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(Saccharomycescerevisiae)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BL2 1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4 6 6 7g L ,葡萄糖的转化率为4 2 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。     

产1,3-丙二醇基因工程菌发酵条件的研究

利用途径工程的方法,将来源于克雷伯氏菌(Klebsiella pneumoniae)的甘油脱水酶基因dhaB和1,3-丙二醇氧化还原酶基因dhaT构建成多顺反子重组质粒pSE-dhaB-dhaT并在大肠杆菌JM 109中进行表达,在大肠杆菌中构建一条新的产1,3-丙二醇代谢途径。研究表明,重组菌株JM 109/pSE-dhaB-dhaT在微好氧条件下,尝试用廉价的乳糖为诱导物、维生素B12为辅酶,可以将甘油转化为1,3-丙二醇,产量达15.34 g/L,甘油转化率为35.7%,对低成本生产1,3-丙二醇作了有益的探索。     

大肠杆菌甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)的敲除及其对甘油产量的影响

利用Red重组系统构建了大肠杆菌JM109甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)缺失的双突变菌株JM109B,然后将表达酿酒酵母3-磷酸甘油脱氢酶基因(GPD1)和3-磷酸甘油酯酶基因(HOR2)的质粒pSE-gpd1-hor2转化到JM109B突变菌株中,在含1%葡萄糖的摇瓶发酵培养基中37℃发酵24 h,甘油的最高产量为5.61 g/L,是原始菌株JM109/pSE-gpd1-hor2甘油产量的1.59倍;在30 L发酵罐中发酵28 h,甘油的最高产量为103.12 g/L,是原始菌株JM109/pSE-gpd1-hor2甘油产量的1.59倍,是原始菌株BL21/pSE-gpd1-hor2甘油产量的1.41倍,葡萄糖转化率为50.39%。     

利用荧光蛋白研究产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因CgGPD启动子

[目的]克隆产甘油假丝酵母(Candida glycerinogenes)胞浆3-磷酸甘油脱氢酶基因CgGPD的启动子(PCggpd),并通过报告基因gfp的差异表达来研究葡萄糖浓度对PCggpd在酿酒酵母(Saccharomyces cerevisiae)中的诱导特性.[方法]采用PCR扩增的方法分别从产甘油假丝酵母基因组和pCAMBIA1302载体中克隆出CgGPD的启动序列PCggpd和绿色荧光蛋白基因gfp.将两个基因同时构建到酿酒酵母表达载体pYX212-zeocin中,构建时将绿色荧光蛋白基因gfp置于CgGPD的启动序列下游,获得重组质粒pYX212-zeocin-PCggpd-gfp.通过电击转化酿酒酵母W303-lA.将重组酿酒酵母S.cerevisiae W303-1A-GFP置于不同葡萄糖浓度培养基中进行培养,利用荧光显微技术对其进行荧光检测.[结果]重组酿酒酵母能产生稳定的荧光,当葡萄糖浓度为2%时,重组酿酒酵母在YEPD培养基中产生较弱的荧光,随着葡萄糖浓度的升高,荧光强度有明显的增强.[结论]PCggpd属于环境胁迫诱导型启动子,高浓度的葡萄糖能诱导PCggpd启动绿色荧光蛋白的高水平表达,这对完善产甘油假丝酵母的遗传背景研究,阐明其高产甘油的机理具有重要意义.     

黑曲霉α-葡萄糖苷酶cDNA的克隆及表达

研究黑曲霉(Aspergillus niger)M-1菌株的α-葡萄糖苷酶基因在大肠杆菌中的克隆及表达。以M-1菌株的总RNA为模板,利用RT-PCR扩增α-葡萄糖转苷酶的cDNA,重组到Trc启动子控制下的表达载体pSE380中,构建重组质粒pSE-αtg,转入大肠杆菌BL21(DE3)进行IPTG诱导表达。初步研究表明:重组蛋白具有葡萄糖苷酶活性,最适pH为6.0,最适温度为45℃,金属离子Cu2 和Mn2 对酶活力有明显的促进作用。添加1.6mmol/L IPTG对重组菌的诱导作用最大,在培养中添加麦芽糖,对重组菌产酶有显著的促进作用。     

