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。     

大肠杆菌PTS系统改造及重组菌生长性能测定

利用Red重组系统对野生大肠杆菌Escherichia coli磷酸烯醇式丙酮酸-糖磷酸转移酶系统(Phosphoenolpyruvate:carbohydrate phosphotransferase system,PTS)进行修饰改造,敲除PTS系统中关键组分EⅡCBGlc的编码基因(ptsG),磷酸组氨酸搬运蛋白HPr的编码基因(ptsI),同时敲入来源于运动发酵单胞菌Zymomonas mobilis的葡萄糖易化体(Glucose facilitator)编码基因(glf),构建重组大肠杆菌,比较测定并系统评价了基因敲除和敲入对细胞的生长、葡萄糖代谢和乙酸积累的影响。敲除基因ptsG和ptsI造成大肠杆菌PTS系统部分功能缺失,细胞生长受到一定限制,敲入glf基因后,重组大肠杆菌能够利用Glf-Glk(葡萄糖易化体-葡萄糖激酶)途径,消耗ATP将葡萄糖进行磷酸化并转运进入细胞。通过该途径转运葡萄糖能够提高葡萄糖利用效率,降低副产物乙酸生成,同时能够使更多的碳代谢流进入后续相关合成途径,预期能够提高相关产物产量。     

基于PTS缺陷型大肠杆菌构建莽草酸生产菌

对大肠杆菌芳香族氨基酸合成途径进行代谢流改造, 以实现高效的生物制备莽草酸。以磷酸烯醇式丙酮酸-糖磷酸转移酶系统(PTS系统)敲除菌DH5α△ptsHIcrr (DHP)为基础, 特异性敲除aroL、ydiB基因并转入受阿拉伯糖诱导表达的T7-RNA聚合酶基因, 最终构建一系列产莽草酸宿主菌。再将aroE、aroB、tktA、glk、aroFfbr组成的系列基因串联起来置于质粒上, 在T7启动子控制下表达, 经摇瓶培养检测得知, 不同重组菌产莽草酸能力与对照相比均有明显提高, 其中DHPYA-T7/pAOC-TGEFB菌株产量最高, 可达到392 mg/L。为进一步构建高表达莽草酸工程菌奠定基础。     

3-脱氢莽草酸生物合成的代谢工程

3-脱氢莽草酸,是芳香族氨基酸生物合成代谢途径中一种重要的中间产物,可作为一些化学合成制剂和药物中间原料。这样以无毒可再生物质为起始原料的合成方法与传统的有机合成化学制剂的方法相比,对环境更加有利。此外,它还是一种十分有效的抗氧化剂。工业上一般采用化学合成法和发酵法来生产3-脱氢莽草酸,随着代谢工程的兴起,使得更加理性改造菌株成为可能,这更加促进了发酵法的广泛应用。本文主要介绍了代谢工程在生物合成3-脱氢莽草酸生产菌改造中的应用情况,其中涉及3-脱氢莽草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3-脱氢莽草酸合成支路的修饰等,并探讨了将来的发展前景。     

途径工程改造谷氨酸棒杆菌产莽草酸

莽草酸是一种芳香族中间代谢产物,也是合成抗禽流感药物磷酸奥司他韦的前体。目前,国内外莽草酸的生产主要依靠成本较高,周期较长的植物提取法。微生物发酵法合成莽草酸具有生产成本低、周期短等优势成为研究的热点。为了构建产莽草酸的重组谷氨酸棒杆菌,此次研究从基因组水平上对谷氨酸棒杆菌体内的莽草酸代谢途径进行代谢工程改造。通过阻断莽草酸分解代谢途径、解除反馈抑制以及阻断竞争性代谢途径的策略,实现了莽草酸产量的大幅提升。结果显示,所构建的重组谷氨酸棒杆菌SKA06经72 h摇瓶发酵,莽草酸产量达到7.61 g/L,相较出发菌种提升了68倍。并且,基于染色体工程的遗传改造策略克服了引入质粒带来传代不稳定、需要添加抗生素等问题,可以为莽草酸工程菌种的选育提供重要参考。     

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。     

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。     

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.     

