理性设计和构建过量合成莽草酸的大肠杆菌代谢工程菌

利用代谢工程手段理性改造野生大肠杆菌的莽草酸(Shikimic acid,SA)合成途径及相关代谢节点,以构建高产莽草酸的工程菌株.根据细胞代谢网络分析,利用Red-Xer重组系统连续删除了野生型大肠杆菌CICIMB0013的莽草酸激酶基因(aroL、aroK),葡萄糖磷酸转移酶系统(PTS)的关键组分EIICBglc的编码基因(ptsG)以及奎宁酸/莽草酸脱氢酶基因(ydiB)并系统评价了基因删除对细胞的生长、葡萄糖代谢和莽草酸积累的影响.aroL、aroK的删除阻断了莽草酸进一步转化成为莽草酸-3-磷酸,初步提高莽草酸的累积.删除ptsG基因使大肠杆菌PTS系统部分缺失,细胞通过GalP-glk(半乳糖透性酶-葡萄糖激酶)途径,利用ATP将葡萄糖磷酸化后进入细胞.利用该途径运输葡萄糖能够减少PEP的消耗,使得更多的碳代谢流进入莽草酸合成途径,从而显著提高了莽草酸的产量.在此基础上删除ydiB基因,阻止了莽草酸合成的前体物质3-脱氢奎宁酸转化为副产物奎宁酸(Quinic acid,QA),进一步提高了莽草酸的累积.初步发酵显示4个基因缺失的大肠杆菌代谢工程菌生产莽草酸的能力比原始菌提高了90多倍.     

利用代谢工程改善大肠杆菌的3-脱氢莽草酸生产

3-脱氢莽草酸是芳香族氨基酸合成代谢途径中的一种重要中间产物。除可作为一种高效的抗氧化剂,还可用于合成己二酸、香草醛等一些重要的化工产品,具有重要的应用价值。相关研究证明具有去酪氨酸反馈抑制的3-脱氧-D-阿拉伯庚酮糖-7-磷酸合成酶基因aroFFBR以及转酮醇酶基因tktA可以有效影响3-脱氢莽草酸的过量合成。通过增加aroFFBR和tktA串联过量表达的拷贝数,可使工程菌株在摇瓶发酵条件下3-脱氢莽草酸产量提高2.93倍。通过同源重组无痕基因敲除技术依次敲除出发菌大肠杆菌Escherichia coli AB2834的乳酸、乙酸、乙醇等副产物合成途径中的重要基因ldhA、ackA-pta和adhE,可使工程菌株的3-脱氢莽草酸产量进一步提高,达到了1.83 g/L,是初始出发菌株大肠杆菌E.coli AB2834产量的6.7倍。利用5 L发酵罐进行分批补料发酵,62 h后工程菌株3-脱氢莽草酸产量达到了25.48 g/L。本研究可为构建有应用前景的3-脱氢莽草酸生产菌株提供重要参考。     

莽草酸生物合成途径的调控

莽草酸是合成生物碱等许多其它物质的前体.近年来,莽草酸作为临床上惟一有效的抗禽流感药物--达啡的原料而倍受关注.简述了莽草酸生物合成的途径,着重从基因工程角度分析了莽草酸合成过程中的关键酶及其对莽草酸产量的影响,旨在为研究和开发微生物工程菌生产莽草酸提供参考.     

微生物发酵生产莽草酸及其树脂分离性能的探讨

莽草酸是一种具有较高药用价值的化合物,在医药、食品、工业等领域具有重要作用。莽草酸的生产方法很多,包括植物提取法、化学合成法、微生物发酵法等,从植物中提取时使用中草药植物八角茴香,化学合成时以环戊二烯和苯醌为原料,微生物发酵生产时以大肠杆菌、枯草芽孢杆菌等微生物为工程菌。本文就莽草酸的微生物发酵生产方法做简单介绍,阐述了莽草酸树脂分离性能等的研究,并对莽草酸未来发展应用做出展望。     

