利用重组大肠杆菌合成几丁寡糖的研究

提取根瘤菌Mesorhizobium.loti基因组,克隆编码N-乙酰氨基葡萄糖转移酶nodC基因,插入质粒pUC19的lac启动子的下游,构建并筛选出能够合成几丁寡糖的重组大肠杆菌DCL-3。利用优化的MMYNG培养基,重组大肠杆菌DCL-3在10L发酵罐中培养26h后,培养液菌体浓度测定OD560=10.8,几丁寡糖得率达到526mg/L。收集重组细菌的细胞并煮沸破碎,利用活性炭的吸附和P4凝胶层析对几丁寡糖产物进行分离纯化。纯化产物的液质分析(LC-ESI-MS)结果表明主要寡糖产物为几丁四糖(m/z,831[M H] )和几丁五糖(m/z,1034[M H] )。     

壳寡糖镧配合物的抑菌活性及其与牛血清白蛋白的相互作用

合成了壳寡糖和稀土离子La3+的配合物,利用红外光谱、紫外光谱和差热-热重手段对其结构和性质进行了表征。采用抑菌圈法考察了壳寡糖、壳寡糖-La对革兰氏阳性菌金黄色葡萄球菌和革兰氏阴性菌大肠杆菌的抑菌活性。此外,紫外光谱、荧光光谱和循环伏安曲线法研究壳寡糖-La与牛血清白蛋白(BSA)相互作用。结果表明壳寡糖、壳寡糖-La对两种细菌均具有较强的抑菌活性,且壳寡糖-La的抑菌活性强于壳寡糖;壳寡糖-La使BSA的内源荧光猝灭,猝灭机制为静态猝灭,并计算了室温下壳寡糖-La与BSA的结合常数和结合位点数分别为6.35×104L/mol和1.29。     

细菌不饱和脂肪酸合成研究进展

脂肪酸不仅是细菌细胞膜组分，还是许多生物活性物质的合成原料。不饱和脂肪酸(unsaturated fatty acid, UFA)具有更低的相变温度，是细菌调节细胞膜流动性的重要分子，因此UFA合成途径是重要的抗菌药物筛选靶点。细菌可利用厌氧途径合成UFA，其中模式生物大肠杆菌利用经典的FabA-FabB途径合成UFA，但不同细菌中UFA合成的厌氧途径具有多样性，相关催化酶类也不尽相同；细菌还可以利用需氧途径合成UFA，利用脂肪酸脱饱和酶直接将饱和脂肪酸(saturated fatty acid, SFA)转化为不饱和脂肪酸，而不同脱饱和酶会生成不同结构的UFA，在逆境耐受、致病力等多方面发挥重要作用；细菌还可以利用单加氧酶，将脂肪酸合成途径中癸酰酰基载体蛋白(acyl carrier protein, ACP)转化为顺-3-癸烯酰ACP，并最终合成UFA。细菌脂肪酸合成相关的其他酶类在UFA合成或不同种类UFA调节中也发挥着重要作用。本文系统地总结了细菌UFA合成途径与相关酶类的多样性研究进展，旨在为进一步了解细菌UFA合成机制，并以此为靶点开发抗菌药物等方面提供理论支撑。     

甲羟戊酸途径限速步骤研究及其在产番茄红素重组大肠杆菌中的应用

甲羟戊酸途径(MVA途径)被引入重组大肠杆菌中,能够提高重组大肠杆菌中萜类化合物的合成能力。但因重组大肠杆菌中萜类化合物合成途径中间产物积累,导致细胞生长和萜类化合物合成受到限制。本研究在稳定表达MVA途径以及优化2-甲基-D-赤藻糖醇-4-磷酸途径(MEP途径)、番茄红素合成途径关键基因表达的重组大肠杆菌LYC103中,用质粒高表达MVA途径和番茄红素合成途径关键基因,挖掘该途径的限速步骤。结果表明,ispA、crtE、mvaK1、idi和mvaD基因过表达后,细胞生长没有明显变化,番茄红素产量依次提高了13.5%、16.5%、17.95%、33.7%和61.1%,说明这几个基因可能是合成番茄红素的限速步骤。mvaK1、mvaK2、mvaD三个基因在同一操纵子上,用mRNA稳定区(RNA stabilizing region)进行启动子文库(mRSL)调控mvaK1,相当于对3个基因同时调控。用高效基因组编辑技术(CAGO)对mvaK1基因的mRNA稳定区进行启动子文库的调控,得到菌株LYC104。番茄红素产量与对照菌株LYC103相比增加了2倍,细胞生长提高了32%。然后,利用CRISPR-Cas9技术在染色体lacZ位点整合idi基因,得到LYC105菌株。与出发菌株LYC103相比,细胞生长提高了147%,番茄红素产量增加了2.28倍。本研究在染色体上具有完整MVA途径的基础上,利用质粒高表达单个基因挖掘限速步骤,用同源重组方法整合限速基因、解除限速,为代谢工程构建高产菌株提供新策略。     

