大豆异黄酮代谢途径在大肠杆菌中的构建及表达

自然界异黄酮合成途径主要存在于豆科植物中。以微生物为宿主研究异黄酮代谢,则需要将整个相关代谢途径的多酶体系组装到工程菌种,从而进行表达及代谢研究,这就需要用到多基因的转化和共表达技术。综合应用了多基因单载体和多基因多载体方法,将大豆异黄酮代谢途径中的五个关键酶基因导入到大肠杆菌中,对异黄酮代谢途径在大肠杆菌中的构建和表达进行了研究和探索,获得了含有五个外源基因的重组大肠杆菌;重组菌经IPTG诱导,以L-酪氨酸为底物进行发酵,发酵产物经过HPLC测定,结果表明和空白对照相比有新的代谢产物生成,初步断定为异黄酮类化合物。     

丙酮酸甲酸裂解酶基因在大肠杆菌中的克隆、表达及酶学性质的研究

丙酮酸甲酸裂解酶（pyruvate format-lyas,PFL）是厌养或兼性厌养微生物中,代谢途径的关键酶之一,为了进一步研究其功能,我们以大肠杆菌JM109菌株基因组DNA为模板,进行PCR扩增大肠杆菌中的pfl基因,为测序方便将所得DNA片段连接到pMD18-T载体上,将测序正确后的pfl基因连接到表达载体pET-22b(＋)中,重组表达载体在大肠杆菌BL21(DE3)中诱导表达, 通过SDS-PAGE电泳分析,在分子量为85kDa处出现新生的蛋白条带。利用金属亲和层析对添加了6×组氨酸标签的PFL进行纯化,对PFL的酶学性质进行了研究。结果表明：此酶的最适温度为35 ℃,最适pH为7.5,米氏常数Km＝2.3mmol,Tm＝49.9℃。     

运动发酵单孢菌ZM4-pdc基因的克隆及在大肠杆菌TOP10中的表达与活性分析

利用高保真聚合酶从运动发酵单胞茵中克隆出丙酮酸脱羧酶基因,加A后克隆到pGM-T载体,测序验证无误.经酶切、连接、转化到表达栽体pUC-18.形成重组质粒pUC-18一pdc,转化到大肠杆菌TOPl0中,经定性定量分析丙酮酸脱羧酶基因在大肠杆菌中高效表达,成功构建出乙醛的代谢途径.     

酿酒酵母gpd1和hor2基因在大肠杆菌中的共表达

利用途径工程的方法,在大肠杆菌中构建一条新的产甘油的代谢途径。从酿酒酵母(Saccharomyces cerevisiae)克隆3-磷酸甘油脱氢酶基因(gpd1)和3-磷酸甘油酯酶基因(hor2),并将两个基因串连到启动子trc的下游,构建由trc启动子控制的能高效表达的多顺反子重组质粒pSE-gpd1-hor2,将重组质粒导入大肠杆菌BL21菌株中,构建得到的重组菌株GxB-gh能将葡萄糖转化为甘油。结果表明重组菌株GxB-gh以葡萄糖为底物进行发酵,甘油产量为46．67g／L,葡萄糖的转化率为42.87％。这为利用工程菌绿色生产甘油进行了前期的探索,也为进一步构建能生产1,3-丙二醇的工程菌打下了良好的基础。     

在大肠杆菌中表达酮酸脱羧酶产异戊醇

酮酸脱羧酶作为异戊醇生物合成的关键酶,不存在于大肠杆菌中。以乳酸乳球菌的基因组DNA为模板,经过PCR扩增得到酮酸脱羧酶基因kivD(rbs),插入到大肠杆菌高效表达载体pET-28a(+)上形成pET-kivD(rbs),重组质粒热击转化进大肠杆菌BL21(DE3)中,其成功表达了酮酸脱羧酶。对发酵产物进行分析,检测到了微量的目标产物—异戊醇。     

糖丁基梭菌产丁醇途径在大肠杆菌中的构建及发酵

摘要:【目的】通过克隆来源于糖丁基梭菌(Clostridium saccharobutylicum DSM13864)丁醇合成途径的关键酶基因(thlA,bcs-operon和adhE),构建产丁醇大肠杆菌。【方法】以Clostridium saccharobutylicum DSM13864的基因组为模板,分别扩增丁醇途径关键酶基因thlA,bcs-operon(crt-bcd1-etfB2-fixB2-hbd)和adhE,构建了两个重组质粒pETDuet-bcs和pRSFDuet-thlA-adhE,并成功转入E.coli JM109(DE3)实现异源表达,使大肠杆菌具备产丁醇能力。在半厌氧条件下进行重组菌的发酵,并研究不同培养基对产丁醇的影响。【结果】该重组菌在半厌氧条件下经摇瓶发酵丁醇产量达到25.4 mg/L,通过优化培养基后,在TB发酵培养基中丁醇产量可达到34.1 mg/L。【结论】通过构建重组共表达质粒,将糖丁基梭菌来源的丁醇途径关键酶基因在大肠杆菌中表达,成功构建产丁醇大肠杆菌。该研究提供了一株易于操作的丁醇发酵重组大肠杆菌,避免了传统梭菌发酵丁醇生产中苛刻的厌氧条件、易产孢子等限制问题。     

