在大肠杆菌内引入甲羟戊酸途径高效合成抗疟药青蒿素前体——紫穗槐-4,11-二烯

以青蒿素为基础的联合药物疗法 (ACTs) 被认为是目前治疗恶性疟疾的最有效方法。然而青蒿素供应不足且价格昂贵,限制了ACTs的广泛使用。采用基因工程手段构建异源类异戊二烯生物合成途径,利用大肠杆菌发酵能高效合成抗疟药青蒿素前体——紫穗槐-4,11-二烯。首先在大肠杆菌Escherichia coli DHGT7中引入人工合成的紫穗槐-4,11-二烯合酶基因,利用大肠杆菌内源的法尼基焦磷酸,成功获得了紫穗槐-4,11-二烯。为提高前体供给,引入粪肠球菌的甲羟戊酸途径,紫穗槐-4,11-二烯的产量提高了13     

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。     

代谢工程酵母菌合成紫杉烯的研究

紫杉烯是紫杉醇生物合成的重要中间体,为在酿酒酵母(Saccharomyces cerevisiae)中建立一个生物合成紫杉烯的代谢途径,克隆了酵母的羟甲基戊二酰CoA(3-hydroxy-3-methylglutarylcoenzyme A,HMG-CoA)还原酶基因和=牛儿基=牛儿基二磷酸(geranylgeranyl diphosphate,GGDP)合酶基因,并构建了其融合表达载体pGBT9/HG;同时构建了包含紫杉烯合酶基因的表达载体pADH/TS;将这两个表达载体共转化酵母细胞,通过GC-MS分析检测工程酵母的代谢产物,结果表明获得的工程酵母能够合成紫杉烯,即在酵母细胞中建立了一个合成紫杉烯的代谢途径。     

操纵茶树类黄酮3′-羟基化酶生物合成B环-3′,4′-二羟基黄酮类化合物

【目的】操纵茶树类黄酮3′-羟基化酶,生物合成B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素。【方法】构建了4个茶树类黄酮3′-羟基化酶基因(CsF3′H)和拟南芥的P450还原酶基因(ATR)融合表达质粒:SUMO-CsF3'H[7-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA、SUMO-CsF3'H[7-517]::ATR2[75-711]3 AA和SUMO-CsF3'H[28-517]::ATR2[75-711]3 AA,分别转化大肠杆菌菌株TOP10、DH5α和BL21,获得12个转化菌株S1–S12;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒p YES-Dest52-CsF3′H,转化酵母菌株WAT11,得到转化菌株S13;构建了茶树类黄酮3′-羟基化酶基因CsF3′H表达质粒pES-URA-CsF3′H,及茶树黄烷酮3-羟基化酶基因CsF3H与拟南芥黄酮醇合成酶基因At FLS的融合表达质粒pES-HIS-CsF3H::At FLS 9AA,二者共转化酵母菌株WAT11,获得转化菌株S14。【结果】转化SUMO-CsF3'H[28-517]::ATR1[49-688]3 AA质粒的TOP10菌株S6在25°C条件下发酵,转化效率最高,能将1000μmol/L柚皮素、二氢山奈酚和山奈酚,分别转化生成287.93μmol/L圣草酚、131.76μmol/L二氢槲皮素和188.62μmol/L槲皮素。发酵菌株S13能分别将1000μmol/L柚皮素、二氢山奈酚和山奈酚,最多能转化生成734.32μmol/L圣草酚、446.07μmol/L二氢槲皮素和594.64μmol/L槲皮素。喂食S14发酵菌株5 mmol/L的底物柚皮素,在发酵36–48 h中,最多能生成1412.16μmol/L圣草酚、490.25μmol/L山奈酚、445.75μmol/L槲皮素、66.75μmol/L二氢槲皮素和73.50μmol/L二氢山奈酚。【结论】本研究首次将茶树类黄酮3′-羟基化酶基因应用于B环-3′,4′-二羟基黄酮类化合物圣草酚、二氢槲皮素和槲皮素的生物合成。     

