共查询到20条相似文献,搜索用时 31 毫秒
1.
José L. Caballero Eduardo Martinez Francisco Malpartida David A. Hopwood 《Molecular & general genetics : MGG》1991,230(3):401-412
Summary Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and-ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics. 相似文献
2.
Susumu Ikegami Yutaka Hirose Yuji Kamiya Saburo Tamura 《Bioscience, biotechnology, and biochemistry》2013,77(10):1843-1845
ABSTRACTThis study aimed to investigate the role of serine/threonine kinase PkaE in Streptomyces coelicolor A3(2). Liquid chromatography tandem mass spectrometry was performed for comparative phosphoproteome and proteome analyses of S. coelicolor A3(2), followed by an in vitro phosphorylation assay. Actinorhodin production in the pkaE deletion mutant was lower than that in wild-type S. coelicolor A3(2), and the spores of the pkaE deletion mutant were damaged. Furthermore, phosphoproteome analysis revealed that 6 proteins were significantly differentially hypophosphorylated in pkaE deletion mutant (p < 0.05, fold-change ≤ 0.66), including BldG and FtsZ. In addition, the in vitro phosphorylation assay revealed that PkaE phosphorylated FtsZ. Comparative proteome analysis revealed 362 differentially expressed proteins (p < 0.05) and six downregulated proteins in the pkaE deletion mutant involved in actinorhodin biosynthesis. Gene ontology enrichment analysis revealed that PkaE participates in various biological and cellular processes. Hence, S. coelicolor PkaE participates in actinorhodin biosynthesis and morphogenesis. 相似文献
3.
4.
Nutritional control of actinorhodin production byStreptomyces coelicolor A3(2): suppressive effects of nitrogen and phosphate 总被引:5,自引:0,他引:5
Summary Actinorhodin production inStreptomyces coelicolor A3(2) was relatively insensitive to the carbon source concentration but was elicited by nitrogen or phosphate depletion, or by a decline in the growth rate. In starch-glutamate media with nitrogen limitation, increasing the nitrogen supply delayed the onset of antibiotic synthesis and, at concentrations above 30 mM, decreased its rate. In a similar medium with phosphate limitation, increasing the initial phosphate concentration delayed actinorhodin formation and, above 2.5 mM, reduced the rate of synthesis. Experiments in which actinorhodin synthesis was elicited by phosphate depletion at various nitrogen concentrations demonstrated strong suppression by residual glutamate. Cultures in which actinorhodin biosynthesis was initiated by nitrogen depletion were not similarly suppressed by increasing amounts of residual phosphate. The results suggest that actinorhodin production inS. coelicolor A3(2) responds to interacting physiological controls, notable among which is nitrogen catabolite regulation. 相似文献
5.
Lee HN Kim HJ Kim P Lee HS Kim ES 《Journal of industrial microbiology & biotechnology》2012,39(5):805-811
Along with traditional random mutagenesis-driven strain improvement, cloning and heterologous expression of Streptomyces secondary metabolite gene clusters have become an attractive complementary approach to increase its production titer, of
which regulation is typically under tight control via complex multiple regulatory networks present in a metabolite low-producing
wild-type strain. In this study, we generated a polyketide non-producing strain by deleting the entire actinorhodin cluster
from the chromosome of a previously generated S. coelicolor mutant strain, which was shown to stimulate actinorhodin biosynthesis through deletion of two antibiotic downregulators as
well as a polyketide precursor flux downregulator (Kim et al. in Appl Environ Microbiol 77:1872–1877, 2011). Using this engineered S. coelicolor mutant strain as a surrogate host, a model minimal polyketide pathway for aloesaponarin II, an actinorhodin shunt product,
was cloned in a high-copy conjugative plasmid, followed by functional pathway expression and quantitative metabolite analysis.
Aloesaponarin II production was detected only in the presence of a pathway-specific regulatory gene, actII-ORF4, and its production level was the highest in the actinorhodin cluster-deleted and downregulator-deleted mutant strain, implying
that this engineered polyketide pathway-free and regulation-optimized S. coelicolor mutant strain could be used as a general surrogate host for efficient expression of indigenous or foreign polyketide pathways
derived from diverse actinomycetes in nature. 相似文献
6.
