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。     

1,2,4-丁三醇的双水相萃取

对低分子有机溶剂/无机盐双水相体系萃取分离发酵液中1,2,4-丁三醇（1,2,4-butanetriol,BT）进行了深入研究。通过对不同双水相体系的筛选,最终选定无水乙醇/K₂HPO₄双水相体系来萃取分离BT。使用浊点法对以BT为溶剂的无水乙醇/K₂HPO₄双水相体系进行相图的绘制,发现在K₂HPO₄质量分数为19.83%~46.87%范围内均能成相。通过单因素实验,考察双水相体系中无水乙醇/K₂HPO₄质量分数、pH对BT在两相之间分配系数和萃取效率的影响,得到最佳萃取条件为：系统总量10g、pH 9.5,无水乙醇/K₂HPO₄的质量分数为28%/28%,分配系数和萃取效率分别可以达到18.35和95.87%。在最佳萃取条件下,进一步探究了放大实验对体系萃取效率的影响,发现其对分配系数和萃取效率影响较小,体系稳定性高,为工业提取发酵液中BT提供新思路。     

1,2,4-丁三醇合成工艺述评

介绍了 1 ,2 ,4 丁三醇的基本性能、可能的用途及各种合成工艺路线 ,并评价了各种工艺路线的优劣。     

1,2,4—丁三醇合成工艺述

介绍了１,２,４－丁三醇的基本性能、可能的用途及各种合成工艺路线,并评价了各种工艺路线的优劣。     

微反应器合成1,2,4-丁三醇三硝酸酯的工艺研究

以1,2,4-丁三醇(BT)水溶液为起始原料,发烟硝酸/浓硫酸为硝化剂,采用微反应器连续合成1,2,4-丁三醇三硝酸酯,考察反应温度、物料及混酸比例、停留时间对反应影响。结果表明,优化工艺参数为：1,2,4-丁三醇∶发烟硝酸∶浓硫酸的摩尔比为1∶5∶4,反应温度15℃,停留时间48 s,工艺收率90.5%,产品含量98.8%。与传统间歇釜式合成工艺相比,该工艺实现了1,2,4-丁三醇三硝酸酯合成反应的稳定进行,反应温度接近室温,消除了过程反应热的积累和温度波动,缩短反应时间。     

手性(S)-1,2,4-丁三醇的制备与表征

介绍以L-苹果酸为原料,经甲酯化、还原反应合成了(S)-1,2,4-丁三醇。运用旋光度测试、红外光谱以及核磁共振谱对其结构进行表征,证实目标产物的结构。通过改进分离方法,简化操作过程,使其向工业化迈进了一步。     

甘油/1,2,4-丁三醇混合醇连续喷雾硝化技术研究

报道使用硝硫混酸与甘油／１，２，４－丁三醇连续喷雾硝化制备硝化甘油／１，２，４－丁三醇三硝酸酯的方法和工艺条件。     

硝化甘油和1,2,4-丁三醇三硝酸酯的高效液相色谱分析

提出了甘油和1,2,4-丁三醇混合硝化产物中硝化甘油和1,2,4-丁三醇三硝酸酯的高效液相色谱分析法。用甲醇/水(50/50)作流动相,在MicropakMCH-5柱上进行分离,外标法定量,相对标准偏差小于2％。     

1,2,4-三嗪类化合物的合成

本文合成了3-巯基-5-羟基-6-甲基-1,2,4-三嗪和3-巯基-5,6-二甲基-1,2,4-三嗪,通过熔点和红外光谱确认了这些化合物。     

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.     

产1,3-丙二醇新型重组大肠杆菌JM109(pHsh-dhaB-yqhD)的构建及其转化甘油

利用温控载体pHsh将编码甘油脱水酶的基因dhaB和编码1,3-丙二醇氧化还原酶同工酶的基因yqhD串联构建重组质粒pHsh-dhaB-yqhD, 将其转化大肠杆菌得到新型产1,3-丙二醇重组大肠杆菌JM109(pHsh-dhaB-yqhD), 并对影响该重组菌发酵的营养因子进行研究.实验结果表明：该重组菌转化甘油合成1,3-丙二醇的适宜培养基组成为甘油60 g·L^-1、酵母膏5.0 g·L^-1、维生素B₁₂ 0.05 g·L^-1以及KH₂PO₄ 7.5 g·L^-1; 以此培养基在5 L发酵罐上进行放大, 1,3-丙二醇产量、转化率和生产能力分别达到43.26 g·L^-1、72.2 %和1.55 g·L^-1·h^-1.     

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。     

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...     

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%。     

Enhanced recombinant protein synthesis in batch and fed‐batch Escherichia coli fermentation based on removal of inhibitory acetate by electrodialysis

BACKGROUND: The formation of acetate as a metabolic by‐product in Escherichia coli fermentation is well known to have detrimental effects on cell growth and productivity. Various bioprocess and genetic approaches have previously been made to limit acetate accumulation, however, they tend to be conservative, limiting overall process productivity, or lead to other problems such as a decrease in maximum specific growth rate and decreased product yield on carbon. RESULTS: In this work, the utility of electrodialysis is examined as a potentially generic approach for in situ acetate removal and its impact on recombinant protein production. Using the induced synthesis of recombinant green fluorescent protein (GFP) in E. coli Tg1 (pGLO) as an example, it is shown that in situ removal of acetate to below inhibitory levels (～1 g L^?1) provides significant improvements in cell growth rate as well as specific biomass and recombinant protein yields. Experiments were performed in a 7.5 L stirred‐tank bioreactor using an external single cell‐pair electrodialysis module with an effective ion exchange membrane area of 0.01 m 2 . For this system increases in specific recombinant protein yield of up to 4‐fold have been observed dependent upon the time of induction, the mode of operation and the level to which acetate concentration is reduced in the fermentation broth. CONCLUSIONS: The implementation of ED can significantly increase the level of recombinant protein synthesis in batch and fed‐batch fermentation. The approach is considered to be generic, readily implemented and has wide application for the production of recombinant enzymes and proteins. Copyright © 2009 Society of Chemical Industry     

溶氧探测补料技术发酵生产类人胶原蛋白的研究

在重组大肠杆菌发酵生产类人胶原蛋白(HLC)过程中,快速确定不同反应器中的补料速率,抑制乙酸产生,高水平表达HLC.采用溶氧探测补料技术,在不同规模反应器中,分批-补料培养重组大肠杆菌生产HLC,根据脉冲补料方式时溶氧信号的响应特征来辩识是否产生乙酸并确定补料速率.在实验室规模获得的最终细胞密度和HLC质量浓度分别为6...