天然产物Ectoine与Hydroxyectoine的生物工程及医学应用研究进展

四氢嘧啶及衍生物(Ectoines,Ects)是嗜盐或耐盐细菌胞内合成的一类能够抵抗外界高盐胁迫的有机相容溶质(Compatible solute),主要包括四氢嘧啶(Ectoine)与羟基化四氢嘧啶(5-hydroxyectoine,5-HE)。在高温、干旱、高pH值、高压和高盐等极端环境条件下,Ects不仅能够平衡细胞的渗透压,而且对蛋白质、DNA、细胞膜以及整个细胞提供抗逆协助作用。本文综述了Ects对生物大分子和细胞的保护与稳定作用,以及在临床疾病治疗应用方面的辅助作用,并讨论与展望了Ects在生物医药制剂和生物保健开发等方面的应用前景。     

嗜盐细菌中四氢嘧啶和羟基四氢嘧啶的生物合成及其生物学功能

在嗜盐细菌盐适应中,四氢嘧啶(1,4,5,6-四氢-2-甲基-4-嘧啶羧酸)和羟基四氢嘧啶(1,4,5,6-四氢-2-甲基-5-羟基-4-嘧啶羧酸)发挥着十分重要的作用.四氢嘧啶的生物合成以L-天冬氨酸-β-半醛(ASA)为底物,依次由2,4.二氨基丁酸转氨酶(EctB),2,4--氨基丁酸乙酰基转移酶(EctA)和四...     

中度嗜盐菌四氢嘧啶合成基因的克隆与功能分析

中度嗜盐菌Bacillus alcalophilus DTY1分离自晋西北黄土高原盐碱土壤, 能够产生耐盐相关的相容性溶质四氢嘧啶。为了研究四氢嘧啶的功能, 克隆了DTY1菌株四氢嘧啶合成基因簇ectABC。ectA、ectB和ectC分别编码169、428和132个氨基酸的肽链, 分别与B. halodurans C-125中的二氨基丁酸乙酰基转移酶(EctA)、二氨基丁酸氨基转移酶(EctB)、四氢嘧啶合成酶(EctC)同源性达59%、81%和81%。将携带该基因簇的4.0 kb片段转入蜡质芽孢杆菌B. cereus Z后, 芽孢杆菌的耐盐度显著提高。HPLC检测发现, 在1.0% NaCl浓度下, 转化菌B. cereus Z-E菌株生成70.1 mg/g四氢嘧啶, 而在5.0%的NaCl浓度下四氢嘧啶的产量高达118.6 mg/g, 显著高于B. alcalophilus DTY1的四氢嘧啶产量。而且随着盐浓度的提高, 四氢嘧啶的合成量也随之提高。由此证明四氢嘧啶参与中度嗜盐菌重要的渗透调节, ectABC的表达受盐诱导。     

中度嗜盐菌四氢嘧啶合成基因的克隆与功能分析

中度嗜盐菌Bacillus alcalophilus DTY1分离自晋西北黄土高原盐碱土壤, 能够产生耐盐相关的相容性溶质四氢嘧啶。为了研究四氢嘧啶的功能, 克隆了DTY1菌株四氢嘧啶合成基因簇ectABC。ectA、ectB和ectC分别编码169、428和132个氨基酸的肽链, 分别与B. halodurans C-125中的二氨基丁酸乙酰基转移酶(EctA)、二氨基丁酸氨基转移酶(EctB)、四氢嘧啶合成酶(EctC)同源性达59%、81%和81%。将携带该基因簇的4.0 kb片段转入蜡质芽孢杆菌B. cereus Z后, 芽孢杆菌的耐盐度显著提高。HPLC检测发现, 在1.0% NaCl浓度下, 转化菌B. cereus Z-E菌株生成70.1 mg/g四氢嘧啶, 而在5.0%的NaCl浓度下四氢嘧啶的产量高达118.6 mg/g, 显著高于B. alcalophilus DTY1的四氢嘧啶产量。而且随着盐浓度的提高, 四氢嘧啶的合成量也随之提高。由此证明四氢嘧啶参与中度嗜盐菌重要的渗透调节, ectABC的表达受盐诱导。     

