重组大肠杆菌利用D-木糖合成D-1,2,4-丁三醇

1,2,4-丁三醇(1,2,4-butanetriol, BT)是一种重要的有机合成中间体。通过克隆表达恶臭假单胞菌(Pseudomonas putida ATCC12633)2-酮酸脱羧酶(mdlC)和新月柄杆菌(Caulobacter crescentus CB15)D-木糖脱氢酶(xdh),敲除木糖利用和D-1,2,4-丁三醇合成中间代谢物分解途径中关键基因木糖异构酶(xylA)和2-酮酸醛缩酶(yjhH和yagE),重构大肠杆菌代谢网络,得到了能够将D-木糖转化为D-1,2,4-丁三醇的重组菌株。考察了温度、装液量、pH控制等条件对重组菌株合成D-1,2,4-丁三醇的影响,在适宜条件下发酵36 h后D-1,2,4-丁三醇产量达到3.96 g·L^-1。探讨了葡萄糖利用与丁三醇合成的关系,通过敲除编码酶IICB^Glc的ptsG基因改造重组菌株的磷酸烯醇式丙酮酸葡萄糖转移酶(phosphoenolpyruvate: sugar phosphotransferase system, PTS)系统,菌株可以在利用葡萄糖生长的同时进行木糖的转化,具有更高的合成能力。     

重组大肠杆菌全细胞用于苯乙酮酸的绿色生物合成

苯乙酮酸是化学合成中重要的合成砌块,可用于合成多种药物中间体,探索苯乙酮酸的绿色合成工艺具有重要的意义。以包含D-扁桃酸脱氢酶LhDMDH编码基因的重组大肠杆菌全细胞为催化剂,考察了它在无辅酶和辅底物添加的条件下对D-扁桃酸生物转化的效果,并对催化产物进行了纯化和鉴定。结果表明,本研究成功实现了在无辅酶和辅底物添加条件下苯乙酮酸的生物合成,产物的得率和纯度分别为45%和99%左右。成果也为外消旋扁桃酸的手性拆分及苯乙酮酸的生物合成奠定了基础。     

Production of 11R-hydroxyeicosatetraenoic acid from arachidonic acid by Escherichia coli cells expressing arachidonate 11R-lipoxygenase from Nostoc sp.

Linoleate 9R-lipoxygenase (9R-LOX) from Nostoc sp. SAG 25.82 was identified as arachidonate (ARA) 11R-LOX by the determination of the product obtained from the conversion of ARA. The specific activity and catalytic efficiency (k_cat/K_m) of the enzyme for C20 and C22 polyunsaturated fatty acids followed the order ARA > eicosapentaenoic acid > docosahexaenoic acid. The production of the lipid mediator 11R-hydroxyeicosatetraenoic acid (11R-HETE) was performed using Escherichia coli cells expressing ARA 11R-LOX from Nostoc sp. The reaction conditions, such as pH, temperature, solvent and its concentration, and substrate and cell concentrations, were optimized using the recombinant cells, and the optimal conditions for the production of 11R-HETE from ARA were pH 7.0, 25°C, 10 g L⁻¹ cells, 5.0 mM ARA, 4% (v/v) ethanol, and 10 mM cysteine as a reducing agent. Under these optimized conditions, E. coli cells expressing 11R-LOX converted 5.0 mM ARA into 4.74 mM 11R-HETE in 60 min, with a molar conversion yield of 95% a volumetric productivity of 79 μM min⁻¹ and a specific productivity of 7.9 μM min⁻¹ g⁻¹. To the best of our knowledge, this is the first report on the quantitative biotechnological production of 11R-HETE.     