神经节苷脂氟化寡糖在大肠杆菌中的生物合成

神经节苷脂是一类存在于所有脊椎动物细胞中的糖脂分子,具有重要的生理功能和药用活性。合成生物学为构建神经节苷脂类的人工高效合成体系提供了契机,而其神经节苷脂氟化寡糖中间体的合成是至关重要的步骤。旨在实现神经节苷脂氟化寡糖在大肠杆菌中的人工生物合成途径:通过在大肠杆菌JM107中引入来源于脑膜炎奈瑟氏菌(Neisseria meningitidis)的CMP-NeuAc合成酶(neuA),空肠弯曲杆菌(Campylobacter jejuni)来源的α2,3-NeuAc转移酶(cst)、β1,4-GalNAc转移酶(cgtA)、β1,3-Gal转移酶(cgtB)和UDP-GlcNAc异构酶(gne),成功构建了以氟化乳糖和唾液酸为底物的3种神经节苷脂GM3、GM2、GM1氟化寡糖在大肠杆菌中的生物合成途径;开发了高灵敏度的氟化寡糖柱前衍生-HPLC定量检测方法 ;并通过对工程菌株进行透酶过表达和启动子优化,在摇瓶发酵中GM3、GM2及GM1氟化寡糖的产量分别达到81.5 mg/L、18.1 mg/L及12.3 mg/L,为进一步的代谢工程改造奠定了良好的基础。     

病原菌多糖基因簇在大肠杆菌中的克隆

目的:构建稳定的外源病原菌多糖基因簇克隆载体,为在糖基工程大肠杆菌中利用外源性多糖O-糖基化修饰靶标蛋白奠定基础。方法:PCR扩增大肠杆菌O157、甲型副伤寒沙门菌CMCC50973和铜绿假单胞杆菌CMCC10110的O-多糖合成基因簇,将多糖基因簇与细菌人工染色体p CC1BAC连接后,分别转化O-多糖合成缺陷的大肠杆菌W3110,并用相应多糖抗血清ELISA检测重组大肠杆菌是否利用外源O-多糖生成脂多糖(LPS),从而验证外源多糖基因簇克隆载体在大肠杆菌内是否能够生成相应的O-多糖;在此基础上,将构建的3种外源多糖基因簇克隆载体分别转化表达O-寡糖转移酶和蛋白底物菌毛蛋白Pil E的糖基工程大肠杆菌,用相应的抗血清进行Western印迹检测,以验证克隆的O-多糖能否修饰蛋白底物Pil E。结果:与阴性对照菌相比,带有大肠杆菌O157的O-多糖合成基因簇克隆载体和带有甲型副伤寒沙门菌CMCC50973的O-多糖合成基因簇克隆载体的重组菌ELISA呈阳性,提示大肠杆菌O157和甲型副伤寒沙门菌CMCC50973的O-多糖合成基因簇在大肠杆菌中被利用生成了相应的LPS;而带有铜绿假单胞杆菌CMCC10110的O-多糖合成基因簇克隆载体的重组菌W3110/BAC-10110则ELISA呈阴性。West-ern印迹结果显示,只有带有O157型大肠杆菌O-多糖合成基因簇克隆载体的糖基工程大肠杆菌CLM24/p MMB66EH-pil E-his/p ETtac28-pgl L/BAC-O157在相对分子质量40×103~58×103处出现了特异条带,表明菌毛蛋白Pil E被大肠杆菌O157型O-多糖O-糖基化修饰。结论:建立了大肠杆菌O157、甲型副伤寒沙门菌CMCC50973的O-多糖合成基因簇大片段的克隆载体,克隆的O157型O-多糖合成基因簇可实现O157型多糖对菌毛蛋白Pil E的修饰,从而为在大肠杆菌中建立稳定的利用外源病原菌多糖修饰靶标蛋白的糖基工程大肠杆菌提供了技术基础。     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA.方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径.结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆...     