Halomonas venusta DSM4743渗透压冲击下四氢嘧啶合成与释放

选择耐受较高NaCl浓度,而且四氢嘧啶胞内浓度阈值较高的菌株,研究其四氢嘧啶发酵条件和工艺,对于提高四氢嘧啶的制备效率具有实际意义。考察了碳源、NaCl浓度、酵母膏添加量对Halomonas venustaDSM4743四氢嘧啶合成的影响,考察了优化条件下的四氢嘧啶分批发酵进程,并利用"细菌挤奶"工艺制备四氢嘧啶。结果表明:谷氨酸单钠为唯一碳氮源、NaCl浓度为1.5mol/L、酵母膏添加量为0.5%的条件有利于四氢嘧啶合成。在优化条件下10L发酵罐分批发酵,四氢嘧啶最大合成量为3.2g/L,合成效率为2.7g/(L.d)。通过"细菌挤奶"工艺制备四氢嘧啶,6个渗透压冲击循环后,四氢嘧啶总合成量为14.7g/L,总释放量为14.3g/L,平均释放率为97%,合成效率2.1g/(L.d)。中度嗜盐菌Halomonas venusta DSM4743耐受较高浓度的NaCl,而且四氢嘧啶胞内浓度阈值较高,优化的发酵条件及"细菌挤奶"工艺,获得了较高的四氢嘧啶制备效率。     

古盐井中嗜盐菌的分离及四氢嘧啶的制备

[目的]分离鉴定古盐井中的嗜盐菌,确定相容性溶质,增加相容性溶质的合成。[方法]从盐井卤水中分离嗜盐菌,对菌株进行形态、生理生化和16S rRNA分析,用高效液相色谱法(HPLC)和质谱法(MS)确定相容性溶质,以谷氨酸钠为唯一碳氮源来制备四氢嘧啶。[结果]菌株S2为色盐杆菌属;相容性溶质为四氢嘧啶; 1. 5 mol/L Na Cl时四氢嘧啶达到最适合成浓度,为0. 168 3 mg/mL,单位质量合成量为238. 201 mg/g;以谷氨酸钠为唯一碳氮源时,相同体积发酵液中四氢嘧啶的总合成量与单位质量合成量均增加,四氢嘧啶合成量为0. 184 8 mg/mL,单位质量合成量为390. 389 mg/g。[结论]成功分离出了产四氢嘧啶的嗜盐菌,并且以谷氨酸钠为唯一碳氮源时四氢嘧啶合成量大大增加。     

相容溶质四氢嘧啶的微生物合成研究进展北大核心CSCD

四氢嘧啶(ectoine)作为一种氨基酸的衍生物,是嗜盐微生物胞内重要的天然次级代谢物,具有保护细胞和稳定生物大分子的功能,可广泛应用于药物制备佐剂、器官移植与保存、皮肤创伤修复与新型化妆品研发等生物医学领域。由于四氢嘧啶的医用价值和商业市场需求,文中从野生菌株的筛选与诱变育种、构建基因工程菌株与系统代谢整合工程菌株、四氢嘧啶的优化发酵与生产工艺以及抽提纯化工艺等方面,系统论述了四氢嘧啶高效积聚和过量化生产研究策略,并展望多组学、计算生物学及“细胞工厂”等技术在后续四氢嘧啶高效生产上的应用前景。     

