人工酵母后鲨烯路径基因对7-脱氢胆固醇合成的影响

酵母内源后鲨烯路径中的固醇类物质,是异源甾体类药物合成的重要前体。为了通过微调后鲨烯路径,与异源模块进行适配,以期达到提高异源甾体类化合物表达的目的,以维生素D₃的直接前体-7-脱氢胆固醇(7-DHC)的合成为例,首先在固醇C-24甲基转移酶(ERG6)缺失的酿酒酵母BY4742中,通过导入人源固醇C-24 还原酶DHCR24,并过表达截短的羟甲基戊二酰辅酶A还原酶tHMGR,获得可以合成7-DHC的人工酵母。在此基础上,将后鲨烯路径分割并构建成ERG1、ERG7、ERG11、ERG24-25-26-27和ERG2-3这5个模块,分别在所构建的7-DHC合成菌株中过表达。通过GC-TOF/MS分析7-DHC以及后鲨烯路径中相关代谢中间体的含量,并结合主成分分析发现,过表达不同后鲨烯模块会引起后鲨烯路径上固醇组分的变化而最终影响7-DHC的产量:与出发菌株相比,过表达ERG11模块会显著强化其他固醇物质到酵母固醇的转化;而过表达ERG2-3模块则会减少鲨烯的积累,同时显著增加羊毛固醇及其之后的固醇组分的含量,并获得迄今为止7-DHC在微生物中摇瓶水平的最高产量。因此,对ERG11和ERG2-3的表达优化对7-DHC的合成以及后鲨烯路径代谢流的强化起到了显著的作用,是后续优化人工7-DHC合成酵母的潜在靶点。为研究后鲨烯路径与其他异源甾体合成模块间的适配,提供了可供参考的案例。     

毕赤酵母截短PGK1启动子与不同终止子组合调控外源基因表达

【目的】调控多基因表达对于优化代谢途径和合成生物学应用至关重要,构建不同的启动子和终止子组合,可作为毕赤酵母代谢途径改造和优化外源基因表达的有力分子调控工具。【方法】首先,将毕赤酵母组成型磷酸甘油酸激酶基因的启动子P_PGK1进行截短,构建截短启动子分别调控报告基因（绿色荧光蛋白基因egfp和β-半乳糖苷酶基因lac Z）表达的毕赤酵母重组菌。检测重组菌的报告基团转录水平、荧光强度和β-半乳糖苷酶产量。然后,构建了不同强度启动子和终止子组合（共27种组合）调控egfp表达的重组菌。最后,选取能调控基因高、中、低表达的6个启动子-终止子组合,调控β-呋喃果糖苷酶基因表达,构建β-呋喃果糖苷酶分泌表达的重组菌。【结果】构建的截短启动子(P_PP、P_PE、P_PG和P_PD)的强度是野生型启动子P_PGK1的70%–190%,最强的启动子为P_PD。分别与9个终止子组合时,P_PG、P_PE和P_PD     

甾醇C-24甲基转移酶基因在酿酒酵母中的内源表达及工程菌麦角甾醇的合成

通过高保真PCR克隆到含酿酒酵母甾醇C-24甲基转移酶基因编码序列及终止子序列的DNA片段ERG6, 以大肠杆菌-酿酒酵母穿梭质粒YEp352为载体, 磷酸甘油酸激酶基因PGK1启动子为上游调控元件构建了酵母菌表达质粒pPERG6。通过同源重组, 以铜离子螯合蛋白基因CUP1替换染色体上ERG6基因内部序列获得ERG6破坏菌株YS58-erg6, 其中麦角甾醇的合成被阻断, 同时细胞的生长也受到明显抑制。表达质粒pPERG6转化破坏菌株YS58-erg6后, 不但使细胞恢复了合成麦角甾醇的能力, 细胞生物量也得到明显提高, 这说明表达质粒上的ERG6基因得到了功能性的表达。分别用载体质粒YEp352和表达质粒pPERG6转化酿酒酵母单倍体菌株YS58, 获得对照菌株YS58(YEp352)和重组菌株YS58(pPERG6)。重组菌株YS58(pPERG6) 生物量和麦角甾醇含量分别是对照菌YS58(YEp352)的1.23和1.32倍。可见甾醇C-24甲基转移酶基因的高表达可以增强酵母细胞麦角甾醇的合成能力。     