表达酿酒酵母ALAS的重组大肠杆菌胞外5-氨基乙酰丙酸的产量和纯化

5-氨基乙酰丙酸（5-aminolevulinate,ALA）由5-氨基乙酰丙酸合酶（5-aminolevulinate synthase,ALAS）催化产生。利用重组细菌在大肠杆菌合成ALA已有不少研究。重组真核生物ALAS在大肠杆菌合成ALA的研究没有报道。酿酒酵母ALAS在大肠杆菌重组表达,在摇瓶培养条件下,分析了胞外ALA的产量,重组菌的生长状况和细胞中ALAS的活性,利用两种国产树脂纯化ALA,毛细管电泳分析确定ALA纯度在LB培养基中,初始pH 6.5,含有20mmol/L的酮戊酸、20mmol/L琥珀酸和20mmol/L的甘氨酸,37℃下诱导培养12h,胞外ALA的产量为162mg /L培养基。纯化的ALA纯度达到90%。     

表达酿酒酵母ALAS的重组大肠杆菌胞外5-氨基乙酰丙酸的产量和纯化

5-氨基乙酰丙酸(5-aminolevulinate,ALA)由5-氨基乙酰丙酸合酶(5-aminolevulinate synthase,ALAS)催化产生。利用重组细菌在大肠杆菌合成ALA已有不少研究。重组真核生物ALAS在大肠杆菌合成ALA的研究没有报道。酿酒酵母ALAS在大肠杆菌重组表达,在摇瓶培养条件下,分析了胞外ALA的产量,重组菌的生长状况和细胞中ALAS的活性,利用两种国产树脂纯化ALA,毛细管电泳分析确定ALA纯度在LB培养基中,初始pH6.5,含有20mmol/L的酮戊酸、20mmol/L琥珀酸和20mmol/L的甘氨酸,37℃下诱导培养12h,胞外ALA的产量为162mg/L培养基。纯化的ALA纯度达到90%。     

细菌通过聚酮合成酶途径合成多不饱和脂肪酸的研究进展

细菌利用聚酮合成酶途径合成多不饱和脂肪酸是近年发现的新的脂肪酸合成途径。这种途径与常规的由脂肪酸去饱和酶和脂肪酸延长酶引导的脂肪酸合成途径有着本质上的差别。总结了近些年细菌利用聚酮合成酶合成多不饱和脂肪酸这一新途径的研究状况,重点阐明其分子机制,并对其研究趋势及应用前景进行了展望。     

Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics

The biosynthesis of CoA from pantothenic acid (vitamin B5) is an essential universal pathway in prokaryotes and eukaryotes. The CoA biosynthetic genes in bacteria have all recently been identified, but their counterparts in humans and other eukaryotes remained mostly unknown. Using comparative genomics, we have identified human genes encoding the last four enzymatic steps in CoA biosynthesis: phosphopantothenoylcysteine synthetase (EC ), phosphopantothenoylcysteine decarboxylase (EC ), phosphopantetheine adenylyltransferase (EC ), and dephospho-CoA kinase (EC ). Biological functions of these human genes were verified using a complementation system in Escherichia coli based on transposon mutagenesis. The individual human enzymes were overexpressed in E. coli and purified, and the corresponding activities were experimentally verified. In addition, the entire pathway from phosphopantothenate to CoA was successfully reconstituted in vitro using a mixture of purified recombinant enzymes. Human recombinant bifunctional phosphopantetheine adenylyltransferase/dephospho-CoA kinase was kinetically characterized. This enzyme was previously suggested as a point of CoA biosynthesis regulation, and we have observed significant differences in mRNA levels of the corresponding human gene in normal and tumor cells by Northern blot analysis.     

Isolation and characterization of the constitutive acyl carrier protein from Rhizobium meliloti.

Rhizobium species produce an inducible acyl carrier protein (ACP), encoded by the nodF gene, that somehow functions in an exchange of cell signals between bacteria and specific plant hosts, leading to nodulation of plant roots and symbiotic nitrogen fixation, as well as a constitutive ACP needed for the synthesis of essential cell lipids. The periplasmic cyclic glucans of Rhizobium spp. are also involved in specific rhizobium-plant interaction. These glucans are strongly similar to the periplasmic membrane-derived oligosaccharides (MDO) of Escherichia coli. E. coli ACP is an essential component of a membrane-bound transglucosylase needed for the biosynthesis of MDO, raising the possibility that either or both of the rhizobial ACPs might have a similar function. We have now isolated the constitutive ACP of R. meliloti and determined its primary structure. We have also examined its function, together with those of ACPs from E. coli, Rhodobacter sphaeroides, and spinach, in the MDO transglucosylase system and as substrate for the E. coli ACP acylase enzyme. All four ACPs act as acceptors of acyl residues, but only the E. coli ACP functions in the transglucosylase system.     