混合菌发酵1,3-丙二醇的初步研究

将表达酿酒酵母3-磷酸甘油脱氢酶基因(GPD1)和3-磷酸甘油酯酶基因(HOR2)的质粒PSE-gpd1-hor2转化到甘油激酶基因(glpK)和甘油脱氢酶基因(gldA)双缺失的大肠杆菌JM109C中,构建产甘油的工程菌JM109C/PSE-gpd1-hor2.接种JM109C/pSE-gpd1-hor2和Klebsiella在含1%葡萄糖的摇瓶发酵培养基中37℃发酵56 h,1,3-丙二醇的最高产量为1.28 g/L,葡萄糖摩尔转化率为37.5%;在30 L发酵罐中发酵68 h,1,3-丙二醇的最高产量为24.09 g/L,葡萄糖摩尔转化率为38.0%;5 g/L的乙酸、乳酸,10 g/L的乙醇分别使1,3-丙二醇的产量降低了91.41%、54.68%和51.56%.     

一步法产1,3-丙二醇酿酒酵母基因工程菌的构建

1,3-丙二醇(1,3-PD)是一种重要的化工原料,发酵法生产1,3-PD是一条新颖且具有潜在竞争力的生产途径。本研究在前期工作的基础上,将分别来源于大肠杆菌和肺炎克雷伯氏菌的基因片段yqhD和dhaB串联表达,构建重组表达载体pYX212-zeocin-pGAP-yqhD-pGAP-dhaB;并得到重组酿酒酵母(Saccharomyces cerevisiae)W303-1A/pYX212-zeocin-pGAP-yqhD-pGAP-dhaB。该重组菌和对照S.cerevisiae分别以葡萄糖为底物摇瓶发酵72h后,重组酿酒酵母发酵液中1,3-PD含量约为1.5g/L;而对照菌株不产1,3-PD。以上结果表明本研究在国内首次成功构建了直接以葡萄糖为底物发酵生产1,3-PD的酿酒酵母基因工程菌。为进一步将dhaB、yqhD基因导入其他以葡萄糖为底物高产甘油的酵母宿主中表达,获得以葡萄糖为底物一步法发酵高产1,3-丙二醇工程菌打下了坚实的基础。     

以甘油为底物利用重组大肠杆菌生产聚3-羟基丙酸

【目的】解决前期研究中所构建的以甘油为底物合成聚3-羟基丙酸(P3HP)的代谢途径中存在两个主要的问题——细胞内还原力不平衡和质粒丢失,以提高P3HP的产量。【方法】克隆来源于肺炎克雷伯氏菌的1,3-丙二醇(1,3-PDO)氧化还原酶基因,构建P3HP和1,3-PDO联产的菌株,解决细胞内还原力不平衡的问题。利用自杀性载体系统介导的同源重组技术,将甘油脱水酶及其激活因子的基因整合到大肠杆菌基因组中,提高质粒的稳定性。同时,对发酵条件进行优化。【结果】菌种改造和发酵条件优化显著提高了P3HP产量,在摇瓶条件下到达2.7 g/L,比以前的报道提高2倍,并可同时得到2.4 g/L 1,3-PDO。【结论】该重组大肠杆菌合成P3HP的产量得到提高,具有较好的工业化生产前景。     