多个调控元件调控萜类合成途径基因表达提高β-胡萝卜素的生产

强启动子对于获得目标产物最大代谢流量来说并不一定是最优的;相比之下,使用多个具有不同强度的调控元件对基因表达进行调控更有可能获得最优的表达强度.为了对比使用多个调控元件和使用强启动子调控萜类合成途径基因表达对β-胡萝卜素生产的影响,并通过对关键基因的组合调控提高β-胡萝卜素的生产.文中使用6个强度差异很大的人工调控元件,对萜类合成途径的8个基因进行调控.对于不同的基因,其最适的调控元件强度各不相同.对8个基因的调控使β-胡萝卜素产量提高1.2～3.5倍.和以前报道不一样的是,文中发现用适当强度的调控元件对dxr、ispG和ispH基因进行调控后,也能提高β-胡萝卜素的生产.对dxs和idi基因的组合调控将β-胡萝卜素产量提高了8倍,最终β-胡萝卜素产量达17.59 mg/g干重细胞.结果表明使用多个不同强度的调控元件对基因表达进行调控比仅使用强启动子调控更为有效,为提高目标产品合成能力提供了一种新的基因表达调控方案.     

大肠杆菌糖酵解途径和三羧酸循环启动子的表征及其应用

启动子是控制基因转录的重要元件,也是合成生物学研究和细胞工厂设计的关键环节。糖酵解途径和三羧酸循环是糖类分解代谢的中心代谢,受到包括启动子强度在内的严格调控。为了筛选一系列能满足合成生物学研究和细胞工厂设计需要的不同强度的内源性组成型启动子,利用报告基因——红色荧光蛋白m Cherry和在线分析软件,系统研究了大肠杆菌糖酵解和三羧酸循环中27个启动子的强度和核心结构元件。结果表明:这些启动子的强度范围变化很大,最强启动子Pgap A的强度是最弱启动子Pacn A强度的43. 6倍;启动子的-10序列和-35序列与它们的一致序列也不完全相同,两者之间的距离为17±3bp;但是,启动子的强度和启动子的结构特征基本一致。应用最强启动子Pgap A在重组大肠杆菌DH5αΔpck中分别表达磷酸烯醇式丙酮酸羧化酶基因和丙酮酸激酶基因,它们的酶活性分别提高了0. 32和1. 57倍,柠檬酸产量也提高了124. 7%和75. 5%。这些不同强度的启动子为大肠杆菌的合成生物学研究和细胞工厂设计奠定了一定的基础。     

高效合成番茄红素酿酒酵母菌株的构建

番茄红素作为一种高附加价值的萜类化合物已受到国内外研究者的广泛关注。首先对酿酒酵母Saccharomyces cerevisiae模式菌株S288c和YPH499合成番茄红素的能力进行分析比较,结果表明YPH499更适合作为底盘细胞用于番茄红素的合成。随后比较组成型启动子GPDpr、TEF1pr和诱导型启动子GAL1pr、GAL10pr对番茄红素合成的影响,结果发现以GPDpr、TEF1pr作为番茄红素合成途径基因crtE、crt B和crtI的启动子,摇瓶发酵60 h后,番茄红素产量为15.31 mg/L;以GAL1pr和GAL10pr为启动子时,其产量为123.89 mg/L,提高8.09倍。继续改造甲羟戊酸(MVA)途径,过量表达N-末端截短的关键酶基因t HMG1(3-羟基-3-甲基戊二酸单酰辅酶A还原酶),番茄红素产量为265.68 mg/L,单位菌体产量72.79 mg/g。文中所设计构建的异源表达番茄红素合成途径的酿酒酵母菌株单位细胞产量高,可以进一步改造和优化后用于番茄红素的工业化生产。     

三种酵母启动子在毕赤酵母中的功能比较

启动子是基因表达调控的重要顺式元件,启动子功能的强弱对于目的基因的表达非常重要.为找到一个启动转录功能较好的启动子,以绿色荧光蛋白(GFP)为报告基因,在毕赤酵母中研究不同启动子对外源蛋白表达的影响.首先采用PCR扩增的方法克隆了酿酒酵母甘油合成关键酶3-磷酸甘油脱氢酶基因启动子pScgpd,借助于载体pPIC9K和p...     