改进莽草酸合成代谢的大肠杆菌工程化研究新进展

莽草酸是芳香族氨基酸合成中的重要中间产物,具有广泛的药用价值,是抗流感药物"达菲"的重要合成前体。微生物发酵生产莽草酸具有许多优点,其中大肠杆菌常用于微生物大规模发酵生产应用。通过对大肠杆菌进行代谢工程改造,是构建工业化莽草酸高产菌的主要技术手段。磷酸烯醇式丙酮酸-糖磷酸转移酶系统(phosphoenolpyruvate:carbohydrate phosphotransferase system,PTS)是大肠杆菌细胞内参与葡萄糖从膜间质转运和磷酸化到胞内的主要活性转运系统,影响莽草酸合成前体磷酸烯醇式丙酮酸(PEP)的利用率。通过对PTS系统的定向修饰和改造,调节细胞内代谢流向,提高碳源利用率,增加莽草酸前体合成量,结合对代谢途径中的特定修饰,能够构建出较为理想的莽草酸高产菌。研究显示在10 L放大体系中最佳产率可达0.36 mol/mol,莽草酸浓度可达84 g/L。本文针对代谢改造中莽草酸途径和葡萄糖转运系统的改造方面进行简单概述,并综述了近年来有关此方面的最新研究进展。     

微生物代谢合成法在莽草酸及其衍生物研究中的应用

天然产物莽草酸(Shikimic acid)具有抗肿瘤、抗血栓等药理活性,在生物及化学合成中扮演着重要角色,而倍受关注.本文综述了近年来国内外有关微生物代谢合成法在莽草酸及其衍生物研究中的应用,为莽草酸的开发利用提供参考.     

使用动态分子开关调控大肠杆菌生产莽草酸

莽草酸是大肠杆菌合成芳香族氨基酸的中间代谢物,也是抗流感药物"达菲"的重要合成前体。合成莽草酸需要截断莽草酸途径,导致芳香族氨基酸无法合成,因此面临细胞生长受到抑制的问题。使用动态调控策略通过将细胞生长和莽草酸的合成相互分离,可以提高菌株的生产性能。通过使用生长偶联型启动子和降解决定子(Degrons),组建动态分子开关。利用该动态分子开关实现细胞生长与莽草酸合成分离,在5L发酵罐中经过72h发酵得到了14.33g/L的莽草酸。结果表明,这种动态分子开关可以通过调控靶蛋白丰度来改变碳流量平衡,使菌株获得更优秀的生产性能。     

奎尼酸生物合成的代谢工程

奎尼酸及其衍生物氢醌和苯醌等是一类重要的化工原料,可作为一些化学合成制剂和药物中间原料,且在食品和化学工业中有着广泛的应用。目前奎尼酸的制备方法有植物提取法、化学合成法、酶工程法和微生物发酵法,其中微生物发酵法是近年发展起来的一种十分经济有效的方法。在介绍奎尼酸的制备方法的基础上重点综述了应用代谢工程在生物合成奎尼酸基因工程菌的改造中的研究进展,其中涉及奎尼酸生物合成途径中相关基因及其酶的调控、中心代谢途径的改造和修饰等,并探讨了将来的发展前景。     

芳香族香料化合物生物合成研究进展

芳香族化合物在香料中占很大的比重,传统生产方式有化学合成和植物提取。化学合成依赖于石油资源,并具有环境不友好、反应条件恶劣等缺点。植物提取方法受限于植物资源,且占用耕地。近年来,随着代谢工程和合成生物学技术的发展,利用可再生原料,微生物合成芳香族香料化合物成为一种新的生产方式。文中介绍了大肠杆菌和酵母菌等模式微生物合成芳香族香料的研究进展,包括利用莽草酸途径合成香兰素等,聚酮途径合成覆盆子酮等。综述重点介绍了生物合成途径解析、人工合成途径创建及代谢调控等,为微生物发酵法生产芳香族香料化合物提供参考。     