Biosynthesis of the vitamin E compound delta-tocotrienol in recombinant Escherichia coli cells

The biosynthesis of natural products in a fast growing and easy to manipulate heterologous host system, such as Escherichia coli, is of increasing interest in biotechnology. This procedure allows the investigation of complex natural product biosynthesis and facilitates the engineering of pathways. Here we describe the cloning and the heterologous expression of tocochromanol (vitamin E) biosynthesis genes in E. coli. Tocochromanols are synthesized solely in photosynthetic organisms (cyanobacteria, algae, and higher green plants). For recombinant tocochromanol biosynthesis, the genes encoding hydroxyphenylpyruvate dioxygenase (hpd), geranylgeranylpyrophosphate synthase (crtE), geranylgeranylpyrophosphate reductase (ggh), homogentisate phytyltransferase (hpt), and tocopherol-cyclase (cyc) were cloned in a stepwise fashion and expressed in E. coli. Recombinant E. coli cells were cultivated and analyzed for tocochromanol compounds and their biosynthesis precursors. The expression of only hpd from Pseudomonas putida or crtE from Pantoea ananatis resulted in the accumulation of 336 mg L(-1) homogentisate and 84 microg L(-1) geranylgeranylpyrophosphate in E. coli cultures. Simultaneous expression of hpd, crtE, and hpt from Synechocystis sp. under the control of single tac-promoter resulted in the production of methyl-6-geranylgeranyl-benzoquinol (67.9 microg g(-1)). Additional expression of the tocopherol cyclase gene vte1 from Arabidopsis thaliana resulted in the novel formation of a vitamin E compound-delta-tocotrienol (15 microg g(-1))-in E. coli.     

Construction of a Chimeric Biosynthetic Pathway for the De Novo Biosynthesis of Rosmarinic Acid in Escherichia coli

Hydroxycinnamic acid esters (HCEs) are widely‐distributed phenylpropanoid‐derived plant natural products. Rosmarinic acid (RA), the most well‐known HCE, shows promise as a treatment for cancer and neurological disorders. In contrast to extraction from plant material or plant cell culture, microbial production of HCEs could be a sustainable, controlled means of production. Through the overexpression of a six‐enzyme chimeric bacterial and plant pathway, we show the de novo biosynthesis of RA, and the related HCE isorinic acid (IA), in Escherichia coli. Probing the pathway through precursor supplementation showed several potential pathway bottlenecks. We demonstrated HCE biosynthesis using three plant rosmarinic acid synthase (RAS) orthologues, which exhibited different levels of HCE biosynthesis but produced the same ratio of IA to RA. This work serves as a proof‐of‐concept for a microbial production platform for HCEs by using a modular biosynthetic approach to access diverse natural and non‐natural HCEs.     

利用改性载体固定化大肠杆菌产琥珀酸

采用聚乙烯亚胺（PEI）和戊二醛（GA）对棉纤维进行化学修饰,考察了载体改性后的性能和对固定化大肠杆菌产丁二酸的影响。改性后的载体菌体负载量提高了63.3%。培养基中葡萄糖浓度为43 g/L,添加改性棉纤维120g/L,以MgCO3为缓冲盐,进行批式发酵,丁二酸浓度达到29.6 g/L,比未改性棉纤维提高了11.3%;丁二酸收率达到70.5%,比改性前提高了7.5%;丁二酸生产速率达到0.66 g/(L?h), 比改性前提高了37.5% 。对该材料固载的细胞进行7次重复批式发酵,丁二酸产量、转化率和产率没有下降趋势,具有一定的重复稳定性。     

Biosynthesis of (R)-1,2-phenylethanediol and (R)-4-chloro-1,2-phenylethanediol by using two recombinant cells expressing enantiocomplementary epoxide hydrolases

Enantioconvergent biosynthesis of (R)-phenyl-1,2-ethanediol and (R)-4-chlorophenyl-1,2-ethanediol using two types of recombinant Escherichia coli cells expressing epoxide hydrolase of Caulobacter crescentus (CcEH) and a triple-point mutated EH of Mugil cephalus was investigated. The CcEH gene in pET28b(+) was expressed at 15 °C, and the reaction conditions such as biocatalyst ratio, reaction temperature and detergent addition were optimized to give best enantiopurity and yield. When two recombinant E. coli cells were used in sequential mode, (R)-phenyl-1,2-ethandiol with 90% enantiopurity and 95% yield was obtained in batch reaction. From 100 mM racemic 4-chlorostyrene oxide, (R)-4-chlorophenyl-1,2-ethanediol with 92% enantiopurity and 71% yield could be obtained.     

