长链二元酸代谢中肉毒碱脂酰转移酶的作用

肉毒碱脂酰转移酶包括肉毒碱棕榈酰转移酶、肉毒碱乙酰基转移酶和肉毒碱辛酰基转移酶,它们对脂肪酸的进一步β氧化代谢起到了转运的作用。综述近年来对肉毒碱脂酰转移酶族成员酶学特性的研究进展,探讨在热带假丝酵母(Candidatropicalis)中肉毒碱脂酰转移酶的作用与调控 。     

阻断嗜乙酰乙酸棒杆菌乙酸合成途径对其在缺氧条件下产琥珀酸的影响

为探究和阻断嗜乙酰乙酸棒杆菌乙酸代谢途径,提高缺氧条件下琥珀酸的产率,减少副产物乙酸的合成,以C. acetoacidophilum ΔldhA为出发菌株,利用同源重组的方法分别敲除磷酸乙酰转移酶、乙酸激酶、CoA转移酶和丙酮酸脱氢酶复合体的相关基因pta,ackA,ctfA与aceE,研究突变菌产琥珀酸过程中相关参数的变化。结果表明：敲除pta与ackA基因后,对乙酸浓度,糖耗速率和糖酸转化率影响不大;pta,ackA与ctfA基因的同时失活使得乙酸的浓度和摩尔转化率分别降低81.4%和77.2%,葡萄糖消耗速率下降28.3%,琥珀酸对葡萄糖摩尔转化率提高25.3%;而单独敲除aceE基因后,乙酸几乎不产生,葡萄糖消耗速率下降35.6%,琥珀酸对葡萄糖摩尔转化率提高34.7%。因此,缺氧条件下,嗜乙酰乙酸棒杆菌的乙酸合成几乎全部走乙酰CoA途径,pta,ackA与ctfA是由乙酰CoA合成乙酸途径中最主要的基因;敲除基因aceE, 可以完全阻断乙酸生成,有效提高琥珀酸产率。     

中间载体pBz6102的构建及CAT基因在转化烟草中的表达

来源于细菌的新霉素磷酸转移酶基因(NPT),氯霉素乙酰转移酶基因(CAT)以及来源于昆虫的荧光素酶基因等都是用于研究根癌农杆菌转化植物的良好标记。我们利用氯霉素乙酰转移酶嵌合基因和来源于Ti质粒T-区DNA的tmr基因,构建了中间载体pBZ 6102,并通过植物基因工程载体pGV 3850,将氯霉素乙酰转移酶嵌合基因和tmr基因引入了植物细胞,并测到了表达,在抗氯霉素植物中测到了氯霉素乙酰转移酶活性。中间裁体pBZ 6102上还有Pst Ⅰ,Xba Ⅰ等单一的限制性内切酶位点,外源基因极易插入。转化植物F_1代的种子抗性分析表明,80%左右的种子都能在含氯霉素的培养基上正常萌发,它们的幼苗中都有氯霉素乙酰转移酶活性,证明CAT基因通过了减数分裂稳定地保留在植物细胞的基因组内。     

生技霉素稳定型基因工程菌的构建*

运用同源重组技术将异戊酰基转移酶基因整合至螺旋霉素产生菌(Streptomyces spiramyceticus F21) 的染色体上,构建了稳定的生技霉素基因工程菌。在不加压的情况下传代,菌种携带选择性遗传标记情况、生长、发酵效价及发酵产物的TLC分析均表明此基因工程菌有较好的遗传稳定性,且发酵效价及产物的组分均得到改善。Southern杂交证明外源基因在螺旋霉素产生菌染色体上的整合情况。     

生技霉素稳定型基因工程菌的构建

运用同源重组技术将异戊酰基转移酶基因整合至螺旋霉素产生菌（ＳｔｒｅｐｔｏｍｙｃｅｓｓｐｉｒａｍｙｃｅｔｉｃｕｓＦ２１）的染色体上,构建了稳定的生技霉素基因工程菌。在不加压的情况下传代,菌种携带选择性遗传标记情况、生长、发酵效价及发酵产物的ＴＬＣ分析均表明此基因工程菌有较好的遗传稳定性,且发酵效价及产物的组分均得到改善。Ｓｏｕｔｈｅｒｎ杂交证明外源基因在螺旋霉素产生菌染色体上的整合情况。     

