大肠杆菌生产异戊二烯的代谢工程研究进展

异戊二烯作为一种重要的化工原料,主要用于合成橡胶。此外,还广泛应用于医药或化工中间体、食品、粘合剂及航空燃料等领域。利用微生物法生产异戊二烯因具有环境友好、利用廉价的可再生原料、可持续发展等优势而成为当今研究的热点。这里介绍了大肠杆菌生产异戊二烯的代谢途径及关键酶,从代谢工程的角度出发综述了目前为提高大肠杆菌异戊二烯产量所应用到的方法和策略,并对今后的发展方向进行了展望。     

转几丁质酶和葡聚糖酶双价基因棉花对土壤细菌种群多样性的影响

以转几丁质酶和葡聚糖酶双价基因棉花为研究对象,非转基因受体棉花为对照,通过比较可培养细菌数量和基于16S rRNA克隆文库细菌种群分析,评价外源双价基因的导入在苗期、蕾期、花铃期和吐絮期对棉花根际细菌群落多样性的影响。结果表明,可培养细菌的数量不受外源双价基因的影响,随着棉花生育期的交替而变化,以代谢旺盛的花铃期最多。构建的转基因和非转基因不同生育期根际土壤细菌16S rRNA文库容量为2400个克隆,涵盖了细菌的283个属。其中,Acidobacterium是最大优势类群,共包括624个克隆,其次为未知细菌种群和Flavisolibacter。比较转基因和非转基因棉花根际土壤细菌的种群结构,结果显示,同一生育期内前者种群的多样性显著低于后者,二者的共有类群随着生长发育的进行而增多。研究结果说明几丁质酶基因和葡聚糖酶基因对棉花根际细菌种群多样性有着不同程度的削减作用,但是随着种植时间的延长,该差异呈现逐渐缩小的趋势。     

A rapid procedure for the construction of PCR cDNA libraries from small amounts of plant tissue

We describe a general method for the preparation of λZAP II cDNA libraries from very small amounts (<50 mg) of plant tissue. We have achieved this by combining an efficient method for RNA extraction with a modified PCR protocol for the synthesis and amplification of cDNA. Using this protocol we have found it possible to generate cDNA libraries containing more than 10⁶ clones from as little as 1 μg of total RNA.     

宏基因组学挖掘新型生物催化剂的研究进展

微生物蕴藏着大量具有工业应用潜力的生物催化剂。然而,传统培养方法只能从环境中获得不到1%的微生物。宏基因组学是通过提取某一特定环境中的所有微生物基因组DNA、构建基因组文库并对文库进行筛选,寻找和发现新的功能基因的一种方法。它绕过了微生物分离培养过程,成为研究环境样品中不可培养微生物的有力手段。因此,从宏基因组中挖掘新型生物催化剂一直倍受生物学家的关注。以下主要对宏基因组文库的样品来源、DNA提取方法、文库的构建和筛选策略的选择这4个方面的研究状况进行了综述,列举了近年来利用宏基因组技术所获得的新型生物催化剂,并对其今后的研究方向提出了展望。     

PCR-mediated screening and labeling of DNA from clones

A simplified and economical protocol for DNA library screening and nonradioactive labeling is described. Bacterial clones are lysed in 1% of Triton X-100 and subjected to polymerase chain reaction in the presence of digoxigenin-11-dUTP to screen and simultaneously to label the DNA inserts. Bacteriallysates are stable in storage at −20°C and can be used repeatedly for PCR-mediated labeling. In this protocol, very low concentrations of dNTP, digoxigenin-dUTP, and primers are used in combination with a reduced reaction volume. This will considerably reduce the expense of screening and labeling bacterial clones and facilitate the exchange of DNA probes among laboratories.     

甜菜夜蛾羧酸酯酶基因cDNA的克隆、表达及序列分析

羧酸酯酶是昆虫体内重要的解毒酶系之一,与昆虫抗药性产生相关。利用粉纹夜蛾Trichoplusia ni (Hübner)肠粘蛋白多克隆抗体免疫筛选甜菜夜蛾Spodoptera exigua (Hübner)中肠cDNA表达文库,得到编码羧酸酯酶的全长cDNA克隆。该cDNA克隆全长1 812 bp(GenBank登录号EF580101),开放阅读框长1 605 bp,编码535个氨基酸。该蛋白活性中心包括3个氨基酸残基（催化三联体）： Ser186, Glu319和His443, 3个N-联糖基化位点,具备羧酸酯酶的结构特征,属羧酸酯酶家族（EC: 3.1.1.-）。将该基因与pQE30载体重组,经IPTG诱导,spot-blot鉴定,蛋白获得了表达;以α-醋酸萘酯为底物,检测表达的羧酸酯酶活性为1.3 nmol/100 μL酶液。     

