青海湖盐单胞菌Ectoine合成基因簇ectABC的重组共表达
RECOMBINANT CO-EXPRESSION OF THE ECTOINE BIOSYNTHESIS GENE CLUSTER ectABC IN HALOMONAS FROM QINGHAI LAKE
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摘要: 盐单胞菌属(Halomonas)通过胞内积聚有机相容溶质(Compatible solutes)来抵抗胞外的高盐渗透压。为了探究相容溶质Ectoine合成代谢相关基因的结构特征和异源共表达的可能性, 以青海湖盐单胞菌Halomonas sp. QHL1为材料, 通过高效液相色谱(HPLC)分析不同盐梯度下QHL1胞内Ectoine的积聚量, 并借助于染色体步移技术(Genome walking)捕获QHL1菌株的Ectoine生物合成基因簇ectABC, 利用分子克隆技术分析ectABC基因簇的异源重组表达(E.coli BL21)。研究结果表明: 胞内Ectoine的积聚量随着培养基中Na+浓度的增加而增加, 最大积聚量为167.1 mg/g细胞干重(1.0 mol/L Na+), 但菌体生长却受到高浓度Na+的强烈抑制作用。QHL1的ectABC操纵子全长序列为3580 bp, 结构基因ectA(579 bp)、ectB(1269 bp)与ectC (390 bp)串联排列。基于生物信息学预测分析, 两个启动子(70与38因子控制)和若干未知功能的保守模序(Motifs)存在于QHL1的ect操纵子上游。构建重组表达载体pET-28-ectABC, 并在E.coli BL21中异源表达ectABC基因簇(2438 bp)。SDS-PAGE结果显示EctA、EctB和EctC分别为27.2、52.5 和 20.8 kD, 与预测结果一致, 表明ectA、ectB和ectC基因能在E. coli BL21中实现异源共表达, 为构建Ectoine合成代谢基因整合的系统代谢工程, 并实现低盐发酵控制和过量化生产提供了重要的理论基础。Abstract: Halomonas is capable of synthesizing organic compatible solutes ectoine in response to high osmotic pressure. To reveal the possibility of heterologous co-expression of ectoine biosynthesis genes, intracellular ectoine in Halomonas sp. QHL1 strain was determined by HPLC under different salt gradients. The entire ectABC gene cluster for ectoine synthesis was cloned using genome walking and expressed in the heterologous recombinant E. coli BL21. The results showed that the concentration of ectoine accumulated in the cells had a positive correlation with the extracellular Na+ concentration and reached a maximum value (167.1 mg/g cell dry weight) at 1.0 mol/L Na+, and high concentration of Na+ strongly inhibited the bacteria growth. The entire ectABC gene cluster in QHL1 strain was 3580 bp, containing structural gene ectA (579 bp), ectB (1269 bp) and ectC (390 bp). Based on bioinformatics prediction analysis, two putative promoters (70 and 38-controlled promoter) and several conserved motifs with unknown function were identified in the upstream of ect-operon. The recombinant plasmid pET-28a (+)-ectABC was successfully constructed, and the results of heterologous expression indicated that these three genes could be simultaneously translated to protein EctA (27.2 kD), EctB (52.5 kD) and EctC (20.8 kD). These results contribute further improvements in ectoine high yield and hypohaline biotechnological process optimization, and also provided a framework for future genetic manipulation of systems metabolic engineering.
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Keywords:
- Halomonas /
- Ectoine /
- Gene cluster ectABC /
- Structural gene /
- Co-expression
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[1] Pastor J M, Salvador M, Argandońa M, et al. Ectoines in cell stress protection: uses and biotechnological production [J]. Biotechnology Advances, 2010, 28(6): 782801
[2] Long Q F, Zhu D R, Han R, et al. Recent progress in researches on synthesis and transportation mechanism of compatible solutes in halophilic bacteria [J]. Environmental Science Technology, 2011, 34(9): 6366 [龙启福, 朱德锐, 韩睿, 等. 嗜盐菌相溶物质合成与转运调节机制. 环境科学与技术, 2011, 34(9): 6366]
[3] Louis P, Galinski E A. Characterization of genes for the biosynthesis of the compatible solute ectoine from Marinococcus halophilus and osmoregulated expression in Escherichia coli [J]. Microbiology, 1997, 143(4): 11411149
[4] Cnovas D, Vargas C, Caldern M I, et al. Characterization of the genes for the biosynthesis of the compatible solute ectoine in the moderately halophilic bacterium Halomonas elongata DSM 3043 [J]. Systematic and Applied Microbiology, 1998, 21(4): 487497
[5] Zhao B, Lu W, Yang L, et al. Cloning and characterization of the genes for biosynthesis of the compatible solute ectoine in the moderately halophilic bacterium Halobacillus dabanensis D-8T [J]. Current Microbiology, 2006, 53(3): 183188
[6] Reshetnikov A S, Khmelenina V N, Trotsenko Y A. Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph Methylomicrobium alcaliphilum 20Z [J]. Archives of Microbiology, 2006, 184(5): 286297
[7] Zhang B, Bao X, Wang L, et al. Cloning and characterization of the gene cluster for biosynthesis of ectoine from Nesterenkonia halobia DSM 20541 [J]. Journal of Microbiology, 2008, 46(3): 309318
