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1.
作为生物体必需的营养元素之一,磷在物质代谢、信号传导和能量储存中起着关键作用。【目的】研究丝状真菌Podospora anserina中调控磷酸盐代谢相关转录因子的作用,可进一步阐明真核微生物中磷元素吸收的调控机制。【方法】利用同源重组的方法定点敲除P.anserina中2个磷代谢相关转录因子PaPho1和PaPho2,遗传杂交构建双重突变体ΔPaPho1ΔPaPho2;通过表型分析、无机磷含量测定和酸性磷酸酶活性测定分析各突变菌株的变化;利用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)分析磷代谢相关基因的表达情况。【结果】在无机磷作为唯一磷来源的培养基上,ΔPaPho1ΔPaPho2无法生长;在添加有机磷的培养基中,ΔPaPho1ΔPaPho2和野生型菌株生长无显著性差异。在同时添加有机磷和无机磷的培养基中,ΔPaPho1ΔPaPho2的无机磷含量和酸性磷酸酶活性比野生型菌株的分别下降了25.0%和61.9%,ΔPaPho1ΔPaPho2中无机磷酸盐转运蛋白基因的表达水平显著降低。【结论】在P... 相似文献
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Rupali M. Khadake Prabhakar K. Ranjekar Abhay M. Harsulkar 《Molecular biotechnology》2009,42(2):168-174
The Δ12 desaturase represents a diverse gene family in plants and is responsible for conversion of oleic acid (18:1) to linoleic
acid (18:2). Several members of this family are known from plants like Arabidopsis and Soybean. Using primers from conserved C- and N-terminal regions, we have cloned a novel Δ12 desaturase gene amplified
from flax genomic DNA, denoted as LuFAD2-2. This intron-less gene is 1,149-base pair long encoding 382 amino acids—putative membrane-bound Δ12 desaturase protein. Sequence
comparisons show that the novel sequence has 85% similarity with previously reported flax Δ12 desaturase at amino acid level
and shows typical features of membrane-bound desaturase such as three conserved histidine boxes along with four membrane-spanning
regions that are universally present among plant desaturases. The signature amino acid sequence ‘YNNKL’ was also found to
be present at the N terminus of the protein, which is necessary and sufficient for ER localization of enzyme. Neighbor-Joining
tree generated from the sequence alignment grouped LuFAD2-2 among the other FAD2 sequences from Ricinus, Hevea, Jatropha, and Vernicia. When LuFAD2-2 and LuFAD2 were expressed in Saccharomyces cerevisiae, they could convert the oleic acid to linoleic acid, with an average conversion rate of 5.25 and 8.85%, respectively. However,
exogenously supplied linoleic acid was feebly converted to linolenic acid suggesting that LuFAD2-2 encodes a functional FAD2 enzyme and has substrate specificity similar to LuFAD2. 相似文献
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The Tsc/Rheb signaling pathway plays critical roles in the control of growth and cell cycle. Studies in fission yeast have
also implicated its importance in the regulation of amino acid uptake. Disruption of tsc2
+, one of the tsc
+ genes, has been shown to result in decreased arginine uptake and resistance to canavanine. A similar effect is also seen
with other basic amino acids. We have identified a permease responsible for the uptake of basic amino acids by genetic complementation
and disruption. SPAC869.11 (termed Cat1 for cationic amino acid transporter) contains 12 predicted transmembrane domains and
its overexpression in wild type fission yeast leads to the increased uptake of basic amino acids and sensitivity to canavanine.
Disruption of cat1
+ in the Δtsc2 background interfered with the suppression of the canavanine-resistant phenotype of Δtsc2 mutants by a dominant negative Rheb. In Δtsc2 mutant strains, the amount of Cat1 was not altered, but instead was mislocalized. This mislocalization was suppressed by
the expression of dominant negative Rheb. In addition, we found that the loss of the E3 ubiquitin ligase, Pub1, also restores
proper localization. These results provide a crucial link between Tsc/Rheb signaling and the regulation of the basic amino
acid permease in fission yeast. 相似文献
6.
