代谢工程改造酿酒酵母合成柠檬烯及其衍生物

柠檬烯及其衍生物紫苏酸作为重要的生物活性天然产物，广泛应用于食品、化妆品、保健品和医药等行业。然而，低效率的植物提取与高能耗的化工合成限制了柠檬烯和紫苏酸的工业合成。本研究在酿酒酵母中通过过氧化物酶体区室化表达绿薄荷来源的柠檬烯合酶，构建获得重组菌株，柠檬烯产量为0.038 mg/L。采用模块化工程分步表达参与柠檬烯合成的基因ERG10、ERG13、tHMGR、ERG12、ERG8、IDI1、MVD1、ERG20ww以及tLS，以研究其对柠檬烯产量的影响。通过增加前体模块，柠檬烯产量增加至1.14 mg/L。采用高拷贝数的质粒表达上述关键基因，柠檬烯的产量显著提高，达到86.74 mg/L，提高至初始菌株产量的4 337倍。以构建的柠檬烯生产菌株为出发菌株，通过表达丹参来源的细胞色素P450酶基因，实现了紫苏酸的生成，其产量达4.42 mg/L，为利用酿酒酵母构建高产单萜类天然产物的细胞工厂奠定了基础。     

采用基因敲除和反义技术抑制酿酒酵母ERG7基因表达

酿酒酵母(Saccharomyces cerevisiae)固有的甲羟戊酸(MVA)/麦角甾醇代谢途径生成的中间体2,3-氧化鲨烯是三萜类化合物的合成前体,以酿酒酵母为底盘细胞通过合成生物学技术组建这些化合物的代谢途径时,需要下调2,3-氧化鲨烯流向麦角甾醇的代谢流。在酿酒酵母中由羊毛甾醇合酶(ERG7)催化的2,3-氧化鲨烯环化是麦角甾醇和三萜类化合物生物合成分支形成的关键位点。采用基因敲除和反义RNA 2种技术对ERG7基因的表达进行下调。设计含有与ERG7基因ORF两侧序列同源的长引物,以质粒PUG66为模板进行PCR扩增,构建带有loxP-Marker-loxP的ERG7基因敲除组件,采用LiAc/SS Carrier DNA/PEG方法转化双倍体酿酒酵母INVSc1,通过同源重组的方式获得酿酒酵母ERG7基因单倍体缺失突变株,并对其进行了分子生物学确证。大量培养野生型和突变型菌株,菌体冷干后在碱醇溶液中90℃回流1h,正己烷萃取后旋蒸干溶剂,甲醇溶解残留物麦角甾醇。通过TLC和HPLC方法比较麦角甾醇含量,结果表明:与野生型菌株相比,突变型菌株的麦角甾醇含量明显降低。     

代谢工程改造酿酒酵母提高法尼醇产量

【目的】法尼醇(FOH,C_(15)H_(26)O)是一种具有芳香气味的非环状倍半萜醇,被广泛应用于化妆品和医学药物的工业化生产,也可作为航空燃料的理想替代品。具有食品级安全性的酿酒酵母细胞能够合成内源性法尼醇,但其产量很低,无法满足工业生产的需要。因此,需要采用代谢工程手段,改造法尼醇合成途径,以有效提高法尼醇在酿酒酵母中的产量。【方法】以酿酒酵母工业菌株CEN.PK2-1D为底盘细胞,强化甲羟戊酸途径中关键酶的表达水平和弱化麦角固醇合成分支途径,以提高法尼醇合成所需的直接前体物质法尼基焦磷酸(FPP);并分别表达催化FPP合成法尼醇的五种内源磷酸酶和两种异源合酶,筛选能高效合成法尼醇的磷酸酶或合酶。【结果】通过在CEN.PK2-1D(法尼醇产量0.1mg/L)中强化表达甲羟戊酸途径中截短形式的HMG-CoA还原酶(tHMGR1)和FPP合酶(ERG20),使法尼醇产量提高约50.8倍,达到5.08 mg/L;使用HXT1启动子替换鲨烯合酶编码基因ERG9启动子以下调其表达水平,使法尼醇产量进一步提升47.1倍,达到239.17 mg/L。在此基础上,筛选发现,表达酿酒酵母内源性磷酸酶PAH1时,获得最高产量法尼醇,达到393.13 mg/L。【结论】采用代谢工程策略对酿酒酵母法尼醇合成途径进行改造,有效提高法尼醇产量至393.13 mg/L,为目前报道的在酿酒酵母中摇瓶培养条件下的最高产量。     

