Screening Of High Productivity Coenzyme Q₁₀ Strain And Optimization Of Fermentation Conditions

Coenzyme Q₁₀ (CoQ₁₀, ubiquinone) is an important biochemical compound receiving increased attention as a nutraceutical dietary supplement for its known benefits in the prevention of aging and cardiovascular problems. Extensive attempts have been made to produce CoQ₁₀ to meet the growing demands. The production of CoQ₁₀ follows one of the three routes: extraction from biological tissues, chemical synthesis, and microbial fermentation. In the wake of environmental awareness, the first two options became least desirable due to the inherent uses of solvents and chemicals in the process. Microbial fermentation, on the contrary, offers an environmentally benign option based on the enzymatic catalysis at the cellular level for CoQ₁₀ assembly. Moreover, this approach is attractive to the industry because the process is easy to control at a relatively low production cost. In this study, A. tumefaciens 1.2554 was treated with HHP, UV, and DES, and strain PK38 was isolated using selection marker. The CoQ₁₀ production of PK38 was investigated using batch and fed-batch cultures. The main results are as following:(1) The yield of CoQ₁₀, an intracellular product extracted from Agrobacterium tumefaciens cells, is dependent on the effectiveness of cell lysis post fermentation. Various cell lysis approaches were investigated, including ultrasound, repetitive freezing/thawing, grinding, and acid-heat treatment. The acid-heat combination using hydrochloric acid was found the most effective in releasing CoQ₁₀, followed by lactic, sulfuric, phosphoric, and oxalic acids. The most significant processing parameters, namely the ratio of acid solution volume and bacteria weight (A/B ratio), incubation temperature, and reaction time, were optimized by using the Central Composite Design (CDD) with a quadratic regression model built by Response Surface Methodology (RSM). The highest CoQ₁₀ yield at 1.518 mg/g dry cell was attained using hydrochloric acid (3 mol/L) under optimal A/B ratio, temperature, and time at 10.8 mL/g, 84.2°C, and 35.3 min, respectively.(2) A. tumefaciens 1.2554 was subjected to a series of treatment including the high hydrostatic pressure (HHP) treatment, UV irradiation, and Diethyl sulfate (DES) treatment. Through these treatments, a mutant strain PK38 with high-yield of CoQ₁₀ was isolated, screened by selecting mutants resisting the respiration chain inhibitor (sodium azide) and the structure analogue (ethionine, daunomycin and Vk3). The stability of the mutants was tested and the strain PK38 had specific CoQ₁₀ content between 2.318 and 2.321, which was 51.51% higher than the original strain.(3) Six nutritional factors, including sucrose, corn steep power (CSP), (NH₄)₂SO₄, MgSO₄·7H₂O, biotin and solanesol were optimized for CoQ₁₀ production using response surface methodology (RSM). The optimal medium for CoQ₁₀ production were (g/L): sucrose 37.2 g/L, CSP 7.1 g/L, (NH₄)₂SO₄ 7.0 g/L, MgSO₄·7H₂O 0.4 g/L, biotin 82.9μg/L, solanesol 0.2 g/L, CaCl₂ 140 mg/L, FeSO₄·7H₂O 0.2 mg/L, ZnSO₄·7H₂O 8 mg/L and nicotinic acid 8 mg/L. Under the optimum condition, the CoQ₁₀ yield was 28.44 mg/L, while the biomass reached 9.25 g/L.(4) The optimal initial pH, culture temperature, inoculum dosage and culture time were 7.2, 30°C, 8%, and 72 h, respectively. Based on the kinetic analysis of the batch fermentation process, a two-stage agitation speed or dissolved oxygen (DO) control strategy was performed. When the agitation speed was gradationally controlled (300 rpm for 0-36 h and 100 rpm for 36-72 h) or the DO concentration was gradationally controlled (90¹00% for 0-36 h and 30% for 36-72 h), both CoQ₁₀ production and specific CoQ₁₀ content were promoted. With DO controlled strategy, the maximal CoQ₁₀ yield and specific CoQ₁₀ content reached 56.46 mg/L and 3.94 mg/g-DCW, respectively, which were 11.71% and 6.78% higher than the best result controlled by constant agitation speeds.(5) A kinetic model of CoQ₁₀ production by batch fermentation with PK38 was studied. After observation of experimental data, a model based on the logistic equation of PK38 growth, CoQ₁₀ accumulation combined non-growth-associated and growth-associated contributions, and consumption of sugar for biomass formation and the maintenance of biomass, was developed. The optimal set of parameters was estimated by fitting the model to experimental data. The results predicted by the model were in good agreement with the experimental observations.(6) The metabolic characteristics under three operation ways of fed-batch fermentation, constant feeding rate fermentation, and exponential fed-batch fermentation were studied in a 5-L fermentator. And the influences of different CoQ₁₀ fermentation modes were investigated. The optimum fermentation mode among x them of CoQ₁₀ was exponential feeding fermentation. With this strategy, the final cell biomass, CoQ₁₀ production, and specific CoQ₁₀ production increased 126.11, 173.12, and 22.76% mg/g-DCW, respectively, compared to those of batch culture.