产1,3-丙二醇菌株的诱变和筛选

为提高克雷伯氏肺炎杆菌产1,3-丙二醇的能力,以离子束、紫外线和氯化锂为复合诱变法,建立了产酸圈和产物耐受相结合的平板筛选方法,获得可耐受高浓度1,3-丙二醇并且副产物中乙醇含量较少的优良突变菌株2株。与出发菌株相比,两株高产突变菌株Klebsiella pneumoniae LM 03和Klebsiella pneumoniae LM05的1,3-丙二醇产量分别提高了33% 和30% ,达到66.74 g/L和65.12 g/L;乙醇产量分别降低了38% 和24% ,降低为6.59 g/L和8.05 g/L。同时测定了诱变前后还原途径中甘油脱水酶（GDHt）和1,3-丙二醇氧化还原酶（PDOR）的酶活变化,研究表明诱变对GDHt有明显的促进作用,而对PDOR的影响不明显。该诱变和筛选方法目标明确、易操作、效率高,在1,3-PD工业规模的生物法生产中将具有良好的应用价值,而且对于其他具有工业应用价值的菌株筛选工作也具有一定的借鉴意义。     

克雷伯氏菌甘油脱水酶基因在大肠杆菌中的克隆与表达

利用PCR技术从克雷伯氏菌(Klebsiella pneumoniae ATCC49790)总DNA中扩增得到甘油脱水酶(glycerol dehydratase,DHAB)基因的DNA片段,并将其连接到表达质粒pSE380,携带有重组质粒pSE-dhaB的大肠杆菌JM109实现了dhaB基因的表达;对含有dhaB工程菌进行表达研究,表明工程菌在37℃,以1．0mmol／L IPTG诱导5h酶活力即达到1164．14u／L,比野生菌酶活力(168．69U／L)提高了6．9倍。     

Characterization of glycerol dehydratase expressed by fusing its α- and β-subunits

The gdh and gdhr genes, encoding B₁₂-dependent glycerol dehydratase (GDH) and glycerol dehydratase reactivase (GDHR), respectively, in Klebsiella pneumoniae, were cloned and expressed in E. coli. Part of the β-subunit was lost during GDH purification when co-expressing α, β and γ subunit. This was overcome by fusing the β-subunit to α- or γ-subunit with/without the insertion of a linker peptide between the fusion moieties. The kinetic properties of the fusion enzymes were characterized and compared with wild type enzyme. The results demonstrated that the fusion protein GDHALB/C, constructed by linking the N-terminal of β-subunit to the C-terminal of α subunit through a (Gly₄Ser)₄ linker peptide, had the greatest catalytic activity. Similar to the wild-type enzyme, GDHALB/C underwent mechanism-based inactivation by glycerol during catalysis and could be reactivated by GDHR. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mutations affecting translation of the bacteriophage T4 rIIB gene cloned in Escherichia coli

Summary Mutant ribosome binding sites of the bacteriophage T4 rIIB gene, resident on an 873 bp DNA fragment, were cloned into a plasmid vector as in-frame fusions to a reporter gene, beta-galactosidase. The collection of mutations included changes in the region 5 to the Shine/Dalgarno sequence, a mutation of the Shine/Dalgarno sequence, the alternate initiation codons GUG, AUA and ACG, and mutants in which several closely spaced initiation codons compete with each other on the same mRNA. The results show that the secondary structure variations we have installed 5 to the Shine/Dalgarno sequence have little effect on translation. GUG is essentially as good an initiator of translation as AUG when they are assayed on separate messages, but is outcompeted at least 50-fold in the sequence AUGUG. AUA and ACG are poor start codons, and are temperature sensitive. The initiation codon pair AUGAUA, in which the AUG is only two nucleotides from the Shine/Dalgarno sequence, displays a novel cold-sensitive phenotype.     

克雷伯杆菌产1,3-丙二醇关键酶甘油脱水酶的酶活分析方法研究

有氧条件下,建立了克雷伯杆菌破碎方法及其生产1,3-丙二醇代谢途径中关键酶甘油脱水酶的酶活测定方法。甘油脱水酶酶活测定时在超声时间25min,功率为300w的条件下最适破碎频率为破碎时间1s,停息时间4s;甘油脱水酶酶活测定所用磷酸盐缓冲液的最适浓度为0.045mol/L,最适pH值7.2。甘油脱水酶酶活测定反应的最佳温度为37℃,甘油脱水酶酶活测定反应液的最佳吸收波长为290nm。     

MBP-gldABC融合蛋白基因的原核表达载体的构建

目的：构建可高效生产甘油脱水酶的大肠杆菌工程菌,方法：将编码甘油脱水酶的三个基因gldA、gldB、gldC,分别克隆至克隆载体pMD18-T和pSIM-T中,经测序正确后,再亚克隆至表达融合蛋白的高效表达载体pMAL-c2X上,构建成表达质粒pMAL-gldABC,并转化大肠杆菌E.coli DH5α。结果：成功地将甘油脱水酶基因gldABC以同向串联方式克隆到大肠杆菌融合表达载体pMAL-c2X中,结论：得到了含gldABC基因的MBP融合蛋白表达载体,为研究甘油脱水酶基因（gldABC）的在原核表达载体中的串联表达奠定了基础。     

甘油脱水酶再激活因子提高重组大肠杆菌3-羟基丙酸合成能力

甘油脱水酶是甘油转化3-羟基丙酸生物合成途径中的关键性限速酶,然而底物甘油的存在会抑制该酶的活性,从而引起3-羟基丙酸合成量的下降.因此解除底物甘油对甘油脱水酶活性的抑制作用,是提高生物合成3-羟基丙酸产量的方法之一.克隆来源于克雷伯氏菌(Klebsiella pneumoniae)的甘油脱水酶编码基因dhaB、甘油脱...     

