M Krastanov A Stoilova I Alexieva Z 《Journal of industrial microbiology & biotechnology》2008,35(11):1309-1312
Trametes versicolor 1 was shown to grow on phenol as its sole carbon and energy source. The culture growth and degradation ability dependence
on culture medium pH value was observed. The optimal pH value of a liquid Czapek salt medium was 6.5. The investigated strain
utilized completely 0.5 g/l phenol in 6 days. The dynamics of the phenol degradation process was investigated. The process
was characterized by specific growth rate μmax 0.33 h−1, metabolic coefficient k = 4.4, yield coefficient Y
x/s
= 0.23 and rate of degradation Q = 0.506 h−1. The intracellular activities of phenol hydroxylase (0.333 U/mg protein) and cis,cis-muconate lactonizing enzyme (0.41 U/mg protein) were demonstrated for the first time in this fungus. In an attempt to estimate
the occurrence of gene sequences in T. versicolor 1 related to phenol degradation pathway a dot blot analysis with total DNA isolated from this strain was performed. Two synthetic
oligonucleotides were used as hybridizing probes. One of the probes was homologous to the 5′end of phyA gene coding for phenol hydroxylase in Trichosporon cutaneum ATCC 46490. The other probe was created on the basis of cis,cis-muconate lactonizing enzyme coding gene in T. cutaneum ATCC 58094. The results of these investigations showed that T. versicolor 1 may carry genes similar to those of Trichosporon cutaneum capable to degrade phenol. 相似文献
17.
We report here the isolation of a methionine and cobalamin mutant strain (SVQ336) of Sinorhizobium fredii HH103 obtained by Tn5-lacZ mutagenesis. Sequence analysis showed that the transposon was inserted into a gene homologous to cobO. This gene codes for a cobalamin adenosyltransferase which is involved in the biosynthesis of vitamin B12. Another HH103 cobO mutant (strain SVQ524), was constructed by the insertion of Ω interposon. Both cobO mutants required the addition of methionine because cobalamin acts as a cofactor of the enzyme MetH, which catalyses the
last step of the methionine biosynthesis. Mutant SVQ524 failed to nodulate on Vigna radiate but was able to nodulate on Glycine max cvs. Williams and Peking and Cajanus cajan, although the total number of nodules formed was highly reduced in comparison with that of plants inoculated with the wild-type
strain HH103. The roots of these plants did not seem to secrete enough cobalamin and/or methionine to support growth of cobalamin/methionine
auxotrophs in the rhizosphere. In all cases, the phenotype of SVQ524 was nearly overcome by the addition of methionine or
cobalamin to the plant growth media or by the presence of a copy of the cobO gene in cosmid pMUS756.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
18.
During 2,3,5,6-tetramethylpyrazine production from glucose by Bacillus strains, a novel product was detected and identified as 2,4,5-trimethylimidazole (TMI) by GC/MS. TMI appeared in the culture
medium only after glucose had been depleted and then increased to 0.25–0.31 g l−1 in 90–120 h. When the ammonium source was changed from (NH4)2SO4 to (NH4)2HPO4, only about one tenth of TMI was detected. Although the mechanistic events largely remain unclear, both microbial strains
tested demonstrated similar dynamic processes of TMI production, suggesting that TMI formation is a genuine feature of Bacillus species.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
19.
A. G. Valladares Juárez J. Dreyer P. K. Göpel N. Koschke D. Frank H. Märkl R. Müller 《Applied microbiology and biotechnology》2009,83(3):521-527
Novel thermophilic and alkaliphilic bacteria for the processing of bast fibres were isolated using hemp pectin as substrate.
The strain PB94A, which showed the highest growth rate (μ = 0.5/h) was identified as Geobacillus thermoglucosidasius (DSM 21625). The strain grew optimally at 60°C and pH 8.5. During growth on citrus pectin, the strain produced pectinolytic
lyases, which were excreted into the medium. In contrast to the commercially available pectinase Bioprep 3000 L, the enzymes
from G. thermoglucosidasius PB94A converted pectin isolated from hemp fibres. In addition to hemp pectin, the culture supernatant also degraded citrus,
sugar beet and apple pectin and polygalacturonic acid. When hemp fibres were incubated with the cell-free fermentation broth
of G. thermoglucosidasius PB94A, the fineness of the fibres increased. The strain did not produce any cellulases, which is important in order to avoid
damaging the fibres during incubation. Therefore, these bacteria or their enzymes can be used to produce fine high-quality
hemp fibres. 相似文献