产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因（CgGPD）功能分析

【目的】从高产甘油生产菌株产甘油假丝酵母（Candida glycerinogenes）基因组中克隆了NAD+依赖3-磷酸甘油脱氢酶编码基因（CgGPD）,但是该基因及其上游调控序列具体的功能还是未知的。本文研究了CgGPD基因及其上游调控序列的功能。【方法】本文以酿酒酵母（Saccharomyces cerevisiae）及其渗透压敏感型突变株为宿主,构建3种不同的酵母表达载体导入酵母细胞,研究了不同酵母转化子在渗透压胁迫条件下CgGPD基因表达对细胞的耐高渗透压胁迫应答及其细胞的甘油合成能力的影响。【结果】实验结果表明无论是以来源于S. cerevisiae 的TPI启动子还是来源于CgGPD基因的启动子,过量表达CgGPD基因的转化子均能够显著加速葡萄糖消耗速度和提高甘油合成能力,在gpd1/gpd2突变株中表达CgGPD基因能够消除细胞对外界高渗透压的敏感性,同时转化子胞内甘油大量积累。【结论】CgGPD基因在野生型酵母S. cerevisiae W303-1A表达显著提高细胞的甘油合成能力,在gpd/1gpd2突变株中能够互补GPD1基因的功能,CgGPD基因表达受渗透压诱导 调控。     

合成芳香族化合物的酵母底盘改造策略*

芳香族化合物种类丰富,在多个行业具有广泛的用途,需求量大。通过构建微生物细胞工厂合成芳香族化合物具有独特的优势和工业化应用前景,其中酵母底盘因其清晰的遗传背景、完善的基因操作工具以及成熟的工业发酵体系等优势,常被用于构建细胞工厂。目前改造酵母底盘生产芳香族化合物的研究取得了一系列进展,并针对关键问题提出了一些可行的解决策略。针对酵母合成芳香族化合物的策略与挑战,从芳香族化合物合成路径改造、多样化碳源利用及转运系统改造、基因组多靶点改造、特殊酵母底盘及混菌系统构建、合成生物学高通量技术的应用这五个方面进行系统地梳理和阐述,为生产芳香族化合物的酵母底盘构建与改造提供思路。     

谷氨酸棒杆菌人工合成启动子文库的构建及应用

启动子是实现基因精细表达调控的重要工具,广泛应用于微生物的代谢工程改造。谷氨酸棒杆菌是重要的工业底盘,已报道的启动子文库较少且主要是基于完全人工设计的突变序列构建获得。本研究对谷氨酸棒杆菌odhA基因天然启动子的–10区及附近序列进行随机突变,借助rfp报告基因和荧光成像系统进行高通量筛选,构建了包含57个相对强度为2.4–16.7倍的人工启动子文库,最高强度可达强诱导型启动子P_trc的2.3倍。分析文库的突变序列,发现55个启动子突变体的–10区保守序列“TANNNT”均向3′端移动1–4个碱基,其中移动4个碱基的突变体占68%;同时发现强、中、弱启动子突变体在不同位置还呈现T或G保守碱基。选择5个不同强度的启动子应用于L-脯氨酸合成途径γ-谷氨酰激酶（ProB）的表达调控,结果显示,L-脯氨酸产量随着启动子强度增强逐渐提高,相对强度为9.8倍的启动子达到最高产量（6.4g/L）,更高强度的启动子将不再提高L-脯氨酸的产量。本研究基于谷氨酸棒杆菌天然启动子P_odhA构建文库,成功获得1个强度增强显著、分布均匀的新型启动子文库,可用于谷氨酸棒...     

