多基因在大肠杆菌中的共表达策略

多基因共表达在多领域有重要的应用价值,大肠杆菌共表达系统包括多顺反子系统和双质粒系统,两者各有优缺点。多顺反子系统不需外界2种抗生素的同时存在,但操作较为复杂。通常认为,具有相同复制子的质粒是不相容的,但近来的实验表明,在双抗生素的选择压力下,不相容的双质粒系统也能稳定传代,且操作简单,周期较短。双质粒系统已经应用于生产、医学等各个领域。     

环氧角鲨烯合成通路在大肠杆菌中的构建和优化

三萜类化合物是一类广泛应用于医药、保健和化妆品等行业的天然产物,具有巨大的商业价值.生物合成三萜类化合物依赖于环氧角鲨烯的高效合成.角鲨烯环氧化酶是整个合成途径中的关键酶,其催化NADPH依赖的环氧化反应将角鲨烯转变为环氧角鲨烯.通过筛选不同来源的角鲨烯环氧化酶,截短的大鼠角鲨烯环氧化酶(RnSETC)在大肠杆菌Esc...     

人转化生长因子β1在大肠杆菌中的分泌表达

为了研究双体形式生长因子的原核基因工程,尝试了人转化生长因子β１（ｈＴＧＦβ１）基因的分泌表达．通过缺失突变,构建了能表达具有天然一级结构的ｈＴＧＦβ１单体蛋白的周质分泌表达质粒．采用双顺反子表达系统,使ＴＧＦβ１在周质中获得了高效可溶性表达．研究了改善转运通路对重组蛋白分泌表达的影响,发现共表达σ３２基因和ｄｓｂＡ基因,可促进周质中ＴＧＦβ１双体分子的形成;而共表达ｓｅｃＥ／Ｙ基因对ＴＧＦβ１的分泌表达则没有明显影响．通过共表达ｋｉｌ基因,使ＴＧＦβ１在胞外培养基中获分泌表达,并在胞外折叠、组装形成具有生物活性的双体分子     

利用合成调控RNA技术提高大肠杆菌异源合成红霉素前体6-脱氧红霉内酯B产量

红霉素为代表的聚酮类化合物已经成功的在大肠杆菌中实现了异源合成,但其产量仍然较低(仅~10 mg/L)。本研究基于大肠杆菌全基因组代谢模型iAF1260,利用通量平衡分析预测了红霉素母核6-脱氧红霉内酯(6-Deoxyerythronolide B,6-d EB)生物合成的关键靶点,通过合成调控RNA技术(Synthetic small regulatory RNAs,sRNAs)对预测的靶点进行验证。结果表明,以弱化lsrC(编码LsrABC转运蛋白)和ack A(编码乙酸激酶蛋白)为代表的关键靶点改造可以显著提高6-d EB异源合成,提高幅度可达48.7%。通过弱化靶点的组合,进一步改善了6-d EB的异源合成,产量最终可达22.8 mg/L,比出发菌株产量提高59.9%。本研究发现和确认了6个有效的调控靶点,最终成功地改善了6-d EB在大肠杆菌中的异源合成。研究表明,通量分布比较分析结合sRNAs技术是一种有效的方法提高6-d EB异源合成,也为改善其他代谢产物的异源合成提供了可供借鉴的研究思路。     

双质粒系统共表达大肠杆菌肉碱脱水酶基因caiB及其辅酶合成基因caiE

caiB基因和caiE基因分别编码肉碱脱水酶及其辅因子合成酶 ,两者的共表达可以获得高活性肉碱脱水酶的重组菌。分别用两相容性质粒和不相容质粒共表达肉碱脱水酶及其辅酶合成基因 ,并对两种方法进行了比较。经IPTG诱导 ,相容性双质粒系统中两基因表达量分别占菌体总蛋白质的 17%和 10 % ;不相容性双质粒系统中两基因表达量分别占菌体总蛋白质的 39%和 2 0 %。共转化菌的酶活力比pET2 8caiB单质粒转化菌均提高 2 .3倍左右。两种双质粒转化系统相似 ,都需在外界抗生素选择压力下保持质粒稳定性     

SUMO基因的内在原核启动子活性及其在大肠杆菌蛋白表达系统中的应用

SUMO融合系统已成为目前大肠杆菌重组蛋白生产的重要手段,但在载体构建效率和蛋白可溶性等方面仍有待改进。本研究在PCR克隆酿酒酵母SUMO基因Smt3(Sm) 时意外发现Sm具有组成型原核启动子活性;而且经软莓BPROM程序预测发现大多数物种SUMO基因编码区都具有依赖s70的原核启动子。进一步通过整合Sm启动子和Sm 3￠末端StuⅠ位点特性以及引入His标签和超酸增溶标签,构建了基于Sm’-LacZα融合基因的一系列通用克隆表达载体,并通过蓝白斑筛选和SDS-PAGE分析进行了多个靶蛋白基因的克隆和表     