胆碱能系统大细胞基底核介导认知过程

前脑基底－大细胞基底核－新皮是胆碱类物质作用的中枢。乙酰胆碱在此区域接受刺激并提供信息到大脑皮层和杏仁核,介导认知过程的产生。采用基因转移技术将果蝇胆碱乙锘转移酶基因植入基因工程细胞载体－纤维母细胞,后者经脑内移植作用于大细胞基底核神经元,释放乙酰胆或胆碱。     

基因工程菌中重组质粒的稳定性研究进展

基因工程菌(细胞)是现代生物工程中的微型生物反应器,是基因工程的研究主体之一.获得使外源基因高效稳定表达的基因工程菌或细胞是基因工程的核心步骤与最终目的.基因工程菌重组质粒稳定性问题是基因工程菌工业化生产与实验研究中的最主要问题,就其在基因工程中的重要性、影响因素及提高稳定性策略方面作简要介绍并展开综述.     

乳酸菌风味代谢物质的基因调控

乳酸菌的主要风味代谢物质包括丁二酮,乙醛以及各种氨基酸。利用基因工程和代谢工程的相关技术提高乙醛和丁二酮产量,是当前乳酸菌研究的热点之一。乙醛的代谢调控主要是针对丝氨酸羟甲基转移酶的表达进行调控,或是针对丙酮酸脱羧酶和NADH氧化酶的表达采用联合调控策略;而丁二酮的代谢调控则主要集中于乳酸脱氢酶、NADH氧化酶、α-乙酰乳酸合成酶和α-乙酰乳酸脱羧酶中任意两种关键酶基因间的联合调控,并且存在进行乳酸脱氢酶,α-乙酰乳酸合成酶和α-乙酰乳酸脱羧酶3种关键酶基因联合调控的可行性。     

乙酰氨基葡萄糖转移酶Ⅴ在肿瘤转移中的作用及其分子机理

目前研究表明N-乙酰氨基葡萄糖转移酶Ⅴ在肿瘤转移中有重要作用.在恶性肿瘤中, N-乙酰氨基葡萄糖转移酶Ⅴ活性增高,其催化产物β1,6分支也增加,β1,6分支与肿 瘤的侵袭转移密切相关.本文综述了N-乙酰氨基葡萄糖转移酶Ⅴ催化形成N-糖链 β1,6分支的特点以及在N-糖链生物合成中的重要作用;还介绍了N-乙酰氨基葡萄糖转移酶Ⅴ基因组成和参与其基因调控的转录因子Ets-1,及基因表达组织特异性;着重综述了近年来N-乙酰氨基葡萄糖转移酶Ⅴ与肿瘤侵袭转移相关的分子机理的最新研究进展,包括了粘附分子钙粘蛋白(cadherin)和整合素α5β1的作用,修饰表皮生长因子受体调节信号 转导,及通过对上皮衍生的细胞表面丝氨酸蛋白酶matriptase的β1,6分支修饰促进仲瘤的 侵袭等方面.提示有效抑制N-乙酰氨基葡萄糖转移酶Ⅴ参与作用的位点,为设计抗肿瘤新药提供潜在的治疗靶点.     

共表达SHMT和TPase载体的构建及双酶法合成L-色氨酸

利用重组大肠杆菌表达丝氨酸羟甲基转移酶(SHMT)和色氨酸酶(TPase),并利用双酶法合成L-色氨酸。采用PCR从大肠杆菌K12基因组中扩增上述两种酶的基因,利用pET-28a载体,构建单表达重组质粒pET-SHMT、pET-TPase和共表达重组质粒pET-ST。将上述3种重组质粒转入大肠杆菌BL21(DE3)进行表达。SDS-PAGE结果表明,单表达基因工程菌BL21(DE3)/pET-SHMT和BL21(DE3)/pET-TPase分别在47kDa(SHMT)和50kDa(TPase)处有蛋白表达带;共表达基因工程菌BL21(DE3)/pET-ST在上述两处均有蛋白表达带。与宿主菌相比,单表达SHMT基因工程菌产酶活性提高了6.4倍;单表达TPase基因工程菌产酶活性提高了8.4倍;共表达SHMT和TPase基因工程菌产酶活性分别提高了6.1和6.9倍。利用工程菌所产酶进行双菌双酶法和单菌双酶法合成L-色氨酸。两菌双酶合成L-色氨酸的累积量达到41.5g/L,甘氨酸转化率为83.3%,吲哚转化率为92.5%;单菌双酶合成L-色氨酸的累积量达到28.9g/L,甘氨酸转化率为82.7%,吲哚转化率为82.9%。     