表达3-甾酮-△1-脱氢酶降解植物甾醇合成雄甾-1,4-二烯-3,17-二酮

构建分枝杆菌表达载体pMTac并在分枝杆菌Mycobacterium neoaurum JC-12中加强表达甾醇降解过程中的关键酶3-甾酮-△1-脱氢酶(KSDD)以提高雄甾-1,4-二烯-3,17-二铜(ADD)的产量。将p MF41的启动子pACE替换成tac启动子构建载体pMTac,在分枝杆菌中分别表达报告基因绿色荧光蛋白(GFP)和关键酶KSDD,通过GFP亮度和KSDD酶活验证tac启动子在M.neoaurum JC-12中的效果,并发酵验证加强表达KSDD对产物ADD的影响。荧光显微照片表明两个载体均能在M.neoaurum JC-12表达GFP,但tac启动子的效果比pACE强。酶活测定结果为重组菌M.neoaurum JC-12/pMTac-ksdd破碎细胞上清液中KSDD酶活比原始菌提高了6.53倍,比M.neoaurum JC-12/pMF41-ksdd提高了4.36倍。摇瓶发酵显示重组菌M.neoaurum JC-12/pMTac-ksdd ADD的产量比原始菌提高了22.2%,由4.86 g/L提高到5.94 g/L,而AD的产量由0.92 g/L减少到0.17 g/L,降低了81.5%;与M.neoaurum JC-12/p MF41-ksdd比,ADD产量提高了12.7%,AD降低了71.2%。以20 g/L植物甾醇为底物,5 L发酵罐中重组菌M.neoaurum JC-12/pMTac-ksdd的ADD产量达到10.28 g/L。结果表明,构建的新型表达载体pMTac适用于在M.neoaurum JC-12中加强表达关键酶KSDD,而且在M.neoaurum JC-12中过量表达KSDD有助于ADD产量的提高,为目前报道的发酵法利用新金色分枝杆菌降解植物甾醇合成ADD的最高水平。     

产朊假丝酵母整合表达载体的构建

[目的]构建一个产朊假丝酵母(Candida utilis,C.utilis)整合表达载体.[方法]该载体以质粒pBR322为骨架,包括3-磷酸甘油醛脱氢酶(GAP)启动子和终止子、放线菌酮(CYH)抗性基因和18S rDNA介导的同源整合区.再以木聚糖酶基因为目标基因,插入pGLR9K载体上,构建重组表达载体,电击转化C.utilis.对阳性转化子进行酶活测定,检测其表达情况.[结果]转化子的胞内外都可检测到木聚糖酶酶活,酶活可达60 U/mL.[结论]本实验构建了一个C.utilis载体,并用此载体表达了木聚糖酶基因,本研究将为产朊假丝酵母工程菌在饲料添加剂及食品行业中的应用提供又一个新的实验平台.     

乳酸克鲁维酵母表达型T载体的研发及应用

[目的]构建以乳酸克鲁维酵母表达载体p KLAC2为基础的表达型T载体。[方法]利用定点突变技术突变载体p KLAC2上的XcmⅠ位点,获得表达载体p KL-MUT;PCR扩增含黄色荧光蛋白基因的XcmⅠ酶切盒,通过EcoRⅠ和XhoⅠ位点连接至表达载体p KL-MUT,构建成重组载体p KL-YFP,限制性内切酶XcmⅠ酶切后即产生T载体p KL-T。连接融合基因14-3-3-Zs G转化至乳酸克鲁维酵母GG799。[结果]利用该T载体可成功克隆外源基因14-3-3-Zs G并在乳酸克鲁维酵母中表达14-3-3-Zs G蛋白,荧光和Western blotting验证正确。[结论]乳酸克鲁维酵母表达型T载体已成功构建,具有快速克隆高效分泌表达外源基因的特点,对于促进乳酸克鲁维酵母表达系统表达相关蛋白的产业化具有实际意义。     

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.     

酿酒酵母SRP40基因对细胞耐受性影响的研究

【目的】本论文研究酿酒酵母srp40³⁹突变基因对酵母细胞异丁醇耐受性的影响。【方法】首先,以酿酒酵母野生型W303-1A和突变株EMS39染色体DNA为模板克隆野生型SRP40基因和srp40³⁹突变基因;然后,将野生型SRP40基因和srp40³⁹突变基因分别连接到质粒YCplac22上,构建质粒YCplac22-SRP40和YCplac22-srp40³⁹。将质粒YCplac22-SRP40、YCplac22-srp40³⁹以及YCplac22空质粒分别转化入野生型酿酒酵母W303-1A中,分别得到W303-1A-SRP40工程菌、W303-1A-srp40³⁹工程菌和W303-1A-control工程菌。将3株工程菌分别置于含1.0%异丁醇、1.3%异丁醇、8.0%乙醇和0.5%异戊醇的CM培养基中进行发酵,测定细胞密度(OD₆₀₀)和生长情况,并计算2–10 h的比生长速率（μ）。将3株工程菌于55°C热激4 min后做稀释...     