酿酒酵母异源表达纤维二糖转运蛋白LacY与纤维二糖利用菌株的构建北大核心

【目的】在酿酒酵母体内设计代谢通路,使酿酒酵母能利用纤维素水解产物纤维二糖生产乙醇。【方法】首先,用大肠杆菌DH5α总DNA为模板克隆编码大肠杆菌乳糖透过酶的LacY基因。为过表达LacY基因,以质粒YEplac181作为载体,将酿酒酵母PGK1p强启动子加到LacY基因之前,CYC1t终止子加到LacY基因之后,构建质粒YEplac181-PGK1p-LacY-CYC1t。之后,将纤维二糖转运蛋白LacY表达质粒和β-葡萄糖苷酶(β-glucosidase,BGL)表达质粒pRS316-PGK1p-gh1-1-CYC1t依次转入野生型酿酒酵母W303-1A中,使野生型酿酒酵母W303-1A异源表达可转运纤维二糖的LacY蛋白和β-葡萄糖苷酶GH1-1,构建可利用纤维二糖的酿酒酵母工程菌W303-1A GL。最后,通过发酵测定酿酒酵母工程菌W303-1A GL的纤维二糖利用情况和乙醇产量,并对纤维二糖代谢通路中纤维二糖酶活力进行测定。【结果】本研究构建了纤维二糖转运蛋白LacY和β-葡萄糖苷酶GH1-1协同表达的酿酒酵母工程菌W303-1AGL。W303-1AGL可以有效利用纤维二糖发酵生产乙醇,W303-1A GL发酵24 h时乙醇产量达到3.25 g/L,得率为0.325 g乙醇/g纤维二糖,利用葡萄糖产乙醇理论得率为0.511 g乙醇/g纤维二糖,达到葡萄糖产乙醇理论得率的64%,细胞密度最高在第54 h达到OD600=10.84,胞内β-葡萄糖苷酶的酶活在72 h最高,可达到0.51 U/mg。【结论】本研究成功构建了能有效利用纤维二糖的重组酿酒酵母工程菌W303-1A GL,为提高纤维素乙醇生产效率、降低纤维素乙醇生产成本提供了新思路。     

代谢工程改造Gluconobacter suboxydans生产2-酮基-D-葡萄糖酸

2-酮基-D-葡萄糖酸是重要的抗氧化剂和食品添加剂——D-异抗坏血酸的重要前体。弱氧化葡糖酸杆菌(Gluconobacter suboxydans)具有丰富的周质空间氧化还原酶类,可将葡萄糖氧化为葡萄糖酸再氧化为2-酮基-D-葡萄糖酸。以提高2-酮基-D-葡萄糖酸的产量和减少副产物为目标,采用同源重组染色体修饰策略,将编码甘油脱氢酶的基因gldh置换为编码葡萄糖脱氢酶的基因gdh,将编码山梨醇脱氢酶的基因sdh置换为编码2-酮-D-葡萄糖酸脱氢酶的基因ga-2-dh。经PCR、酶活性显色及发酵产物HPLC检测验证表明:构建的工程菌株gdh和ga-2-dh基因被强化而gldh和sdh被敲除;使用10%的葡萄糖复合培养基,摇瓶发酵72h,工程菌2KGA3发酵液中没有副产物5-酮基-葡萄糖酸,2-酮基-D-葡萄糖酸的含量终浓度达到72.3 g/L,比野生菌株提高42.2g/L,工程菌和野生菌的2-D-KGA质量转化率分别为72.3%和30.1%,工程菌比野生菌提高1.4倍。构建获得的工程菌,不需要外加抗生素,可以保持稳定遗传,对于工业化规模生产具有一定优势,为获得可产业化显示的优势遗传资源打下了基础。     

Production of 3-hydroxypropionic acid via malonyl-CoA pathway using recombinant Escherichia coli strains