产玉米黄质的人工酵母细胞的构建

为了实现微生物异源合成天然类胡萝卜素玉米黄质,以一株产β-胡萝卜素的酿酒酵母为底盘细胞,利用合成生物学技术构建人工酵母细胞。通过在染色体整合玉米黄质生物合成关键酶-β-胡萝卜素羟化酶（CrtZ）,并对其9种来源进行筛选,发现整合欧文氏菌来源的β-胡萝卜素羟化酶的菌株获得玉米黄质的最高产量。法尼基焦磷酸（FPP）作为合成萜烯类天然产物的重要前体,通过敲除 Lpp1和Dpp1 基因,削减法尼基焦磷酸向法呢醇的转化,为玉米黄质的合成提供更多的前体,使玉米黄质的产量提高了1.27倍（从29 mg/L提高到36.8 mg/L）。在此基础上,通过增加欧文氏菌来源CrtZ的基因拷贝数及调节其启动子的强弱来增强β-胡萝卜素羟化酶的表达强度,使得玉米黄质的摇瓶产量达到96.2 mg/L,是目前公开报道中产量最高的。     

甾醇生物合成中的关键酶-环氧角鲨烯环化酶的分子生物学研究

植物体中环氧角鲨烯环化酶催化2,3-环氧角鲨烯形成一系列三萜烯,为甾醇和三萜化合物的生物合成提供前体。这一催化反应被认为是甾醇和三萜化合物生物合成分支形成的关键位点．综述了甾醇和三萜化合物生物合成中的关键酶——环氧角鲨烯环化酶（OSCs）家族的生物学功能,基因克隆与属性,酶的细胞定位与酶活的表达调控等分子生物学研究进展．     

不同植物药提取物对灵芝细胞生长和胞内三萜产物形成的影响

研究了不同植物药的水提和醇提物对灵芝深层发酵过程中菌丝量和胞内三萜产量的影响。将不同植物药的水提物和醇提物分别加入到发酵基础培养基中,培养7d后检测灵芝生物量和胞内三萜含量。结果表明,金银花和枸杞子水提物添加浓度为100mg/L时,可促进灵芝细胞的生长（p＜0.05）。连翘水提物对灵芝生长和胞内三萜的形成都有显著促进作用,当连翘水提物浓度为400mg/L时,胞内三萜产量从对照的（192.54±8.99）mg/L提高到（302.52±3.79）mg/L。金银花和枸杞子醇提物浓度为200mg/L时能显著促进灵芝细胞生长;枸杞子醇提物在同样浓度下还能促进灵芝胞内三萜的形成。但板蓝根和银杏叶水提物和醇提物都对灵芝的细胞生长和胞内三萜形成有较强的抑制作用。     

代谢调节剂对紫杉醇及其相关化合物生物合成的影响

在诱导子添加的基础上研究了氯化氯代胆碱（CCC）和肉桂酸（CA）的浓度和时间因互对红豆杉悬浮培养细胞活力、三尖杉宁碱（Cephlomannine）和紫杉醇（Taxol）生物合成的影响,并进一步探讨了两者与前体的协同作用。实验表明：（1）氯化氯代胆碱、内桂酸对 细胞活力的影响均为：延滞期＞稳定期＞对数期;（2）d18加入氯化氯代胆碱10mg/L,肉桂酸20mg／L可使紫杉醇含量分别提高10,14倍;d18加入乞肥乞采抻填20mg/L可使三尖杉宁碱含量提高2倍,d12加入肉桂酸20mg/L可提高10倍;（3）40mg/L氯化氯代胆碱,20mg/L肉桂酸,5mg／L苯异丝氨酸,40mg/L甲瓦龙酸（MVA）内酯对提高紫杉醇产量较优;而10mg/L氯化氯代胆碱,20mg/L肉桂酸,10mg/L苯异丝氨酸,20mg/L 龙酸（MVA）内酯对三尖杉宁碱合成剂。     