Wenjuan Yao Xiaozhao Deng Hui Zhong Miao Liu Pu Zheng Zhihao Sun Yun Zhang 《Journal of industrial microbiology & biotechnology》2009,36(7):911-921
Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis.
The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC
13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by
the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in
ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production
strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis. 相似文献
7.
A native homoethanol pathway (pyruvate-to-acetyl-CoA-to-acetaldehyde-to-ethanol) was engineered in Escherichia coli B. The competing fermentation pathways were eliminated by chromosomal deletions of the genes encoding for fumarate reductase
(frdABCD), lactate dehydrogenase (ldhA), acetate kinase (ackA), and pyruvate formate lyase (pflB). For redox balance and anaerobic cell growth, the pyruvate dehydrogenase complex (aceEF-lpd, a typical aerobically-expressed operon) was highly expressed anaerobically using a native anaerobic inducible promoter.
The resulting strain SZ420 (ΔfrdBC ΔldhA ΔackA ΔfocA-pflB ΔpdhR::pflBp6-pflBrbs-aceEF-lpd) contains no foreign genes and/or promoters and efficiently ferments glucose and xylose into ethanol with a yield of 90%
under anaerobic conditions. 相似文献
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Wan-Chi Liang Min-Guan Lin Meng-Chun Chi Hui-Yu Hu Huei-Fen Lo Hui-Ping Chang Long-Liu Lin 《Archives of microbiology》2009,191(7):583-593
Bacillus licheniformis DnaK (BlDnaK) is predicted to consist of a 45-kDa N-terminal ATPase domain and a 25-kDa C-terminal substrate-binding domain. In this
study, the full-length BlDnaK and its T86W and three C-terminally truncated mutants were constructed to evaluate the role of up to C-terminal 255 amino
acids of the protein. The steady-state ATPase activity for BlDnaK, T86W, T86W/ΔC120, T86W/ΔC249, and T86W/ΔC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min per mg, respectively.
In vivo, BldnaK, T86W and T86W/ΔC120 genes allowed an E. coli
dnaK756-ts mutant to grow at 44°C. Except for T86W/ΔC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/ΔC120, and T86W/ΔC249 by 16.9-, 13.9-, 33.9-, 9.9-fold, respectively. Measurement of intrinsic tryptophan
fluorescence revealed significant alterations of microenvironment of aromatic amino acids in the C-terminally truncated mutants.
The temperature-dependent signal in the far-UV region for T86W was consistent with that of BlDnaK, but the C-terminally truncated mutant proteins showed a higher sensitivity toward temperature-induced denaturation.
These results suggest that C-terminal truncations alter the ATPase activity and thermal stability of BlDnaK and induce the conformation change of the ATPase domain.
Wan-Chi Liang and Min-Guan Lin contributed equally to this work. 相似文献
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Yun-Tao Li Mao-Teng Li Chu-Hua Fu Peng-Peng Zhou Jian-Min Liu Long-Jiang Yu 《Biotechnology letters》2009,31(7):1011-1017
Genes encoding Δ6 desaturase, Δ6 fatty acid elongase, and Δ5 desaturase from the alga, Phaeodactylum tricornutum, were co-expressed in Pichia pastoris to produce arachidonic acid (ARA; 20:4 Δ5, 8, 11, 14) and eicosapentaenoic acid (EPA; 20:5 Δ5, 8, 11, 14, 17). A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro
multimerization approach. ARA and EPA accumulated up to 0.3 and 0.1% of total fatty acids, respectively, in the recombinant
P. pastoris carrying with double copies of these three heterologous genes, as compared to 0.1 and 0.05%, respectively, in the recombinant
