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.     

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.     

High‐level extracellular production of penicillin acylase by genetic engineering of Escherichia coli

The extracellular production of penicillin acylase (PAC) in genetically engineered Escherichia coli by coexpression of the brp gene encoding bacteriocin release protein (BRP) and the pac gene was demonstrated. Cell physiology was affected while PAC was released into the medium, depending on the strategy for brp expression. The performance for the production and release of PAC was optimized by taking several culture parameters, including host, inducer (mitomycin C) concentration, and induction timing for brp expression, into consideration. The effect of PAC release on inclusion body formation was also investigated. It was observed that the amount of inclusion bodies was significantly affected by brp expression. A reason for the limitation of PAC production and a strategy for resolving this problem are proposed. © 2001 Society of Chemical Industry     

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.     

Metabolic strategies for microbial glycerol overproduction

Glycerol as a platform chemical has wide applications in chemical, pharmaceutical, and food industries and is a useful feedstock for the production of bio‐based chemicals. There is an increasing demand for sustainable glycerol production and microbial glycerol production is a promising alternative. The paper highlighted this new trend and summarized recent research progress on microbial glycerol production, and focused mainly on the efforts achieved in rational design of S. cerevisiae, as well as Escherichia coli, to overcome the limitations of glycerol production in these two microorganisms. More efforts are required for overcoming hurdles in yield and productivity enhancement for large scale production. © 2017 Society of Chemical Industry     

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。     

代谢工程改造大肠杆菌生产琥珀酸

琥珀酸（succinic acid）是一种四碳二羧酸,在食品、医药、塑料和化工行业具有广泛的应用。目前,微生物法生产琥珀酸存在得率低、生产强度低、副产物积累等问题。为此,本研究通过复合诱变（ARTP和⁶⁰Co-γ射线）筛选到一株耐高渗突变株FMME-N-2,其琥珀酸得率为0.70g/g葡萄糖,同时积累18.8g/L乳酸、7.6g/L甲酸和17.3g/L乙酸。为了提高琥珀酸得率,通过敲除乳酸脱氢酶基因（ldhA）、丙酮酸-甲酸裂解酶-甲酸转运蛋白基因（pflB-focA）、磷酸转乙酰基基因（pta）、丙酸激酶基因（tdcD）和a-酮丁酸甲酸酯裂解酶基因（tdcE）,阻断冗余代谢支路减少副产物积累,获得工程菌株FMME-N-13,琥珀酸得率增加到0.92g/g葡萄糖,同时副产物大大降低,积累0.6g/L乳酸、3.6g/L甲酸和12.3g/L乙酸。同时,通过调控RBS强度组合优化来自产琥珀酸放线杆菌的磷酸烯醇式丙酮酸羧激酶基因（AsPCK）和来自博伊丁假丝酵母的甲酸脱氢酶基因（CbFDH）的表达水平,调控胞内ATP和NADH的浓度,最优工程菌FMME-N-26（FMME-N-13-L-AsPCK-L-CbFDH）的琥珀酸得率增加至1.04g/g葡萄糖,仅积累5.5g/L乙酸;最终,对厌氧阶段葡萄糖浓度进行优化,当葡萄糖浓度控制在0~5g/L时,菌株FMME-N-26的琥珀酸浓度增加到111.9g/L,得率为1.11g/g葡萄糖（理论产率的99%）,生产强度为1.76g/L/h,为琥珀酸的工业化生产奠定了良好的基础。     

Optimization of 1,3‐propanediol production by novel recombinant Escherichia coli using response surface methodology

Response surface methodology was used to optimize 1,3-propanediol production by a novel recombinant Escherichia coli JM109 (pHsh-dhaB-yqhD). The optimal fermentation parameters for enhanced 1,3-propanediol yield were found to be: glycerol 61.8 g L⁻¹, yeast extract 6.2 g L⁻¹, Vitamin B₁₂ 0.049 g L⁻¹ and fermentation time 30 h. Subsequent experimental trials confirmed the validity of the model. These optimal fermentation conditions in the cultivation flask culture led to a 1,3-propanediol concentration of 43.1 g L⁻¹ and a conversion rate of 69.7% (g g⁻¹). A maximum 1,3-propanediol concentration of 41.1 g L⁻¹ was achieved in a 5 L fermenter using the optimized parameters. Copyright © 2006 Society of Chemical Industry     

Metabolic engineering of 2‐pentanone synthesis in Escherichia coli

Expanding the chemical diversity of microbial fermentation products enables green production of fuel, chemicals, and pharmaceuticals. In recent years, coenzyme A (CoA) dependent chain elongation, resembling the reversed β‐oxidation pathway, has attracted interest for its use in producing higher alcohols, fatty acids, and polyhydroxyalkanoate. To expand the chemical diversity of this pathway, we metabolically engineered Escherichia coli to produce 2‐pentanone, which is not a natural fermentation product of E. coli. We describe the first demonstration of 2‐pentanone synthesis in E. coli by coupling the CoA‐dependent chain elongation with the acetone production pathway. By bioprospecting for enzymes capable of efficient hydrolysis of 3‐keto‐hexanoyl‐CoA, production of 2‐pentanone increased 20 fold, reaching a titer of 240 mg/L. © 2013 American Institute of Chemical Engineers AIChE J, 59: 3167–3175, 2013     

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.     

产高纯度D-乳酸大杨杆菌的构建及发酵

以可利用蔗糖的Escherichia coli W为出发菌株,敲除产生副产物的相关基因(adhE, frdBC, pta, pflB, aldA),为促进蔗糖利用,还敲除蔗糖启动子的抑制基因(cscR),构建了D-乳酸工程菌WD 206. 结果表明,该菌经72 h发酵可有效将100 g/L蔗糖转化生成88.15 g/L乳酸,产率为84%,占代谢产物的99.5%, D-乳酸的光学纯度达99%.     

The isolation of poly(3‐hydroxybutyrate) from recombinant Escherichia coli XL1‐blue using the digestion method

This study examines the isolation of poly(3‐hydroxybutyrate) (PHB) from recombinant Escherichia coli XL1‐blue pBHB2 OLD‐2 harbouring the PHB biosynthesis gene. Six types of commercial surfactants (Emal 10P, Emal AD‐25, Emal S PASTE, Neopelex S‐S, Triton X‐100 and cetyl trimethyl ammonium bromide (CTAB)) were screened for PHB isolation by solubilising non‐polymer cellular material (NPCM) in the cell. Emal 10P, Emal AD‐25, Emal S PASTE and Triton X‐100 are suitable surfactants for PHB isolation. Factors such as the reaction temperature, reaction time, ratio of NPCM/surfactant and pH were investigated to achieve optimum conditions. For 80% of the PHB content in dry cells, the purity and recovery of the obtained PHB was 98% and 99% when 0.67 of NPCM/Emal S PASTE was used at 70°C for 30 min. The cost of the used surfactant is below 0.5 USD/kg (PHB). The pretreatment and multistage digestion method must be combined when the PHB content is lower than 80%. © 2011 Canadian Society for Chemical Engineering