雪松松针中莽草酸的纯化方法研究

为建立莽草酸的纯化方法,以莽草酸的含量为指标,对雪松松针水提液的醇沉、大孔吸附树脂单用或与不同柱层析合用的纯化方法进行了探索。最终确定的莽草酸的纯化工艺流程为:提取-醇沉-柱层析-重结晶,其中醇沉法可以将莽草酸的含量由12.69%提升到20.08%,大孔吸附树脂柱层析法单用可以将莽草酸的含量由20.08%提升到35.73%,回收率均超过95%;大孔吸附树脂柱结合硅胶柱层析法可得到纯度为80.05%的莽草酸近白色固体,回收率达60.04%,再经二氯甲烷和甲醇重结晶得到纯度为96.54%的莽草酸白色粉末,表明该纯化方法简单可行,重复性好,易于实施工业化生产。     

L-苯丙氨酸生产的代谢工程研究

L-苯丙氨酸是一种重要的食品和医药中间体。工业上一般采用酶法和发酵法来生产L-苯丙氨酸。代谢工程的兴起,使得更加理性的改造菌株成为可能,这更加促进了发酵法的广泛应用。主要介绍了代谢工程在L-苯丙氨酸生产菌的改造中的应用情况,其中涉及苯丙氨酸生物合成途径中相关基因及其酶的调控、中央代谢途径的改造和芳香族氨基酸生物合成支路的修饰。并探讨了将来的发展前景。     

Protocatechuate overproduction by Corynebacterium glutamicum via simultaneous engineering of native and heterologous biosynthetic pathways

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is a natural bioactive phenolic acid potentially valuable as a pharmaceutical raw material owing to its diverse pharmacological activities. Corynebacterium glutamicum forms PCA as a key intermediate in a native pathway to assimilate shikimate/quinate through direct conversion of the shikimate pathway intermediate 3-dehydroshikimate (DHS), which is catalyzed by qsuB-encoded DHS dehydratase (the DHS pathway). PCA can also be formed via an alternate pathway extending from chorismate by introducing heterologous chorismate pyruvate lyase that converts chorismate into 4-hydroxybenzoate (4-HBA), which is then converted into PCA catalyzed by endogenous 4-HBA 3-hydroxylase (the 4-HBA pathway). In this study, we generated three plasmid-free C. glutamicum strains overproducing PCA based on the markerless chromosomal recombination by engineering each or both of the above mentioned two PCA-biosynthetic pathways combined with engineering of the host metabolism to enhance the shikimate pathway flux and to block PCA consumption. Aerobic growth-arrested cell reactions were performed using the resulting engineered strains, which revealed that strains dependent on either the DHS or 4-HBA pathway as the sole PCA-biosynthetic route produced 43.8 and 26.2 g/L of PCA from glucose with a yield of 35.3% and 10.0% (mol/mol), respectively, indicating that PCA production through the DHS pathway is significantly efficient compared to that produced through the 4-HBA pathway. Remarkably, a strain simultaneously using both DHS and 4-HBA pathways achieved the highest reported PCA productivity of 82.7 g/L with a yield of 32.8% (mol/mol) from glucose in growth-arrested cell reaction. These results indicated that simultaneous engineering of both DHS and 4-HBA pathways is an efficient method for PCA production. The generated PCA-overproducing strain is plasmid-free and does not require supplementation of aromatic amino acids and vitamins due to the intact shikimate pathway, thereby representing a promising platform for the industrial bioproduction of PCA and derived chemicals from renewable sugars.     

Metabolic engineering of edible plant oils]

Plant seed oil is the major source of many fatty acids for human nutrition, and also one of industrial feedstocks. Recent advances in understanding of the basic biochemistry of seed oil biosynthesis, coupled with cloning of the genes encoding the enzymes involved in fatty acid modification and oil accumulation, have set the stage for the metabolic engineering of oilseed crops that produce "designer" plant seed oils with the improved nutritional values for human being. In this review we provide an overview of seed oil biosynthesis/regulation and highlight the key enzymatic steps that are targets for gene manipulation. The strategies of metabolic engineering of fatty acids in oilseeds, including overexpression or suppression of genes encoding single or multi-step biosynthetic pathways and assembling the complete pathway for the synthesis of long-chain polyunsaturated fatty acids (e.g. arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid) are described in detail. The current "bottlenecks" in using common oilseeds as "bioreactors" for commercial production of high-value fatty acids are analyzed. It is also discussed that the future research focuses of oilseed metabolic engineering and the prospects in creating renewable sources and promoting the sustainable development of human society and economy.     

Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli.

3-Dehydroshikimic acid (DHS), in addition to being a potent antioxidant, is the key hydroaromatic intermediate in the biocatalytic conversion of glucose into aromatic bioproducts and a variety of industrial chemicals. Microbial synthesis of DHS, like other intermediates in the common pathway of aromatic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Escherichia coli constructs is examined in a fed-batch fermentor where glucose availability, oxygenation levels, and solution pH are controlled. DHS yields and titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. This enzyme's expression levels, sensitivity to feedback inhibition, and the availability of its substrates, phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), dictate its in vivo activity. By combining fed-batch fermentor control with amplified expression of a feedback-insensitive isozyme of DAHP synthase and amplified expression of transketolase, DHS titers of 69 g/L were synthesized in 30% yield (mol/mol) from D-glucose. Significant concentrations of 3-dehydroquinic acid (6.8 g/L) and gallic acid (6.6 g/L) were synthesized in addition to DHS. The pronounced impact of transketolase overexpression, which increases E4P availability, on DHS titers and yields indicates that PEP availability is not a limiting factor under the fed-batch fermentor conditions employed.     

微生物发酵法生产L-色氨酸的代谢工程研究

L-色氨酸作为人体内的一种必需氨基酸,广泛应用于医药、食品与饲料等行业.工业上采用的色氨酸生产方法有化学合成法、转化法及微生物发酵法.近年来,随着代谢工程在色氨酸菌种选育中的成功运用,微生物发酵法逐渐成为主要的色氨酸生产方法.系统综述了微生物发酵法生产色氨酸所涉及的代谢工程策略,包括生物合成色氨酸的代谢调控机制以及途径...     

Strategies for enhancing fermentative production of acetoin: A review

Acetoin is a volatile compound widely used in foods, cigarettes, cosmetics, detergents, chemical synthesis, plant growth promoters and biological pest controls. It works largely as flavour and fragrance. Since some bacteria were found to be capable of vigorous acetoin biosynthesis from versatile renewable biomass, acetoin, like its reduced form 2,3-butanediol, was also classified as a promising bio-based platform chemical. In spite of several reviews on the biological production of 2,3-butanediol, little has concentrated on acetoin. The two analogous compounds are present in the same acetoin (or 2,3-butanediol) pathway, but their production processes including optimal strains, substrates, derivatives, process controls and product recovery methods are quite different. In this review, the usages of acetoin are reviewed firstly to demonstrate its importance. The biosynthesis pathway and molecular regulation mechanisms are then outlined to depict the principal network of functioning in typical species. A phylogenetic tree is constructed and the relationship between taxonomy and acetoin producing ability is revealed for the first time, which will serve as a useful guide for the screening of competitive acetoin producers. Genetic engineering, medium optimization, and process control are effective strategies to improve productivity as well. Currently, downstream processing is one of the main barriers in efficient and economical industrial acetoin fermentation. The future prospects of microbial acetoin production are discussed in light of the current progress, challenges, and trends in this field.     

Metabolic engineering for higher alcohol production

Engineering microbial hosts for the production of higher alcohols looks to combine the benefits of renewable biological production with the useful chemical properties of larger alcohols. In this review we outline the array of metabolic engineering strategies employed for the efficient diversion of carbon flux from native biosynthetic pathways to the overproduction of a target alcohol. Strategies for pathway design from amino acid biosynthesis through 2-keto acids, from isoprenoid biosynthesis through pyrophosphate intermediates, from fatty acid biosynthesis and degradation by tailoring chain length specificity, and the use and expansion of natural solvent production pathways will be covered.     