Engineering the acetyl-CoA transportation system of candida tropicalis enhances the production of dicarboxylic acid

Dicarboxylic acids (DCAs) can be obtained by oxidizing alkanes by Candida tropicalis. Through alpha-monocarboxylic acids (MCAs), alpha- and omega-oxidation yield alpha- or omega-DCAs, respectively. However, both MCAs and DCAs may be degraded to acetyl-CoA by beta-oxidation, resulting in a limited DCA yield. Acetyl-CoA can be transported into the mitochondrion for the TCA cycle by carnitine acetyltransferase (CAT), by which the energy generation and beta-oxidation are connected. In this paper, we present a method to reconstruct the metabolic pathway by inhibiting the acetyl-CoA transportation system. Metabolic engineering is applied on the acetyl-CoA transportation system, but not the key enzymes in beta-oxidation. Starting with the original strain W10-1, cat heterozygote CZ-15 and cat homozygote CKC-11 were obtained by gene knockout. The CAT specific activity in CZ-15 was about 50% lower than that in W10-1, resulting in a 21.0% increase of the DCA concentration, and a 12% increase of the molar conversion of alkane, reaching 61.6%. However, no CAT activity was detected in CKC-11, and CKC-11 could not grow on alkane. These results indicate that inhibition of beta-oxidation via reconstruction of the transportation process between organelles can facilitate DCA production, but that totally blocking the & betagr;-oxidation would be harmful for energy supply. We thus provide a novel insight into regulation of the beta-oxidation system and metabolic flux. Further understanding of beta-oxidation and the acetyl-CoA transportation system in Candida tropicalis is reached through examination of fermentation data by metabolic flux analysis.     

Repression of fatty-acyl-CoA oxidase-encoding gene expression is not necessarily a determinant of high-level production of dicarboxylic acids in industrial dicarboxylic-acid-producing Candida tropicalis

The synthesis of dicarboxylic acids (DCAs) in Candida tropicalis is thought to be induced by a decrease in fatty acyl-CoA-oxidase activity. However, in the present study we demonstrate that repression of the POX4 gene, encoding fatty acyl-CoA oxidase, does not directly lead to high-level production of DCAs. No fatty acyl-CoA-oxidase activity was detected if the POX4 gene of C. tropicalis strain 1098 (wild-type strain) was disrupted. Furthermore, introduction of the POX4 gene from C. tropicalis strain M1210A3, which is a mutant derived from strain 1098 and is used as an industrial DCA-producing strain, still exhibited low-level fatty acyl-CoA-oxidase activity. Nevertheless, production of DCA was not observed in either case. Furthermore, the increase in acyl-CoA-oxidase activity by expression of the POX4 gene in strain M1210A3 did not reduce high-level production of DCA. These results suggest that alterations in acyl-CoA-oxidase activity are not necessarily related to production of DCA in industrial DCA-producing C. tropicalis M1210A3.     

Carnitine-dependent transport of acetyl coenzyme A in Candida albicans is essential for growth on nonfermentable carbon sources and contributes to biofilm formation

In eukaryotes, acetyl coenzyme A (acetyl-CoA) produced during peroxisomal fatty acid beta-oxidation needs to be transported to mitochondria for further metabolism. Two parallel pathways for acetyl-CoA transport have been identified in Saccharomyces cerevisiae; one is dependent on peroxisomal citrate synthase (Cit), while the other requires peroxisomal and mitochondrial carnitine acetyltransferase (Cat) activities. Here we show that the human fungal pathogen Candida albicans lacks peroxisomal Cit, relying exclusively on Cat activity for transport of acetyl units. Deletion of the CAT2 gene encoding the major Cat enzyme in C. albicans resulted in a strain that had lost both peroxisomal and mitochondrion-associated Cat activities, could not grow on fatty acids or C(2) carbon sources (acetate or ethanol), accumulated intracellular acetyl-CoA, and showed greatly reduced fatty acid beta-oxidation activity. The cat2 null mutant was, however, not attenuated in virulence in a mouse model of systemic candidiasis. These observations support our previous results showing that peroxisomal fatty acid beta-oxidation activity is not essential for C. albicans virulence. Biofilm formation by the cat2 mutant on glucose was slightly reduced compared to that by the wild type, although both strains grew at the same rate on this carbon source. Our data show that C. albicans has diverged considerably from S. cerevisiae with respect to the mechanism of intracellular acetyl-CoA transport and imply that carnitine dependence may be an important trait of this human fungal pathogen.     