β-甘露聚糖酶和木聚糖酶基因在大肠杆菌中共表达

【目的】β-甘露聚糖酶和木聚糖酶都属于半纤维素酶,它们已经同时运用于工农业生产的许多领域。构建β-甘露聚糖酶和木聚糖酶共表达菌株并进行相关评价。【方法】通过设计一个共同的酶切位点,将菌株Bacillus subtilis BE-91中的β-甘露聚糖酶和木聚糖酶基因串联到表达载体pET28a(+)上,转化大肠杆菌构建了一株能够共表达β-甘露聚糖酶和木聚糖酶的菌株B.pET28a-man-xyl。【结果】菌株诱导21 h后,发酵液中β-甘露聚糖酶和木聚糖酶的酶活分别为713.34 U/mL和1455.83 U/mL,是胞内酶活的11.8倍和2.53倍。【结论】SDS-PAGE分析、水解圈活性检测和胞外酶与胞内酶酶活检测表明:两个酶均以功能蛋白独立分泌到胞外。此外,与β-甘露聚糖酶和木聚糖酶单独酶解半纤维素相比,复合酶的酶解效果更好。菌株的成功构建为复合酶制剂(半纤维素酶制剂)的研究和生产奠定基础。     

应用硫氧还蛋白促进外源蛋白在大肠杆菌的可溶性表达

为了观察硫氧还蛋白（ＴｒｘＡ）促进外源蛋白在大肠杆菌中可溶性表达的作用,我们从质粒ｐＥＴ－３２ａ（＋）上克隆了ｔｒｘＡ基因,构建了ＴｒｘＡ表达质粒ｐＴ－ＴｒｘＡ。将该质粒与其它蛋白基因的表达质粒共同转化Ｅ．ｃｏｌｉ并同时获得表达。结果表明,共表达ＴｒｘＡ可以明显促进外源蛋白,如甲状旁腺激素相关蛋白（ＰＴＨｒＰ）、ＰＴＨｒＰ受体（ＰＴＨｒＰ－Ｒ）的血管内皮生长因子（ＶＥＧＦ）的可溶性表达。说明共表达     

红球菌Rhodococcus sp.DS-3 dszABC基因和dszD基因在大肠杆菌中的共表达

二苯并噻吩(DBT)及其衍生物微生物脱硫的4S途径需要4个酶(DszA,DszB,DszC and DszD)参与催化。其中DBT单加氧酶(DszC or DBT-MO)和DBT-砜单加氧酶(DszA or DBTO2-MO)都是黄素依赖型氧化酶,它们的催化反应需要菌体中还原型的黄素单核苷酸(FMNH2),FMNH2由辅酶黄素还原酶(DszD)再生。因此,共表达DszA,DszB,DszC和DszD可以提高整个脱硫途径的速率。构建了两个不相容性表达载体pBADD和paN2并在大肠杆菌中实现了4个脱硫酶基因的共表达。DszA,DszB,DszC和DszD的可溶性蛋白表达量分别占菌体总蛋白质的7.6%,3.5%,3.1%和18%。共表达时的脱硫活性是单独用paN2表达时的5.4倍,并对工程菌休止细胞脱除模拟柴油中DBT的活性进行了研究。     

(R)-专一性羰基还原酶与甲酸脱氢酶基因在大肠杆菌中的共表达

【目的】通过研究(R)-专一性羰基还原酶和甲酸脱氢酶基因在大肠杆菌中的共表达,解决较高底物浓度下不对称转化反应的辅酶限制性问题。【方法】分别以近平滑假丝酵母(Candida parapsilosis CCTCC M203011)和博伊丁假丝酵母(Candida boidinii)基因组为模板,采用PCR方法扩增得到(R)-专一性羰基还原酶基因（rcr）和甲酸脱氢酶基因（fdh）,克隆到共表达载体pETDuetTM-1中进行表达。共表达质粒pETDuet-rcr-fdh转化稀有密码子优化型菌株E. coli Rosetta,获得重组菌E. coli Rosetta/pETDuet-rcr-fdh。【结果】在30℃条件下,经1 mmol/L IPTG诱导表达8 h后,SDS-PAGE结果表明(R)-专一性羰基还原酶和甲酸脱氢酶均有明显的表达,其相对分子质量分别为37 kDa和 40 kDa。以高浓度(6 g/L)2-羟基苯乙酮为底物时,0.1 g重组菌细胞催化产生(R)-苯基乙二醇,产物光学纯度为100% e.e.,产率为85.9%。与无甲酸脱氢酶参与辅酶再生循环的重组菌E. coli Rosetta/pETDuet-rcr相比,产物光学纯度和产率分别提高了1.3和2.7倍。【讨论】该重组菌的构建为基因工程法生物合成(R)-苯基乙二醇的工业应用奠定了基础。     

