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1.
中链脂肪酸(C6-C12)衍生的化学品包括脂肪醇、脂肪烃、中链酯、ω-修饰脂肪酸等,这些化合物是生物燃料、聚合物、日用化学品、特种化学品的重要组分。天然微生物底盘不能合成中链脂肪酸,而通过操纵脂肪酸合成、逆β-氧化等碳链延伸途径,尤其是表达生成游离中链脂肪酸的硫酯酶,可使大肠埃希菌、酿酒酵母等微生物细胞合成超过1 g/L的中链脂肪酸。引入脂肪酸衍生反应,如羧基还原、脱羧、ω-氧化等,可合成许多中链化学品。本文综述了中链化学品合成的酶学基础以及代谢工程策略,为中链化学品高效生物制造提供参考和思路。 相似文献
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热带假丝酵母转化烷烃过程中P450酶活的研究 总被引:6,自引:2,他引:4
a-、ω-长链二元酸(α-、ω-Long Chin Dicarboxylic Acid,DCA)是一种重要的化工原料,是合成工程塑料、香料、耐寒性增塑剂、涂料和液晶等物质的主要原料.目前,人们主要通过热带假丝酵母(Candidatropicalis)代谢烷烃来生产从DCA11到DCA18等不同碳链长的二元酸[1,2].多年来在各种微生物,尤其是假丝酵母的烷烃氧化途径方面有大量的研究[3,4].在假丝酵母转化烷烃生成长链二元酸的代谢过程中[5-7],烷烃被吸引进入细胞后,首先经过细胞色素P450酶(Cy-tochrome P450)氧化生成a-一元醇,再进一步被氧化生成a-一元酸,引过程称为a-氧化. 相似文献
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热带假丝酵母代谢烷烃过程中的β-氧化和代谢调控 总被引:5,自引:0,他引:5
热带假丝酵母 (Candidatropicalis)能利用烷烃作唯一碳源和能源。当以烷烃或脂肪酸为碳源时 ,在细胞内可形成大量的过氧化物酶体 (peroxisome) ,同时诱导生成脂肪酸 β 氧化酶系 ,当以葡萄糖为碳源时 ,则极少有过氧化物酶体形成[1] ,一些C .tropicalis能氧化烷烃生成长链二元酸 (longchaindicarboxylicacid ,DCA)。由于这些特征 ,人们从酶学、分子生物学和实际应用等方面对这种酵母进行了深入研究 ,并阐述了C .tropicalis代谢烷烃的途径、脂肪酸β 氧化酶系… 相似文献
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热带假丝酵母(Candida tropicalis)能利用烷烃作唯一碳源和能源.当以烷烃或脂肪酸为碳源时,在细胞内可形成大量的过氧化物酶体(peroxisome),同时诱导生成脂肪酸β-氧化酶系,当以葡萄糖为碳源时,则极少有过氧化物酶体形成[1],一些C.tropicalis能氧化烷烃生成长链二元酸(long chain dicarboxylicacid,DCA).由于这些特征,人们从酶学、分子生物学和实际应用等方面对这种酵母进行了深入研究,并阐述了C.tropicalis代谢烷烃的途径、脂肪酸β-氧化酶系的生理功能及其几种重要酶的基因结构和酶活性的调控,阐明了它与哺乳动物细胞在脂肪酸代谢及调控方面上的差异;通过C.tropicalis突变株的筛选和发酵工艺的优化,使长链二元酸发酵技术实现了产业化[2,3]. 相似文献
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哺乳动物因为缺乏Δ-12和ω-3脂肪酸脱氢酶,不能自身合成必需的多不饱和脂肪酸.目前,通过转基因技术在哺乳动物体内表达ω-3脂肪酸脱氢酶,能将长链的n-6多不饱和脂肪酸转化成n-3多不饱和脂肪酸,造成体内长链的n-6多不饱和脂肪酸含量显著减低.本研究通过自我剪切2A肽介导Δ-12和ω-3脂肪酸脱氢酶(FAT-2和FAT-1)以及人过氧化氢酶(human catalase,hCAT)在小鼠的肌肉同时表达.结果表明,转基因小鼠肌肉中长链n-3多不饱和脂肪酸含量提高2.6倍,长链n-6多不饱和脂肪酸含量没有显著变化,而n-6/n-3比例显著降低(P < 0.01).同时蛋白质印迹检测到人过氧化氢酶hCAT在小鼠的肌肉组织中表达,且过氧化氢酶活性比野生型小鼠显著提高(P < 0.01). 相似文献
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长链脂肪酸在哺乳动物体内具有广泛的生理功能,特别是在生物膜的形成和动态特性维持中发挥着不可或缺的作用,同时,作为能量产生的重要原料,长链脂肪酸在保持心脏和骨骼肌正常功能方面也具有极其重要的作用.脂肪酸转运蛋白家族(fatty acid transport proteins,FATPs)是一组膜蛋白,在心脏、肝脏、肌肉和小肠等脂肪酸代谢活跃的组织器官中均有表达.已有研究表明,FATPs在长链脂肪酸的摄取和代谢调节中发挥着重要作用,现对FATPs的组织分布、结构特点、功能、作用机制及其与人类疾病的关系等方面进行综述. 相似文献
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Vanhanen S West M Kroon JT Lindner N Casey J Cheng Q Elborough KM Slabas AR 《The Journal of biological chemistry》2000,275(6):4445-4452
The yeast Candida cloacae is capable of growing on alkanes and fatty acids as sole carbon sources. Transfer of cultures from a glucose medium to one containing oleic acid induced seven proteins of M(r) 102,000, 73,000, 61,000, 54,000, and 46,000 and two in the region of M(r) 45,000 and repressed a protein of M(r) 64,000. The induction of the M(r) 73,000 protein reached a 7-fold maximum 24 h after induction. The protein was confirmed by its enzyme activity to be a long-chain fatty-acid alcohol oxidase (LC-FAO) and purified to homogeneity from microsomes by a rapid procedure involving hydrophobic chromatography. An internal peptide of 30 amino acids was sequenced. A 1100-base pair