牻牛儿基牻牛儿基焦磷酸合酶和紫杉二烯合酶基因在灰盖鬼伞中的组合表达

紫杉二烯是紫杉醇合成途径中的前体物质。紫杉醇是红豆杉的一种重要的次级代谢产物,是一种重要的新型抗癌药物。然而,紫杉醇在植物中含量低且难提取,限制了高效应用。利用基因工程手段,借助担子菌类真菌灰盖鬼伞具有的内源类异戊二烯合成途径,构建含有牻牛儿基牻牛儿基焦磷酸(Geranylgeranyl diphosphate,GGPP)合酶和紫杉二烯合酶的融合基因表达载体p Bg GGTS和独立表达盒表达载体p Bg GGg TS,并分别转入灰盖鬼伞LT2菌株中,经过选择性筛选、PCR鉴定、Southern blotting杂交验证,分别获得了5株融合表达的灰盖鬼伞工程菌和5株独立表达盒的灰盖鬼伞工程菌株。各随机挑选了1株工程菌株,分别提取菌丝体和发酵液分析。GC-MS分析表明,两种工程菌株与原出发菌株的菌丝提取物无明显差异峰,而与出发菌株的发酵液提取物相比,两种转基因灰盖鬼伞的发酵液中均出现了明显的差异峰,采用GC-MS特征质量离子分析方法判定为紫杉二烯,分别为44 ng/L(转化p Bg GGg TS)和30 ng/L(转化p Bg GGTS)。结果表明,通过在灰盖鬼伞融合基因或各自独立表达的形式共表达ggpps和ts基因,可以生物合成紫杉二烯。     

Improvement of amorpha-4,11-diene production by a yeast-conform variant of Vitreoscilla hemoglobin

Amorpha-4,11-diene is the precursor of the antimalarial compound artemisinin. The effect of Vitreoscilla hemoglobin (VHb) and its yeast-conform variant (VHbm) on amorpha-4,11-diene production in engineered Saccharomyces cerevisiae was investigated. First, the VHb gene was mutated to the yeast-conform variant VHbm based on step-by-step extension of a short region of the gene through a series of polymerase chain reactions (PCR). The artificial VHbm gene contained codons preferred by the yeast translation machinery. Two yeast expression vectors containing VHb or VHbm gene were constructed and introduced into the amorpha-4,11-diene-producing strain S. cerevisiae WK1 to form WK1[VHb] and WK1[VHbm], respectively. Western blot and CO-difference spectrum absorbance assay showed that VHb and VHbm were successfully expressed. In shake flasks, VHbm expression conferred higher cell growth than VHb expression. GC-MS results indicated the amorpha-4,11-diene production in WK1[VHbm] and WK1[VHb] was 3- and 2-fold higher than that in WK1, respectively. This suggests that VHb might improve the amorpha-4,11-diene production in engineered S. cerevisiae.     

The improvement of amorpha-4,11-diene production by a yeast-conform variant

Aims:  To investigate the effect of the yeast-conform variant of the Artemisia annua gene encoding for amorpha-4,11-diene synthase (ADS) on the production of amorpha-4,11-diene in a transformed yeast.
Methods and Results:  The ADS gene was mutated to the yeast-conform variant ADSm . The ADSm synthesis was performed based on step-by-step extension of a short region of the gene through a series of polymerase chain reactions (PCR). The artificial ADSm gene contained codons preferred by the yeast translation machinery. The sequence was then integrated into a yeast expression vector pYeDP60. The fusion construct was active and the transformed yeast cells produced higher level of amorpha-4,11-diene compared with the plant gene-transformed yeast cells.
Conclusions:  Strains transformed with the yeast-conform allele ( ADSm ) were more efficient in terms of production of amorpha-4,11-diene than those transformed with the plant gene.
Significance and Impact of the Study:  We demonstrated that yeast-conform allele of foreign genes by serial PCR reactions can be a solution to low efficiency of heterologous gene expression in Saccharomyces cerevisiae cells.     