Knockout of ptsG Gene in Escherichia coli and Its Influence on Production of L-phenylalanine

L-phe 是重要的食品和医药中间体,用大肠杆菌发酵葡萄糖生成 phe 时,对葡糖糖转运起重要作用的磷酸烯醇丙酮酸糖磷酸转移酶系统(PTS)对 phe 产量合成有很大影响,在大肠杆菌 PTS 系统中,葡糖糖主要由 ptsG 基因编码的葡萄糖特异性转运蛋白酶ⅡCBGlc转运入细胞,通过基因敲除技术获取ptsG缺陷菌株,可以减少菌株对葡糖糖的摄取,减少乙酸的生成,利于菌株的高密度发酵和相关代谢中间物获得.利用 Red 同源重组技术将大肠杆菌染色体上的 ptsG 基因进行敲除,得到 PTS 缺陷菌株 MD-ptsG-.该菌株在以葡萄糖为惟一碳源的培养基中摇瓶培养,菌密度为对照菌株的3.5倍,L-phe 产量提高12％.     

大肠杆菌ptsG基因的敲除及其对L-phe生产的影响

L-phe是重要的食品和医药中间体,用大肠杆菌发酵葡萄糖生成phe时,对葡糖糖转运起重要作用的磷酸烯醇丙酮酸糖磷酸转移酶系统(PTS)对phe产量合成有很大影响,在大肠杆菌PTS系统中,葡糖糖主要由ptsG基因编码的葡萄糖特异性转运蛋白酶ⅡCBGlc转运入细胞,通过基因敲除技术获取ptsG缺陷菌株,可以减少菌株对葡糖糖的摄取,减少乙酸的生成,利于菌株的高密度发酵和相关代谢中间物获得。利用Red同源重组技术将大肠杆菌染色体上的ptsG基因进行敲除,得到PTS缺陷菌株MD-ptsG-。该菌株在以葡萄糖为惟一碳源的培养基中摇瓶培养,菌密度为对照菌株的3.5倍,L-phe产量提高12%。     

Combinatorial pathway analysis for improved L-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression

Combinatorial overexpression of aromatic amino acid biosynthesis (AAAB) genes in the L-tyrosine producing Escherichia coli strains T1 and T2 was employed to search for AAAB reactions limiting L-tyrosine production. All AAAB genes except aroG and tyrA, which were substituted by their feedback resistant derivatives in the host strains, were cloned and overexpressed. A total of 72 different strains overexpressing various AAAB gene combinations were generated and from those strains with improved phenotype, enzymatic bottlenecks of the AAAB pathway could be inferred. The two major gene overexpression targets for increased L-tyrosine production in E. coli were ydiB and aroK, coding for a shikimate dehydrogenase and a shikimate kinase, respectively, and the combination of ydiB and aroK for overexpression resulted in the best L-tyrosine producing strains in this study, yielding 45% for strain T1 and 26% for strain T2, respectively, higher L-tyrosine titers. Interestingly, overexpression studies with combinations of more than one gene revealed that new gene targets could be identified when overexpessed together with other genes but not alone as single gene overexpression. For example, tyrB encoding the last enzyme of the AAAB pathway, an aromatic amino acid transaminase, improved L-tyrosine production significantly when co-overexpressed together with ydiB or aroK, but not when overexpressed alone. It is also noteworthy that E. coli T1, which generally yielded less L-tyrosine, was amenable to greater improvements than strain T2, i.e. E. coli T1 exhibited generally more space for phenotype improvement.     

Elucidating and reprogramming Escherichia coli metabolisms for obligate anaerobic n-butanol and isobutanol production