7.
Genetics of actinorhodin biosynthesis by Streptomyces coelicolor A3(2) 总被引:19,自引:0,他引:19
A series of 76 mutants of Streptomyces coelicolor A3(2) specifically blocked in the synthesis of the binaphthoquinone antibiotic actinorhodin were classified into seven phenotypic classes on the basis of antibiotic activity, accumulation of pigmented precursors or shunt products of actinorhodin biosynthesis, and cosynthesis of actinorhodin in pairwise combinations of mutants. The polarity of cosynthetic reactions, and other phenotypic properties, allowed six of the mutant classes to be arranged in the most probable linear sequence of biosynthetic blocks. One member of each mutant class was mapped unambigiguously to the chromosomal linkage map in the short segment between the hisD and guaA loci, suggesting that structural genes for actinorhodin biosynthesis may form an uninterrupted cluster of chromosomal genes. 相似文献
8.
Andy Hesketh Holger Kock Saraspadee Mootien Mervyn Bibb 《Molecular microbiology》2009,74(6):1427-1444
The availability of zinc was shown to have a marked influence on the biosynthesis of actinorhodin in Streptomyces coelicolor A3(2). Production of actinorhodin and undecylprodigiosin was abolished when a novel pleiotropic regulatory gene, absC, was deleted, but only when zinc concentrations were low. AbsC was shown to control expression of the gene cluster encoding production of coelibactin, an uncharacterized non‐ribosomally synthesized peptide with predicted siderophore‐like activity, and the observed defect in antibiotic production was found to result from elevated expression of this gene cluster. Promoter regions in the coelibactin cluster contain predicted binding motifs for the zinc‐responsive regulator Zur, and dual regulation of coelibactin expression by zur and absC was demonstrated using strains engineered to contain deletions in either or both of these genes. An AbsC binding site was identified in a divergent promoter region within the coelibactin biosynthetic gene cluster, adjacent to a putative Zur binding site. Repression of the coelibactin gene cluster by both AbsC and Zur appears to be required to maintain appropriate intracellular levels of zinc in S. coelicolor. 相似文献
9.
10.
M A Fernández-Moreno E Martínez L Boto D A Hopwood F Malpartida 《The Journal of biological chemistry》1992,267(27):19278-19290
A 5.3-kb region of the Streptomyces coelicolor actinorhodin gene cluster, including the genes for polyketide biosynthesis, was sequenced. Six identified open reading frames (ORF1-6) were related to genetically characterized mutations of classes actI, VII, IV, and VB by complementation analysis. ORF1-6 run divergently from the adjacent actIII gene, which encodes the polyketide synthase (PKS) ketoreductase, and appear to form an operon. The deduced gene products of ORF1-3 are similar to fatty acid synthases (FAS) of different organisms and PKS genes from other polyketide producers. The predicted ORF5 gene product is similar to type II beta-lactamases of Bacillus cereus and Bacteroides fragilis. The ORF6 product does not resemble other known proteins. Combining the genetical, biochemical, and similarity data, the potential activities of the products of the six genes can be postulated as: 1) condensing enzyme/acyl transferase (ORF1 + ORF2); 2) acyl carrier protein (ORF3); 3) putative cyclase/dehydrase (ORF4); 4) dehydrase (ORF5); and 5) "dimerase" (ORF6). The data show that the actinorhodin PKS consists of discrete monofunctional components, like that of the Escherichia coli (Type II) FAS, rather than the multifunctional polypeptides for the macrolide PKSs and vertebrate FASs (Type I). 相似文献
11.
Neutral lipid accumulation during early growth phase of Streptomyces coelicolor A3(2) was essential for the actinorhodin production during later growth. The activities of lipase and isocitrate dehydrogenase were increasing and decreasing, respectively, suggesting that the degradation products of neutral lipids serve actinorhodin biosynthesis as precursors. 相似文献
12.