cDNA fragment containing the LC-FAO peptide coding sequence was used to isolate a single exon genomic clone containing the full-length coding sequence of an LC-FAO (fao1). The fao1 gene product was expressed in Escherichia coli and was translated as a functional long-chain alcohol oxidase, which was present in the membrane fraction. In addition, full-length coding sequences for a Candida tropicalis LC-FAO (faoT) and a second C. cloacae LC-FAO (fao2) were isolated. The DNA sequences obtained had open reading frames of 2094 (fao1), 2091 (fao2), and 2112 (faoT) base pairs. The derived amino acid sequences of fao2 and faoT showed 89.4 and 76.2% similarities to fao1. The fao1 gene is much more highly induced on alkane than is fao2. Although this study describes the first known DNA sequences encoding LC-FAOs from any source, there are unassigned Arabidopsis sequences and an unassigned Mycobacterium sequence in the GenBank(TM) Data Bank that show strong homology to the described LC-FAO sequences. The conservation of sequence between yeast, plants, and bacteria suggests that an as yet undescribed family of long-chain fatty-acid oxidases exists in both eukaryotes and prokaryotes. 相似文献
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Cheng Q Sanglard D Vanhanen S Liu HT Bombelli P Smith A Slabas AR 《Biochimica et biophysica acta》2005,1735(3):192-203
The industrial yeasts Candida tropicalis or Candida cloacae are able to grow on a variety of long chain alkanes and fatty acids as the sole carbon source. The complete oxidation of these substrates involves two sequential oxidative pathways: omega-oxidation, comprising the P450 alkane oxidase, a flavin-dependent membrane-bound long chain fatty alcohol oxidase [FAO] and a possible separate aldehyde oxidase [F.M. Dickinson, C. Wadforth, Purification and some properties of alcohol oxidase from alkane-grown Candida tropicalis, Biochem. J. 282 (1992) 325-331], and the beta-oxidation pathway, which utilises acylCoA substrates. We recently purified the membrane-bound long chain fatty alcohol oxidase FAO1 and confirmed it is also a c-type haemoprotein. Multiple isoforms may exist for many of these long chain fatty alcohol oxidases and the in vivo requirements for individual genes with respect to specific substrates are still being elucidated. In vitro reconstitution experiments have demonstrated that in Candida maltosa, the cytochrome P450 52A3 gene product can completely oxidise alkanes to dicarboxylic acids [U. Scheller, T. Zimmer, D. Becher, F. Schauer, W. Schunck, Oxygenation Cascade in Conversion of n-Alkanes to, -Dioic Acids Catalyzed by Cytochrome P450 52A3, J. Biol. Chem. 273 (1998) 32528-32534], potentially obviating requirements for a long chain alcohol oxidase. Here, we directly determine in vivo the role of the long chain alcohol oxidase (FAOT) in C. tropicalis, grown on a variety of substrates, followed by gene deletion. The faot double knockout has no detectable faot activity and is incapable of growth on octadecane, but it grows well on oleic acid, palmitic acid and shorter chain alkanes/fatty acids. A spontaneous mutation[s] may have occurred in the faot double gene knockout of C. tropicalis resulting in its inability to grow on oleic acid and hexadecane. The mutations demonstrate that different pathways of octadecane, hexadecane, oleic acid and palmitic acid utilisation exist in C. tropicalis. 相似文献