渗透压冲击下中度嗜盐菌Halomonas sp. Y四氢嘧啶类相容性溶质的合成与释放

【背景】四氢嘧啶类物质在高温、冷冻和干燥等逆境条件下,对酶、蛋白质、核酸及整个细胞具有良好的保护作用,已经应用于酶制剂、生物医药及护肤品等相关领域。目前此类物质只能依赖中度嗜盐菌采用细菌泌乳工艺进行商业化生产,因此四氢嘧啶类高产菌株及其发酵技术的研究日益受到国内外研究者关注。【目的】分离获得高产合成四氢嘧啶类相容性溶质的中度嗜盐细菌,研究渗透压冲击对其胞内四氢嘧啶合成与释放的影响,探索细菌泌乳法制备四氢嘧啶的可行性。【方法】采用涂布平板法分离中度嗜盐菌,对分离菌株进行形态、生理生化和16S rRNA基因序列分析,鉴定其种属;采用高效液相色谱法(HPLC)和质谱法(MS)分析四氢嘧啶类物质,细菌泌乳法制备四氢嘧啶类物质。【结果】从盐池土样中分离到一株以四氢嘧啶类物质为主要相容性溶质的中度嗜盐菌Y,鉴定为盐单胞菌(Halomonas sp.)Y。盐单胞菌Y能在NaCl质量浓度为10-250 g/L的培养基中生长,最适生长的NaCl浓度为100 g/L;HPLC-MS测试结果证明盐单胞菌Y可同时合成四氢嘧啶和羟基四氢嘧啶2种相容性溶质,在最适生长的盐浓度下其合成量分别达175.5 mg/g和47.9 mg/g;在NaCl质量浓度为0-30 g/L的低渗溶液中胞内四氢嘧啶类物质经5 min即可达到最大释放率,而细菌泌乳工艺中最适合诱导四氢嘧啶释放的低渗溶液为质量浓度为10 g/L的NaCl溶液;采用细菌泌乳工艺制备四氢嘧啶,经连续11轮的高渗/低渗冲击,四氢嘧啶总合成量为6.0 g/L,总释放量为5.7 g/L,平均释放率为64.5%,底物转化率为128.9 mg/g。【结论】盐单胞菌Y是一株较高产合成四氢嘧啶类的中度嗜盐菌,能够耐受反复的渗透压冲击,采用细菌泌乳工艺显著提高了四氢嘧啶的制备效率。     

盐单胞菌属BYS1四氢嘧啶合成基因ectABC克隆及其盐激表达

利用SEFA-PCR技术从中度嗜盐菌Halomonassp.BYS-1总DNA中克隆了四氢嘧啶合成基因ectABC及其上游序列(GenBank accession number DQ017757);OMIGA软件分析结果显示ectA、ectB、ectC位于同一个操纵子上,大小分别为573bp1、251bp和387bp,预测编码的DAT(L-二氨基丁酸转氨酶)、DAA(L-二氨基丁酸乙酰转移酶)和ES(四氢嘧啶合酶)大小分别为21.1kDa(191 amino acid)、45.7kDa(417 amino acid)和14.5kDa(129 amino acid);将包含ectABC基因及其上游1000bp序列的片段克隆到pUC19中并转化E.coliDH5α,转化子E.coli(pUC19ECT)能够在盐激条件下合成四氢嘧啶,但其耐盐能力没有得到显著改善。     

四氢嘧啶吸收缺陷突变株高效制备四氢嘧啶

【目的】为进一步提高四氢嘧啶(1,4,5,6-四氢-2-甲基-4-嘧啶羧酸;1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid;ectoine)合成效率,【方法】利用步移PCR方法克隆了Halomonas salina DSM 5928T四氢嘧啶特异性转运蛋白(ectoine-specific transporter;TeaABC)编码基因teaABC,Red重组技术构建了四氢嘧啶吸收缺陷突变株H.salina DSM 5928T(teaABC-)。【结果】H.salina DSM 5928T(teaABC-)10 L发酵罐的四氢嘧啶发酵,四氢嘧啶总浓度9.10(±0.08)g/L,合成效率为9.93(±0.09)g/L.d。【结论】四氢嘧啶吸收缺陷突变株H.salina DSM 5928T(teaABC-),解除了四氢嘧啶吸收对其合成的负反馈调节,从而显著提高了四氢嘧啶合成效率。     

Unexpected property of ectoine synthase and its application for synthesis of the engineered compatible solute ADPC

A new cyclic amino acid was detected in a deletion mutant of the moderately halophilic bacterium Halomonas elongata deficient in ectoine synthesis. Using mass spectroscopy (MS) and nuclear magnetic resonance (NMR) techniques, the substance was identified as 5-amino-3,4-dihydro-2H-pyrrole-2-carboxylate (ADPC). We were able to demonstrate that ADPC is the product of a side reaction of lone ectoine synthase (EC 4.2.1.108), which forms ADPC by cyclic condensation of glutamine. This reaction was shown to be reversible. Subsequently, a number of ectoine derivatives, in particular 4,5-dihydro-2-methylimidazole-4-carboxylate (DHMICA) and homoectoine, were also shown to be cleaved by ectoine synthase, which is classified as a hydro-lyase. This study thus reports for the first time that ectoine synthase accepts more than one substrate and is a reversible enzyme able to catalyze both the intramolecular condensation into and the hydrolytic cleavage of cyclic amino acid derivatives. As ADPC supports growth of bacteria under salt stress conditions and stabilizes enzymes against freeze-thaw denaturation, it displays typical properties of compatible solutes. As ADPC has not yet been described as a natural compound, it is presented here as the first man-made compatible solute created through genetic engineering.     