Analysis of the maximum theoretical yield for the synthesis of erythromycin precursors in Escherichia coli

The heterologous biosynthesis of complex polyketides in Escherichia coli was recently achieved through metabolic engineering. However, it was observed that less than 10% of the propionate carbon source is transformed into the erythromycin precursor, 6-deoxyerythronolide B (6dEB), resulting in a 1.4% molar yield. Therefore, metabolic flux analysis was performed using a model of the Escherichia coli metabolism with the addition of the enzymes required for 6dEB synthesis. The analysis shows that the maximum theoretical yield for 6dEB synthesis in E. coli is 11%. The maintenance energy requirement of E. coli and limitations in the specific oxygen uptake rate can further decrease the yield, suggesting that the observed 6dEB yield of 1.4% can be the result of these two factors. In addition, the results suggest that an increase in the specific carbon and oxygen uptake rates will increase the yield of 6dEB. The use of glucose as an alternative carbon source was also evaluated using metabolic flux analysis and the results suggest that the choice of glucose as the carbon source will allow a small improvement in performance relative to a propionate-based process.     

Deoxysugar pathway interchange for erythromycin analogues heterologously produced through Escherichia coli

The overall erythromycin biosynthetic pathway can be sub-divided into macrocyclic polyketide formation and polyketide tailoring to produce the final bioactive molecule. In this study, the native deoxysugar tailoring reactions were exchanged for the purpose of demonstrating the production of alternative final erythromycin compounds. Both the d-desosamine and l-mycarose deoxysugar pathways were replaced with the alternative d-mycaminose and d-olivose pathways to produce new erythromycin analogues through the Escherichia coli heterologous system. Both analogues exhibited bioactivity against multiple antibiotic-resistant Bacillus subtilis strains. Besides demonstrating an intrinsic flexibility for the biosynthetic system to accommodate alternative tailoring pathways, the results offer an initial attempt to leverage the E. coli platform for erythromycin analogue production.     

LC-MS/MS quantification of short-chain acyl-CoA's in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity

Aims: This paper utilized quantitative LC‐MS/MS to profile the short‐chain acyl‐CoA levels of several strains of Escherichia coli engineered for heterologous polyketide production. To further compare and potentially expand the levels of available acyl‐CoA molecules, a propionyl‐CoA synthetase gene from Ralstonia solanacearum (prpE‐RS) was synthesized and expressed in the engineered strain BAP1. Methods and Results: Upon feeding propionate, the engineered E. coli strains had increased the levels of both propionyl‐ and methylmalonyl‐CoA of 6‐ to 30‐fold and 3·7‐ to 6·8‐fold, respectively. Expression of prpE‐RS resulted in no significant increases in acetyl‐, butyryl‐ and propionyl‐CoA when fed the corresponding substrates (sodium acetate, butyrate or propionate). More interesting, however, were the results from strain BAP1 engineered for native prpE overexpression, which indicated increases in the same range of acyl‐CoA formation. Conclusions: The increased acyl‐CoA levels across the strains profiled in this study reflect the genetic modifications implemented for improved polyketide production and also indicate flexibility of the native PrpE. Significance and Impact of the Study: The results provide direct evidence of enhanced acyl‐CoA levels correlating to those strains engineered for polyketide biosynthesis. This information and the inherent flexibility of the native PrpE enzyme support future efforts to characterize, engineer and extend acyl‐CoA precursor supply for additional heterologous biosynthetic attempts.     

The deoxycytidine pathway for thymidylate synthesis in Escherichia coli

When thymidylate production is diminished by a mutation affecting dCTP deaminase, Escherichia coli is known to use an alternate pathway involving deoxycytidine as an intermediate. The pathway requires the gene for any of three nucleoside diphosphate kinases (ndk, pykA, or pykF) and the gene for a 5′-nucleotidase (yfbR).     