Production of the Artemisinin Precursor Amorpha-4,11-diene by Engineered Saccharomyces cerevisiae

The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN·PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced 600 and 100 μg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days’ batch cultivation. Revisions requested 14 November 2005; Revisions received 17 January 2006     

Amorpha-4,11-diene synthase: cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin

The sesquiterpenoid artemisinin, isolated from the plant Artemisia annua L., and its semi-synthetic derivatives are a new and very effective group of antimalarial drugs. A branch point in the biosynthesis of this compound is the cyclisation of the ubiquitous precursor farnesyl diphosphate into the first specific precursor of artemisinin, namely amorpha-4,11-diene. Here we describe the isolation of a cDNA clone encoding amorpha-4,11-diene synthase. The deduced amino acid sequence exhibits the highest identity (50%) with a putative sesquiterpene cyclase of A. annua. When expressed in Escherichia coli, the recombinant enzyme catalyses the formation of amorpha-4,11-diene from farnesyl diphosphate. Introduction of the gene into tobacco (Nicotiana tabacum L.) resulted in the expression of an active enzyme and the accumulation of amorpha-4,11-diene ranging from 0.2 to 1.7 ng per g fresh weight. Received: 8 June 2000 / Accepted: 21 August 2000     

Enhanced artemisinin production from engineered yeast precursors upon biotransformation

Abstract

Production of artemisinin in genetically modified microorganisms is an attractive option to enable sufficient supply of the effective antimalarial agent. Although a sundry of artemisinin precursors are available from engineered bacteria or yeast, no artemisinin has been manufactured by engineering any microbial platforms due to inaccessibility to unidentified steps. To this end, it is essential to consider how to convert artemisinin precursors to artemisinin, either biochemically or chemically. To establish a novel procedure of artemisinin production, we incubate the mixture of artemisinin precursors from engineered Sacchromyces cerevisiae with the cell-free enzyme extract of Artemisia annua. For the single gene-expressing strain INVScI (pYES-ADS), amorpha-4,11-diene accumulation within 48 h or 14 days led to higher artemisinin content than the control. In the multiple gene-expressing strain YPH501 (pYES-ADS:: pESC-CYP71AV1-DBR2), artemisinin accumulation from the 14-day-induced yeast precursor mixture was nearly equivalent between the single gene-transferred strain and the multiple gene-transferred strain. Alternatively, biotransformation of 48-hour-induced yeast amorpha-4,11-diene mixture by the cold-acclimated A. annua cell-free extract that possesses the abundant enzymes relevant to artemisinin biosynthesis gave rise to considerable elevation of artemisinin content up to 0.647% in maximum, accounting to 15-folds increase as the A. annua cell-free extract without cold-acclimation (0.045%), thereby providing a practical protocol for artemisinin overproduction through the interplay of engineered microbial artemisinin precursors with upregulated plant enzymes.     

Expression of a synthetic Artemesia annua amorphadiene synthase in Aspergillus nidulans yields altered product distribution

A gene encoding a plant terpene cyclase, Artemisia annua amorpha-4,11-diene synthase (ADS), was expressed in Aspergillus nidulans under control of a strong constitutive promoter, (p)gpdA. The transformants produced only small amounts of amorphadiene, but much larger amounts of similar sesquiterpenes normally produced as minor by-products in planta. In contrast, expression of ADS in Escherichia coli produced almost exclusively amorpha-4,11-diene. These results indicate that the host environment can greatly impact the terpenes produced from terpene synthases.     

A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene.     

Expression, purification, and characterization of recombinant amorpha-4,11-diene synthase from Artemisia annua L

A cDNA clone encoding amorpha-4,11-diene synthase from Artemisia annua was subcloned into a bacterial expression vector in frame with a His6-tag. Recombinant amorpha-4,11-diene synthase was produced in Escherichia coli and purified to apparent homogeneity. The enzyme showed pH optimum at pH 6.5, and a minimum at pH 7.5. Substantial activity was observed in the presence of Mg2+, Mn2+ or Co2+ as cofactor. The enzyme exhibits a low activity in the presence of Ni2+ and essentially no activity with Cu2+ or Zn2+. The sesquiterpenoids produced from farnesyl diphosphate in the presence of Mg2+ were analyzed by GC-MS. In addition to amorpha-4,11-diene, 15 sesquiterpenoids were produced. Only small quantitative differences in product pattern were observed at pH 6.5, 7.5, or 9.5. Amorpha-4,11-diene synthase showed significant increased product selectivity in the presence of Mn2+ or Co2+. Km for farnesyl diphosphate was 3.3, 8.0, and 0.7 microM in the presence of Mg2+, Mn2+ or Co2+, respectively. The corresponding kcat-values were 6.8, 15.0, and 1.3 x 10(-3) s(-1), respectively. Km and kcat for geranyl diphosphate were 16.9 microM and 7.0 x 10(-4) s(-1), respectively, at pH 6.5, in the presence of Mn2+.