Chang-Young Kim Hyun-Joo Park Eung-Soo Kim 《Biotechnology and Bioprocess Engineering》2005,10(3):248-253
AfsR2, originally identified fromStreptomyces lividans, is a global regulatory protein which stimulates antibiotic biosynthesis. Through its stable chromosomal integration, the
high level of gene expression ofafsR2 significantly induced antibiotic production as well as the sporulation ofS. lividans, implying the presence of yet-uncharacterized AfsR2-target proteins. To identify and evaluate the putative AfsR2-target proteins
involved in antibiotic regulation, the proteomics-driven approach was applied to the wild-typeS. lividans and theafsR2-integrated actinorhodin overproducing strain. The 2D gel-electrophoresis gave approximately 340 protein spots showing different
protein expression patterns between these twoS. lividans strains. Further MALDI-TOF analysis revealed several AfsR2-target proteins, including glyceraldehyde-3-phosphate dehydrogenase,
putative phosphate transport system regulator, guanosine pentaphosphate synthetase/polyribonucleotide nucleotidyltransferase,
and superoxide dismutase, which suggests that the AfsR2 should be a pleiotropic regulatory protein which controls differential
expressions of various kinds of genes inStreptomyces species. 相似文献
13.
The downstream gene controlled by promoter--PTH4 which is related to Streptomyces differentiation was cloned, and its sequence was determined by the dideoxy chain termination method. The results indicated that the 1597 bp of DNA fragment conferred a complete open reading frame (ORF). In searches of databases, the deduced product of the ORF was not homologous with any known proteins; it may be a new protein. The function of the gene was studied using the strategy of gene disruption; the actinorhodin could not be produced when this gene was disrupted. Therefore, this gene may be related to actinorhodin biosynthesis in Streptomyces coelicolor, and the result also shows that this gene may play a role in multiple level regulation of differentiation genes in Streptomyces. 相似文献
14.
15.
Haomiao Ouyang Yuanming Luo Lei Zhang Yanjie Li Cheng Jin 《Molecular biotechnology》2010,46(3):317-319
We attempted to identify membrane proteins associated with the glycoconjugates and cell wall biosynthesis in the total membrane
preparations of Aspergillus fumigatus. The total membrane preparations were first run on 1D gels, and then the stained gels were cut and submitted to in-gel digestion
followed by 2D LC-MS/MS and database search. A total of 530 proteins were identified with at least two peptides detected with
MS/MS spectra. Seventeen integral membrane proteins were involved in N-, O-glycosylation or GPI anchor biosynthesis. Nine
membrane proteins were involved in cell wall biosynthesis. Eight proteins were identified as enzymes involved in sphingolipid
synthesis. In addition, the proteins involved in cell wall and ergosterol biosynthesis can potentially be used as antifungal
drug targets. Our method, for the first time, clearly provided a global view of the membrane proteins associated with glycoconjugates
and cell wall biosynthesis in the total membrane proteome of A. fumigatus. 相似文献
16.
A collection of actinomycin-producing Streptomycesstrains, their variants with different levels of antibiotic biosynthesis, and recombinant strains were screened in order to select new strains that produce polyketide antibiotics. Screening with the use of the cloned actgene encoding a component of actinorhodin polyketide synthase (PKS) multienzyme complex from Streptomyces coelicolorrevealed that many strains tested can synthesize polyketide antibiotics along with actinomycins. A relationship between the biosynthetic pathways of actinomycins and polyketides is discussed. 相似文献
17.