原核生物核糖核酸酶HII基因的克隆及在大肠杆菌中的高效表达

核糖核酸酶HII (RNaseHII)能有效降解RNA和DNA杂交链中的RNA链。为进一步研究其功能 ,利用大肠杆菌XL1blue为模板 ,相应的寡聚脱氧核苷酸为引物 ,PCR扩增大肠杆菌RNaseHII(rnh 2 )基因 ,并将目的基因连接到克隆载体 pUC18上 ,经测序确认无误 ,分别亚克隆到能够进行IPTG诱导的表达载体pTrcHisC和进行温度诱导的表达载体pBV2 2 0上。重组质粒转化到大肠杆菌DH5α细胞中获得高效表达。在载体pTrcHisC和 pBV2 2 0中目的蛋白RNaseHII的表达量均超过菌体总蛋白的 2 0 % ,且表达产物以稳定的包涵体形式存在。此项工作为以后目的蛋白的纯化提供了有利条件 ,并为研究其结构和功能奠定了基础。     

用P. pastoris的pAOX1表达系统表达木糖异构酶

目的:应用P. pastoris的pAOX1表达系统分泌表达重组木糖异构酶.方法:用PCR法从大肠杆菌基因组中扩增木糖异构酶基因(xi).用EcoRⅠ和NotⅠ双酶切将其基因克隆进P. pastoris表达载体.通过电转法将其木糖异构酶基因重组于P. pastoris基因组,筛选G418抗性700μg/ml的重组子作为工程菌GS115(pPIC9K-xi).在摇瓶中发酵用甲醇诱导表达重组木糖异构酶.用SDS-PAGE分析重组蛋白的表达情况,用糖酵解法对表达产物进行活性分析.结果:木糖异构酶基因在pAOX1的调控下,在P. pastoris中经甲醇诱导能分泌表达,摇瓶发酵2d表达量为35mg/L,表达产物具有代谢木糖的作用.结论:成功地克隆了大肠杆菌的木糖异构酶基因,并实现用pAOX1系统在P. pastoris中表达中木糖异构酶,为用P. pastoris规模化生产重组木糖异构酶奠定了基础.     

树干毕赤酵母木糖还原酶基因的克隆及在大肠杆菌中的表达

根据NCBI中的木糖还原酶基因序列设计引物,利用高保真聚合酶克隆树干毕赤酵母木糖还原酶基因,加A后克隆到质粒pGM-T中,测序验证.然后将目的基因克隆到舍有强启动子的穿梭表达载体p424GPD中,构建含有XYL1基因的重组质粒p424GPD-XYL1.将p424GPD-XYL1转化到大肠杆菌中,提取总蛋白,聚丙烯酰胺凝胶电泳分析.酶活测定确定木糖还原酶基因XYL1在大肠杆菌中得到活性表达,表明表达载体构建成功.表达载体的成功构建为后续构建重组酿酒酵母利用木糖发酵奠定基础.     

丙酮丁醇梭菌ldhL基因的克隆及其在大肠杆菌中的表达

首次从丙酮丁醇梭菌(Clostridium acetobutylicum ATCC824)中克隆得到L-乳酸脱氢酶(L-lactate dehydrogenase,ldhL)基因,并将其连接到pSE380表达载体上,得到重组质粒pSE380ldhL,将重组质粒转化到乳酸脱氢酶和丙酮酸裂解酶缺陷的Escherichia coli FMJl44大肠杆菌中进行表达。SDS-PAGE分析表达产物的分子量约为34kD,摇瓶发酵后用HPLC检测分析L-乳酸产量为2.4g/L,纯度达到99.9%,不需要再进行手性分离,为以后在工业上生物法生产高纯度的L-乳酸打下基础。     