产瓦伦西亚烯酿酒酵母的表达载体适配及发酵碳氮源优化

瓦伦西亚烯是一种倍半萜类化合物,广泛应用于香水、香皂、食品和饮料等工业制造上。但由于其自然含量极低,且目前获取瓦伦西亚烯的方法较为麻烦且花费高,因而构建细胞工厂进行瓦伦西亚烯的生物合成是更为高效和环保的方法。选取酿酒酵母(Saccharomyces cerevisiae)作为宿主构建细胞工厂,先在酿酒酵母基因组上引入黄扁柏的瓦伦西亚烯合成酶(Valencene synthase from Callitropsis nootkatensis,CnVS),实现瓦伦西亚烯的初步合成,初始产量为4.16 mg/L。随后利用CRISPR/Cas9系统对酿酒酵母中Mevalonate(MVA)途径的erg9和rox1基因进行敲除,提高通往瓦伦西亚烯合成的碳流量。不同碳氮源浓度发酵的结果表明,细胞生长积累过高可能不利于瓦伦西亚烯的积累。最后探究了不同CnVS表达载体对瓦伦西亚烯产量的影响,并获得17.54 mg/L的最高产量,是出发菌株的4.2倍。     

9,10-环甲基十七烷酸诱导灵芝三萜酸合成的信号转导和基因表达分析

为探究9,10-环甲基十七烷酸（9,10-CMA）诱导灵芝三萜酸合成的相关信号机制,分析了NO信号的介导作用。结果表明,在NO信号分子介导下,9,10-CMA可有效地刺激灵芝菌丝体中三萜合成关键酶细胞色素CYP450和苯丙氨酸解氨酶（PAL）的活化以及三萜酸的合成。NO可作为灵芝菌丝体中CYP450产生和PAL活化的上游分子发挥作用。在9,10-CMA诱导下,通过荧光定量PCR分析了6个关键酶基因在灵芝三萜酸合成过程的动态表达。结果表明,在9,10-CMA诱导下与灵芝三萜酸合成相关的角鲨烯合成酶基因sqs、细胞色素P450单加氧酶CYP5150L8基因和3‐羟基‐3‐甲基戊二酰辅酶A还原酶基因hmgr的上调最显著,提示这3个酶在三萜诱导过程中具有重要作用。     

利用基因工程改造的大肠杆菌合成蒎烯

目的:以大肠杆菌为底盘细胞,利用合成生物学手段导入外源杂合蒎烯合成代谢途径,构建高效合成蒎烯的微生物工厂。方法:将来源于酵母和粪肠球菌的异源杂合甲羟戊酸(MVA)代谢途径和来源于北美巨冷杉的牻牛儿焦磷酸合酶(GPPS)和蒎烯合成酶(PS)基因序列共同导入大肠杆菌,构建催化蒎烯合成的大肠杆菌工程菌。首先优化合成GPPS、PS基因,再分别以pETDuet-1和pET-24a(+)载体为基础构建GPPS、PS共表达和融合表达载体,并分别转化大肠杆菌获得工程菌E.hzh01和E.hzh02,摇瓶培养后利用GC-MS技术检测E.hzh01和E.hzh02的蒎烯产量。进一步获取MVA代谢途径相关酶基因mvaE、mvaS、ERG12、ERG8、ERG19、IDI序列,分别利用多顺反子模型和BioBrick方法构建2种不同方案的异源MVA代谢途径,再将异源MVA代谢途径和GPPS、PS融合表达基因共同转化大肠杆菌,构建完整蒎烯合成代谢工程菌株E.hzh03和E.hzh05。结果:摇瓶培养E.hzh01和E.hzh02的蒎烯产量分别为0.85和1.86 mg/L,结果表明融合表达更有利于蒎烯的合成。E.hzh03和E.hzh05摇瓶培养得到的蒎烯产量分别为6.32和19.26 mg/L。结论:利用融合表达载体和多顺反子模型导入异源蒎烯代谢途径构建大肠杆菌工程菌,可以显著提高蒎烯的产量,为工业生物合成蒎烯奠定了一定的基础。     