Biosynthesis of the anti‐diabetic metabolite montbretin A: glucosylation of the central intermediate mini‐MbA

Type 2 diabetes (T2D) affects over 320 million people worldwide. Healthy lifestyles, improved drugs and effective nutraceuticals are different components of a response against the growing T2D epidemic. The specialized metabolite montbretin A (MbA) is being developed for treatment of T2D and obesity due to its unique pharmacological activity as a highly effective and selective inhibitor of the human pancreatic α‐amylase. MbA is an acylated flavonol glycoside found in small amounts in montbretia (Crocosmia × crocosmiiflora) corms. MbA cannot be obtained in sufficient quantities for drug development from its natural source or by chemical synthesis. To overcome these limitations through metabolic engineering, we are investigating the genes and enzymes of MbA biosynthesis. We previously reported the first three steps of MbA biosynthesis from myricetin to myricetin 3‐O‐(6′‐O‐caffeoyl)‐glucosyl rhamnoside (mini‐MbA). Here, we describe the sequence of reactions from mini‐MbA to MbA, and the discovery and characterization of the gene and enzyme responsible for the glucosylation of mini‐MbA. The UDP‐dependent glucosyltransferase CcUGT3 (UGT703E1) catalyzes the 1,2‐glucosylation of mini‐MbA to produce myricetin 3‐O‐(glucosyl‐6′‐O‐caffeoyl)‐glucosyl rhamnoside. Co‐expression of CcUGT3 with genes for myricetin and mini‐MbA biosynthesis in Nicotiana benthamiana validated its biological function and expanded the set of genes available for metabolic engineering of MbA.     

产鼠李糖脂生物表面活性剂大肠杆菌的构建与优化

目前鼠李糖脂生物表面活性剂主要由条件致病的铜绿假单胞菌生产获得,从而影响工业应用。为了开发一种相对安全的鼠李糖脂生产菌,将带有不同强度组成型合成启动子的鼠李糖基转移酶基因(Rhamnosyltransferase gene,rhl AB)以单、中、高3种拷贝数分别在大肠杆菌ATCC 8739中异源表达,实现了不同产量的鼠李糖脂异源合成。对rhl AB基因和rha BDAC基因簇(TDP-L-鼠李糖合成的基因簇)进一步利用合成启动子进行组合调控,筛选获得了最优生产鼠李糖脂工程菌——大肠杆菌TIB-RAB226。对大肠杆菌TIB-RAB226进行发酵温度优化,鼠李糖脂产量达到124.3 mg/L,是优化前的1.17倍。通过分批补料发酵,12h时鼠李糖脂产量达到209.2 mg/L。对发酵产物进行高效液相色谱-质谱联用技术分析,共检出相对含量变化的5类质核比不同的鼠李糖脂同系物。本研究可为异源合成产鼠李糖脂提供重要参考。     

An immunodominant site of γ-zein1 is in the region of tandem hexapeptide repeats

The immunochemical data from studies with polyclonal antisera to -zein₁, the 27 kD component of the maize prolamin, indicated that the region containing 8 tandem repeats of the sequence PPPVHL is an immunodominant site. In one case, the entire antibody repertoire of an antiserum recognized epitope(s) within this region. Three 17-mer oligopeptides corresponding to the predicted antigenic epitopes of -zein₁ were synthesized and reacted with three different anti--zein₁ sera in order to map antigenic sites in the intact protein. These antisera yielded positive reactions with a 17-mer peptide (peptide 37), which was not in a hydrophilic maximum but derived from the repeat region. The same antisera gave little or no reaction with other peptides (peptides 38 and 39), both of which were in a hydrophilic maximum. In addition, an antiserum to peptide 37 reacted strongly with both the homologous antigen and the intact -zein₁. Peptide 37 also blocked the binding of antisera to -zein₁ in competition assays. Subsequently, the shorter 6-mer (peptide 82) and 12-mer (peptide 80) versions of peptide 37 were synthesized, and both reacted with anti-peptide 37 serum and also with each of the three anti--zein₁ sera. In these reactions and in competition assays, the reactivity and the blocking ability increased in proportion to the length of the peptide. Based on these data, it was concluded that the repeat region of -zein₁ is the site of one or more continuous immunodominant epitopes. The data also suggest that the repeat region is exposed on the surface of the folded protein and probably occur as a mobile, random coil.     

人脑髓鞘碱性蛋白cDNA体外扩增、克隆和鉴定

采用聚合酶链反应(PCR)从人脑cDNA文库中扩增出600bp的髓鞘碱性蛋白(MBP)cDNA片段,与载体pGEM-3Zf(+)平端连接.重组质粒DNA转化宿主菌JM109,在含X-gal和IPTG的平板上直接筛选阳性克隆.限制性内切酶分析和成套引物扩增鉴定证明,该克隆含有7个外显子的21.5kD人脑MBP全长编码序列.