红霉素大环内酯合成通路在大肠杆菌中的重建

研究在大肠杆菌中重建了红霉素大环内酯(6-脱氧-红霉内酯B,6dEB)合成通路。先将参与6dEB合成所必需的基因分别克隆于多基因串联共表达载体中,获得单基因重组质粒;再利用载体中XbaⅠ/SpeⅠ互为同尾酶的特性实现相关基因的串联组合,获得多基因重组质粒pBJ130和pBJ144。将多基因重组质粒共转化BAP1,获得含6dEB合成通路的工程菌株BAP1(pBJ130/pBJ144),SDS-PAGE检测结果显示通路中各基因均有明显的表达;进行低温发酵,产物粗提后质谱检测到6dEB,其产量约10 mg/L。表明成功实现了6dEB合成通路在大肠杆菌中的重建,为红霉素大环内酯的改造和修饰提供了平台,也为红霉素合成通路在大肠杆菌中的完整重建以及聚酮类抗生素的组合性生物合成提供了参考。     

大肠杆菌多基因共表达策略

利用基因工程手段实现多个基因在同一宿主菌中共表达是大肠杆菌细胞发育调节研究和代谢途径改造的有效手段。介绍了单一转录单元的多基因共表达载体、多重转录单元的多基因共表达和单基因载体的构建原理、特点、优势及转化策略,并着重介绍了利用LIC衔接子实现基因在多基因载体上定位连接的原理和方法。     

Biosynthetic pathway for poly(3-Hydroxypropionate) in recombinant Escherichia coli

Poly(3-hydroxypropionate) (P3HP) is a biodegradable and biocompatible thermoplastic. In this study, we engineered a P3HP biosynthetic pathway in recombinant Escherichia coli. The genes for malonyl-CoA reductase (mcr, from Chloroflexus aurantiacus), propionyl-CoA synthetase (prpE, from E. coli), and polyhydroxyalkanoate synthase (phaC1, from Ralstonia eutropha) were cloned and expressed in E. coli. The E. coli genes accABCD encoding acetyl-CoA carboxylase were used to channel the carbon into the P3HP pathway. Using glucose as a sole carbon source, the cell yield and P3HP content were 1.32 g/L and 0.98% (wt/wt [cell dry weight]), respectively. Although the yield is relatively low, our study shows the feasibility of engineering a P3HP biosynthetic pathway using a structurally unrelated carbon source in bacteria.     

Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis

We attempted to optimize the production of zeaxanthin in Escherichia coli by reordering five biosynthetic genes in the natural carotenoid cluster of Pantoea ananatis. Newly designed operons for zeaxanthin production were constructed by the ordered gene assembly in Bacillus subtilis (OGAB) method, which can assemble multiple genes in one step using an intrinsic B. subtilis plasmid transformation system. The highest level of production of zeaxanthin in E. coli (820 microg/g [dry weight]) was observed in the transformant with a plasmid in which the gene order corresponds to the order of the zeaxanthin metabolic pathway (crtE-crtB-crtI-crtY-crtZ), among a series of plasmids with circularly permuted gene orders. Although two of five operons using intrinsic zeaxanthin promoters failed to assemble in B. subtilis, the full set of operons was obtained by repressing operon expression during OGAB assembly with a p(R) promoter-cI repressor system. This result suggests that repressing the expression of foreign genes in B. subtilis is important for their assembly by the OGAB method. For all tested operons, the abundance of mRNA decreased monotonically with the increasing distance of the gene from the promoter in E. coli, and this may influence the yield of zeaxanthin. Our results suggest that rearrangement of biosynthetic genes in the order of the metabolic pathway by the OGAB method could be a useful approach for metabolic engineering.     

产L-苏氨酸重组大肠杆菌的构建和发酵性能

【背景】大肠杆菌由于生长性能优良、遗传背景清晰,常被用作苏氨酸生产菌。【目的】敲除大肠杆菌Escherichia coli THR苏氨酸合成途径的非必需基因,并异源表达苏氨酸合成必需的关键酶,构建一株苏氨酸高产菌株。【方法】利用FLP/FRT重组酶系统,敲除E. coli THR中lysC、pfkB和sstT,同时进行谷氨酸棒杆菌中lysC~(fbr)、thrE和丙酮丁醇梭菌中gapC的重组质粒构建并转化到宿主菌中。【结果】以E. coli THR为出发菌株,敲除其苏氨酸合成途径中表达天冬氨酸激酶Ⅲ (AKⅢ)的基因lysC、磷酸果糖激酶Ⅱ基因pfkB及苏氨酸吸收蛋白表达基因sstT,使菌株积累苏氨酸的产量达到75.64±0.35g/L,比出发菌株增加9.9%。随后异源表达谷氨酸棒杆菌中解除了反馈抑制的天冬氨酸激酶(lysC~(fbr))、苏氨酸分泌转运蛋白(thrE)及丙酮丁醇梭菌中由gapC编码的NADP+依赖型甘油醛-3-磷酸脱氢酶,获得重组菌株E. coli THR6菌株。该菌株积累苏氨酸的产量提高到105.3±0.5 g/L,糖酸转化率提高了43.20%,单位产酸能力提高到5.76 g/g DCW,最大生物量为18.26 g DCW/L。【结论】单独敲除某个基因或改造某个途径不能使苏氨酸大量合成和积累,对多个代谢途径共同改造是构建苏氨酸工程菌的最有效方法。     

Enhanced production of arginine and urea by genetically engineered Escherichia coli K-12 strains.

