北京棒杆菌转酮酶:基因克隆、序列分析与表达

【目的】转酮酶是非氧化磷酸戊糖途径中的关键酶。从北京棒杆菌(Corynebacterium pekinense PD-67)中克隆转酮酶(transketolase,EC2.2.1.1,TK)基因,并将转酮酶基因在C.pekinense PD-67中进行表达,研究增加转酮酶活性对C.pekinense PD-67生理特性的影响。【方法】分别以C.pekinense野生株AS1.299和突变株PD-67的基因组为模板,用PCR方法扩增tkt的全基因序列和前端控制序列;通过pAK6载体提高tkt基因在C.pekinense PD-67中的拷贝数,从而提高C.pekinense PD-67中转酮酶的活性。【结果】tkt基因核苷酸序列及其编码的氨基酸序列与结构分析结果表明,C.pekinense突变株PD-67与野生株AS1.299相比较,二者调控序列及结构基因核苷酸序列完全一致。与谷氨酸棒杆菌ATCC13032相比较,突变株PD-67的氨基酸序列有5个氨基酸差异,其中4个位于与辅因子硫胺素焦磷酸结合的结构域内。突变株PD-67来源的tkt基因在北京棒杆菌PD-67中得到了表达,重组菌转酮酶比活力比对照菌株提高了2倍。C.pekinense PD-67(pTK3)与对照菌株PD-67(pAK6)相比,生长加快,L-色氨酸的最终积累量也较高。【结论】本工作从C.pekinense1.299和PD-67中克隆到tkt基因,并实现tkt基因的同源表达。适当提高菌株转酮酶活力,有助于菌体生长和色氨酸积累。     

北京棒杆菌DAHP合成酶Ⅰ基因的克隆﹑序列分析及表达

摘要：【目的】从北京棒杆菌(Corynebacterium pekinense)中克隆DAHP合成酶 (EC 2.5.1.54,3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, DS)Ⅰ基因,对其进行功能验证;并将DAHP合成酶Ⅰ基因在C. pekinensePD-67进行同源表达,研究该酶的比活力与生长的相关性。【方法】分别以C. pekinense野生株AS1.299和突变株PD-67的基因组为模板,用PCR方法扩增了DAHP合成 酶Ⅰ的全基因序列aroⅠ和前端控制序列;通过pAK6载体提高DAHP合成酶Ⅰ基因在C. pekinen-sePD-67中的拷贝数实现其同源表达。【结果】核苷酸序列分析结果表明,C. pekinense 野生株AS1.299与突变株PD-67相比较,DAHP合成酶Ⅰ基因序列完全一样;通过PCR方法得到的DAHP合成酶Ⅰ基因结构功能完整,能与DAHP合成酶完全缺陷的E.coli 3257实现异源互补。突变株PD-67来源的DAHP合成酶Ⅰ基因在重组菌PD-67( pAD1)中进行了表达,在稳定期初期重组菌PD-67( pAD1)的DAHP合成酶Ⅰ的酶比活力比同期的对照菌株PD-67( pAK6)中的该酶酶比活力提高了约5倍。【结论】本工作首次证实了 C. pekinense 1.299和PD-67中存在DAHP合成酶Ⅰ基因,异源互补试验证明扩增得到的DNA片段编码DAHP合成酶Ⅰ,酶学性质研究表明DAHP合成酶Ⅰ基因在C. pekinensePD-67中的同源表达将有助于提高该菌的色氨酸积累。     