P. pastoris with single copy.
Yun-Tao Li and Mao-Teng Li contributed equally to this work. 相似文献
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【背景】精胺在植物应对逆境胁迫、动物抵抗疲劳和衰老、真菌生长代谢等过程中发挥重要作用,但目前在昆虫病原真菌中的研究未见报道。【目的】在分子水平上探究罗伯茨绿僵菌精胺合成关键酶——精胺合成酶在昆虫血腔定殖中的作用机制。【方法】显微注射法测定Mrsps敲除株ΔMrsps的致病力变化,并观察血腔中ΔMrsps生长状态;收集ΔMrsps和野生型WT注射侵染30 h后的大蜡螟血淋巴进行转录组测序,分别与罗伯茨绿僵菌和大蜡螟参考基因组进行比对分析,并结合定量PCR进行验证。【结果】与WT和回补株ΔMrsps-cp相比较,ΔMrsps致病力显著下降,而且随着注射浓度的降低,ΔMrsps致病力下降越显著。侵染36 h后WT和ΔMrsps孢子都能正常萌发且开始以类酵母状态生长,60 h后,相较于WT,ΔMrsps的生长繁殖数量较少。转录组共检测到3 202个罗伯茨绿僵菌基因,其中1 769个基因在ΔMrsps中表达上调,922个基因表达下调;差异表达基因涉及碳水化合物代谢、运输、分解代谢、翻译和氨基酸代谢等多条途径;筛选出28个血腔致病相关基因全部在ΔMrsps中表达下调;定量PCR检测发现在整个血腔定殖阶段免疫逃避蛋白Mcl1基因和血腔定殖Colonization of hemocoel 1基因在WT和ΔMrsps-cp中的表达量高于ΔMrsps。共检测到13 249个大蜡螟基因,其中4 026个差异表达基因;KEGG注释分析显示大量差异表达基因富集到内分泌系统和免疫系统等途径;深入分析发现22个差异表达基因归属于Toll和Imd信号通路,其中18个基因在ΔMrsps侵染的大蜡螟中表达上调,表明ΔMrsps侵染大蜡螟过程中更易引起免疫系统的激活。【结论】揭示了Mrsps在罗伯茨绿僵菌血腔定殖阶段作用的分子机制,为进一步揭示精胺在真菌中的作用机理提供了理论基础。 相似文献
11.
Dong-Hoon Kang Periasamy Anbu Won-Ho Kim Byung-Ki Hur 《Biotechnology and Bioprocess Engineering》2008,13(4):483-490
Thraustochytrium aureum ATCC 34304 produces a high level of polyunsaturated fatty acids (PUFAs), which are typically synthesized by strings of reactions
catalyzed by desaturase and elongase enzymes. In this study, the genes related to the biosynthesis of PUFAs were investigated
and targeted to enable optimization of the production of PUFAs. To the best of our knowledge, this is first study to evaluate
the co-expression of genes TaElo, Tad5, and Tad4genes derived from T. aureum. We found that C22 PUFAs such as docosapentaenoic acid (DPA, C22:5n–6) and docosahexaenoic acid (DHA, 22:6n–3) were synthesized
from γ-linolenic acid (GLA, C18:3n–6) and stearidonic acid (SDA, C18:4n–3), respectively, as exogenous substrates via a series of
reactions catalyzed by an Elo-like enzyme and Δ5, Δ4-desaturase enzymes. In addition, the results of this study revealed that
the TaElo gene could synthesize the Δ6-and Δ5-elongation products. Taken together, these results confirmed that the Elo-like enzyme
was involved in multiple reactions leading to the production of PUFAs and that the TaElo, Tad5, and Tad4 genes were capable of functioning together to produce DPA and DHA using GLA and SDA. 相似文献
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In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae
relA
+ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of
(p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp0 strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient
poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial
growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae.
Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007. 相似文献
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Krzysztof Kur Iwona Gabriel Joachim Morschhäuser Francesco Barchiesi Elisabetta Spreghini Sławomir Milewski 《Mycopathologia》2010,170(6):397-402
Two genes, LYS21 and LYS22, encoding isoforms of homocitrate synthase, an enzyme catalysing the first committed step in the lysine biosynthetic pathway,
were disrupted in Candida albicans using the SAT1 flipper strategy. The double null lys21Δ/lys22Δ mutant lacked homocitrate synthase activity and exhibited lysine auxotrophy in minimal media that could be fully rescued
by the addition of 0.5–0.6 mM l-lysine. On the other hand, its virulence in vivo in the model of disseminated murine candidiasis appeared identical to that
of the mother, wild-type strain. These findings strongly question a possibility of exploitation of homocitrate synthase and
possibly also other enzymes of the lysine biosynthetic pathway as targets in chemotherapy of disseminated fungal infections. 相似文献
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Zhong-peng Guo Liang Zhang Zhong-yang Ding Zheng-Xiang Wang Gui-Yang Shi 《Applied microbiology and biotechnology》2009,82(2):287-292
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. 相似文献
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Photobacterium damsela α2,6-sialyltransferase was cloned as N- and C- His-tagged fusion proteins with different lengths (16–497 aa or 113–497 aa). Expression and activity assays indicated that
the N-terminal 112 amino acid residues of the protein were not required for its α2,6-sialyltransferase activity. Among four truncated
forms tested, N-His-tagged Δ15Pd2,6ST(N) containing 16–497 amino acid residues had the highest expression level. Similar to the Δ15Pd2,6ST(N),
the shorter Δ112Pd2,6ST(N) was active in a wide pH range of 7.5–10.0. A divalent metal ion was not required for the sialyltransferase
activity, and the addition of EDTA and dithiothreitol did not affect the activity significantly.
Mingchi Sun and Yanhong Li contributed equally to this work. 相似文献
16.
Jae-Cheol Lee Periasamy Anbu Won-Ho Kim Myung-Ju Noh Su-Jin Lee Jeong-Woo Seo Byung-Ki Hur 《Biotechnology and Bioprocess Engineering》2008,13(5):524-532
The Δ9-elongase isolated from Thraustochytrium aureum, which contains a high level of polyunsaturated fatty acids (PUFAs), was demonstrated to be associated with the synthesis
of C20 PUFAs. The TaELO gene contains a 825 bp ORF that encodes a protein of 274 amino acids that shares a high similarity with other PUFA elongases.
The expression of the TaELO gene in Pichia pastoris resulted in the elongation of linoleic acid (LA, C18:2; n-6) and α-linolenic acid (ALA, C18:3; n-3) to eicosadienoic acid
(EDA, C20:2; n-6) and eicosatrienoic acid (ETrA, C20:3; n-3), respectively. The endogenous conversion rate of LA and ALA to
EDA and ETrA was 32.68 and 38.57%, respectively. In addition, TaELO was also able to synthesize eicosenoic acid (C20:1; n-9) from oleic acid (OA, C18:1; n-9), even though the conversion level
was low (2.81%). Furthermore, TaELO was able to carry out the 6Δ-elongation of γ-linolenic acid (GLA, C18:3; n-6) to dihomo-γ-linolenic acid (DGLA, C20:3; n-6)
and Δ5-elongation of eicosapentaenoic acid (EPA, C20:5; n-3) to docosapentaenoic acid (DPA, C22:5; n-3). The conversion rate
of GLA to DGLA and EPA to DPA were 93 and 28.36%, respectively. The TaELO protein was confirmed to have multifunctional activities, such as Δ9, Δ6, and Δ5-elongations as well as the elongation of
monounsaturated fatty acid. 相似文献
17.
The genes involved in biosynthesis of the major compatible solute ectoine (1,4,5,6-tetrahydro-2-methylpyrimidine carboxylic acid) in halotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z” were studied. The complete nucleotide sequences of the structural genes encoding l-aspartokinase (Ask), l-2,4-diaminobutyric acid transaminase (EctB), l-2,4-diaminobutyric acid acetyltransferase (EctA), and l-ectoine synthase (EctC) were defined and shown to be transcribed as a single operon ectABCask. Phylogenetic analysis revealed high sequence identities (34–63%) of the Ect proteins to those from halophilic heterotrophs with the highest amino acid identities being to Vibrio cholerae enzymes. The chromosomal DNA fragment from “M. alcaliphilum 20Z” containing ectABC genes and putative promoter region was expressed in Escherichia coli. Recombinant cells could grow in the presence of 4% NaCl and synthesize ectoine. The data obtained suggested that despite the ectoine biosynthesis pathway being evolutionary well conserved with respect to the genes and enzymes involved, some differences in their organization and regulation could occur in various halophilic bacteria.Dedicated to the 70th birthday of Professor Gerhard Gottschalk who inspired our studies on methylotrophic haloalkaliphiles. 相似文献
18.