Occurrence of chloroplast ribosome recognition sites within conserved elements of the RNA genomes of carlaviruses

The nucleotide sequences upstream from the carlavirus open reading frames were examined for direct sequence homology. Blocks of homology were evident upstream from the 25 K ORFs of potato virus S (PVS), potato virus M (PVM) and lily symptomless virus (LSV), and upstream from the coat protein initiation codons of PVS, PVM, LSV, carnation latent virus and Helenium virus S. These blocks, which correspond to the 5′-terminal regions of the subgenomic RNAs, were shown to contain potential ribosome recognition sequences. The distances between the binding sites and initiation codons ranged from 20 to 40 nucleotides on the viral RNAs. Whilst the majority of chloroplasts mRNAs have a distance of 8 nucleotides between binding site and initiation codon, the remaining have a distance of 23 nucleotides which is similar to that reported here for the carlaviruses.     

从甘油富集菌宏基因组中克隆甘油脱水酶基因

采用一种简便快速的方法从经甘油富集培养的土样中提取出质量较好宏基因组DNA。然后以此DNA为模板,以扩增肺炎克雷伯氏菌、弗氏柠檬酸菌和丁酸梭菌甘油脱水酶基因的引物进行PCR,分别扩增出目的条带,并将其克隆至T载体中。进行测序分析显示,PCR扩增出来的片段与其引物相应的甘油脱水酶序列同源性分别达到99％,90％和99％。这表明克隆出来的这3个基因为相应的甘油脱水酶基因。     

重组依赖辅酶B12的甘油脱水酶基因表达系统构建

采用PCR方法从肺炎克雷伯氏杆菌基因组中分别扩增得到了编码依赖辅酶B12的甘油脱水酶α、β、γ三个亚基的基因gldA、gldB、gld将gldBC基因克隆至pSIM—T载体上,经测序正确后亚克隆至pGEX-4T-1载体中。将gldA进行T-A克隆后测定核苷酸序列正确,亚克隆至连有gldBC基因的pGEX-4T-1载体中。从而成功的构建了gldABC基因的同向串联原核融合表达载体pGEX—gldABC。     

利用交错延伸剪接PCR技术扩增油菜花粉育性基因MS2Bnap全长cDNA序列

交错延伸剪接PCR是一种用于分子进化研究的DNA改组技术。本实验利用其原理,将已获得的油菜花粉育性MS2Bnap有部分序列重叠的三段PCR产物作为模板进行扩增,获得了花粉育性基因MS2Bnap的全长cDNA序列。     

重组肺炎克雷伯氏菌转化甘油为聚3-羟基丙酸

聚3-羟基丙酸[Poly(3-hydroxypropionate),P3HP]是一种生物可降解及生物相容的新型聚羟基脂肪酸酯。目前已鉴定的生物均不能天然合成P3HP。采用PCR克隆鼠伤寒沙门氏菌的丙醛脱氢酶(Pdu P)基因及罗尔斯通氏菌的聚羟基脂肪酸酯合成酶(Pha C)基因,构建共表达载体,转化肺炎克雷伯氏菌后获得两株重组菌。以甘油为唯一碳源进行摇瓶发酵,pdu P和pha C共用tac启动子的工程菌K.p(p ET-tac-pdu P-pha C)产生0.054 g/L的P3HP,而pdu P和pha C各自独用tac启动子的工程菌K.p(p ET-tac-pdu P-tac-pha C)产生0.091 g/L的P3HP。     

弗氏柠檬酸菌甘油脱水酶激活因子基因的克隆、表达与功能鉴定

1,3-丙二醇是一种重要的化工原料,其生物法生产的研究逐渐受到的关注。研究以弗氏柠檬酸菌的总DNA为模板,通过PCR分别扩增出约1.8kb（dhaF）和0.4kb（dhaG）的两个基因片段分别编码甘油脱水酶激活因子大、小亚基, 连接于pMD-18T载体,测序分析显示与GenBank中相关基因的相似性最高为86%。将两基因以多顺反子的方式与pSE380连接构建表达载体,并在大肠杆菌中进行高效表达,表达量占总蛋白的30%。将高效表达的激活因子用金属亲合层析和分子筛进行了纯化,得到电泳纯级的甘油脱水酶激活因子,SDS-PAGE分析显示：大、小亚基分子量约为63kDa和12kDa;非变性胶分析显示：全酶的分子量约为150kDa,经扫描分析推测甘油脱水酶激活因子很有可能是以α2β2方式结合的。以弗氏柠檬酸菌甘油脱水酶为研究对象,进行激活实验,结果证实该激活因子具备甘油脱水酶激活因子的功能,为进一步阐明甘油脱水酶的激活机制及1,3-丙二醇的高效生产奠定了基础。     

Common themes and differences in SAM recognition among SAM riboswitches

The recent discovery of short cis-acting RNA elements termed riboswitches has caused a paradigm shift in our understanding of genetic regulatory mechanisms. The three distinct superfamilies of S-adenosyl-l-methionine (SAM) riboswitches are the most commonly found riboswitch classes in nature. These RNAs represent three independent evolutionary solutions to achieve specific SAM recognition. This review summarizes research on 1) modes of gene regulatory mechanisms, 2) common themes and differences in ligand recognition, and 3) ligand-induced conformational dynamics among SAM riboswitch families. The body of work on the SAM riboswitch families constitutes a useful primer to the topic of gene regulatory RNAs as a whole.