代谢工程改善野生酵母利用木糖产乙醇的性能

从256个自然样品中筛选得到1株可高效转化D-木糖的酵母。通过生理生化和分子生物学方法鉴定, 证实该菌株是属于Candida tropicalis。以该酵母为研究对象, 增加木糖醇脱氢酶表达量, 通过改变代谢流以达到提高酒精产率的目的。以pXY212-XYL2质粒为基础载体, 构建了含有潮霉素抗性的pYX212-XYL2-Hygro, 电击转化进入野生型C. tropicalis, 潮霉素抗性筛选, 得到含高拷贝木糖醇脱氢酶基因的重组菌株C. tropicalis XYL2-7。重组菌的比酶活达到0.5 u/mg protein, 比原始菌株提高了3倍。实验表明, 重组菌木糖醇得率比原始菌株降低了3倍, 酒精得率提高了5倍。首次通过实验验证了热带假丝酵母利用木糖产乙醇的可行性, 这对研究酵母利用秸秆、麦糠、谷壳等纤维质农业废弃物生产燃料乙醇具有重要启示。     

代谢工程改善野生酵母利用木糖产乙醇的性能

从256个自然样品中筛选得到1株可高效转化D-木糖的酵母。通过生理生化和分子生物学方法鉴定, 证实该菌株是属于Candida tropicalis。以该酵母为研究对象, 增加木糖醇脱氢酶表达量, 通过改变代谢流以达到提高酒精产率的目的。以pXY212-XYL2质粒为基础载体, 构建了含有潮霉素抗性的pYX212-XYL2-Hygro, 电击转化进入野生型C. tropicalis, 潮霉素抗性筛选, 得到含高拷贝木糖醇脱氢酶基因的重组菌株C. tropicalis XYL2-7。重组菌的比酶活达到0.5 u/mg protein, 比原始菌株提高了3倍。实验表明, 重组菌木糖醇得率比原始菌株降低了3倍, 酒精得率提高了5倍。首次通过实验验证了热带假丝酵母利用木糖产乙醇的可行性, 这对研究酵母利用秸秆、麦糠、谷壳等纤维质农业废弃物生产燃料乙醇具有重要启示。     

A new oxygen-regulated promoter for the expression of proteins in Escherichia coli

Several properties of a new oxygen-regulated promoter, OXYPRO, were tested in small-scale Escherichia coli cultures. Using OXYPRO, maximal activity of a reporter gene encoding chloramphenicol acetyltransferase (CAT) occurred in cultures that were tightly capped immediately after inoculation. This is probably a result of the reduced oxygen concentration attained in capped cultures, a condition known to be required for OXYPRO induction. CAT levels were significantly higher when the cells were grown in a glycerol-based medium. Similar levels of CAT expression were obtained when OXYPRO was compared to the trp-lac (tac) promoter. In addition, regulated expression of CAT occurred in a wild type strain of E. coli, suggesting that OXYPRO will be useful in most E. coli strains. Thus, OXYPRO provides a simple, inexpensive, and unobtrusive method to achieve high levels of cloned protein expression in most strains of E. coli. OXYPRO is available in a high copy plasmid with a convenient multiple cloning site for the insertion of genes for direct expression in E. coli.     