对甲苯磺酸催化乙二醇合成1，4-二氧六环

以对甲苯磺酸作催化剂，对乙二醇脱水制备1，4-二氧六环的反应进行研究。考察了催化剂用量、反应时间和反应温度等因素对收率的影响，得出最佳反应条件：催化剂用量为乙二醇质量的4.3%，反应时间50 min，收率可达84.82%。     

甲烷磺酸催化合成7-羟基-4-甲基香豆素

以甲烷磺酸为催化剂,乙酰乙酸乙酯和间苯二酚为原料,通过Pechmann缩合反应合成7-羟基-4-甲基香豆素。考察了原料配比、催化剂用量和反应温度对产品收率的影响,得到了较适宜的反应条件。在120 ℃,催化剂用量为间苯二酚物质的量的7.5%和n(乙酰乙酸乙酯)∶n(间苯二酚)=1.25∶1条件下,反应（9.5~10） min,产品平均收率为91.5%,并对产品进行了IR表征。     

微波辐射对甲苯磺酸催化合成对羟基苯甲酸乙二醇单酯

在微波辐射下,研究了以对甲苯磺酸为催化剂,对羟基苯甲酸和乙二醇为原料合成对羟基苯甲酸乙二醇单酯的工艺条件。用红外光谱分析和核磁共振光谱等方法对产物的结构进行表征,考察微波辐射功率、催化剂用量、酸醇物质的量比以及微波辐射时间对酯化率的影响。结果表明,合成对羟基苯甲酸乙二醇单酯的优化条件为：微波辐射功率500 W,酸醇物质的量比为1∶3.5,催化剂用量0.28 g,微波辐射时间20 min,此条件下,酯化率达92.1%。     

Recovery of alpha-ketoglutaric acid from model fermentation broth using electrodialysis with bipolar membrane

ABSTRACT

A bipolar (BP) membrane electrodialysis (EDBM) was used to recover the alpha-ketoglutaric acid (AKG) from the model broth. A two-chamber EDBM membrane stack consisting of an anion exchange membrane and a BP membrane was used. The effect of the initial composition, applied current density, and pH of diluate on the efficiency of EDBM processes was investigated. The obtained results showed that the used membrane stack configuration allows complete separation of AKG from glucose and ethanol and simultaneous conversion of AKG salts to the acidic form. The scale-up of the EDBM process for model fermentation broth was also carried out.     

Preparation of LiNiVO4 nano-powder using tartaric acid as a complexing agent

LiNiVO₄ was prepared from Li₂CO₃, Ni(CH₃COO)₂·4H₂O and NH₄VO₃ using tartaric acid as a complexing agent with 1:1–1:4 mole ratios of metals:tartaric acid and subsequent calcination at 350–700 °C for 6–12 h. Inverse spinel LiNiVO₄ was detected using XRD. FTIR and Raman analyses revealed the presence of stretching band of VO₄ tetrahedrons. Only Ni, V and O were detected by EDX. The 1:4 mole ratio for the product with 450 °C calcination for 6 h analyzed by SEM, TEM and electron diffraction (ED) composes of LiNiVO₄ nano-powder with 10–30 nm in diameter.     

