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。     

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.     

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。     

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。     

基于莽草酸途径微生物合成芳香族化合物及其衍生物的研究进展

芳香族化合物是一类重要的天然产物,在自然界中广泛存在,应用于食品、医药、化工等多个领域,主要通过化学合成、植物提取等方式获得。近年来,随着石化资源减少、人类环保意识的加强,微生物合成芳香族化合物及其衍生物成为热点。莽草酸途径合成的芳香族化合物及其衍生物多种多样。现重点综述通过莽草酸途径合成的"达菲"药物前体莽草酸、大宗化学品己二酸前体顺,顺-粘康酸、芳香族氨基酸及其他高附加值芳香族氨基酸衍生物的微生物合成研究进展,为建立生产高附加值化合物的细胞工厂提供参考。     

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.     

大肠杆菌莽草酸途径限速酶多基因盒的构建及基因替换

优化大肠杆菌芳香族氨基酸生物合成代谢途径 ,构建莽草酸代谢途径限速酶的多基因盒PtacaroAaroCaroBkan .利用Red重组系统 ,在破坏整体调控基因csrA时 ,替换多基因盒 .Southern印迹证实 ,基因破坏和基因替换是成功的 .摇瓶发酵表明 ,构建的基因工程菌株比原始菌株基础产酸率提高了 4 5 3倍     

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.     

八角茴香中莽草酸提取和纯化工艺的研究

本文对八角茴香中莽草酸的提取和纯化工艺进行优化研究.结果表明,莽草酸的最佳提取工艺条件为:常规水浸提,温度80℃,固液比1:15,提取时间100 min,莽草酸的提取量达到0.1795 g.最佳纯化工艺:莽草酸浸膏搅拌硅胶,柱层析,乙酸乙酯洗脱,莽草酸洗脱量达到0.1742 g,真空浓缩至干,甲醇和水混合结晶,得含量98%的纯品莽草酸.     

Rewiring carbon flux in Escherichia coli using a bifunctional molecular switch

The unbalanced distribution of carbon flux in microbial cell factories can lead to inefficient production and poor cell growth. Uncoupling cell growth and chemical synthesis can therefore improve microbial cell factory efficiency. Such uncoupling, which requires precise manipulation of carbon fluxes, can be achieved by up-regulating or down-regulating the expression of enzymes of various pathways. In this study, a dynamic turn-off switch (dTFS) and a dynamic turn-on switch (dTNS) were constructed using growth phase-dependent promoters and degrons. By combining the dTFS and dTNS, a bifunctional molecular switch that could orthogonally regulate two target proteins was introduced. This bifunctional molecular switch was used to uncouple cell growth from shikimic acid and D-glucaric acid synthesis, resulting in the production of 14.33 g/L shikimic acid and the highest reported productivity of D-glucaric acid (0.0325 g/L/h) in Escherichia coli MG1655. This proved that the bifunctional molecular switch could rewire carbon fluxes by controlling target protein abundance.     

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.     

Some properties of rat-liver glucose–adenosine triphosphate phosphotransferases

1. Bacilysin, a peptide which yields l-alanine and l-tyrosine on acid hydrolysis, was produced by a strain of Bacillus subtilis (A 14) in a chemically defined medium containing glucose, ammonium acetate or ammonium chloride, potassium phosphate and other inorganic salts, and ferric citrate. 2. Under the conditions used growth was diphasic. Bacilysin was formed during the second phase of slower growth, and there was little production during the stationary phase. Nevertheless, bacilysin production occurred when protein synthesis was inhibited by chloramphenicol. It thus appears that there is no obligatory coupling of protein synthesis and bacilysin synthesis. 3. When dl-[1-(14)C]alanine was added to a growing culture of B. subtilis, (14)C was incorporated into bacilysin, which contains an N-terminal alanine residue. 4. Under similar conditions virtually no (14)C was incorporated into bacilysin from dl-[2-(14)C]tyrosine, l-[U-(14)C]tyrosine or [1-(14)C]acetate, although these compounds were used by the cell for the biosynthesis of other substances. These results indicate that neither tyrosine nor acetate is a precursor of the fragment of bacilysin which yields tyrosine on hydrolysis with hot 6n-hydrochloric acid. 5. The tyrosine-yielding fragment of bacilysin was labelled with (14)C from [1,6-ring-(14)C(2)]shikimic acid. The biosynthesis of bacilysin thus appears to involve a diversion from the pathway leading to aromatic amino acids at the shikimic acid stage, or a subsequent one.     