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We report the isolation and nucleotide (nt) sequence determination of a cDNA encoding the peroxisomal trifunctional beta-oxidation enzyme hydratase-dehydrogenase-epimerase (HDE) from the yeast Candida tropicalis pK233. Poly(A)+RNA isolated from C. tropicalis cells grown in oleic acid medium was used to construct a cDNA library in lambda gt11. The library was screened with a polyclonal antiserum against HDE. A recombinant was confirmed to encode HDE by hybridization-selection translation and immunoprecipitation. The HDE cDNA (HDE) has a single open reading frame of 2718 nt, encoding a protein of 905 amino acids, not including the initiator methionine. The Mr of the protein is 99,350. A partial gene duplication is believed to have occurred in the evolution of the HDE gene. Codon utilization in the gene is not random, with 86.0% of the amino acids specified by 23 preferentially used codons, a situation similar to that found in genes encoding peroxisomal catalase and the various fatty acyl-CoA oxidases from C. tropicalis. The increase in HDE activity in C. tropicalis cells grown in oleic acid medium as opposed to glucose medium is due, at least in part, to increased HDE-specific mRNA levels. 相似文献
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嗜铬粒蛋白(CGA)是存在于分泌细胞的由439个氨基酸组成的可溶性蛋白。近年的研究发现CGA的N端具有抗血管收缩、抗细菌和抗真菌的功能。为了寻找高效低毒的抗真菌片段,利用PCR技术扩增了编码人嗜铬粒蛋白N端1-76位氨基酸(CGA1-76)的DNA片段,并将之克隆进本实验构建的枯草杆菌诱导型表达载体pSBPTQ,获得含CGA1-76基因的重组质粒pSVTQ,转化蛋白酶三缺陷的枯草杆菌DB403。经蔗糖诱导后,CGA1-76片段在枯草杆菌DB403(pSVTQ)中获得表达,产物分泌到细胞外,分泌量约为5mg/L,占总分泌蛋白的133% 。测试了表达产物对几种丝状真菌和酵母的抑制作用,发现在4μmol/L的浓度下,枯草杆菌表达的重组CGA1-76对镰刀菌、链格孢霉及白假丝酵母有明显的抑制作用。 相似文献
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Occurrence of fatty alcohol oxidase in alkane-and fatty-acid-utilising yeasts and moulds 总被引:1,自引:0,他引:1
Glen D. Kemp F. Mark Dickinson Colin Ratledge 《Applied microbiology and biotechnology》1994,40(6):873-875
Preparations of membrane fractions from 16 yeasts and three moulds were assayed for long-chain fatty alcohol oxidase (FAOD) activities after being grown on hexadecane or glucose and, in nine cases, on oleic acid. The enzyme was usually repressed in glucose-grown cells but in Candida bombicola ATCC 22 214 and Debaryomyces hansenii NCYC 33 appeared to be constitutive. Highest activities occurred in C. tropicalis and D. polymorphus (about 0.8 unit/mg protein) grown on hexadecane. Growth of yeasts on oleic acid partially induced FAOD activity but not with the moulds. In two strains of Yarrowia lipolytica (DSM 3286 and CBS 2076) no activity of FAOD was found but this could have been due to the known photo-lability of the enzyme. FAOD from different species shared similar characteristics with respect to substrate specificity and pH optimum.