Interactions of oxidosqualene cyclase (Erg7p) with 3-keto reductase (Erg27p) and other enzymes of sterol biosynthesis in yeast

In Saccharomyces cerevisiae and Candida albicans, two enzymes of the ergosterol biosynthetic pathway, oxidosqualene cyclase (Erg7p) and 3-keto reductase (Erg27p) interact such that loss of the 3-keto reductase also results in a concomitant loss of activity of the upstream oxidosqualene cyclase. This interaction wherein Erg27p has a stabilizing effect on Erg7p was examined to determine whether Erg7p reciprocally has a protective effect on Erg27p. To this aim, three yeast strains each lacking the ERG7 gene were tested for 3-ketoreductase activity by incubating either cells or cell homogenates with unlabeled and radiolabeled 3-ketosteroids. In these experiments, the ketone substrates were effectively reduced to the corresponding alcohols, providing definitive evidence that oxidosqualene cyclase is not required for the 3-ketoreductase activity. This suggests that, in S. cerevisiae, the protective relationship between the 3-keto reductase (Erg27p) and oxidosqualene cyclase (Erg7p) is not reciprocal. However, the absence of the Erg7p, appears to affect other enzymes of sterol biosynthesis downstream of lanosterol formation. Following incubation with radiolabeled and non-radiolabeled 3-ketosteroids we detected differences in hydroxysteroid accumulation and ergosterol production between wild-type and ERG7 mutant strains. We suggest that oxidosqualene cyclase affects Erg25p (C-4 sterol oxidase) and/or Erg26p (C-3 sterol dehydrogenase/C-4 decarboxylase), two enzymes that, in conjunction with Erg27p, are involved in C-4 sterol demethylation.     

The ERG28-encoded protein, Erg28p, interacts with both the sterol C-4 demethylation enzyme complex as well as the late biosynthetic protein, the C-24 sterol methyltransferase (Erg6p)

In Saccharomyces cerevisiae, the C-24 sterol methyltransferase (Erg6p) converts zymosterol to fecosterol, an enzymatic step following C-4 demethylation of 4,4-dimethylzymosterol. Our previous study showed that an endoplasmic reticulum (ER) transmembrane protein, Erg28p, functions as a scaffold to tether the C-4 demethylation enzymatic complex (Erg25p-Erg26p-Erg27p) to the ER. To determine whether Erg28p also interacts with other ergosterol biosynthetic proteins, we compared protein levels of Erg3p, Erg6p, Erg7p, Erg11p and Erg25p in three pairs of erg28 and ERG28 strains. In erg28 strains, the Erg6p level in the ER fraction was decreased by about 50% relative to the wild-type strain, while ER protein levels of the four other ergosterol proteins showed no significant differences. Co-immunoprecipitation experiments, using an erg28 strain transformed with the epitope-tagged plasmid pERG28-HA and proteins detected with anti-HA and anti-Erg6p antibodies, indicated that Erg6p and Erg28p reciprocally co-immunoprecipitate. Further, the split ubiquitin yeast membrane two-hybrid system designed to detect protein interactions between membrane bound proteins also indicated an Erg28p-Erg6p interaction when pERG6-Cub was used as the bait and pERG28-NubG was used as the prey. We conclude that Erg28p may not only anchor the C-4 demethylation enzyme complex to the ER but also acts as a protein bridge to the Erg6p enzyme required for the next ergosterol biosynthetic step.     

Closing the gap: identification of human 3-ketosteroid reductase,the last unknown enzyme of mammalian cholesterol biosynthesis