重组大肠杆菌的分批补料培养方法

在重组大肠杆菌的培养过程中,存在着菌体的高浓度与外源蛋白的高表达这一矛盾,使得重组菌的比生长速率通常远远低于宿主菌,限制了基因工程菌由实验室规模向工业化规模的转变。要实现重组大肠杆菌的高密度培养,最常用和最有效的方法就是分批补料流加培养。     

New FadB homologous enzymes and their use in enhanced biosynthesis of medium-chain-length polyhydroxyalkanoates in FadB mutant Escherichia coli

Recombinant Escherichia coli harboring the medium-chain-length (MCL) polyhydroxyalkanoate (PHA) synthase gene has been shown to accumulate MCL-PHAs from fatty acids when FadB is inactive. However, the enzymes in fadB mutant E. coli responsible for channeling the beta-oxidation intermediates to PHA biosynthesis have not been fully elucidated. Only recently, two enzymes encoded by yfcX and maoC have been found to be partially responsible for this. In this study, we identified five new FadB homologous enzymes in E. coli: PaaG, PaaF, BhbD, SceH, and YdbU, by protein database search, and examined their roles in the biosynthesis of MCL-PHAs in an fadB mutant E. coli strain. Coexpression of each of these genes along with the Pseudomonas sp. 61-3 phaC2 gene did not allow synthesis of MCL-PHA from fatty acid in recombinant E. coli W3110, which has a fully functional beta-oxidation pathway, but allowed MCL-PHA accumulation in an fadB mutant E. coli WB101. In particular, coexpression of the paaG, paaF, and ydbU genes resulted in a MCL-PHA production up to 0.37, 0.25, and 0.33 g/L, respectively, from 2 g/L of sodium decanoate, which is more than twice higher than that obtained with E. coli WB101 expressing only the phaC2 gene (0.16 g/L). These results suggest that the newly found FadB homologous enzymes, or at least the paaG, paaF, and ydbU genes, are involved in MCL-PHA biosynthesis in an fadB mutant E. coli strain and can be employed for the enhanced production of MCL-PHA.     

In situ recovery of lycopene during biosynthesis with recombinant Escherichia coli

Lycopene is produced by recombinant Escherichia coli expressing genes to encode for the lycopene biosynthesis. However, the productivity of lycopene seemed to be limited by many factors including product toxicity. In the present study, we have investigated physiology of recombinant E. coli during biosynthesis and in situ recovery of lycopene based on an organic/aqueous two-phase system. Lycopene, the 40-carbon molecule product, was little extracted from recombinant E. coli cells to octane or decane phase. However, partial digestion of cell walls with lysozyme promoted extraction of lycopene into the organic phases. Engineering of an organic/aqueous two-phase system allowed recombinant E. coli cells to produce ca. 40% larger amount of lycopene compared to that in a conventional aqueous single-phase system. Optimization of the in situ product recovery process will lead to further increase of product concentration and productivity.     

Mapping of a locus (mdoA) that affects the biosynthesis of membrane-derived oligosaccharides in Escherichia coli.

Mutants of Escherichia coli defective in the newly discovered mdoA locus are blocked at an early stage in the biosynthesis of membrane-derived oligosaccharides. The mutation has now been mapped and found to be located near 23 min on the E. coli chromosome between putA and pyrC. The mdoA mutants are defective in the membrane-localized component of the glucosyl transferase system described by Weissborn and Kennedy (A. C. Weissborn and E. P. Kennedy, Fed. Proc. 42:2122, 1983).     

Methodologies for screening of bacteria-carbohydrate interactions: anti-adhesive milk oligosaccharides as a case study

Many studies have demonstrated the capacity of glycan-based compounds to disrupt microbial binding to mucosal epithelia. Therefore, oligosaccharides have potential application in the prevention of certain bacterial diseases. However, current screening methods for the identification of anti-adhesive oligosaccharides have limitations: they are time-consuming and require large amounts of oligosaccharides. There is a need to develop analytical techniques which can quickly screen for, and structurally define, anti-adhesive oligosaccharides prior to using human cell line models of infection. Considering this, we have developed a rapid method for screening complex oligosaccharide mixtures for potential anti-adhesive activity against bacteria. Our approach involves the use of whole bacterial cells to "deplete" free oligosaccharides from solution. As a case study, the free oligosaccharides from the colostrum of Holstein Friesian cows were screened for interactions with whole Escherichia coli cells. Reductions in oligosaccharide concentrations were determined by High pH Anion Exchange Chromatography and Hydrophilic Interaction Liquid Chromatography (HILIC-HPLC). Oligosaccharide structures were confirmed by a combination of HILIC-HPLC, exoglycosidase digestion and off-line negative ion mode MS/MS. The depletion assay confirmed selective bacterial interaction with certain bovine oligosaccharides which in previous studies, by other methodologies, had been shown to interact with E. coli. In particular, the bacterial cells depleted the following oligosaccharides in a population dependent manner: 3'-sialyllactose, disialyllactose, and 6'-sialyllactosamine. The assay methodology was further validated by studies in which we demonstrated the inhibitory activity of 3'-sialyllactose, and a mixture of bovine colostrum oligosaccharides, on E. coli adhesion to differentiated HT-29 cells.     

