Study On Building Coenzyme Q₁₀ High Yield Strain By Protoplast Fusion

The coenzyme Q₁₀ is a prenylated benzoquinone lipid, and it is also named ubiquinone. The chemical name is 2,3-dimethoxy-5-methy-6-decaprenyl-1,4-benzoquinone. Members of the coenzyme Q₁₀ are found in microorganisms, plants, and the heart, kidney, liver of animals, where they function are redox carriers in mitochondria or in association with respiratory processes of electron transport chains. It is an antioxidant produced naturally in the cells. Coenzyme Q₁₀ exhibited excellent medical and physiological activities against various diseases such as heart diseases, cancer, hepatitis, Parkinson and so on. In this paper, we studied the protoplast preparing and fusion conditions of Agrobacterium tumefaciens mutant AT-N10 and Rhodopseudomonas rubrum 1.5005, and screened a CoQ₁₀ high yield strain A-Rh15 to provide basic statistic and make experimental instruction for further extending coenzyme Q₁₀ production. The main research contents and results are as follows:(1)We determined the proper protoplast preparing and regeneration conditions of AT-N10 and Rh1.5005 seperately. The proper conditions for AT-N10: 13.5h aged, hypertonic solution 0.8mol/L NaCl, enzymatic time 75min, enzymatic temperature 35℃, enzymatic concentration 0.2mg/ml; the proper conditions for Rh1.5005: 45h aged, hypertonic solution 0.5mol/L sucrose and 0.02mol/L MgCl2·6H2O, enzymatic time 60min, enzymatic temperature 37℃, enzymatic concentration 3mg/L.(2)We determined the proper inactivation parameter of AT-N10 and Rh1.5005. After Rh1.5005 was inactivated 20min at 60℃and AT-N10 was inactivated 30min at 55℃, they could be fused by PEG at 32°C. And after UV treated 320s and 400s separately, AT-N10 and Rh1.5005 could be fused by PEG at 32°C.(3) Using AT-N10 and Rh1.5005 as parent strains, after protoplast inactivation, fusion, primary screening and ferment repeating screening, we screened a performance markable raised strain A-Rh15. It can yield CoQ₁₀ 15.0278mg/L, which was 156.46% higher than the parent strain. And the coenzyme Q₁₀ content in the dried cells reached 2.8145mg/g, which was 69.53% higher than the parent strain. A reduction of only 0.3% and 0.4% in the coenzyme Q₁₀ yield of A-Rh15 was observed after five and ten times of subculture. Therefore, the obtained mutant is stable strain. (4) The optimizing medium for coenzyme Q₁₀ biosynthesis was formulated vas Plackett -Burman design and Box-Behnken response surface analysis method (RSM). The optimum fermentation medium consisted of yeast extract 14.585g, CH₃COONa·3H₂O1.448g, KH₂PO₄0.802g, NaCl0.292g, glucose 10g, peptone 5g, Na₂C₄H₄O₆1g, Na₂S₂O₃·5H₂O0.1g, K₂H_PO₄1g, (NH₄)₂SO₄1g, MgSO₄·7H₂O0.7g, CaCl₂·2H2O0.05g, vitamin mixture 1mL, trace element solution 1mL, distilled water 1L, pH6.7. The biomass of A-Rh15 was 8.5415g/L under the optimum condition, which was 59.97% higher than that of non-optimized medium which was 5.34g/L.