Joon-Ryeol Yang Eunjung Song Byung-Gee Kim Eung-Soo Kim Jae-Kyung Sohng Min-Kyu Oh 《Biotechnology and Bioprocess Engineering》2008,13(6):738-744
Doxorubicin (DXR) and daunorubicin (DNR) are anthracycline antibiotics produced by Streptomyces peucetius and widely used as cancer chemotherapeutic agents. To improve their productivity, regulation of DXR/DNR synthesis genes as
well as central metabolic pathway genes must be understood more clearly. So far, studies have focused on DXR/DNR gene regulation.
To investigate the correlation between the central metabolic pathway genes and DXR/DNR productivity, we selected 265 genes
involved in glycolysis, fermentation, the citric acid cycle, butanoate metabolism, etc., and searched for their sequences in the S. peucetius genome by comparing gene sequences to those of Streptomyces coelicolor. The homologous genes were amplified by PCR and arrayed on glass microarray slides. Gene expression was monitored under two
different growth media conditions, R2YE and NDYE. Genes involved in the production of malonyl-CoA and propionyl-CoA, the main
precursors for doxorubicin synthesis, were mainly upregulated in NDYE media. Genes related to acetyl-CoA and the urea cycle
were also upregulated. These changes in gene expression were confirmed by real-time RT-PCR. 相似文献
19.
Okino S Suda M Fujikura K Inui M Yukawa H 《Applied microbiology and biotechnology》2008,78(3):449-454
In mineral salts medium under oxygen deprivation, Corynebacterium glutamicum exhibits high productivity of l-lactic acid accompanied with succinic and acetic acids. In taking advantage of this elevated productivity, C. glutamicum was genetically modified to produce d-lactic acid. The modification involved expression of fermentative d-lactate dehydrogenase (d-LDH)-encoding genes from Escherichia coli and Lactobacillus delbrueckii in l-lactate dehydrogenase (l-LDH)-encoding ldhA-null C. glutamicum mutants to yield strains C. glutamicum ΔldhA/pCRB201 and C. glutamicum ΔldhA/pCRB204, respectively. The productivity of C. glutamicum ΔldhA/pCRB204 was fivefold higher than that of C. glutamicum ΔldhA/pCRB201. By using C. glutamicum ΔldhA/pCRB204 cells packed to a high density in mineral salts medium, up to 1,336 mM (120 g l−1) of d-lactic acid of greater than 99.9% optical purity was produced within 30 h. 相似文献
20.
ω3-fatty acid desaturase and Δ12-fatty acid desaturase of Pichia pastoris with distinguishable regioselectivity and high degree of sequence similarity were chosen for regioselectivity research. Chimeras
were constructed in which Histidine-rich boxes 1, 2 and the carboxyl terminal region of ω3-fatty acid desaturase were replaced with corresponding region of Δ12-fatty acid desaturase. The replacement was found to result in a change of regioselectivity from ωy to x + 3 by functionally characterizing these chimeric enzymes in Saccharomyces cerevisae strain INVScI. Using site-directed mutagenesis, we further demonstrated that seven conserved amino acids of ω3-fatty acid desaturase within the first two Histidine-rich regions are responsible for the regioselectivity switch. Therefore,
the regioselectivity of fatty acid desaturases may be better understood by investigating the evolutionary relationships of
different fatty acid desaturases.
Dongsheng Wei is the partake of first-author’s profits. 相似文献