北京棒杆菌DAHP合成酶Ⅰ基因的克隆、序列分析及表达

[目的]从北京棒杆菌(Corynebacterium pekinense)中克隆DAHP合成酶(EC 2.5.1.54,3-deoxy-D-arabino-heptulosonate-7-phosphate synthase,DS)Ⅰ基因,对其进行功能验证;并将DAHP合成酶Ⅰ基因在C.pekinensePD-67进行同源表达,研究该酶的比活力与生长的相关性.[方法]分别以C.pekinense野生株ASl.299和突变株PD-67的基因组为模板,用PCR方法扩增了DAHP合成酶Ⅰ的全基因序列aro Ⅰ和前端控制序列;通过pAK6载体提高DAHP合成酶Ⅰ基因在C.pekinen-sePD-67中的拷贝数实现其同源表达.[结果]核苷酸序列分析结果表明,C. pekinense野生株AS1.299与突变株PD-67相比较,DAHP合成酶Ⅰ基因序列完全一样;通过PCR方法得到的DAHP合成酶Ⅰ基因结构功能完整,能与DAHP合成酶完全缺陷的E.coli 3257实现异源互补.突变株PD-67来源的DAHP合成酶Ⅰ基因在重组菌PD-67(pAD1)中进行了表达,在稳定期初期重组菌PD-67(pAD1)的DAHP合成酶Ⅰ的酶比活力比同期的对照菌株PD-67(pAK6)中的该酶酶比活力提高了约5倍.[结论]本工作首次证实了C. pekinense 1.299和PD-67中存在DAHP合成酶Ⅰ基因,异源互补试验证明扩增得到的DNA片段编码DAHP合成酶Ⅰ,酶学性质研究表明DAHP合成酶Ⅰ基因在C. pekinensePD-67中的同源表达将有助于提高该菌的色氨酸积累.     

磷酸烯醇式丙酮酸羧化酶基因的敲除对北京棒杆菌PD-67的生理代谢的影响

【目的】为了优化L-色氨酸合成的前体供应,构建北京棒杆菌PD-67(Corynebacterium pekinense PD-67)磷酸烯醇式丙酮酸羧化酶(EC:4.1.1.31,phosphoenolpyruvate carboxylase,PEPCx)基因ppc敲除的菌株,并研究ppc基因敲除对菌株生理特性的影响。【方法】运用PCR技术扩增ppc基因的上游和下游序列,构建带有目标基因内部缺失的基因整合载体。通过同源重组技术将C.pekinense PD-67的ppc基因敲除,构建ppc基因缺陷突变株C.pekinense PD-67-Δppc。通过摇瓶发酵研究突变株的生理特性,并测定突变株丙酮酸激酶和丙酮酸羧化酶的活性。【结果】PCR验证和PEPCx活性分析结果表明,筛选到ppc缺陷的突变株。摇瓶发酵结果表明,与出发菌株相比,突变株的生长速率下降,生物量降低20%,L-色氨酸积累降低62%,丙酮酸激酶活力提高,而丙酮酸羧化酶活力下降。【结论】C.pekinense PD-67的ppc基因敲除以后,对菌株的代谢影响较大。仅通过阻断PEPCx催化的回补途径,减少磷酸烯醇式丙酮酸的分支代谢,不能提高该菌株L-色氨酸的积累。     

钝齿棒杆菌N-乙酰谷氨酸激酶基因的克隆、序列分析及表达

以钝齿棒杆菌(Corynebacterium crenatum)野生株AS 1.542及产精氨酸突变株971.1的基因组为模板,用PCR方法扩增出N-乙酰谷氨酸激酶基因(argB)片段。核酸序列分析结果表明,该片段全长1505bp,包含一个ORF,推测此ORF区编码一条317个氨基酸的多肽,分子量为33.6kDa。C.crenatum野生株AS 1.542与突变株971.1的argB基因序列比较,发现只在结构区有一个核苷酸的差别但没有引起氨基酸变化。野生株AS 1.542argB基因的编码区核苷酸序列与C.glutamicumATCC 13032、Corynebacterium efficiensYS-314和Escherichia colik12的同源性分别是99.89%、76.62%和37.94%,而氨基酸同源性分别是100%、78.55%和25.25%。在C.crenatum argB基因上游存在启动子区域。经IPTG诱导该基因在棒杆菌中得到有效表达,野生株AS 1.542为宿主的重组子酶活明显提高。突变株971.1为宿主的重组菌酶活提高一倍,精氨酸积累提高约25%。     