Studies on the production of shikimic acid using the <Emphasis Type="Italic">aroK</Emphasis> knockout strain of <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Shikimic acid has various pharmaceutical and industrial applications. It is the sole chemical building block for the antiviral drug oseltamivir (Tamiflu^®) and one of the potent pharmaceutical intermediates with three chiral centres. Here we report a modified strain of Bacillus megaterium with aroK (shikimate kinase) knock out to block the aromatic biosynthetic pathway downstream of shikimic acid. Homologous recombination based gene disruption approach was used for generating aroK knock out mutant of B. megaterium. Shake flask cultivation showed shikimic acid yield of 2.98 g/L which is ~6 times more than the wild type (0.53 g/L). Furthermore, the shikimate kinase activity was assayed and it was 32 % of the wild type. Effect of various carbon sources on the production of shikimic acid was studied and fructose (4 %, w/v) was found to yield maximum shikimic acid (4.94 g/L). The kinetics of growth and shikimic acid production by aroK knockout mutant was studied in 10 L bioreactor and the yield of shikimic acid had increased to 6 g/L which is ~12 fold higher over the wild type. It is evident from the results that aroK gene disruption had an immense effect in enhancing the shikimic acid production.     

Stimulation of some litter-decomposing basidiomycetes by shikimic acid

Shikimic acid, which constitutes 1.5-2.5% of the dry matter in needles of Scots pine, was found to stimulate the growth of various litter-decomposing basidiomycetes of the genera Marasmius, Mycena and Xeromphalina in a synthetic nutrient medium. Out of eighteen litter-decomposing species, ten were stimulated by shikimic acid, wheras the eight mycorrhizal and four wood-rotting species tested were not affected. Maximal effect was obtained at a concentration of ca 2 m M . Growth experiments at varying pH-values indicated active uptake of shikimic acid. Even in the presence of aromatic amino acids, shikimic acid stimulated the growth of the fungi. In certain species the strong inhibiting effect of phenylalanine, tyrosine and tryptophan, when added simultaneously, was reversed in the presence of shikimic acid. Fungi which were stimulated by shikimic acid were also able to use this compound as their sole carbon source. Maximal stimulating effect of shikimic acid occurred when glucose had been added at optimal concentration.     

Production of shikimic acid

Shikimic acid is a key intermediate for the synthesis of the antiviral drug oseltamivir (Tamiflu®). Shikimic acid can be produced via chemical synthesis, microbial fermentation and extraction from certain plants. An alternative production route is via biotransformation of the more readily available quinic acid. Much of the current supply of shikimic acid is sourced from the seeds of Chinese star anise (Illicium verum). Supply from star anise seeds has experienced difficulties and is susceptible to vagaries of weather. Star anise tree takes around six-years from planting to bear fruit, but remains productive for long. Extraction and purification from seeds are expensive. Production via fermentation is increasing. Other production methods are too expensive, or insufficiently developed. In the future, production in recombinant microorganisms via fermentation may become established as the preferred route. Methods for producing shikimic acid are reviewed.     

Transport and utilization of the biosynthetic intermediate shikimic acid in Escherichia coli.