The protein encoded by the HSD17B7 gene was originally described as a prolactin receptor-associated protein and as 17beta-hydroxysteroid dehydrogenase (HSD) type 7. Its ability to synthesize 17beta-estradiol in vitro has been reported previously. However, we demonstrate that HSD17B7 is the ortholog of the yeast 3-ketosteroid reductase Erg27p and converts zymosterone to zymosterol in vitro, using reduced nicotinamide adenine dinucleotide phosphate as cofactor. Expression of human and murine HSD17B7 in an Erg27p-deficient yeast strain complements the 3-ketosteroid reductase deficiency of the cells and restores growth on sterol-deficient medium. A fusion of HSD17B7 with green fluorescent protein is located in the endoplasmic reticulum, the site of postsqualene cholesterogenesis. Further critical evidence for a role of HSD17B7 in cholesterol metabolism is provided by the observation that its murine ortholog is a member of the same highly distinct embryonic synexpression group as hydroxymethyl-glutaryl-coenzyme A reductase, the rate-limiting enzyme of sterol biogenesis, and is specifically expressed in tissues that are involved in the pathogenesis of congenital cholesterol-deficiency disorders. We conclude that HSD17B7 participates in postsqualene cholesterol biosynthesis, thus completing the molecular cloning of all genes of this central metabolic pathway. In its function as the 3-ketosteroid reductase of cholesterol biosynthesis, HSD17B7 is a novel candidate for inborn errors of cholesterol metabolism.     

In yeast sterol biosynthesis the 3-keto reductase protein (Erg27p) is required for oxidosqualene cyclase (Erg7p) activity

In Saccharomyces cerevisiae, the 3-keto reductase (Erg27p) encoded by ERG27 gene is one of the key enzymes involved in the C-4 demethylation of the sterol intermediate, 4,4-dimethylzymosterol. The oxidosqualene cyclase (Erg7p) encoded by the ERG7 gene converts oxidosqualene to lanosterol, the first cyclic component of sterol biosynthesis. In a previous study, we found that erg27 strains grown on cholesterol- or ergosterol-supplemented media did not accumulate lanosterol or 3-ketosterols but rather squalene, oxidosqualene, and dioxidosqualene intermediates normally observed in ERG7 (oxidosqualene cyclase) mutants. These results suggested a possible interaction between these two enzymes. In this study, we present evidence that Erg27p interacts with Erg7p, facilitating the association of Erg7p with lipid particles (LPs) and preventing digestion of Erg7p both in the endoplasmic reticulum (ER) and LPs. We demonstrate that Erg27p is required for oxidosqualene cyclase (Erg7p) activity in LPs, and that Erg27p co-immunoprecipitates with Erg7p in LPs but not in microsomal fractions. While Erg27p is essentially a component of the ER, it can also be detected in LPs. In erg27 strains, a truncated Erg7p mislocalizes to microsomes. Restoration of Erg7p enzyme activity and LPs localization was achieved in an erg27 strain transformed with a plasmid containing a wild-type ERG27 allele. We suggest that the physical interaction of Erg27p with Erg7p is an essential regulatory tool in yeast sterol biosynthesis.     

Targeting of proteins involved in sterol biosynthesis to lipid particles of the yeast Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, three enzymes of the sterol biosynthetic pathway, namely Erg1p, Erg6p and Erg7p, are located in lipid particles. Whereas Erg1p (squalene epoxidase) is also present in the endoplasmic reticulum (ER) to a significant amount, only traces of Erg6p (sterol C-24 methyltransferase) and Erg7p (lanosterol synthase) are found in the ER. We have chosen these three Erg-proteins as typical representatives of lipid particle proteins to study targeting to their destination. Lipid particle proteins do not contain obvious targeting motifs, but the only common structural feature is the presence of one or two hydrophobic domains near the C-termini. We constructed truncated versions of Erg1p, Erg6p and Erg7p to test the role of these hydrophobic domains in subcellular distribution. Our results demonstrate that lack of the hydrophobic domains prevents at least in part the association of the proteins with lipid particles and causes their retention to the ER. This result strongly supports the view that ER and lipid particles are related organelles.     

Genetic analyses involving interactions between the ergosterol biosynthetic enzymes, lanosterol synthase (Erg7p) and 3-ketoreductase (Erg27p), in the yeast Saccharomyces cerevisiae