URA3基因影响青蒿酸酿酒酵母工程菌中试发酵产量

CRISPR/Cas9基因编辑技术已经被广泛应用于工程酿酒酵母的基因插入、基因替换和基因敲除,通过使用选择标记进行基因编辑具有简单高效的特点。前期利用CRISPR/Cas9系统敲除青蒿酸生产菌株酿酒酵母(Saccharomyces cerevisiae) 1211半乳糖代谢负调控基因GAL80,获得菌株S. cerevisiae 1211-2,在不添加半乳糖诱导的情况下,青蒿酸摇瓶发酵产量达到了740 mg/L。但在50 L中试发酵实验中,S. cerevisiae 1211-2很难利用对青蒿酸积累起到决定性作用的碳源-乙醇,青蒿酸的产量仅为亲本菌株S.cerevisiae 1211的20%–25%。我们推测因遗传操作所需的筛选标记URA3突变,影响了其生长及青蒿酸产量。随后我们使用重组质粒pML104-KanMx4-u连同90 bp供体DNA成功恢复了URA3基因,获得了工程菌株S. cerevisiae 1211-3。S. cerevisiae 1211-3能够在葡萄糖和乙醇分批补料的发酵罐中正常生长,其青蒿酸产量超过20g/L,与亲本菌株产量相当。研究不但获得了不加半乳糖诱导的青...     

Engineering Saccharomyces cerevisiae for high yield production of α-amyrin via synergistic remodeling of α-amyrin synthase and expanding the storage pool

α-Amyrin is a plant-originated high-valued triterpene that is highly effective against several pathological ailments. α-Amyrin production by engineered Saccharomyces cerevisiae has been achieved by introducing α-amyrin synthase (αAS). However, the low yield of α-amyrin highly limits its industrial application; the low catalytic activity of αAS and the toxic effect of α-amyrin have been considered key elements. In this study, the highest yield of α-amyrin was obtained in engineered S. cerevisiae by remodeling α-amyrin synthase MdOSC1 and expanding the storage pool. The yield of α-amyrin was increased to 11-fold higher than that of the control by the triple mutant MdOSC1^{N11T/P250H/P373A} obtained based on the modeling analysis. Furthermore, key genes of MVA pathway were overexpressed to provide sufficient precursors, and DGA1 (Diacylglycerol acyltransferase) was overexpressed to expand the intracellular storage capacity. Finally, the as-constructed aAM12 strain produced 213.7 ± 12.4 mg/L α-amyrin in the shake flask and 1107.9 ± 76.8 mg/L in fed-batch fermentation; the fermentation yield was 106-fold higher than that of the original aAM1 strain under the same conditions, representing the highest α-amyrin yield in yeast reported to date. Microbial production of α-amyrin with over 1 g/L will be suitable for commercialization and can accelerate the industrial production of α-amyrin in yeast.     

过表达乙酰辅酶A乙酰基转移酶2基因(RKAcat2)对红冬孢酵母产类胡萝卜素的影响

[背景] 乙酰辅酶A乙酰基转移酶（Acetyl Coenzyme A Acyltransferase,Acat）是硫解酶家族的一员,分为I型和II型,而II型作为甲羟戊酸（Mevalonate,MVA）途径的第一个限速酶,其表达水平和催化活性会影响萜类及其衍生物的合成量。[目的] 分析Acat II型基因的过表达对红冬孢酵母产类胡萝卜素的影响。[方法] 从红冬孢酵母YM25235菌株中克隆编码Acat II型的基因RKAcat2,将其回转到红冬孢酵母YM25235菌株中,构建一株RKAcat2基因过表达菌株进行分析。[结果] 与对照菌株相比,RKAcat2基因过表达使YM25235菌株中类胡萝卜素含量提高了50.53%,而菌株中油脂含量降低了22.80%,脂肪酸组成中油酸含量显著下降了17.78%,而且菌株中乙酰辅酶A （Coenzyme A,CoA）的含量也下降了13.64%。[结论] 过表达RKAcat2基因促进更多乙酰CoA进入MVA途径中,从而提高了类胡萝卜素的合成水平,这与部分MVA途径和类胡萝卜素合成途径中基因的转录分析结果一致。研究结果可为进一步通过代谢工程手段提高产油红酵母中类胡萝卜素及其特定组分含量的研究提供参考。     

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant (Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. α-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.     