肺炎克雷伯菌KP1＿4563基因突变株的构建及其生物膜表型分析

KP1_4563基因是肺炎克雷伯菌NTUH-K2044中假设的蛋白编码基因,与Ⅲ型菌毛的功能有关。本实验首先采用同源重组基因敲除方法构建肺炎克雷伯菌KP1_4563基因缺失的突变株(Kp-△4563),然后PCR扩增KP1_4563基因片段,克隆到质粒p BAD33上,将重组质粒导入Kp-△4563获得回补株(Kpc-△4563)。分别测定野生株、突变株、回补株用普通LB培养基,改良Minka培养基以及含胆汁盐的LB培养基培养时生物膜形成能力,以此来探讨KP1_4563基因以及不同培养基对肺炎克雷伯菌体外生物膜形成的影响。我们成功构建KP1_4563基因缺失的突变株和回补株Kpc-△4563。与野生株相比,突变株Kp-△4563生物膜形成能力减弱,回补株介于野生株和突变株之间。使用改良Minka培养基使菌株菌毛化以及加入胆汁盐可以增加生物膜的形成能力。这些分析表明肺炎克雷伯菌KP1_4563基因能正调控细菌生物膜的形成。体外培养使细菌菌毛化以及加入胆汁盐可以促进生物膜的形成。     

Determination of Candida tropicalis acyl coenzyme A oxidase isozyme function by sequential gene disruption.

A recently developed transformation system has been used to facilitate the sequential disruption of the Candida tropicalis chromosomal POX4 and POX5 genes, encoding distinct isozymes of the acyl coenzyme A (acyl-CoA) oxidase which catalyzes the first reaction in the beta-oxidation pathway. The URA3-based transformation system was repeatedly regenerated by restoring the uracil requirement to transformed strains, either through selection for spontaneous mutations or by directed deletion within the URA 3 coding sequence, to permit sequential gene disruptions within a single strain of C. tropicalis. These gene disruptions revealed the diploid nature of this alkane- and fatty acid-utilizing yeast by showing that it contains two copies of each gene. A comparison of mutants in which both POX4 or both POX5 genes were disrupted revealed that the two isozymes were differentially regulated and displayed unique substrate profiles and kinetic properties. POX4 was constitutively expressed during growth on glucose and was strongly induced by either dodecane or methyl laurate and to a greater extent than POX5, which was induced primarily by dodecane. The POX4-encoded isozyme demonstrated a broad substrate spectrum in comparison with the narrow-spectrum, long-chain oxidase encoded by POX5. The absence of detectable acyl-CoA oxidase activity in the strain in which all POX4 and POX5 genes had been disrupted confirmed that all functional acyl-CoA oxidase genes had been inactivated. This strain cannot utilize alkanes or fatty acids for growth, indicating that the beta-oxidation pathway has been functionally blocked.     

啤酒酵母DNA修复基因RAD24温度敏感突变株的分离及其特性的初步研究

以ＹＣｐｌａｃ系列带Ｔｒｐ、Ｈｉｓ和Ｕｒａ标志基因的载体为骨架构建含野生型和经羟胺处理的突变型的啤酒酵母ＲＡＤ２４基因质粒,用质粒替换方法分离ＲＡＤ２４基因温度敏感突变株（ｒａｄ２４－ｔｓ３）．紫外生存试验发现,ｒａｄ２４－ｔｓ３对紫外线敏感;同位素（３Ｈ－ＴｄＲ,３Ｈ－ＵＲ,３Ｈ－Ｌｅｕ）参入试验表明,该突变株ＤＮＡ、ＲＮＡ及蛋白质合成均较野生型明显降低．     

头状轮生链霉菌中丝裂霉素C抗性基因的克隆及功能研究

头状轮生链霉菌(\%Streptoverticillium caespitosus\%)ATCC27422是抗肿瘤药物丝裂霉素的主要产生菌,实验通过诱变筛选获得不产生丝裂霉素同时对丝裂霉素C敏感的阻断变种S6,并以它为受体宿主,以质粒pIJ699为载体,建立野生型头状轮生链霉菌菌株ATCC27422的基因库。采用鸟枪法克隆技术,从库中筛选获得含有丝裂霉素C抗性基因的62kb外源片段的克隆子。将含此外源片段的质粒pLX5导入变铅青链霉菌(\%Streptomyces lividans\%)获得表达。并且首次成功地运用电穿孔法将pLX5导入野生型菌株中,使其对丝裂霉素C的抗性大幅度提高：最低抑制浓度(MIC)由原来的200μg/mL上升至1000μg/mL以上。摇瓶发酵实验表明：单位菌量的ATCC27422(pLX5)的丝裂霉素产量高于野生菌株ATCC27422,因此丝裂霉素C抗性与产量之间存在一定的相关性。