多策略代谢工程大肠杆菌高效生产辅酶Q10

Escherichia coli BW25113 was metabolically engineered for CoQ10 production by replacing ispB with ddsA from Gluconobacter suboxydans.Effects of precursor balance and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) availability on CoQ10 production in E.coli were investigated.The knockout of pykFA along with pck overexpression could maintain a balance between glyceraldehyde 3-phosphate and pyruvate,increasing CoQ10 production.Replacement of native NAD-dependent gapA with NADP-dependent gapC from Clostridium acetobutylicum,together with the overexpression of gapC,could increase NADPH availability and then enhanced CoQ10 production.Three effects,overexpressions of various genes in CoQ biosynthesis and central metabolism,different vectors and culture conditions on CoQ10 production in E.coli,were all investigated.The investigation of different vectors indicated that low copy number vector may be more beneficial for CoQ10 production in E.coli.The recombinant E.coli (△ispB::ddsA,△pykFA and △gapA::gapC),harboring the two plasmids encoding pck,dxs,idi and ubiCA genes under the control of PT5 on pQE30,ispA,ddsA from Gluconobacter suboxydans and gapC from Clostridium acetobutylicum under the control of PBAD on pBAD33,could produce CoQ10 up to 3.24 mg·g-1 dry cell mass simply by changing medium from M9YG to SOB with phosphate salt and initial culture pH from 7.0 to 5.5.The yield is unprecedented and 1.33 times of the highest production so far in E.coli.     

硫氧化还原酶热稳定性的结构基础(英文)

The thermostability of three sulfur oxygenase reductases(SORs) was investigated from thermoaci-dophilic achaea Acidianus tengchongensis(SORAT) and Sulfolobus tokodaii(SORST) as well as the moderately thermophilic bacterium Acidithiobacillus sp.SM-1(SORSB).The optimal temperatures for catalyzing sulfur oxida-tion were 80 °C(SORAT),85 °C(SORST),and 70 °C(SORSB),respectively.The half-lives of the three SORs at their optimal catalytic conditions were 100 min(SORAT),58 min(SORST),and 37 min(SORSB).In order to reveal the structural basis of the thermostability of these SORs,three-dimensional structural models of them were generated by homology modeling using the previously reported high-resolution X-ray structure of SORAA(from Acidianus ambivalens) as a template.The results suggest that thermostability was dependent on:(a) high number of the charged amino acid glutamic acid and the flexible amino acid proline,(b) low number of the thermolabile amino acid glutamine,(c) increased number of ion pairs,(d) decreased ratio of hydrophobic accessible solvent surface area(ASA) to charged ASA,and(e) increased volumes of the cavity.The number of cavities and the number of hydro-gen bonds did not significantly affect the thermostability of SORs,whereas the cavity volumes increased as the thermal stability increased.     

重组大肠杆菌流加发酵中短暂溶氧休克促进5-氨基乙酰丙酸生成(英文)

5-Aminolevulinic acid （ALA） is a common precursor for tetrapyrrole compounds in all kinds of organ isms and has wide applications in agriculture and medicines. In this study, a new strategy, i.e. short-term dissolved oxygen （DO） shock during aerobic fermentation, was introduced to produce 5-aminolevulinic acid with a recombi-nant E. coli. Effects of duration time of DO shock operation on plasmid concentration, intracellular ALA synthase （ALAS） activity and ALA production were investigated in Erlenmeyer shake flasks. The results indicated that both ALAS activity and ALA yield were enhanced in an anaerobic operation of 45 rain in the early exponential phase during fermentation, while they decreased when the anaerobic operation time was further increased to 60 rain. The DO shock protocol was confirmed with the fed-batch fermentation in a 15 L fermenter and the ALA production achieved 9.4 g.L-1 （72 mmol.L-1）, which is the highest yield in the fermentation broth reported up to now.     

大颗粒超高纯度硅溶胶的制备及其表征(英文)

采用新工艺路线制备四乙氧基硅烷（tetraethoxysilane,TEOS）,以TEOS为原料制备超纯度大颗粒硅溶胶。TEOS是烷氧基硅烷法制备甲硅烷的副产物,经过精馏提纯以后,加入盐酸或氨水催化剂,采用溶胶-凝胶法制备硅溶胶。结果表明：TEOS和硅溶胶金属杂质离子总含量都低于0.3mg/L。用盐酸催化TEOS水解制备溶胶,胶粒直径达到129nm;用氨水催化,胶粒直径达272nm。当物质的量比n（C2H5OH）/n（TEOS）=6,n（H2O）/n（TEOS）=5,搅拌速度为250 r/min,制备的硅溶胶胶粒均匀度最好。加水不搅拌,胶粒很容易发生团聚,但是,当搅拌速率高于500 r/min时,胶粒出现团聚现象。     