Metabolic Engineering for Production of β-Carotene and Lycopene in Saccharomyces cerevisiae

We have engineered a conventional yeast, Saccharomyces cerevisiae, to confer a novel biosynthetic pathway for the production of β-carotene and lycopene by introducing the bacterial carotenoid biosynthesis genes, which are individually surrounded by the promoters and terminators derived from S. cerevisiae. β-Carotene and lycopene accumulated in the cells of this yeast, which was considered to be a result of the carbon flow for the ergosterol biosynthetic pathway being partially directed to the pathway for the carotenoid production.     

Engineering heterologous molybdenum-cofactor-biosynthesis and nitrate-assimilation pathways enables nitrate utilization by Saccharomyces cerevisiae

Metabolic capabilities of cells are not only defined by their repertoire of enzymes and metabolites, but also by availability of enzyme cofactors. The molybdenum cofactor (Moco) is widespread among eukaryotes but absent from the industrial yeast Saccharomyces cerevisiae. No less than 50 Moco-dependent enzymes covering over 30 catalytic activities have been described to date, introduction of a functional Moco synthesis pathway offers interesting options to further broaden the biocatalytic repertoire of S. cerevisiae. In this study, we identified seven Moco biosynthesis genes in the non-conventional yeast Ogataea parapolymorpha by SpyCas9-mediated mutational analysis and expressed them in S. cerevisiae. Functionality of the heterologously expressed Moco biosynthesis pathway in S. cerevisiae was assessed by co-expressing O. parapolymorpha nitrate-assimilation enzymes, including the Moco-dependent nitrate reductase. Following two-weeks of incubation, growth of the engineered S. cerevisiae strain was observed on nitrate as sole nitrogen source. Relative to the rationally engineered strain, the evolved derivatives showed increased copy numbers of the heterologous genes, increased levels of the encoded proteins and a 5-fold higher nitrate-reductase activity in cell extracts. Growth at nM molybdate concentrations was enabled by co-expression of a Chlamydomonas reinhardtii high-affinity molybdate transporter. In serial batch cultures on nitrate-containing medium, a non-engineered S. cerevisiae strain was rapidly outcompeted by the spoilage yeast Brettanomyces bruxellensis. In contrast, an engineered and evolved nitrate-assimilating S. cerevisiae strain persisted during 35 generations of co-cultivation. This result indicates that the ability of engineered strains to use nitrate may be applicable to improve competitiveness of baker's yeast in industrial processes upon contamination with spoilage yeasts.     

Evaluation of pyruvate decarboxylase‐negative Saccharomyces cerevisiae strains for the production of succinic acid

Dicarboxylic acids are important bio‐based building blocks, and Saccharomyces cerevisiae is postulated to be an advantageous host for their fermentative production. Here, we engineered a pyruvate decarboxylase‐negative S. cerevisiae strain for succinic acid production to exploit its promising properties, that is, lack of ethanol production and accumulation of the precursor pyruvate. The metabolic engineering steps included genomic integration of a biosynthesis pathway based on the reductive branch of the tricarboxylic acid cycle and a dicarboxylic acid transporter. Further modifications were the combined deletion of GPD1 and FUM1 and multi‐copy integration of the native PYC2 gene, encoding a pyruvate carboxylase required to drain pyruvate into the synthesis pathway. The effect of increased redox cofactor supply was tested by modulating oxygen limitation and supplementing formate. The physiologic analysis of the differently engineered strains focused on elucidating metabolic bottlenecks. The data not only highlight the importance of a balanced activity of pathway enzymes and selective export systems but also shows the importance to find an optimal trade‐off between redox cofactor supply and energy availability in the form of ATP.