北京棒杆菌芳香族氨基酸转运蛋白基因敲除对L-色氨酸积累的影响

[目的]为了减少北京棒杆菌PD-67(Corynebacterium pekinense PD-67)从细胞外吸收色氨酸,降低细胞内色氨酸库的浓度,从而使色氨酸的反馈控制作用减弱,增加胞外L-色氨酸的积累量,构建北京棒杆菌PD-67的芳香族氨基酸转运蛋白基因aroP敲除的菌株,研究aroP基因敲除对菌株L-色氨酸积累的影响.并进一步研究在aroP敲除菌株中表达邻氨基苯甲酸合成酶(AS)基因对L-色氨酸积累的影响.[方法]运用PCR技术扩增aroP基因,与整合质粒连接后,用限制性内切酶法构建带有内部片段缺失的aroP基因的敲除载体.利用同源重组技术,敲除北京棒杆菌PD-67的aroP基因,构建菌株PD-67 ΔaroP,并用带有aroP基因的表达载体对PD-67ΔaroP进行互补验证.采用PCR技术扩增AS基因,与表达载体连接构建重组质粒.将重组质粒转入菌株PD-67ΔaroP,构建工程菌株PD-67 ΔaroP/pXAS.通过摇瓶发酵研究PD-67 AaroP和PD-67 ΔaroP/pXAS的发酵特性.[结果]经PCR验证获得了aroP基因缺陷的菌株.摇瓶发酵结果表明,与出发菌株相比,PD-67ΔaroP的L-色氨酸的积累量提高了65％.酶活分析结果表明,AS基因在菌株PD-67 △aroP中得到表达.AS基因表达使工程菌单位菌体产酸率提高了25.6％.[结论]北京棒杆菌PD-67中芳香族氨基酸转运蛋白基因arop的敲除能够提高胞外L-色氨酸的积累量.在arop基因敲除菌中表达AS基因,可以进一步提高工程菌的产酸率.     

过表达pps基因和aroG~(fbr)基因对北京棒杆菌L-色氨酸合成的影响

【目的】通过增加北京棒杆菌(Corynebacterium pekinense)PD-67芳香族氨基酸合成的前体物质磷酸烯醇式丙酮酸(PEP)的供应,解除终产物对芳香族氨基酸合成途径中第一个酶同时也是关键酶3-脱氧-D-阿拉伯庚酮糖-7-磷酸合酶(DS)的反馈抑制并提高抗反馈抑制的DS的活力,使碳流更多地流向芳香族氨基酸合成途径,从而积累更多L-色氨酸。【方法】运用PCR技术扩增北京棒杆菌PD-67磷酸烯醇式丙酮酸合酶基因pps,与表达载体连接构建重组质粒pXPS;运用重叠PCR技术定点突变大肠杆菌(Escherichia coli)受苯丙氨酸调控的DS基因aroG,使相应的编码氨基酸序列发生突变:Leu175Asp,新的基因命名为aroGfbr,与表达载体连接构建重组质粒pXA;构建pps和aroGfbr的共表达重组质粒pXAPS。将3个重组质粒分别转入菌株PD-67,构建工程菌株PD-67/pXPS、PD-67/pXA和PD-67/pXAPS。通过摇瓶发酵研究工程菌株的发酵特性。【结果】酶活分析结果表明,pps基因和aroGfbr基因在北京棒杆菌PD-67中均实现了表达。工程菌株PD-67/pXA粗酶液DS抗反馈抑制分析表明,AroGfbr已解除酪氨酸和苯丙氨酸的反馈抑制。过表达pps基因和aroGfbr基因分别使工程菌L-色氨酸产量提高12.1%和26.8%,双基因共表达可使工程菌的产酸量提高35.9%。【结论】北京棒杆菌PD-67pps基因的过表达以及大肠杆菌来源的解除反馈抑制的aroGfbr的过表达均有助于增加PD-67 L-色氨酸的合成,而双基因的共表达可以进一步提高L-色氨酸的积累量。     