Escherichia coli strains capable of enhanced synthesis of arginine and urea were produced by derepression of the arginine regulon and simultaneous overexpression of the E. coli carAB and argI genes and the Bacillus subtilis rocF gene. Plasmids expressing carAB driven by their natural promoters were unstable. Therefore, E. coli carAB and argI genes with and without the B. subtilis rocF gene were constructed as a single operon under the regulation of the inducible promoter ptrc. Arginine operator sequences (Arg boxes) from argI were also cloned into the same plasmids for titration of the arginine repressor. Upon overexpression of these genes in E. coli strains, very high carbamyl phosphate synthetase, ornithine transcarbamylase, and arginase catalytic activities were achieved. The biosynthetic capacity of these engineered bacteria when overexpressing the arginine biosynthetic enzymes was 6- to 16-fold higher than that of controls but only if exogenous ornithine was present (ornithine was rate limiting). Overexpression of arginase in bacteria with a derepressed arginine biosynthetic pathway resulted in a 13- to 20-fold increase in urea production over that of controls with the parent vector alone; in this situation, the availability of carbamyl phosphate was rate limiting.     

Functional expression and extension of staphylococcal staphyloxanthin biosynthetic pathway in Escherichia coli

The biosynthetic pathway for staphyloxanthin, a C(30) carotenoid biosynthesized by Staphylococcus aureus, has previously been proposed to consist of five enzymes (CrtO, CrtP, CrtQ, CrtM, and CrtN). Here, we report a missing sixth enzyme, 4,4'-diaponeurosporen-aldehyde dehydrogenase (AldH), in the staphyloxanthin biosynthetic pathway and describe the functional expression of the complete staphyloxanthin biosynthetic pathway in Escherichia coli. When we expressed the five known pathway enzymes through artificial synthetic operons and the wild-type operon (crtOPQMN) in E. coli, carotenoid aldehyde intermediates such as 4,4'-diaponeurosporen-4-al accumulated without being converted into staphyloxanthin or other intermediates. We identified an aldH gene located 670 kilobase pairs from the known staphyloxanthin gene cluster in the S. aureus genome and an aldH gene in the non-staphyloxanthin-producing Staphylococcus carnosus genome. These two putative enzymes catalyzed the missing oxidation reaction to convert 4,4'-diaponeurosporen-4-al into 4,4'-diaponeurosporenoic acid in E. coli. Deletion of the aldH gene in S. aureus abolished staphyloxanthin biosynthesis and caused accumulation of 4,4'-diaponeurosporen-4-al, confirming the role of AldH in staphyloxanthin biosynthesis. When the complete staphyloxanthin biosynthetic pathway was expressed using an artificial synthetic operon in E. coli, staphyloxanthin-like compounds, which contained altered fatty acid acyl chains, and novel carotenoid compounds were produced, indicating functional expression and coordination of the six staphyloxanthin pathway enzymes.     

黄鳍金枪鱼抗菌肽YFGAP在大肠杆菌中融合表达及其活性检测

【目的】抗菌肽YFGAP由32个氨基酸组成,分子量为3.4 kD,对革兰氏阳性菌(G+)和革兰氏阴性菌(G?)表现出强效的抑制作用,不具有溶血活性。在大肠杆菌中表达抗菌肽YFGAP,分离纯化抗菌肽并鉴定其生物学活性。【方法】化学合成EK-YFGAP和L-EK-YFGAP基因序列,构建表达载体pET22b-ELP20-EK-YFGAP、pET22b-ELP40-EK-YFGAP和pET22b-ELP40-L-EK- YFGAP,分别转化至大肠杆菌BL21(DE3)中诱导表达,可逆相变循环纯化融合蛋白。肠激酶酶切,经Vivaspin Turbo纯化柱纯化,测定重组抗菌肽的抑菌活性和溶血活性。【结果】纯化出两种融合蛋白ELP40-EK-YFGAP和ELP40-L-EK-YFGAP,肠激酶酶切纯化后获得重组抗菌肽YFGAP,对4种病原菌均有抑制效果,溶血活性较低。【结论】以ELPs作为非色谱纯化标签,实现了抗菌肽YFGAP的融合表达,具有操作简单、成本低、易于扩大的优势,为重组抗菌肽的量化制备及应用提供了理论基础和技术支持。