Malonyl-CoA is an intermediary compound that is produced during fatty acid metabolism. Our study aimed to produce the commercially important platform chemical 3-hydroxypropionic acid (3-HP) from its immediate precursor malonyl-CoA by recombinant Escherichia coli strains heterologously expressing the mcr gene of Chloroflexus aurantiacus DSM 635, encoding an NADPH-dependent malonyl-CoA reductase (MCR). The recombinant E. coli overexpressing mcr under the T5 promoter showed MCR activity of 0.015 U mg?1 protein in crude cell extract and produced 0.71 mmol/L of 3-HP in 24h in shake flask cultivation under aerobic conditions with glucose as the sole source of carbon. When acetyl-CoA carboxylase and biotinilase, encoded by the genes accADBCb (ACC) of E. coli K-12 were overexpressed along with MCR, the final 3-HP titer improved by 2-fold, which is 1.6 mM. Additional expression of the gene pntAB, encoding nicotinamide nucleotide transhydrogenase that converts NADH to NADPH, increased 3-HP production to 2.14 mM. The strain was further developed by deleting the sucAB gene, encoding α-ketoglutarate dehydrogenase complex in tricarboxylic acid (TCA) cycle, or blocking lactate and acetate production pathways, and evaluated for the production of 3-HP. We report on the feasibility of producing 3-HP from glucose through the malonyl-CoA pathway.     

Design of a recombinant Escherichia coli for producing l-phenylalanine from glycerol

A recombinant Escherichia coli was engineered to produce the commercially important amino acid L: -phenylalanine (L: -Phe) using glycerol as the carbon source. Compared to the conventionally used glucose and sucrose, glycerol is a less expensive carbon source. As phenylalanine dehydrogenase (PheDH) activity is involved in the last step of L: -Phe synthesis in E. coli, a phenylalanine dehydrogenase gene (phedh) from the thermotolerant Bacillus lentus was cloned into pRSFDuet-1 (pPheDH) and expressed in E. coli BL21(DE3). The resulting clone had a limited ability to produce L: -Phe from glycerol, possibly because of a poor glycerol uptake by the cell, or an inability to excrete L: -Phe, or both. Therefore, yddG gene encoding an aromatic amino acid exporter and glpF gene encoding a glycerol transport facilitator were coexpressed with the phedh in a reengineered E. coli. In a glycerol medium, the maximum L: -Phe production rates of the clones pPY (phedh and yddG genes) and pPYF (phedh, yddG and glpF genes) were 1.4- and 1.8-fold higher than the maximum production rate of the pPheDH clone. The better producing pPYF clone was further evaluated in a 5?l stirred-tank fermenter (37?°C, an aeration rate of 1 vvm, an agitation speed of 400?rpm). In the fermenter, the maximum concentration of L: -Phe (366?mg/l) was achieved in a much shorter period compared to in the shake flasks. In the latter, the highest titer of L: -Phe was only 76?% of the maximum value attained in the fermenter.     

含甘油脱水酶激活因子编码基因的产1,3-丙二醇新型重组菌的构建

利用PCR技术扩增来源于弗氏柠檬杆菌(Citrobacter freundii)的甘油脱水酶编码基因dhaB以及甘油脱水酶激活因子编码基因dhaGdhaF,将其与1,3-丙二醇氧化还原酶同工酶的编码基因yqhD串联在温控表达载体pHsh上,构建重组菌E.coliJM109(pHsh-dhaB-dhaG-dhaF-yqhD)。SDS-PAGE分析显示,融合表达产物的分子量同核酸序列测定的推导值相符。与未串联甘油脱水酶激活因子编码基因的重组菌E.coliJM109(pHsh-dhaB-yqhD)相比,1,3-丙二醇的产量提高了28%。     

产1，3-丙二醇重组大肠杆菌JM109（pEtac—dhaB—tac—yqhD）的构建

在生物柴油的生产过程中,最高可得到约10％的副产物甘油,副产物甘油的去向将成为生物柴油大规模产业化发展所面临的严峻问题。以生物柴油副产物甘油为原料耦合生产1,3-丙二醇,不仅解决了生物柴油副产物甘油的出路问题,同时降低了1,3-丙二醇的生产成本。本研究在前期工作的基础上,分别获得了来源于肺炎克雷伯氏茵的甘油脱水酶编码基因dhaB和来源于大肠杆菌的1,3-PD氧化还原酶同工酶编码基因yqhD,利用表达载体pEtac串联构建了重组质粒pEtac—dhaB—tac—yqhD,将其转化大肠杆菌得到产1,3-丙二醇重组大肠杆菌JM109（pEtac—dhaB-tac—yqhD）,降低了代谢中间产物3-羟基丙醛的积累,提高了1,3-丙二醇的产量。     