钝齿棒杆菌argR基因克隆、表达及其重组菌发酵产精氨酸研究

钝齿棒杆菌(Corynebacterium crenatum)AS.M7是筛选获得的一株高产精氨酸生产菌株。ArgR是精氨酸合成过程中的一种调控蛋白。为进一步验证其在钝齿棒杆菌中对精氨酸合成量的影响,利用特异性引物,分别扩增标准菌C. creantum AS 1.542和诱变菌C. creantum AS.M7的argR全长基因,测序后比较二者的差异;结果表明标准菌argR基因ORF全长516 bp,编码一个含172个氨基酸残基的蛋白;而诱变菌argR基因的109位碱基由C替换为T,导致ArgR蛋白在钝齿诱变菌中表达被提前终止。同时,将来源于标准菌的argR基因连接到穿梭表达载体pXMJ19中,电击转化至诱变菌C. crenatum AS.M7 得到重组菌株,用摇瓶发酵的方法观测重组菌产精氨酸量的变化。SDS-PAGE和Western blot分析证明标准菌的argR基因在诱变菌中得到了表达。对重组诱变菌产精氨酸量进行了测定,结果显示:产精氨酸能力由原来7.8 mg/ml下降至2.5 mg/ml,下降了约67.9%。     

钝齿棒杆菌天冬氨酸激酶基因的克隆和序列分析

运用PCR方法,从野生型钝齿棒杆菌株(Corynebacterium crenatum)AS1542及具有AEC抗性的突变株CD945染色体上分别扩增出天冬氨酸激酶(AK)基因(ask),构建了重组质粒。核苷酸序列分析表明,C.crenatum AS1542AK基因与C.crenatum CD945相比,第1199位的碱基由T变为C,引起酶蛋白β亚基第80位氨基酸从亮氨酸变成脯氨酸。该氨基酸的突变在蛋白结构上位于ACT结构域内,该区受赖氨酸调控。C.crenatum AS1542的AK基因的编码区核苷酸序列与C.glutamicum\,C.flavum及B.lactofermentum相比,同源性分别为97.23%、97.55%和97.62%,酶蛋白氨基酸序列的同源性分别为99.76%、99.52%和99.76%。但在AK基因的启动子上游序列部分与其它棒杆菌相比有较大差异。     

体外定点突变法构建凝血因子Ⅶ C329G突变型真核表达载体

以野生型凝血因子Ⅶ（FⅦ）真核表达载体为模板,通过定点突变方法构建FⅦC329G突变型表达载体;序列分析鉴定C329G突变重组子。重组子目的基因的序列分析结果显示FⅦ基因编码的第329个半胱氨酸残基被甘氨酸残基替换（C329G）,即成功构建了FⅦC329G突变型真核表达载体,为下一步在哺乳类细胞中表达该突变型FⅦ,并对其进行功能性研究提供了条件。     

Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications.

Two anthranilate synthase gene pairs have been identified in Pseudomonas aeruginosa. They were cloned, sequenced, inactivated in vitro by insertion of an antibiotic resistance gene, and returned to P. aeruginosa, replacing the wild-type gene. One anthranilate synthase enzyme participates in tryptophan synthesis; its genes are designated trpE and trpG. The other anthranilate synthase enzyme, encoded by phnA and phnB, participates in the synthesis of pyocyanin, the characteristic phenazine pigment of the organism. trpE and trpG are independently transcribed; homologous genes have been cloned from Pseudomonas putida. The phenazine pathway genes phnA and phnB are cotranscribed. The cloned phnA phnB gene pair complements trpE and trpE(G) mutants of Escherichia coli. Homologous genes were not found in P. putida PPG1, a non-phenazine producer. Surprisingly, PhnA and PhnB are more closely related to E. coli TrpE and TrpG than to Pseudomonas TrpE and TrpG, whereas Pseudomonas TrpE and TrpG are more closely related to E. coli PabB and PabA than to E. coli TrpE and TrpG. We replaced the wild-type trpE on the P. aeruginosa chromosome with a mutant form having a considerable portion of its coding sequence deleted and replaced by a tetracycline resistance gene cassette. This resulted in tryptophan auxotrophy; however, spontaneous tryptophan-independent revertants appeared at a frequency of 10(-5) to 10(6). The anthranilate synthase of these revertants is not feedback inhibited by tryptophan, suggesting that it arises from PhnAB. phnA mutants retain a low level of pyocyanin production. Introduction of an inactivated trpE gene into a phnA mutant abolished residual pyocyanin production, suggesting that the trpE trpG gene products are capable of providing some anthranilate for pyocyanin synthesis.     