Auxotrophic mutants of Escherichia coli W or K12 blocked before shikimic acid in the aromatic biosynthetic pathway grew poorly on shikimic acid as sole aromatic supplement. This poor growth response was correlated with a relatively poor ability to transport shikimic acid. If citrate was present in the growth medium (as it is in some commonly used basal media) the growth of some of the E. coli K12 mutants on shikimate was further reduced. Mutants were derived from pre-shikimate auxotrophs which grew rapidly on media containing shikimic acid. These derivatives all had an increased ability to transport shikimic acid. Thus, it is proposed that the growth on shikimate observed in the parent cells is restricted by their relatively poor uptake of shikimate from the medium and that this restriction may be removed by a mutation which enhances shikimate transport. Transduction analysis of the mutations which enhanced utilization and transport of shikimic acid by E. coli K12 strains indicated at least two classes. Class 1 was about 20% cotransduced with the histidine region of the E. coli K12 chromosome and appeared to be coincident with a known shikimate transport locus, shiA. Class 2 was not cotransduced with his. The locus (or loci) of this class is unknown. Kinetic measurements suggested that both classes had shikimate uptake systems derived from the wild-type system. Two class 1 mutants had increased levels of otherwise unaltered wild-type transport while one class 2 mutant had an altered Michaelis constant (Km) for shikimate transport.     

Fermentative production of shikimic acid: a paradigm shift of production concept from plant route to microbial route

Different physiological and nutritional parameters affect the fermentative production of shikimic acid. In our study, Citrobacter freundii initially produced 0.62 g/L of shikimic acid in 72 h. However, when process optimization was employed, 5.11 g/L of shikimic acid was produced in the production medium consisting of glucose (5.0 %), asparagine (4.5 %), CaCO₃ (2.0 %), at pH 6.0, when inoculated with 6 % inoculum and incubated at 30 ± 1 °C, 200 rpm for 60 h. Preliminary fed-batch studies have resulted in the production of 9.11 g/L of shikimic acid on feeding the production medium by 20 g/L of glucose at 24 h of the fermentation run. Production of similar amount of shikimic acid was observed when the optimized conditions were employed in a 10-L bioreactor as obtained in shake flask conditions. A total of 9.11 g/L of shikimic acid was produced in 60 h. This is approximately 14.69-fold increase in shikimic acid production when compared to the initial un-optimized production conditions. This has also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.     

Improvement of shikimic acid production in <Emphasis Type="Italic">Escherichia coli</Emphasis> with growth phase-dependent regulation in the biosynthetic pathway from glycerol

Shikimic acid is an important metabolic intermediate with various applications. This paper presents a novel control strategy for the construction of shikimic acid producing strains, without completely blocking the aromatic amino acid biosynthesis pathways. Growth phase-dependent expression and gene deletion was performed to regulate the aroK gene expression in the shikimic acid producing Escherichia coli strain, SK4/rpsM. In this strain, the aroL and aroK genes were deleted, and the aroB, aroG*, ppsA, and tktA genes were overexpressed. The relative amount of shikimic acid that accumulated in SK4/rpsM was 1.28-fold higher than that in SK4/pLac. Furthermore, a novel shikimic acid production pathway, combining the expression of the dehydroquinate dehydratase-shikimate dehydrogenase (DHQ-SDH) enzyme from woody plants, was constructed in E. coli strains. The results demonstrated that a growth phase-dependent control of the aroK gene leads to higher SA accumulation (5.33 g/L) in SK5/pSK6. This novel design can achieve higher shikimic acid production by using the same amount of medium used by the current methods and can also be widely used for modifying other metabolic pathways.     

Characterization of a meta-Fluorotyrosine-Tolerant Cell Culture of Eschscholtzia californica Cham

A cell line of Eschscholtzia californica selected for meta-fluorotyrosine (MFT) tolerance was found to have 10-fold increased levels of phenylalanine and tyrosine compared to the parent line, while most other amino acids were only increased 2-fold. Tracer experiments with shikimic acid in the presence of MFT showed that the biosynthesis of the aromatic amino acids was not impaired in the tolerant line. Feeding experiments with phenylalanine, tyrosine, or shikimic acid also revealed a reduced turnover of the pools of the aromatic amino acids in the variant. Thus undisturbed de novo biosynthesis of the aromatic amino acids and dilution of toxic effects of MFT by the enlarged pool sizes seemed to be the main reason for the acquired tolerance. Despite the enlarged availability of the precursor tyrosine, formation of the benzophenanthridine alkaloids was enhanced neither in the growth nor in the production medium.