Production of 1,3-Propanediol from Glucose by Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21(DE3)

A range of recombinant strains of Escherichia coli were developed to produce 1,3-propanediol (1,3-PDO), an important C3 diol, from glucose. Two modules, the glycerol-producing pathway converting dihydroxyacetone phosphate to glycerol and the 1,3-PDO-producing pathway converting glycerol to 1,3-PDO, were introduced into E. coli. In addition, to avoid oxidative assimilation of the produced glycerol, glycerol oxidative pathway was deleted. Furthermore, to enhance the carbon flow to the Embden- Meyerhof-Parnas pathway, the Entner-Doudoroff pathway was disrupted by deleting 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase. Finally, the acetate production pathway was removed to minimize the production of acetate, a major and toxic by-product. Flask experiments were carried out to examine the performance of the developed recombinant E. coli. The best strain could produce 1,3-PDO with a yield of 0.47 mol/mol glucose. Along with 1,3-PDO, glycerol was produced with a yield of 0.33 mol/mol glucose.     

以葡萄糖为唯一碳源合成聚-4-羟基丁酸的重组大肠杆菌的构建

为实现重组大肠杆菌以葡萄糖为唯一碳源合成均聚的P( 4HB) ,PCR扩增大肠杆菌编码谷氨酸:琥珀酰半缩醛转氨基酶基因(gabT) ,谷氨酸脱羧酶基因(gadA)以及富养罗尔斯通氏菌(Ralstoniaeutropha)H16的4_羟基丁酸脱氢酶基因(gadB) ,并组装到携带富养罗尔斯通氏菌(Ralstoniaeutropha)H16的PHA聚合酶基因(phaC)和克氏梭菌(Clostridiumkluyveri)中编码4_羟基丁酸:CoA转移酶基因(orfZ)的重组质粒pKESS5 3上,形成一个大的操纵元。携带重组质粒的大肠杆菌获得从三羧酸循环的中间物———α_酮戊二酸到P( 4HB)的代谢途径。结果表明,重组大肠杆菌可以以葡萄糖为唯一碳源合成均聚的P( 4HB) ,当向以葡萄糖为唯一碳源的无机培养基添加蛋白胨、酵母提取物、酪蛋白水解物时,P( 4HB)的含量可以高达菌体干重的30 %。     

Construction of glycerol synthesis pathway in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> for bioconversion of glucose into 1,3-propanediol

1,3-Propanediol (1,3-PDO) is an important three-carbon compound widely used in new polyester polymer materials. Natural organisms that can produce 1,3-PDO from glycerol were well studied. However, no natural microorganisms found could directly convert glucose to 1,3-PDO due to its insufficient glycerol synthesis pathway. In this study, two essential glycerol synthesis genes, CgGPD gene (encoding glycerol-3-phosphate dehydrogenase from Candida glycerinogenes) and ScGPP2 gene (encoding glycerol-3-phosphatase from Saccharomyces cerevisiae), were expressed in wild-type Klebsiella pneumoniae, a natural 1,3-PDO producers with reduction pathway for 1,3-PDO synthesis from glycerol. The results of fermentation, key enzyme activities, and metabolites analysis confirmed that recombinant K. pneumoniae now possessed a metabolic pathway capable of converting glucose to 1,3-PDO. The strain could produce 1,3-PDO from glucose with a final titer of 17.27 g/L with 40 g/L glucose in the medium, showing a 1.26-fold increase compared with 30 g/L glucose. Also, adding certain concentrations of glycerol could quickly initiate the 1,3-PDO synthetic pathway and promote the accumulation of 1,3-PDO, which could shorten the fermentation cycle. These results have important implications for further studies involving the use of one strain for bioconversion of glucose to 1,3-PDO.