Bruheim P Sletta H Bibb MJ White J Levine DW 《Journal of industrial microbiology & biotechnology》2002,28(2):103-111
Streptomyces lividans 1326 usually does not produce the red/blue colored polyketide actinorhodin in liquid culture even though it carries the entire
actinorhodin biosynthesis gene cluster. The bacterium can be forced to produce this secondary metabolite by introducing actII-ORF4, the actinorhodin pathway-specific activator gene from Streptomyces coelicolor, on a multicopy plasmid. The production of actinorhodin by such a strain has been optimized by medium and process manipulations
in fed-batch cultures. With high-yield cultivation conditions, 5 g actinorhodin/l are produced during 7 days of cultivation;
or approximately 0.1 g actinorhodin/g dry weight (DW)/day in the production phase. The yield in this phase is 0.15 Cmol actinorhodin/Cmol
glucose, which is in the range of 25% to 40% of the maximum theoretical yield. This high-level production mineral medium is
phosphate limited. In contrast, nitrogen limitation resulted in low-level production of actinorhodin and high production of
α-ketoglutaric acid. Ammonium as nitrogen source was superior to nitrate supporting an almost three times higher actinorhodin
yield as well as a two times higher specific production rate. The wild-type strain lacking the multicopy plasmid did not produce
actinorhodin when cultivated under any of these conditions. This work examines the actinorhodin-producing potential of the
strain, as well as the necessity to improve the culture conditions to fully utilize this potential. The overexpression of
biosynthetic pathway-specific activator genes seems to be a rational first step in the design of secondary metabolite overproducing
strains prior to alteration of primary metabolic pathways for redirection of metabolic fluxes. Journal of Industrial Microbiology & Biotechnology (2002) 28, 103–111 DOI: 10.1038/sj/jim/7000219
Received 04 April 2001/ Accepted in revised form 30 October 2001 相似文献
18.
19.
【目的】以基因组信息为指导,定向激活海洋来源真菌Arthrinium arundinisZSDS1-F3中沉默的聚酮合成酶-非核糖体肽合成酶(PKS-NRPS)类生物合成基因簇,鉴定次级代谢产物结构。【方法】通过启动子工程和异源表达的策略激活实验室培养条件下沉默或低表达的生物合成基因簇,实现目标化合物的分离,通过HR-ESI-MS和NMR数据分析鉴定产物结构,结合基因重组和生物信息学分析结果推导化合物的生物合成途径。【结果】依据基因组生物信息学分析,从海洋来源真菌A. arundinis ZSDS1-F3中选取一个编码PKS-NRPS类次级代谢产物的生物合成基因簇开展研究,在宿主Aspergillus nidulansA1145中实现了基因簇的异源表达,从中分离到2个新化合物,并推导了其生物合成途径。【结论】基因组信息指导下的天然产物挖掘,可以目标明确地分离产物,加快真菌中新颖天然产物的发现步伐。 相似文献
20.
Dong-Dong Wang Hua Bai Wei-Qi Chen Hai Lu Xiang-Ning Jiang 《Journal of Plant Biology》2009,52(5):482-491
A cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzyme involved in lignin biosynthesis, was cloned from
Populus tomentosa (Chinese white poplar). At the same time, a 4CL1 gene was cloned from P. tomentosa, too. The two genes were subcloned in pQE31 vector and expressed in Escherichia coli M15. Both of them were purified by Ni-NTA. Purified CCR protein was digested by trypsin and analyzed by HPLC-MS; the peptide
segments had 27% similarity with the sequence of the CCR protein. 4CL was thought to be a neighbor enzyme of CCR in lignin
biosynthesis. In this paper, a 4CL1 from P. tomentosa was cloned, and its enzyme reaction products were extracted for the substrates of CCR. Three 4CL1 enzyme reaction products
were monitored by HPLC-MS and then the CCR enzyme reaction was detected by GC-MS. In the CCR reaction, the three corresponding
aldehyde (p-coumaraldehyde, caffealdehyde, and coniferaldehyde) were detected and identified by Frontier3 software. The results showed
that the CCR that we cloned from P. tomentosa had affinities with 4CL1 enzyme reaction products and a ptCCR that was cloned from aspen (Li et al., Plant Cell Physiol 46(7):1073–1082, 2005) only had affinity with feruloyl-CoA. The different results maybe depend on the different study method. The method of exacting
4CL enzyme products as the substrates of CCR in the paper was reliable and can be used in lignin biosynthesis network to detect
the enzymes in the neighborhood that depended on the polarity of the substrates and products. This CCR gene had eight homology
sequence CCR gene when a BLAST was conducted in Populus trichocarpa genome database. The CCR homology genes in Populus suggested that some CCRs may take part in the lignin biosynthesis, too. The gene family would be the hot spot in the future
study. 相似文献