重组大肠杆菌不对称还原2-羟基苯乙酮合成(R)-苯基乙二醇

通过对近平滑假丝酵母(R)-专一性羰基还原酶（rCR）进行氨基酸序列分析并根据其序列的保守性设计PCR引物,以近平滑假丝酵母基因组为模板利用PCR技术得到目的基因rcr。该基因全长1011bp,共编码336个氨基酸。将该基因与表达载体pET21c连接后构建重组质粒pETRCR,并转化入Escherichia coli BL21（DE3）中进行表达。重组大肠杆菌具有(R)-专一性羰基还原酶活性,可催化不对称还原2-羟基苯乙酮合成(R)-苯基乙二醇。研究发现,在重组菌培养过程中,同添加IPTG诱导培养的情况相比,不添加IPTG诱导培养的酶活及不对称转化(R)-苯基乙二醇的效果较好。在反应过程中,转化48 h及在pH值 8～9的条件下更有利于不对称反应的进行。另外,较高的底物浓度会对反应产生抑制作用,通过提高重组菌细胞浓度可显著提高转化效果。在5 g/L的底物浓度条件下,利用0.3 g/mL重组菌细胞可还原2-羟基苯乙酮得到(R)-苯基乙二醇,产物光学纯度为95.5% e.e,产率为92.6%。     

邻二甲苯-4-磺酸从猪血粉水解液中沉淀提取L-亮氨酸的工艺

考察了pH值、邻二甲苯–4–磺酸与L–亮氨酸的物质的量比、氯化铵浓度和温度等因素对邻二甲苯–4–磺酸反应沉淀L–亮氨酸的影响。结果表明：邻二甲苯–4–磺酸沉淀L-亮氨酸的适宜pH值为不高于1.5;邻二甲苯–4–磺酸与L-亮氨酸的最适物质的量比为3∶1;溶液中氯化铵浓度增大使L–亮氨酸的沉淀率缓慢下降;温度升高L–亮氨酸的沉淀率迅速降低,反应应于低温下进行,实际生产中可选择在0 ℃。按上述最佳工艺条件从除酸后猪血粉水解液中提取L–亮氨酸,沉淀率达到90.62%,经精制L-亮氨酸产品达到《中华人民共和国药典》(2000年版二部)的标准。     

Recovery of chlorine from dilute hydrochloric acid by electrolysis using a chlorine resistant anion exchange membrane

A new chlorine resistant anion exchange membrane enables innovative possibilities for hydrochloric acid electrolysis for recovery of chlorine. This is of interest for hydrochloric acid that is neutralized in the chemical industry because purity and concentration are not sufficiently high for recycling. In the common electrolysis process hydrochloric acid is fed into the anode compartment and needs a satisfactory HCl concentration for supplying the anode with chloride ions. Using an anion exchange membrane as a cell separator the feed flows into the cathode chamber where a low HCl concentration is acceptable because Cl⁻ ions at the anode can be supplied by addition of a salt which is not consumed. Experimental data of the membrane and the process are presented: membrane permselectivity improved up to above 97% using CaCl₂ as added salt, chlorine current efficiency up to 98% and oxygen content as low as 0.5 vol%, cell voltage at 4 kA m⁻² 2.3 V, equivalent to 1740 kWh per t produced chlorine, even at low HCl concentrations. Thus, the power consumption is comparable with the common process. A problem of the new process is the high water transport through the membrane. Therefore, experiments for two process alternatives were carried out. Disadvantages of water transport can be avoided by using a high concentrated CaCl₂ solution as anolyte and catholyte and as absorption medium for diluted HCl gas streams. Additionally, a cell design was investigated where the anode is directly connected to the membrane in an empty (gas filled) anode compartment.     

应用KMnO_4滴定法快速检测米根霉发酵液中富马酸含量

应用KMnO4滴定法快速检测米根霉发酵液中富马酸含量,通过研究富马酸滴定体系下的酸度以及发酵培养基中的葡萄糖和代谢副产物如乳酸、苹果酸、乙醇、草酰乙酸、丁二酸等对KMnO4滴定结果的影响,发现应用KMnO4滴定法测定米根霉发酵液中富马酸的含量,与HPLC检测法测定的含量误差在3%以内,平均回收率为96.86%,相对标准偏差(RSD)为0.27%.以上结论表明,KMnO4滴定法可有效应用于快速检测米根霉发酵液中富马酸的含量.