Correspondence to: C. Ratledge 相似文献
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The thermotolerant yeast Candida thermophila SRY-09 isolated from Thailand produces an extracellular lipase that hydrolyses various triglycerides. To clone the gene encoding the lipase, Saccharomyces cerevisiae was transformed with a C. thermophila genomic library and screened for lipase activity on medium containing olive oil emulsion and rhodamine B. One C. thermophila lipase gene (CtLIP) was found that contained an ORF of 1317 bp encoding a deduced polypeptide of 438 amino acids. Candida thermophila lipase contained a Gly-Asp-Ser-Gln-Gly motif which matched the consensus Gly-X-Ser-X-Gly conserved among lipolytic enzymes. Heterologous expression of the cloned CtLIP under the control of the alcohol oxidase gene (AOX1) promoter in the methylotrophic yeast Pichia pastoris, and enzymatic measurements confirmed the function of the respective protein as a lipase. The recombinant CtLIP could hydrolyse various substrates at high temperature (55 degrees C) with higher efficiency than at 37 or 45 degrees C and preferentially hydrolysed two-positional ester bonds. As with C. thermophila, the heterologously expressed lipase was secreted into the medium by Pichia pastoris. 相似文献
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Functional identification of AtFao3, a membrane bound long chain alcohol oxidase in Arabidopsis thaliana 总被引:2,自引:0,他引:2
The Arabidopsis thalina genome database was searched for homologues of the Candida cloacae fao1 gene which encodes a membrane bound, flavin-containing, hydrogen peroxide generating, long chain alcohol oxidase. This gene has not been isolated from plants or animals. Four putative candidates were found in the database but their function has not been proven. The cDNAs for two of them were cloned by RT-PCR from Arabidopis suspension culture and one of them [AtFAO3] was overexpressed in Escherichia coli and shown to functionally express long chain alcohol oxidase activity. The protein has been solubilised and retains biological activity thereby preparing the way for crystallographic studies. This is the first functional proof identifying a long chain alcohol oxidase in higher plants. 相似文献
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烟实夜蛾脂肪酸结合蛋白基因的克隆、序列分析与表达 总被引:1,自引:0,他引:1
应用RT-PCR技术,从烟实夜蛾Helicoverpa assulta幼虫脂肪体组织和血细胞总RNA中反转录扩增脂肪酸结合蛋白(fatty-acid binding protein,FABP)基因的cDNA片段,克隆到原核表达载体pGEX-4T-2上,转化大肠杆菌BL21(DE3),用IPTG进行诱导表达并进行检测。结果表明:扩增得到的片段全长399 bp(GenBank登录号为DQ299942),编码132个氨基酸残基,预测分子量15.0 kD,等点电5.83。FABP融合了GST。原核表达后经电泳检测到约41 kD大小的外源蛋白,Western blot检测表明是目的蛋白。 相似文献
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Acyl-CoA oxidase contains two targeting sequences each of which can mediate protein import into peroxisomes. 总被引:31,自引:4,他引:27 下载免费PDF全文
Acyl-CoA oxidase is a major induced enzyme in peroxisomes of Candida tropicalis grown on fatty acids. The gene, POX4, encoding acyl-CoA oxidase was expressed in vitro, and the resulting polypeptide was imported into purified peroxisomes in a temperature-dependent fashion. Plasmids containing fragments of POX4 were prepared, expressed and the polypeptides tested for import into peroxisomes. We identified two regions of acyl-CoA oxidase (amino acids 1-118 and 309-427) that contained information that specifically targeted fragments of acyl-CoA oxidase to peroxisomes. The corresponding regions of the gene were fused to cDNA encoding the cytosolic enzyme dihydrofolate reductase (DHFR), and the expressed fusion proteins were likewise imported into peroxisomes. DHFR itself neither bound to, nor was imported into peroxisomes. Thus, there are at least two regions of peroxisomal targeting information in the acyl-CoA oxidase gene. 相似文献
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Degradation of microbiodies in the methanolutilizing yeastCandida boidinii was mainly studies by electron microscopical observation. The yeast cells precultured on methanol medium contained five to six microbodies per section and showed high activities of alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase. When the precultured cells were transferred into an ethanol medium the number of microbodies and concomitantly the activities of alcohol oxidase and catalase decreased. After 6 h of cultivation microbodies were hardly detected. Also the activity of alcohol oxidase was not measurable and catalase activity was reduced to one tenth, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase decreased only to about 70%. Experiments with methanol-grown cells transferred into an ethanol medium without nitrogen source indicated that the inactivation of alcohol oxidase and catalase does not require protein synthesis. However, the reappearance of these enzymes is presumably due to de novo protein synthesis as shown by experiments with cycloheximide. 相似文献