重组大肠杆菌的构建及利用木糖生产木糖醇的研究

xylB基因失活可以降低木酮糖的磷酸化,使木糖还原为木糖醇而不进入PPP途径。利用Red重组技术构建xylB缺失大肠杆菌DxylB/E.coli BL21(DE3)可以提高木糖醇的产率。通过PCR克隆得到来自Neurospora crassa的木糖还原酶基因xr,将该基因与载体p ET30a(+)连接构建表达质粒p ET30a-xr;PCR克隆得到来自E.coli K-12的6-磷酸葡萄糖酸脱氢酶(6-phosphogluconate dehydrogenase,6-PGDH)基因gnd和葡萄糖6-磷酸脱氢酶(glucose-6-phosphate dehydrogenase,G6PDH)基因zwf,依次与双启动子原核表达载体p CDFDute-1连接构建表达质粒p CDFDuet-gnd-zwf;将上述构建好的两个重组质粒同时转化到DxylB/E.coli BL21(DE3)宿主体内。经异丙基-?-D-硫代半乳糖苷(IPTG)诱导表达获得分子量约为38、51、54 k D的三种蛋白,酶活测定显示,重组菌种XR的比酶活为7.25 U×mg-1,6-PGDH的比酶活为2.26 U×mg-1,G6PDH的比酶活为1.31 U×mg-1。5 L发酵罐发酵结果显示,xylB基因敲除可以提高木糖醇的产率,含NADPH再生系统的菌株木糖醇的体积产率是1.04 g×(L×h)-1,比只含有木糖还原酶的重组菌株0.88 g×(L×h)-1高24.32%,为后续工业化利用大肠杆菌生产木糖醇奠定了基础。     

由二氧化硅直接合成含硅聚氨酯及其表征(英文)

二氧化硅与乙二醇在催化剂作用下直接合成了五配位硅酸酯 ,然后与氯乙醇反应制得了双羟基含硅化合物 ,该化合物与 2 ,4 -甲苯二异氰酸酯反应得到含硅聚氨酯。采用元素分析、FTIR ,1H -NMR表征了双羟基含硅化合物。结果表明 ,含硅聚氨酯的相对分子质量不高 ,相对分子质量分布比较宽 ,其数均相对分子质量、重均相对分子质量和分布宽度指数分别为 1790 ,6 2 0 0 ,3.4 6。含硅聚氨酯的玻璃化转变温度为 115 .1℃。     

木糖醇的生产应用与开发前景

本文介绍了木糖醇生产方法、工艺条件及理化性质 概述了木糖醇在食品、医药、化工等行业的应用及开发前景。     

大肠杆菌E.coli HB101(pBR322) 高密度培养过程质粒的稳定性

通过正交实验考察了大肠杆菌E.coli HB101(pBR322)高密度培养过程中,不同培养条件对质粒的稳定性和β-内酰胺酶活力的影响. 结果表明,当温度在33~39℃、pH为6.4~7.2、DO为 40%~80%、 补料速率在5.4~10.8 g/h范围内变化,以及细胞密度达到27.3 g/L、比生长速率达到0.73 h-1时,质粒没有发生丢失现象,但在培养过程中β-内酰胺酶的活力降低.     

木糖醇的功能及其食品应用

木糖醇是自然界广泛存在的天然物质与山梨醇、麦芽糖醇等六元醇不同,它只有五个碳原子（五个羟基）,为五元醇,分子式C5H12O5,分子量152．15。木糖醇广泛存在于天然物质中,但含量较低,如表1。     

木糖醇的制备方法及其应用

介绍了木糖醇的特性、几种制备方法以及在医药、食品、化工等方面的应用     

Characterization of promoters in Escherichia coli and application for xylitol synthesis

Promoters are the most important tools to control and regulate the gene expression in synthetic biology and met-abolic engineering. The expression of target genes in Escherichia coli is usually control...     

大肠杆菌耐热元器件的构建及其应用

以提高大肠杆菌耐热性为目的,基于腾冲嗜热菌（Thermoanaerobacter tengcongensis MB4）热激蛋白基因 T.te-HSP20 构建了诱导型耐热元器件T7-T.te-HSP20 和组成型耐热元器件gapA-T.te-HSP20,转入大肠杆菌（Escherichia coli）获得工程菌 E. coli-TH 和 E. coli-GH。工程菌E. coli-TH在30℃和IPTG诱导下,目标蛋白呈可溶性表达,经50℃热激30 min后,存活率提高了3.2倍。高温发酵表明gapA-T.te-HSP20扩宽了工程菌E. coli-GH的最适生长温度的范围（37～43℃）,较大程度提高了大肠杆菌的耐热性。抗逆性分析还发现工程菌E. coli-GH具备了耐热与耐丁醇的双重功能,并有一定的抗乙酸和乙醇能力。为工业梯度升温发酵生产生物基产品的高效制造、节省成本提供了新思路。     

外源蛋白在大肠杆菌中的可溶性表达策略

长期以来,大肠杆菌一直是表达外源蛋白的首选表达系统. 但由于外源蛋白在表达过程中容易被宿主细胞蛋白酶降解或者形成包涵体,其应用受到了限制. 本文综述了在大肠杆菌中表达可溶外源蛋白的策略和进展,以期提高具有生物活性的外源基因的表达水平.     