Biochemical Properties of Ectoine Hydroxylases from Extremophiles and Their Wider Taxonomic Distribution among Microorganisms

Ectoine and hydroxyectoine are well-recognized members of the compatible solutes and are widely employed by microorganisms as osmostress protectants. The EctABC enzymes catalyze the synthesis of ectoine from the precursor L-aspartate-β-semialdehyde. A subgroup of the ectoine producers can convert ectoine into 5-hydroxyectoine through a region-selective and stereospecific hydroxylation reaction. This compatible solute possesses stress-protective and function-preserving properties different from those of ectoine. Hydroxylation of ectoine is carried out by the EctD protein, a member of the non-heme-containing iron (II) and 2-oxoglutarate-dependent dioxygenase superfamily. We used the signature enzymes for ectoine (EctC) and hydroxyectoine (EctD) synthesis in database searches to assess the taxonomic distribution of potential ectoine and hydroxyectoine producers. Among 6428 microbial genomes inspected, 440 species are predicted to produce ectoine and of these, 272 are predicted to synthesize hydroxyectoine as well. Ectoine and hydroxyectoine genes are found almost exclusively in Bacteria. The genome context of the ect genes was explored to identify proteins that are functionally associated with the synthesis of ectoines; the specialized aspartokinase Ask_Ect and the regulatory protein EctR. This comprehensive in silico analysis was coupled with the biochemical characterization of ectoine hydroxylases from microorganisms that can colonize habitats with extremes in salinity (Halomonas elongata), pH (Alkalilimnicola ehrlichii, Acidiphilium cryptum), or temperature (Sphingopyxis alaskensis, Paenibacillus lautus) or that produce hydroxyectoine very efficiently over ectoine (Pseudomonas stutzeri). These six ectoine hydroxylases all possess similar kinetic parameters for their substrates but exhibit different temperature stabilities and differ in their tolerance to salts. We also report the crystal structure of the Virgibacillus salexigens EctD protein in its apo-form, thereby revealing that the iron-free structure exists already in a pre-set configuration to incorporate the iron catalyst. Collectively, our work defines the taxonomic distribution and salient biochemical properties of the ectoine hydroxylase protein family and contributes to the understanding of its structure.     

Feedback inhibition of ecdysone production by 20-hydroxyecdysone during pupal—adult metamorphosis of Mamestra configurata wlk

Large peaks of ecdysone (E, 2,875 ng/g live wt) and 20-hydroxyecdysone (20-HE, 2,150 ng/g live wt) occur on days 8 and 12, respectively, of postdiapause pupal-adult metamorphosis at 20°C in the bertha armyworm, Mamestra configurata, and then decline to low levels (< 100 ng/g live wt) prior to eclosion of the moth (50% eclosion at day 31.8). These peaks of E and 20-HE can be suppressed by treating the developing pupae with a physiological dose (2,500 ng/g live wt) of 20-HE. Suppression of E and 20-HE by 20-HE treatment was dose dependent, rapid (within 24 h of treatment) and permanent. The peaks of E and 20-HE were suppressed by 20-HE treatment on days 1, 3, 5, and 7 but the 20-HE peak was not suppressed by treatment on days 9 or 11. It is proposed that the mechanism by which 20-HE suppresses the production of E and thereby its own production forms a negative feedback loop that operates during the first 0.4 units of pupal-adult development in M. configurata. The function of the transitory peaks of E and 20-HE that form this feedback loop is currently unknown. Since most adults from pupae that had their ecdysteroid levels experimentally suppressed by 20-HE treatment were morphologically normal, it seems that the peaks of E and 20-HE have little or no function in controlling morphological development in pupae of M. configurata.     