Identification and nucleotide sequence of the Leptospira biflexa serovar patoc trpE and trpG genes.

Leptospira biflexa is a representative of an evolutionarily distinct group of eubacteria. In order to better understand the genetic organization and gene regulatory mechanisms of this species, we have chosen to study the genes required for tryptophan biosynthesis in this bacterium. The nucleotide sequence of the region of the L. biflexa serovar patoc chromosome encoding the trpE and trpG genes has been determined. Four open reading frames (ORFs) were identified in this region, but only three ORFs were translated into proteins when the cloned genes were introduced into Escherichia coli. Analysis of the predicted amino acid sequences of the proteins encoded by the ORFs allowed us to identify the trpE and trpG genes of L. biflexa. Enzyme assays confirmed the identity of these two ORFs. Anthranilate synthase from L. biflexa was found to be subject to feedback inhibition by tryptophan. Codon usage analysis showed that there was a bias in L. biflexa towards the use of codons rich in A and T, as would be expected from its G + C content of 37%. Comparison of the amino acid sequences of the trpE gene product and the trpG gene product with corresponding gene products from other bacteria showed regions of highly conserved sequence.     

Structure and regulation of the anthranilate synthase genes in Pseudomonas aeruginosa: I. Sequence of trpG encoding the glutamine amidotransferase subunit

We have determined the DNA sequence of the distal 148 codons of trpE and all of trpG in Pseudomonas aeruginosa. These genes encode, respectively, the large and small (glutamine amidotransferase) subunits of anthranilate synthase, the first enzyme in the tryptophan synthetic pathway. The sequenced region of trpE is homologous with the distal portion of E. coli and Bacillus subtilis trpE, whereas the trpG sequence is homologous to the glutamine amidotransferase subunit genes of a number of bacterial and fungal anthranilate synthases. The two coding sequences overlap by 23 bp. Codon usage in these Pseudomonas genes shows a marked preference for codons ending in G or C, thereby resembling that of trpB, trpA, and several other chromosomal loci from this species and others with a high G + C content in their DNA. The deduced amino acid sequence for the P. aeruginosa trpG gene product differs to a surprising extent from the directly determined amino acid sequence of the glutamine amidotransferase subunit of P. putida anthranilate synthase (Kawamura et al. 1978). This suggests that these two proteins are encoded by loci that duplicated much earlier in the phylogeny of these organisms but have recently assumed the same function. We have also determined 490 bp of DNA sequence distal to trpG but have not ascertained the function of this segment, though it is rich in dyad symmetries.      

Evolutionary differences in chromosomal locations of four early genes of the tryptophan pathway in fluorescent pseudomonads: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC.

Pseudomonas putida possesses seven structural genes for enzymes of the tryptophan pathway. All but one, trpG, which encodes the small (beta) subunit of anthranilate synthase, have been mapped on the circular chromosome. This report describes the cloning and sequencing of P. putida trpE, trpG, trpD, and trpC. In P. putida and Pseudomonas aeruginosa, DNA sequence analysis as well as growth and enzyme assays of insertionally inactivated strains indicated that trpG is the first gene in a three-gene operon that also contains trpD and trpC. In P. putida, trpE is 2.2 kilobases upstream from the trpGDC cluster, whereas in P. aeruginosa, they are separated by at least 25 kilobases (T. Shinomiya, S. Shiga, and M. Kageyama, Mol. Gen. Genet., 189:382-389, 1983). The DNA sequence in P. putida shows an open reading frame on the opposite strand between trpE and trpGDC; this putative gene was not characterized. Evidence is also presented for sequence similarities in the 5' untranslated regions of trpE and trpGDC in both pseudomonads; the function of these regions is unknown, but it is possible that they play some role in regulation of these genes, since all the genes respond to repression by tryptophan. The sequences of the anthranilate synthase genes in the fluorescent pseudomonads resemble those of p-aminobenzoate synthase genes of the enteric bacteria more closely than the anthranilate synthase genes of those organisms; however, no requirement for p-aminobenzoate was found in the Pseudomonas mutants created in this study.     

