Isolation And Identification Of Basophilic Actinomycetes And Halophilic Actinomycetes In The Soil Of Jilantai Saline Lake And Construction Of Ectoine Engineering Strains

1,4,5,6-Tetrahydro-2-methyl-4-pyrimidine carboxylic acid is known as ectoine which is a kind of osmotic pressure compensating solute. The molecular formula is C6H10N2O2 and the molecular weight is 142.2g/mol, and the ectoine is a derivative of cyclic amino acids. The characteristics of balancing osmotic pressure and the function of stabilizing or protecting macromolecule, such as proteins, DNA, and enzyme, make it a higher exploitation and utilization value.29 strains bisophilic actinomycetes and halophilic acrinomycetes were isolated from the soils of Jilantai Salt Lake in this paper. Then analyzed the acrinomycetes with the method of enzyme digestion polymorphism in 16 S rDNA and determined the 16 S rDNA sequence of representative strains. The 3 strains of the obtained strains from the soils of Jilantai Salt Lake were Nocardia sp. Extracted the intracellular ectoine of all isolated strains from the soils of Jilantai Salt Lake by using 80% ethanol and detected by TLC. It a quickly method to screen out the actinomycetes strains which could product ectoine.Streptomyces pactum Act12 is a multifunctional actinomycetes strain, and TLC qualitative detection showed that it could produce ectoine which was related to salt tolerance. Streptomyces pactum Act12 could grow in the range of 0~10.0% NaCl concentration. And in the 2.5% NaCl concentration, the intracellular ectoine accumulation reached the maximum, 72.39mg/L. EctA, ectB and ectC were located in the same operon of Streptomyces pactum Act12 by bioinformatics analysis, and the length of the sequencing were 516 bp,1272bp and 399 bp respectively. Prediction of ectA, ectB, and ectC, these were coded for EctA, EctB, and EctC and the length of the amino acids were 18.8kDa(171 amino acid)、46.6kDa(423 amino acid)、14.9kDa(132 amino acid)respectively.The ectABC gene cluster which was 2408 bp was cloned into the expression vector pET-28 a and transformed into E coli BL21(DE3) by using the high fidelity enzyme Pfu. E. coli BL21(DE3) containing pET28a-ectABC plasmid was able to synthesize ectoine by the inducing of 0.5mmol/L IPTG after 6h at 37 in LB culture medium.However, its salt tolerance was ℃not significantly improved.The precursor of L-aspartic-β-semialdehyde was generated under the catalysis of Aspartokinase(Ask) and Aspartate-semialdehyde dehydrogenase(Asd), and Ask and Asd were the key rate limiting enzymes in the synthesis of ectoine. Only about 30%(132 microorganisms) of the 440 species microbial genomes which containing the ectoine or hydroxyectoine synthesis gene existed a Ask gene nearby the ectABC/D gene cluster.. In the whole genome of Streptomyces pactum Act12, Ask and Asd genes were single copied.In this paper, the key rate limiting enzyme Ask and Asd in the synthesis of ectoine were over expressed in Streptomyces pactum Act12 in order to improve the salt tolerance of Streptomyces pactum Act12 and the yield of ectoine.