大肠杆菌偏利共培养系统合成大豆苷元

大豆苷元是一种植物雌激素,具有多种生物活性,但在大肠杆菌中的生物全合成还未见报道。基于大豆苷元合成途径的三个模块（对香豆酸、甘草素和大豆苷元模块）,构建大肠杆菌共培养系统从头合成大豆苷元。将对香豆酸和甘草素模块分配到两株大肠杆菌中构建双菌共培养系统,合成甘草素。在此基础上,探索了三种共培养模式合成大豆苷元,结果显示,三菌共培养系统比其他两种双菌共培养系统的产量更高,达到27.8 mg/L。共培养菌株间通过苯丙氨酸的单向流动形成了偏利共生的关系,有助于平衡代谢途径,提高大豆苷元产量。该共培养系统在大肠杆菌中实现大豆苷元的从头合成,为其他黄酮类化合物的生物合成提供了即插即用的平台。     

大豆苷元的合成

大豆异黄酮具有保健,抗肿瘤等作用,本文合成了其主要成分大豆苷元,经元素分析,核磁氢谱,质谱验证结构正确。本文使用2,4-二羟基苯基-4′-羟基苄基酮在新蒸的BF3／Et2O溶液催化下脱氧安息香反应合成了大豆苷元,此方法原料易得、收率较高。     

大豆苷元合成

报道了以间苯二酚和对羟基苯乙酸为原料,经过缩合,环合两步反应获得大豆苷元新方法。合成收率可达43．O％,产品质量符合药典标准。     

大豆苷元的水溶性改性研究进展

大豆苷元来源于自然界,是异黄酮化合物中很重要的一种,其具有重要的药理活性。近年来,国内外学者通过化学改性以提高其应用性进行了深入的研究。综述了大豆苷元的水溶性改性的最新研究进展,以促进对其的研究和应用。     

大豆甙元的合成方法

利用化学合成方法制造大豆活动物质大豆甙元,大豆中大豆甙元含量很低,而大豆甙元在农业,畜牧业,医药上用途日益广泛,本合成路线具有工序短,成本低的优势。     

生物活性物质大豆苷元的量子化学研究

采用PM3-MO方法，对大豆苷元进行了量子化学计算，给出了分子轨道及其能级、电荷密度、键长、二面角参数等。结果表明：大豆苷元分子中苯并吡喃环（A环和C环）带较强的正电荷，易与受体的负电荷中心结合；B环是负电子区域，易与受体的正电荷中心结合；其他部分通过氢键与受体发生作用，从而发挥生物活性。     

大豆苷元在混合有机溶剂中溶解度的测定和关联

采用动态法利用激光监视技术测定了大豆苷元在乙醇和N,N-二甲基甲酰胺混合有机溶剂中配比为0.000、0.100、0.181、0.273、0.358、0.460、0.547、0.649、0.812、1.000,温度范围在280~340 K的溶解度;将溶解度实验数据用Apelblat方程进行关联。结果表明,大豆苷元在混合有机溶剂中的溶解度随温度的升高增大,随乙醇比例的增大而降低,关联结果良好,Apelblat方程关联溶解数据的平均相对误差小于1.652%,实验结果和关联方程为大豆苷元分离结晶过程提供基础数据。     

灯盏花乙素苷元的制备研究进展

综述了标题化合物的制备方法及最新研究进展。其中,植物提取方法主要是从灯盏花或野黄芩药材中提取分离得到。化学合成方法有全合成和半合成方法,全合成方法以5,6,7,4'-四甲氧基黄酮为起始原料,采用不同的脱甲基试剂合成标题化合物;半合成方法是用灯盏花乙素经水解断糖苷键合成标题化合物。     

大豆甙元两锅法合成工艺研究

为解决天然大豆甙元提取设备复杂、存在溶剂残留的问题,本研究以羟基苯乙酸和间苯二酚为原料,采用两锅法化学合成工艺制备大豆甙元,分别探讨了物质的量比、反应温度、反应时间对缩合反应和合环反应产率的影响,并采用傅里叶变换红外光谱仪对缩合反应产品脱氧安息香和合环反应产品大豆甙元相应官能团进行了定性分析,确定缩合反应最佳反应条件为物质的量比3∶1∶1.3,反应温度90℃,反应时间4 h,产率56.2%,合环反应最佳反应条件为物质的量比1∶2.1,反应温度90～95℃,反应时间3 h,产率72%,为大豆甙元的工业化生产应用提供了依据。     

甲基营养型大肠杆菌构建策略的研究进展

利用微生物细胞工厂实现原料转化和产品合成是绿色生物制造的核心。然而,当前生物制造仍以富含糖类的谷物粮食为主要原料,存在“与民争粮”的争议,亟需开发非粮原料。甲醇作为煤化工产业中的重要产品,具有来源广、价格低、还原性强等优势,有替代粮食原料的潜力。天然甲基营养菌可利用甲醇生产单细胞蛋白和各种氨基酸,但存在理论收率低、遗传改造工具不足等缺点。随着合成生物学的发展,以大肠杆菌等模式生物作为底盘细胞构建人工甲基营养菌,实现甲醇到各种化学品的生产已成为研究热点。本文总结了多种甲基营养型大肠杆菌的构建策略,明确了影响天然路径代谢的关键因素与代谢过程中的重要中间产物,概括了各种天然路径在大肠杆菌中的优化策略与人工新路径的构建方法,并对工程菌株的优化提出了展望。     

大肠杆菌生产琥珀酸的代谢工程研究进展

琥珀酸是一种重要的化工原料,具有广阔的市场. 微生物发酵法生产琥珀酸可以解决常规化学合成法对石油的依赖. 代谢工程的兴起使重组大肠杆菌生产琥珀酸变为可能,也取得了一定的成效,但是其发酵强度还不够高,且过程中伴随着其他有机酸的积累,因此还不适于工业化生产. 代谢工程以系统生物学为基础,为重组大肠杆菌的进一步改造提供了更合理的依据. 本工作以大肠杆菌生产琥珀酸所涉及的关键酶、代谢途径及其改造为对象,系统综述了大肠杆菌生产琥珀酸所涉及的代谢工程技术及其最新研究进展,并探讨了将来的发展前景.     