Molecular Dynamics Simulations and Structure-Guided Mutagenesis Provide Insight into the Architecture of the Catalytic Core of the Ectoine Hydroxylase

Many bacteria amass compatible solutes to fend-off the detrimental effects of high osmolarity on cellular physiology and water content. These solutes also function as stabilizers of macromolecules, a property for which they are referred to as chemical chaperones. The tetrahydropyrimidine ectoine is such a compatible solute and is widely synthesized by members of the Bacteria. Many ectoine producers also synthesize the stress protectant 5-hydroxyectoine from the precursor ectoine, a process that is catalyzed by the ectoine hydroxylase (EctD). The EctD enzyme is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily. A crystal structure of the EctD protein from the moderate halophile Virgibacillus salexigens has previously been reported and revealed the coordination of the iron catalyst, but it lacked the substrate ectoine and the co-substrate 2-oxoglutarate. Here we used this crystal structure as a template to assess the likely positioning of the ectoine and 2-oxoglutarate ligands within the active site by structural comparison, molecular dynamics simulations, and site-directed mutagenesis. Collectively, these approaches suggest the positioning of the iron, ectoine, and 2-oxoglutarate ligands in close proximity to each other and with a spatial orientation that will allow the region-selective and stereo-specific hydroxylation of (4S)-ectoine to (4S,5S)-5-hydroxyectoine. Our study thus provides a view into the catalytic core of the ectoine hydroxylase and suggests an intricate network of interactions between the three ligands and evolutionarily highly conserved residues in members of the EctD protein family.     

Hormonal regulation of Drosophila microRNA let-7 and miR-125 that target innate immunity

The steroid 20-hydroxy-ecdysone (20-HE) and the sesquiterpenoid Juvenile Hormone (JH) coordinate insect life stage transitions. 20-HE exerts these effects by the sequential induction of response genes. In the nematode Caenorhabditis elegans hormones also play a role in such transitions, but notably, microRNA such as let-7 and lin-4 have likewise been found to help order developmental steps. Little is known about the corresponding function of homologous microRNA in Drosophila melanogaster, and the way microRNA might be regulated by 20-HE in the fly is ambiguous. Here we used Drosophila S2 cells to analyze the effects of 20-HE on D. melanogaster microRNA let-7 and miR-125, the homolog of lin-4. The induction by 20-HE of let-7 and miR-125 in S2 cells is inhibited by RNAi knockdown of the ecdysone receptor and, as previously shown, by knockdown of its cofactor broad-complex C. To help resolve the currently ambiguous role of 20-HE in the control of microRNA, we show that nanomolar concentrations of 20-HE primes cells to subsequently express microRNA when exposed to micromolar levels of 20-HE. We then explore the role microRNA plays in the established relationship between 20-HE and the induction of innate immunity. We show that the 3'UTR of the antimicrobial peptide diptericin has a let-7 binding site and that let-7 represses translation from this site. We conclude that 20-HE facilitates the initial expression of innate immunity while it simultaneously induces negative regulation via microRNA control of antimicrobial peptide translation.     

Synthesis of osmoprotectors by halophilic and alkalophilic methanotrophs

The 1H-NMR analysis of methanol extracts of halophilic and halotolerant alkaliphilic methanotrophs isolated from the soda lakes of Southern Transbaikal and Tuva showed that bacterial cells grown at an optimum salinity accumulated mainly sucrose and 5-oxo-1-proline, whereas cells adapted to 0.5-1.0 M NaCl additionally synthesized ectoine. A more detailed study showed that nitrogen deficiency in the growth medium of Methylobacter alcaliphilus 20Z decreased the synthesis of nitrogen-containing osmoprotectants, ectoine and 5-oxo-1-proline. M. alcaliphilus 20Z cells exhibited activities of UDP-glucose pyrophosphorylase and sucrose-phosphate synthase involved in sucrose synthesis. Glutamine synthetase in vitro did not require NH4+ ions, which implies that this enzyme is involved in 5-oxo-1-proline synthesis. Cells grown at high salinity exhibited elevated levels of aspartate kinase, aspartate-semialdehyde dehydrogenase, and ectoine synthase. This suggests that ectoine is synthesized via aspartate and aspartate-semialdehyde, i.e., via the route earlier established for extremely halophilic bacteria.     

Ecdysteroid mediated fat body acid phosphatase activity during larval development of rice moth, Corcyra cephalonica (Lepidoptera)

20-hydroxyecdysone (20-HE) stimulates acid phosphatase activity in the fat body of ligated late-last instar larvae. This effect is time dependent and the specific activity of enzyme increases significantly in hormone treated insects. 20-HE also stimulates general protein synthesis. Cycloheximide treatment either in conjunction with 20-HE or after hormone treatment blocks the increase in enzyme activity as well as increase in protein content. However, actinomycin D treatment does not alter the enzyme activity while it blocks the increase in total RNA as well as increase in protein content.