Protein-protein interaction studies in the Saccharomyces cerevisiae ergosterol biosynthetic pathway suggest that enzymes in this pathway may act as an integrated multienzyme complex. The yeast sterol 3-ketoreductase (Erg27p) required for C-4 demethylation of sterols has previously been shown to also be required for the function of the upstream oxidosqualene cyclase/lanosterol synthase (Erg7p); thus, erg27 mutants accumulate oxidosqualenes as precursors rather than 3-ketosterones. In the present study, we have created various mutations in the ERG27 gene. These mutations include 5 C-terminal truncations, 6 internal deletions, and 32 point mutants of which 14 were obtained by site-directed mutagenesis and 18 by random mutagenesis. We have characterized these ERG27 mutations by determining the following: Erg27 and Erg7 enzyme activities, presence of Erg27p as determined by western immunoblots, ability to grow on various sterol substrates and GC sterol profiles. Mutations of the predicted catalytic residues, Y202F and K206A, resulted in the endogenous accumulation of 3-ketosterones rather than oxidosqualenes suggesting retention of Erg7 enzyme activity. This novel phenotype demonstrated that the catalytic function of Erg27p can be separated from its Erg7p chaperone ability. Other erg27 mutations resulted in proteins that were present, as determined by western immunoblotting, but unable to interact with the Erg7 protein. We also classify Erg27p as belonging to the SDR (short-chain dehydrogenase/reductase) family of enzymes and demonstrate the possibility of homo- or heterodimerization of the protein. This study provides new insights into the role of Erg27p in sterol biosynthesis.     

Characterization of a mutation that results in independence of oxidosqualene cyclase (Erg7) activity from the downstream 3-ketoreductase (Erg27) in the yeast ergosterol biosynthetic pathway

In yeast, deletion of ERG27, which encodes the sterol biosynthetic enzyme, 3-keto-reductase, results in a concomitant loss of the upstream enzyme, Erg7p, an oxidosqualene cyclase (OSC). However, this phenomenon occurs only in fungi, as mammalian Erg27p orthologues are unable to rescue yeast Erg7p activity. In this study, an erg27 mutant containing the mouse ERG27 orthologue was isolated that was capable of growing without sterol supplementation (FGerg27). GC/MS analysis of this strain showed an accumulation of squalene epoxides, 3-ketosterones, and ergosterol. This strain which was crossed to a wildtype and daughter segregants showed an accumulation of squalene epoxides as well as ergosterol indicating that the mutation entailed a leaky block at ERG7. Upon sequencing the yeast ERG7 gene an A598S alteration was found in a conserved alpha helical region. We theorize that this mutation stabilizes Erg7p in a conformation that mimics Erg27p binding. This mutation, while decreasing OSC activity still retains sufficient residual OSC activity such that the strain in the presence of the mammalian 3-keto reductase enzyme functions and no longer requires the yeast Erg27p. Because sterol biosynthesis occurs in the ER, a fusion protein was synthesized combining Erg7p and Erg28p, a resident ER protein and scaffold of the C-4 demethyation complex. Both FGerg27 and erg27 strains containing this fusion plasmid and the mouse ERG27 orthologue showed restoration of ergosterol biosynthesis with minimal accumulation of squalene epoxides. These results indicate retention of Erg7p in the ER increases its activity and suggest a novel method of regulation of ergosterol biosynthesis.     

Interaction of the GTS1 gene product with glyceraldehyde- 3-phosphate dehydrogenase 1 required for the maintenance of the metabolic oscillations of the yeast Saccharomyces cerevisiae.

We previously reported that GTS1 is involved in regulating ultradian oscillations of the glycolytic pathway induced by cyanide in cell suspensions as well as oscillations of energy metabolism in aerobic continuous cultures. Here, we screened a yeast cDNA library for proteins that bind to Gts1p using the yeast two-hybrid system and cloned multiple TDH cDNAs encoding the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found that the zinc-finger and dimerization sites of Gts1p were required for full ability to bind GAPDH, and Gts1ps mutated at these sites lost the ability to regulate both aerobic and unaerobic ultradian oscillations of energy metabolism. Of the three TDH genes, only TDH1 fluctuated at the mRNA level in continuous culture and its deletion resulted in the disappearance of the oscillation without any affect on growth rate. This loss of biological rhythms in the TDH1-deleted mutant was rescued by the expression of TDH1 but not of TDH2 or TDH3 under the control of the TDH1 promoter. Thus, we hypothesized that Gts1p plays a role in the regulation of metabolic oscillation by interacting with the TDH1 product, GAPDH1, in yeast.