| Objective In order to provide an excellent source of fermentation bacteria for engineering production of Ectoine,Halomonas sp.XH26 from the Xiao Chaidan Saline lake,was mutated by UV radiation to obtain the mutant strain with high yield of Ectoine,providing an excellent source of fermentation bacteria for engineering production of Ectoine.Methods Based on the great application value and research significance of Ectoine,Halomonas sp.XH26 from Xiao Chaidan Salt Lake was selected as the research object in this study.Morphological observation,physiological and biochemical tests and 16 S rRNA gene sequencing were used to determine the classification status of the strain.The mutants with high yield of Ectoine were selected by UV mutagenesis.The positive mutants with more than two times of the original strain were screened by UV mutagenesis.The intracellular production of the mutants was determined by HPLC,and the mutants were cultured continuously to study the stability of the production of the mutants.Finally,through morphological comparison,physiological and biochemical identification,nutrient demand comparison and molecular difference analysis of the synthesis gene of Ectoine,the mechanism of the surge in the production of mutant strain Ectoine was preliminarily explored,providing reliable experimental basis and strains for subsequent experiments,and providing excellent fermentation sources for the engineering production of Ectoine.Results The original strain XH26(the optimized yield of Ectoine is 456.82 ± 15.72 mg/L,CDW is 0.71 ± 0.04 g/L),after 9 rounds of UV cycle mutagenesis(50 s is the best mutagenesis time),7 strains with high yield of Ectoine were screened,and the yield of Ectoine was greatly increased(about 200 %).Among them,HU09-32,the strain with the largest accumulation,has a yield of 1351.09 ± 17.69 mg/L(an increase of 290 %).Seven mutants were cultured continuously for 40 days(inoculated for 2 days/time).The yield of intracellular Ectoine is stable,and its growth characteristics,proliferation rate and accumulation are significantly better than those of the original strain XH26,which has a good potential for fermentation production and industrial application.The optimal fermentation conditions were determined by single factor analysis.Under the optimized fermentation conditions,combined with single factor experiment,Plackett-buman experiment and response surface method,the optimal fermentation medium scheme was determined as follows: Na+ 1.42 mol/L,K+ 0.5 mol/L,L-monosodium glutamate 0.039 mol/L,Gasein enzymatic hydrolysates 19.7 g/L,Mg SO4.7H2 O 0.10 mol/L,Sodium citrate 0.012 mol/L,Ca Cl2 0.2 g/L and Yeast extract 2.0 g/L.Through the model validation experiment,the actual yield of the mutant strain Ectoine is 1615.94 mg/L,and the cell dry weight(CDW g/L)is 0.73 g/L,which is basically consistent with the yield predicted by response surface analysis.The yield of Ectoine is increased by 19.70 % after the optimization of the medium.Conclusion Through electron microscopy,it was found that the mutant HU09-32 was significantly shorter than XH26,and could not use Inositol,Lactose,Rhamnose,soluble starch and D-galactose;the demand for KCl,L-monosodium glutamate and Gasein enzymatic hydrolysates was significantly increased.In the accumulation of Ecotoine,it showed a better ability of continuous synthesis.By NCBI blast comparison,the similarity of ect ABC gene between XH26 strain and mutant strain was 100%,indicating that there was no difference in the synthesis gene fragment of econine,and the preliminary analysis was not related to the increase of the yield of Econine. |
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