Nucleotide sequence and analysis of a gene encoding anthranilate synthase component I in Spirochaeta aurantia.

A Spirochaeta aurantia DNA fragment containing the trpE gene and flanking chromosomal DNA was cloned, and the sequence of the trpE structural gene plus 870 bp upstream and 1,257 bp downstream of trpE was determined. The S. aurantia trpE gene codes for a polypeptide of 482 amino acid residues with a predicted molecular weight of 53,629 that showed sequence similarity to TrpE proteins from other organisms. The S. aurantia TrpE polypeptide is not more closely related to the other published spirochete TrpE sequence (that of Leptospira biflexa) than to TrpE polypeptides of other bacteria. Two additional complete open reading frames and one partial open reading frame were identified in the sequenced DNA. One of the complete open reading frames and the partial open reading frame are upstream of trpE and are encoded on the DNA strand opposite that containing trpE. The other open reading frame is downstream of trpE and on the same DNA strand as trpE. On the basis of the results of a protein sequence data base search, it appears that trpE is the only tryptophan biosynthesis gene in the sequenced DNA. This is in contrast to L. biflexa, in which trpE is separated from trpG by only 64 bp.     

Molecular cloning and nucleotide sequence of Thermus thermophilus HB8 trpE and trpG

The trpE gene of Thermus thermophilus HB8 was cloned by complementation of an Escherichia coli tryptophan auxotroph. The E. coli harboring the cloned gene produced the anthranilate synthase I, which was heat-stable and enzymatically active at higher temperature. The nucleotide sequence of the trpE gene and its flanking regions was determined. The trpE gene was preceded by an attenuator-like structure and followed by the trpG gene, with a short gap between them. No other gene essential for tryptophan biosynthesis was observed after the trpG gene. The amino-acid sequences of the T. themophilus anthranilate synthase I and II deduced from the nucleotide sequence were compared with those of other organisms.     

Transcriptional network analysis of the tryptophan-accumulating rice mutant during grain filling

In a previous study, we selected a high tryptophan (Trp)-accumulating rice (Oryza sativa L.) mutant line by in vitro mutagenesis using gamma rays. To obtain detailed information about the Trp biosynthetic pathway during the grain-filling in rice, we investigated the gene expression profiles in the wild-type (cv. Dongan) and the high-level Trp-accumulating mutant line (MRVII-33) at five different grain-filling stages using microarray analysis. The mutant line showed approximately 6.3-fold higher Trp content and 2.3-fold higher amino acids compared with the original cultivar at the final stage (stage V). The intensity of gene expression was analyzed and compared between the wild-type and mutant line at each of the five grain-filling stages using the Rice 4?×?44K oligo DNA microarray. Among the five stages, stage III showed the highest gene expression changes for both up- and down-regulated genes. Among the Trp biosynthesis-related genes, trpG showed high expression in the mutant line during stages I to IV and trpE showed higher at stage III. Gene clustering was performed based on the genes of KEGG's amino acid metabolism, and a total of 276 genes related to amino acid metabolism were placed into three clusters. The functional annotation enrichment analysis of the genes classified into the three clusters was also conducted using ClueGO. It was found that cluster 3 uniquely included biological processes related to aromatic amino acid metabolism. These results suggest that gene analysis based on microarray data is useful for elucidating the biological mechanisms of Trp accumulation in high Trp-accumulating mutants at each of the grain-filling stages.