Metabolic engineering strategies for D‐lactate over production in Escherichia coli

D‐lactate is an important chiral intermediate and a substrate for polylactic acid (PLA) production. Escherichia coli accumulate D‐lactate as the primary fermentative product, and synthesis is directly controlled by the activity of lactate dehydrogenase, the efficiency of the carbon resource utilization, and the redox state. D‐lactate accumulation is complicated by the flux through the competing metabolic routes and the indirect regulation of energy metabolism, as well as by the unexpected interconnectivities of the cellular components in the remote metabolic routes. These effects have been extensively studied, and a number of metabolic engineering strategies have been applied to overproduce D‐lactate with high titers, yields and productivities in E. coli that have been able to reduce the production costs and precisely control the fermentation process. This review summarizes the related strategies and suggests directions for further study; these directions provide guidelines for the development of other metabolic products using E. coli as an industrial platform. © 2015 Society of Chemical Industry     

大肠杆菌乙酰辅酶A代谢调控及其应用研究进展

利用生物法合成生物基化学品具有高效、绿色、可持续发展等优势。乙酰辅酶A作为细胞内物质代谢的重要中间产物,是利用生物转化法合成许多生物基化学品的重要前体,在微生物碳代谢过程中发挥着枢纽作用。本文综述了大肠杆菌乙酰辅酶A的合成、代谢调控策略及其重要应用,重点总结了乙酰辅酶A的合成途径及近期发展的提高乙酰辅酶A胞内通量的代谢调控策略,包括乙酸途径的代谢调控、丙酮酸合成乙酰辅酶A途径的代谢调控、中心碳代谢途径的代谢调控、β氧化合成乙酰辅酶A途径的代谢调控和乙酰辅酶A合成新途径的发掘,进一步展望了提高乙酰辅酶A供给的策略,利用基因组编辑技术构建合成乙酰辅酶A为前体化学品细胞工厂的方法。     

Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli

N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.     

重组大肠杆菌生产极端耐热木聚糖酶

将含有来自于嗜热网球菌(Dictyoglomus thermophilum)Rt46B.1编码极端耐热木聚糖酶基因xynB的表达载体pET-DBc转化大肠杆菌(Escherichia coli)BL21(DE3),获得重组菌E. coli DB1,目的基因可表达出有活性且耐90oC的木聚糖酶. 初步优化的E. coli DB1发酵培养基的组成为(g/L)：葡萄糖50,NH4Cl 3,MgSO4 0.5,CaCl2 0.6,Na2HPO4×7H2O 12.8,KH2PO4 3.0,NaCl 0.5. 重组菌E. coli DB1木聚糖酶的耐热特性有利于木聚糖酶的下游回收和提取.     

大肠杆菌及其耐乙酸突变菌的连续培养和代谢流比较

在氮源限制基本培养基中连续培养大肠杆菌DH5α及其乙酸耐受突变株DA19。与DH5α相比,突变株DA19降低了葡萄糖的消耗,减少了乙酸和丙酮酸的生成,提高了菌体关于葡萄糖的得率。利用物料平衡和代谢反应的化学计量关系,分析了二者在比生长速率0.13 h^-1下的稳态代谢流分布。与DH5α相比,DA19的三羧酸循环流量高,酵解途径的流量低,从而减少了乙酸等副产物的分泌,反映了能量代谢效率的明显提高。比较了二者异柠檬酸裂解酶、6-磷酸葡萄糖脱氢酶、磷酸果糖激酶、异柠檬酸脱氢酶和乙酸激酶的活性,酶活变化与代谢流结果基本一致。     

Improvement of the riboflavin production by engineering the precursor biosynthesis pathways in Escherichia coli

3,4-Dihydroxy-2-butanone 4-phosphate (DHBP) and GTP are the precursors for riboflavin biosynthesis. In this research, improving the precursor supply for riboflavin production was attempted by overexpressing ribB and engineering purine pathway in a riboflavin-producing Escherichia coli strain. Initially, ribB gene was overexpressed to increase the flux from ribulose 5-phosphate (Ru-5-P) to DHBP. Then ndk and gmk genes were overexpressed to enhance GTP supply. Subsequently, a R419L mutation was introduced into purA to reduce the flux from IMP to AMP. Finally, co-overexpression of mutant purF and prs genes further increased riboflavin production. The final strain RF18S produced 387.6 mg riboflavin · L?1 with a yield of 44.8 mg riboflavin per gram glucose in shake-flask fermentations. The final titer and yield were 72.2%and 55.6%higher than those of RF01S, respectively. It was concluded that simultaneously engineering the DHBP synthase and GTP biosynthetic pathway by rational metabolic engineering can efficiently boost riboflavin production in E. coli.