大肠杆菌角鲨烯合成途径的构建与调控

角鲨烯因其具有良好的抗氧化功能而被广泛应用于食品、医药、化妆品、工业应用等领域。本实验在大肠杆菌中构建角鲨烯合成途径,通过对其合成途径中关键限速酶(1-脱氧-D-木酮糖-5-磷酸合酶和异戊烯基二磷酸异构酶)过表达的方法进行初步调控,使角鲨烯的产量提升了近三倍。之后采用单因素试验对其发酵培养基和培养条件进行优化,以此来提高角鲨烯的产量。优化发酵条件后,使用最优发酵培养基——TB培养基,在最佳发酵条件:37℃,220r/min培养至OD600约为1.2时加入终浓度为0.1mmol/L的IPTG诱导剂,25℃条件下诱导48h,角鲨烯产量可达73.88mg/L。     

Monoterpene biosynthesis pathway construction in Escherichia coli

Four genes encoding sequential steps for the biosynthesis of the spearmint monoterpene ketone (-)-carvone from the C(5) isoprenoid presursors isopentenyl diphosphate and dimethylallyl diphosphate were installed in Escherichia coli. Inducible overexpression of these genes in the bacterial host allowed production of nearly 5 mg/l of the pathway intermediate (-)-limonene, which was mostly excreted to the medium such that products of the downstream steps, (-)-carveol and (-)-carvone, were not detected. Assay of pathway enzymes and intermediates indicated that flux through the initial steps catalyzed by geranyl diphosphate synthase and limonene synthase was severely limited by the availability of C(5) isoprenoid precursors in the host. Feeding studies with (-)-limonene, to overcome the flux deficiency, demonstrated the functional capability of limonene-6-hydroxylase and carveol dehydrogenase to produce the end-product carvone; however, uptake and trafficking restrictions greatly compromised the efficiency of these conversions.     

Identification of phosphatidylserylglutamate: a novel minor lipid in Escherichia coli

Advances in mass spectrometry have facilitated the identification of novel lipid structures. In this work, we fractionated the lipids of Escherichia coli B and analyzed the fractions using negative-ion electrospray ionization mass spectrometry to reveal unknown lipid structures. Analysis of a fraction eluting with high salt from DEAE cellulose revealed a series of ions not corresponding to any of the known lipids of E. coli. The ions, with m/z 861.5, 875.5, 887.5, 889.5, and 915.5, were analyzed using collision-induced dissociation mass spectrometry (MS/MS) and yielded related fragmentation patterns consistent with a novel diacylated glycerophospholipid. Product ions arising by neutral loss of 216 mass units were observed with all of the unknowns. A corresponding negative product ion was also observed at m/z 215.0. Additional ions at m/z 197.0, 171.0, 146.0, and 128.0 were used to propose the novel structure phosphatidylserylglutamate (PSE). The hypothesized structure was confirmed by comparison with the MS/MS spectrum of a synthetic standard. Normal phase liquid chromatography-mass spectrometry analysis further showed that the endogenous PSE and synthetic PSE eluted with the same retention times. PSE was also observed in the equivalent anion exchange fractions of total lipids extracted from the wild-type E. coli K-12 strain MG1655.     

Metabolic pathway structures for recombinant protein synthesis in Escherichia coli

Escherichia coli is a valuable commercial host for the production of heterologous proteins. We used elementary mode analysis to identify all possible genetically independent pathways for the production of three specific recombinant proteins, green fluorescent protein, savinase and an artificial protein consisting of repeating units of a five-amino-acid cassette. Analysis of these pathways led to the identification of the most efficient pathways for the production of each of these proteins. The results indicate that the amino acid composition of expressed proteins has a profound effect on the number and identity of possible pathways for the production of these proteins. We show that several groups of elementary modes produce the same ratio of biomass and recombinant protein. The pattern of occurrence of these modes is dependent on the amino acid composition of the specific foreign protein produced. These pathways are formed as systemic combinations of other pathways that produce biomass or foreign protein alone after the elimination of fluxes in specific internal reversible reactions or the reversible carbon dioxide exchange reaction. Since these modes represent pathway options that enable the cell to produce biomass and protein without utilizing these reactions, removal of these reactions would constrain the cells to utilize these modes for producing biomass and foreign protein at constant ratios.     

Facile biocatalytic synthesis of a macrocyclic lactone in sub- and supercritical solvents

Abstract

High yield and selectivity in the intramolecular synthesis of the macrocyclic lactone oxacyclohexadecan-2-one were achieved via a straightforward biocatalytic synthesis utilising non-conventional solvents: supercritical carbon dioxide (scCO₂), ethane, fluoroform and 1,1,1,2-tetrafluoroethane. Batch and continuous syntheses were demonstrated with a higher yield attained in a continuous synthesis using in-situ product extraction by scCO₂. A remarkably high yield was obtained in 1,1,1,2-tetrafluoroethane at batch conditions.