The Breeding Of Rhodobaber Sphaeroides Train With High Production Coenzyme Q₁₀ By Genome Shuffling

The strain NM-FZ-47producing Coenzyme Q10was screened from strains persevered in the laboratory; its fermentation level of Coenzyme Q10was up to63.4mg/L First, three determination methods of Coenzyme Q10were studied and compared, the ultraviolet spectrophotometric method showed maximum error, the error of the improved method ultraviolet spectrophotometric was relatively small, it could be used for primary strains screening process because of its speedy. The method of high performance liquid chromatography was accurate and reproducible, adapt for strains rescreening process.Rhodobater sphaeroides NM-FZ-47being a starting strain was mutagenized by UV. By screening the mutants resisting PHB、sodium azide, roxithromeycin and kannamy the positive mutant library was set up. From library the six mutant, namely UV-LH-37UV-NaN3-44、NTG-LH-28、NTG-NaN3-17、NTG-PHB-54、NTG-LN-67, whose fermentation level were15%higher than that of original strain, were choosed as the strarting strains for genome shuffling.The protoplast preparation and regeneration methods of Rhodobater sphaeroides were studied. The optimized conditions were as follows:the strain being cultured for18h; lysozyme concentration0.75mg/mL;enzymolysis time30minutes, enzymolysis temperature35℃, osmotic stabilizers0.6mol/L NaCl and the regeneration medium0.6mol/L NaCl of medium. Under the aboved conditions the rate of protoplast preparation and regeneration reached92.1%and25.1%respectively.The conditions optimizations of protoplast fusion of Rhodobacter sphaeroides were studied. The protoplast inactivation methods were determined to be UV irradiation for lOmin and heat irradiation for8min and optimized fusion conditions were as follows:35%PEG, O.Olmol/L Ca2+, temperature30℃, fusion for5min. The last fusion rate could reach9.1×10-3. A high production Coenzyme Q10strain named F3-40was obtained by three rounds of genome shuffling from parent strains namely the mutant strains UV-LH-37、UV-NaN3-44、NTG-LH-28、NTG-NaN3-17、NTG-PHB-54、NTG-LN-67being. The production of Coenzyme Q10was118.26mg/L, increased86.52%more than that of NM-FZ-47.The fermentation optimization of F3-40was studied. Through single factor test the results showed that the optimum fermentation conditions were as follows:the inoculation amount15%, the initial pH6.4and the liquid volume40mL in250mL flask. The results of culture medium optimization showed that the best carbon was glucose; the optimum nitrogrn was yeast powder. A Plackett-Burman design was used to select3important factors from9impact factors which affected Coenzyme Q10fermentation in culture medium; they were glucose, glutamic acid and ammonium sulfate. The optimum compositions of three factors obtained by response surface analysis method were as follows:glutamate6.98g/L, ammonium chloride5.15g/L, glucose24.05g/L. After these optimizations, the fermentation level of F3-42reached213.25mg/L, which was80.32%higher than that of original fermentation.