Modular pathway engineering of key carbon‐precursor supply‐pathways for improved N‐acetylneuraminic acid production in Bacillus subtilis

N‐acetylneuraminic acid (NeuAc) is widely used as a nutraceutical for facilitating infant brain development, maintaining brain health, and enhancing immunity. Currently, NeuAc is mainly produced by extraction from egg yolk and milk, or via chemical synthesis. However, its low concentration in natural resources and its non‐ecofriendly chemical synthesis result in insufficient NeuAc production and environmental pollution, respectively. In this study, improved NeuAc production was attained via modular pathway engineering of the supply pathways of two key precursors—N‐acetylglucosamine (GlcNAc) and phosphoenolpyruvate (PEP)—and by balancing NeuAc biosynthesis and cell growth in engineered Bacillus subtilis. Specifically, we used a previously constructed GlcNAc‐producing B. subtilis as the initial host for NeuAc biosynthesis. First, we constructed a de novo NeuAc biosynthetic pathway utilizing glucose by coexpressing glucosamine‐6‐phosphate acetyl‐transferase (GNA1), N‐acetylglucosamine 2‐epimerase (AGE), and N‐acetylneuraminic acid synthase (NeuB), resulting in 0.33 g/l NeuAc production. Next, to balance the supply of the two key precursors for NeuAc biosynthesis, modular pathway engineering was performed. The optimal strategy for balancing the GlcNAc module and PEP supply module involved the use of an engineered, unique glucose and malate coutilization pathway in B. subtilis, supplied with both glucose (for the GlcNAc moiety) and malate (for the PEP moiety) at high strength. This led to 1.65 g/L NeuAc production, representing a 5.0‐fold improvement over the existing methods. Furthermore, to enhance the NeuAc yield on cell, glucose and malate coutilization pathways were engineered to balance NeuAc biosynthesis and cell growth via the blocking of glycolysis, the introduction of the Entner–Doudoroff pathway, and the overexpression of the malic enzyme YtsJ. NeuAc titer reached 2.18 g/L, with 0.38 g/g dry cell weight NeuAc yield on cell, which represented a 1.32‐fold and 2.64‐fold improvement over the existing methods, respectively. The strategy of modular pathway engineering of key carbon precursor supply pathways via engineering of the unique glucose‐malate coutilization pathway in B. subtilis should be generically applicable for engineering of B. subtilis for the production of other important biomolecules. Our study also provides a good starting point for further metabolic engineering to achieve industrial production of NeuAc by a Generally Regarded As Safe bacterial strain.     

A Novel Muconic Acid Biosynthesis Approach by Shunting Tryptophan Biosynthesis via Anthranilate

Muconic acid is the synthetic precursor of adipic acid, and the latter is an important platform chemical that can be used for the production of nylon-6,6 and polyurethane. Currently, the production of adipic acid relies mainly on chemical processes utilizing petrochemicals, such as benzene, which are generally considered environmentally unfriendly and nonrenewable, as starting materials. Microbial synthesis from renewable carbon sources provides a promising alternative under the circumstance of petroleum depletion and environment deterioration. Here we devised a novel artificial pathway in Escherichia coli for the biosynthesis of muconic acid, in which anthranilate, the first intermediate in the tryptophan biosynthetic branch, was converted to catechol and muconic acid by anthranilate 1,2-dioxygenase (ADO) and catechol 1,2-dioxygenase (CDO), sequentially and respectively. First, screening for efficient ADO and CDO from different microbial species enabled the production of gram-per-liter level muconic acid from supplemented anthranilate in 5 h. To further achieve the biosynthesis of muconic acid from simple carbon sources, anthranilate overproducers were constructed by overexpressing the key enzymes in the shikimate pathway and blocking tryptophan biosynthesis. In addition, we found that introduction of a strengthened glutamine regeneration system by overexpressing glutamine synthase significantly improved anthranilate production. Finally, the engineered E. coli strain carrying the full pathway produced 389.96 ± 12.46 mg/liter muconic acid from simple carbon sources in shake flask experiments, a result which demonstrates scale-up potential for microbial production of muconic acid.