Biosynthesis of phospholipids in Bacillus megaterium.

Information on the biosynthesis of phospholipids in bacteria has been derived principally from the study of Escherichia coli and other gram-negative organisms. We have now carried out a detailed study of the pathways of phospholipid biosynthesis in the gram-positive organism Bacillus megarterium KM in relation to investigations on the biogenesis of lipid asymmetry in membranes. Radioactive precursors such as 32Pi and [3H]palmitate initially label phosphatidylethanolamine much more than phosphatidylglycerol. This raised the possibility that phosphatidylglycerol may be the precursor of phosphatidylethanolamine in a pathway different from that in E. coli. Phosphatidylglycerol is known to be highly reactive metabolically, since it functions as a donor of phosphatidyl residues in the synthesis of cardiolipin and as a donor of glycerophosphate residues in the synthesis of teichoic acids and of membrane-derived oligosaccharides. The large pool of phosphatidylglycerol would dilute the radioactive isotope, slowing the initial rate of incorporation of label into phosphatidylethanolamine. However, assays of cell-free extracts revealed no evidence for such a novel pathway. Instead, phosphatidylserine synthase (cytidine 5'-diphosphate-diglyceride:L-serine phosphatidyl transferase) and phosphatidylserine decarboxylase were detected, although at low levels. These results suggest that the pathway in B. megaterium is the same as that in E. coli in which phosphatidylserine, derived from cytidine 5'-diphosphate-diglyceride, is the precursor of phosphatidylethanolamine. The lag in the appearance of label in phosphatidylethanolamine appears to be the effect of a considerable pool of phosphatidylserine (ca. 5 to 10% of the total phospholipid) in certain strains of B. megaterium. The lag in labeling can be correlated with the size of the pool of phosphatidylserine. Pulse-chase experiments in vivo support the conclusion that in B. megaterium phosphatidylserine is not derived from phosphatidylglycerol. Rates of turnover of the membrane phospholipids of B. megaterium have also been studied.     

Identification of UDP-glucose as an intermediate in the biosynthesis of the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides of Escherichia coli constitute a closely related family of oligosaccharides containing approximately 9 glucose units variously substituted with sn-glycero-1-phosphate and phosphoethanolamine residues derived from the head groups of membrane phospholipids, and also with succinate in O-ester linkage (Kennedy, E.P., Rumley, M.K., Schulman, H., and van Golder, L.M.G. (1976) J. Biol. Chem. 251, 4208-4213). Studies with mutant strains defective in the synthesis of various nucleoside diphosphate sugars have now revealed that UDP-glucose is an essential intermediate in the biosynthesis of these oligosaccharides. Mutants unable to synthesize UDP-glucose do not contain significant amounts of the membrane-derived oligosaccharides. In contrast, a strain unable to synthesize ADP-glucose, the glucosyl donor for glycogen synthesis in E. coli, contained normal amounts of the membrane-derived oligosaccharides, although with a somewhat different pattern of distribution of the various subspecies. In confirmation of these genetic studies, pulse-label isotope tracer studies have been carried out with glucose of high specific activity, under conditions in which UDP-glucose comprises a large fraction of the total radioactivity in the low molecular weight pool. Subsequent "chase" experiments clearly revealed the conversion of UDP-glucose to the higher molecular weight membrane-derived oligosaccharides.     

Recombinant interleukin-3 expression system in E. coli]

Plasmid pTOTE2IL3 (III) has been constructed, expressing an artificial human interleukin-3 (hIL3) gene under conditions of the induced protein biosynthesis. Levels of the recombinant protein synthesis have been compared in several E. coli strains containing expression plasmids pTE2IL3 (I) (constitutive biosynthesis) and (III) (induced biosynthesis). Optimal combinations of the expression plasmids and the bacterial strains are of importance. A simple and effective method has been elaborated for isolation, purification and renaturation of the recombinant protein accumulated in inclusion bodies.