[8] He J, Huang X, Gu L F, et al. Cloning of the ectoine biosynthesis gene ectABC from Halomonas sp. BYS-1 and salt stressed expression in Escherichia coli [J]. Acta Microbiologica Sinica, 2006, 46(1): 2832 [何健, 黄星, 顾立锋, 等. 盐单胞菌属BYS-1四氢嘧啶合成基因 ectABC 克隆及其盐激表达. 微生物学报, 2006, 46(1): 2832]
[9] Zhang W, Wei H L, Gao H W, et al. Cloning and Characterization of ectABC Cluster from Bacillus alcalophilus DTY1 [J]. Chinese Journal of Biotechnology, 2008, 25, 24(3): 395400 [张薇, 魏海雷, 高洪文, 等. 中度嗜盐菌四氢嘧啶合成基因的克隆与功能分析. 生物工程学报, 2008, 24(3): 395400]
[10] Rajan L A, Joseph T C, Thampuran N, et al. Characterization and phylogenetic analysis of ectoine biosynthesis genes from Bacillus halodurans [J]. Archives of Microbiology, 2008, 190(4): 481487
[11] Bursy J, Kuhlmann A U, Pittelkow M, et al. Synthesis and uptake of the compatible solutes ectoine and 5-hydroxyectoine by Streptomyces coelicolor A3(2) in response to salt and heat stresses [J]. Applied and Environmental Microbiology, 2008, 74(23): 72867296
[12] Reshetnikov A S, Khmelenina V N, Trotsenko Y A. Identification of ectoine synthesis genes in a moderate halophilic alphaproteobacterium Methylarcula marina [J]. Microbiology, 2010, 79(6): 856857
[13] Seip B, Galinski E A, Kurz M. Natural and engineered hydroxyectoine production based on the Pseudomonas stutzeri ectABCD-ask gene cluster [J]. Applied and Environmental Microbiology, 2011, 77(4): 13681374
[14] Kunte H J, Lentzen G, Galinski E A. Industrial production of the cell protectant ectoine: protection mechanisms, processes, and products [J]. Current Biotechnology, 2014, 3(1): 1025
[15] Jrg-Kunte H, Galinski E A, Trper H G. A modified FMOC-method for the detection of amino acid-type osmolytes and tetrahydropyrimidines (ectoines) [J]. Journal of Microbiological Methods, 1993, 17(2): 129136
[16] Li Y D, Long Q F, Li W J, et al. Detection of intracellular compatible solute ectoine in halophilic bacteria from Qinghai Lake by a high performance liquid chromatography (HPLC) [J]. Environmental Chemistry, 2013, 32(9): 16871692 [李耀东, 龙启福, 李文军, 等. 高效液相色谱检测青海湖嗜盐菌胞内积聚的相溶物质四氢嘧啶. 环境化学, 2013, 32(9): 16871692]
[17] Ausubel F M, Brent R, Kingston R E, et al. Short Protocols in Molecular Biology [M]. Beijing: Science Press. 2001, 378397
[18] Yang H, Li F G, Lan Q J, et al. Cloning, identification and expression of ferritin heavy chain from chinese giant salamanders, Andrias davidianus [J]. Acta Hydrobiologica Sinica, 2014, 38(1): 2734 [杨辉, 李锋刚, 蓝青景, 等. 大鲵铁蛋白重链FTH基因的克隆、鉴定及表达分析. 水生生物学报, 2014, 38(1): 2734]
[19] Reshetnikov A S, Khmelenina V N, Mustakhimov I I, et al. Diversity and phylogeny of the ectoine biosynthesis genes in aerobic, moderately halophilic methylotrophic bacteria [J]. Extremophiles, 2011, 15(6): 653663
[20] Schwibbert K, Marin-Sanguino A, Bagyan I, et al. A blueprint of ectoine metabolism from the genome of the industrial producer Halomonas elongata DSM 2581T [J]. Environmental Microbiology, 2011, 13(8): 19731994
[21] Ma R, Xu H, Ding R, et al. The strategy of gene coexpression in Escherichia coli [J]. China Biotechnology, 2012, 32(4): 117122 [马蓉, 徐昊, 丁锐, 等. 大肠杆菌多基因共表达策略. 中国生物工程杂志, 2012, 32(4): 117122]
[22] Bestvater T, Louis P, Galinski E A. Heterologous ectoine production in Escherichia coli: by-passing the metabolic bottle-neck [J]. Saline Systems, 2008, 4(1): 1214
[23] Stveken N, Pittelkow M, Sinner T, et al. A Specialized aspartokinase enhances the biosynthesis of the osmoprotectants ectoine and hydroxyectoine in Pseudomonas stutzeri A1501 [J]. Journal of Bacteriology, 2011, 193(17): 44564468
[24] Eilert E, Kranz A, Hollenberg C P, et al. Synthesis and release of the bacterial compatible solute 5-hydroxyectoine in Hansenula polymorpha [J]. Journal of Biotechnology, 2013, 167(2): 8593
[25] Rodrguez M J, Argandoa M, Iglesias G F, et al. Temperature-and salinity-decoupled overproduction of hydroxyectoine by Chromohalobacter salexigens [J]. Applied and Environmental Microbiology, 2013, 79(3): 10181023
[26] Becker J, Schfer R, Kohlstedt M, et al. Systems metabolic engineering of Corynebacterium glutamicum for production of the chemical chaperone ectoine [J]. Microbial Cell Factories, 2013, 12(1): 110112
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