Elementary mode (EM) analysis based on the constraint-based metabolic network modeling was applied to elucidate and compare complex fermentative metabolisms of Escherichia coli for obligate anaerobic production of n-butanol and isobutanol. The result shows that the n-butanol fermentative metabolism was NADH-deficient, while the isobutanol fermentative metabolism was NADH redundant. E. coli could grow and produce n-butanol anaerobically as the sole fermentative product but not achieve the maximum theoretical n-butanol yield. In contrast, for the isobutanol fermentative metabolism, E. coli was required to couple with either ethanol- or succinate-producing pathway to recycle NADH. To overcome these "defective" metabolisms, EM analysis was implemented to reprogram the native fermentative metabolism of E. coli for optimized anaerobic production of n-butanol and isobutanol through multiple gene deletion (~8-9 genes), addition (~6-7 genes), up- and downexpression (~6-7 genes), and cofactor engineering (e.g., NADH, NADPH). The designed strains were forced to couple both growth and anaerobic production of n-butanol and isobutanol, which is a useful characteristic to enhance biofuel production and tolerance through metabolic pathway evolution. Even though the n-butanol and isobutanol fermentative metabolisms were quite different, the designed strains could be engineered to have identical metabolic flux distribution in "core" metabolic pathways mainly supporting cell growth and maintenance. Finally, the model prediction in elucidating and reprogramming the native fermentative metabolism of E. coli for obligate anaerobic production of n-butanol and isobutanol was validated with published experimental data.     

Metabolic engineering for microbial production of shikimic acid

Shikimic acid is a high valued compound used as a key starting material for the synthesis of the neuramidase inhibitor GS4104, which was developed under the name Tamiflu for treatment of antiviral infections. An excellent alternative to the isolation of shikimic acid from fruits of the Illicium plant is the fermentative production by metabolic engineered microorganisms. Fermentative production of shikimic acid was most successfully carried out by rational designed Escherichia coli strains by blocking the aromatic amino acid pathway after the production of shikimic acid. An alternative is to produce shikimic acid as a result of dephosphorylation of shikimate-3-phosphate. Engineering the uptake of carbon, the regulatory circuits, central metabolism and the common aromatic pathway including shikimic acid import that have all been targeted to effect higher productivities and lower by-product formation are discussed.     

Growth recovery on glucose under aerobic conditions of an Escherichia coli strain carrying a phosphoenolpyruvate:carbohydrate phosphotransferase system deletion by inactivating arcA and overexpressing the genes coding for glucokinase and galactose permease

In Escherichia coli the phosphotransferase system (PTS) consumes one molecule of phosphoenolpyruvate (PEP) to phosphorylate each molecule of internalized glucose. PEP bioavailability into the aromatic pathway can be increased by inactivating the PTS. However, the lack of the PTS results in decreased glucose transport and growth rates. To overcome such drawbacks in a PTS(-) strain and reconstitute rapid growth on glucose phenotype (Glc(+)), the glk and galP genes were cloned into a plasmid and the arcA gene was inactivated. Simultaneous overexpression of glk and galP increased the growth rate and regenerated a Glc(+) phenotype. However, the highest growth rate was obtained when glk and galP were overexpressed in the arcA(-) background. These results indicated that the arcA mutation enhanced glycolytic and respiratory capacities of the engineered strain.     

Shikimic acid production by a modified strain of E. coli (W3110.shik1) under phosphate-limited and carbon-limited conditions

Shikimic acid is one of several industrially interesting chiral starting materials formed in the aromatic amino acid pathway of plants and microorganisms. In this study, the physiology of a shikimic acid producing strain of Escherichia coli (derived from W3110) deleted in aroL (shikimic acid kinase II gene), was compared to that of a corresponding control strain (W3110) under carbon- and phosphate-limited conditions. For the shikimic acid producing strain (referred to as W3110.shik1), phosphate limitation resulted in a higher yield of shikimic acid (0.059 +/- 0.012 vs. 0.024 +/- 0.005 c-mol/c-mol) and a lower yield of by-products from the shikimate pathway, when compared to carbon-limited condition. The yield of the by-product 3-dehydroshikimic acid (DHS) decreased from 0.076 +/- 0.028 to 0.022 +/- 0.001 c-mol/c-mol. Several other by-products were only detected under carbon-limited conditions. The latter group included 3-dehydroquinic acid (0.021 +/- 0.021 c-mol/c-mol), quinic acid (0.012 +/- 0.005 c-mol/c-mol), and gallic acid (0.002 +/- 0.001 c-mol/c-mol). For both strains, more acetate was produced under phosphate than the carbon-limited case. Considerable cell lysis was found for both strains but was higher for W3110.shik1, and increased for both strains under phosphate limitation. The advantages of the latter condition in terms of an increased shikimic acid yield was thus counteracted by an increased cell lysis, which may make downstream processing more difficult.