重组大肠杆菌1,2,4-丁三醇合成途径的平衡优化

1,2,4-丁三醇(1,2,4-butantriol,BT)属于非天然化学品,是军工材料1,2,4-丁三醇三硝酸酯的前体。在重组大肠杆菌中,木糖经脱氢、脱水、脱羧和还原合成BT。但途径各反应不平衡使得中间代谢物积累限制菌体生长和产物合成。本研究首先通过CRISPR/Cas9敲除基因yjh G和yqh D构建无本底表达宿主菌,随后利用不同启动子组合调节BT合成途径中基因xdh、yjh G和yqh D的表达。结果发现,以P_inv表达醇脱氢酶基因yqh D使BT产量达到14.4g/L;以P_tac表达脱氢酶基因xdh和P_{rrn HP1}表达脱水酶基因yjh G的组合方式,BT产量达到15.6g/L,比对照菌株KXW3009分别增加5.9%和14.7%。本研究通过对中间代谢物木糖酸合成和代谢的组合优化,促进了BT的合成,为后续研究提供了借鉴。     

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.     

重组大肠杆菌共表达D-海因酶和N-氨甲酰基-D-氨基酸酰胺水解酶

通过PCR分别扩增得到N-carbamoylase基因和上游带有RBS区的D-hydantoinase基因,并将其依次克隆入pBAD/His A质粒中,得到呈多顺反子结构的双酶表达质粒pBAD-CRH,转化E. coli Top10F￠得到重组菌TaraCRH. 实验证明,E. coli TaraCRH对外源蛋白的表达控制严紧,而加入阿拉伯糖诱导后可以成功表达两酶基因. 诱导条件优化结果表明,诱导温度采用29℃,培养基中阿拉伯糖浓度0.05 g/L可达到最高酶活. 进一步尝试采用玉米浆培养基代替LB培养基,在经过优化的玉米浆培养基中培养TaraCRH菌株,经阿拉伯糖诱导,海因酶和水解酶酶活分别达到3.52和4.21 U/mL.     

大肠埃希菌和肺炎克雷伯菌临床分离株的耐药性分析

目的分析临床分离的大肠埃希菌和肺炎克雷伯菌的分布情况及耐药性。方法收集吉林大学中日联谊医院临床标本中分离的221株大肠埃希菌和152株肺炎克雷伯菌,采用纸片扩散法检测抗菌素的敏感性;双纸片协同试验和纸片表型确证试验筛选并确证产超广谱β-内酰胺酶的菌株;按照美国国家临床实验室标准化委员会(NCCLS)2005年版的标准判断结果。结果大肠埃希菌和肺炎克雷伯菌对亚胺培南和美罗培南的敏感性最高,均达100%;对头孢他啶、哌拉西林/他唑巴坦和头孢西丁的敏感性也较高,均大于70%,而对氨苄西林的耐药性均大于90%。共检出产超广谱β-内酰胺酶的大肠埃希菌118株,检出率为53.4%;肺炎克雷伯菌21株,检出率为13.8%;产超广谱β-内酰胺酶的两种菌株与非产超广谱β-内酰胺酶的同种菌株相比,对抗菌素的耐药性均明显增加。结论大肠埃希菌和肺炎克雷伯菌仍是产超广谱的β-内酰胺酶的主要菌株,且对常用抗菌素产生了较高的耐药性。     

Efficient fuzzy control strategies for the application of pH-stat to fed-batch cultivation of genetically engineered Escherichia coli

In the cultivation of genetically engineered Escherichia coli it is very important to control the substrate concentration at an appropriate level in order to avoid the accumulation of acetate, thereby elevating the expression level of plasmid-encoded protein. In this paper, a pH-stat mode of fuzzy control was considered for the overexpression of β-galactosidase in the fed-batch cultivation of recombinant E. coli. In the simple pH-stat fuzzy control, the response of pH change in the culture broth to the feeding rate of glucose was used to estimate the glucose consumption rate. In the modified pH-stat fuzzy control, the glucose consumption rate was accurately estimated by using pH change and the change in the carbon dioxide content of the exhaust gas. With this control strategy, the cell density could be increased to 72 g DCW dm^?3, which was twofold higher than that attained in the cultivation with the simple pH-stat fuzzy control. The bulk β-galactosidase concentration was increased to 4150 U cm^?3, which was threefold higher than when the simple pH-stat control was used.     

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。