Screening High Gibberellin Acid Producing Strains From Gibberella Fujikuroi And Optimization On The Fermentation Conditions

Gibberellic acid (GA3) is a commercially important plant growth hormone, which has effect on the plant seed germination and root, stem growth. Industrially it is produced by submerged fermentation using the ascomycetous fungus Gibberella fujikuroi recently named Fusarium fujikuroi. Due to its effective use in agriculture, forestry, horticulture, food brewing and many other areas, the market potential is huge. In China, many manufacturers of producing GA3 had been constructed, but the fermentation level was staged about 1800 mg/L. The cost of GA3 has restricted its use to preclude application. This research will focus on how to improve the yield and quality of GA3, the strain screening, fermentation process optimization, metabolic pathway analysis, fermentation process modeling and optimization feeding strategies and resin adsorption coupling fermentation process were studied systematically, in order to reduce the production cost, promote the development of related industry fields. The main results are as follows:1. Directional screening of high yield GA3-producing strain.With industrial production strain Gibberella fujikuroi 978# as the parent strain, according to the characteristics of filamentous mycelium about multi-nuclear, no sporulation and pigment, the biochemical reasoning breeding (lipase activity as screening index) and resistant selection of the resistance to antifungal agents (nystatin and terbinafine) had been carried out. The lipase-producing mutants were screened and four mutants were obtained by the UV (30 W,20 cm distance,60 s dosage), after hypha were cut mechanically. Moreover, a high-titer strain GL-2 was studied. Adjusted the feeding oil technology in the fermentative experiments, the productivity of GL-2 was increased by 36.4% than the strain 978. About resistance screen, mycelium single cell suspension was combined mutation and screened resistant using the antifungal agents of nystatin and terbinafine respectively. The potential fermentation of mutant ZNL-1, the resistance to nystatin, reached 2170 mg/L, increased 1.167 times compared with the parent strain. Another mutant strain ZNL13-3, Resistance to 120 μg/L of terbinafine, was obtained and preservation. Its producing gibberellin titer was 2 215±35 mg/L, increased in 1.119 times than the parent strain. The corresponding relation between gibberellin yield and terbinafine resistance was revealed. The production performance and stability of above strains were investigated. After slant subculturing repeatedly, the production potential of the strain could be above 93.2% of the primary strains, with good genetic stability and production application prospect.2. Optimization of fermentation process of GA3.Combined single factor and orthogonal analysis, fermentation medium components and seed quality were optimized. The most suitable GA3 shake flask fermentation conditions:seed culture age 48 h, inoculation 10%, loading liquid rate 20%, culture temperature 28 C, the initial pH5.5; and fermentation medium ratio (g/L): peanut powder 10, starch 50, bean powder 2, anhydrous Magnesium sulfate 0.7, potassium dihydrogen phosphate 0.4, ammonium sulfate 0.01. Based on the above results, the factors of pH, T and DO in the fermentation process were optimized, amplified in 5L fermentation bioreactor. Through mathematics differential treatment in the fermentation process, the trend to the specifically growth rate and specifically production rate was obtained. By the multi-combination and segmentation processing, the optimal profile of fermentation pH, T, DO were obtained and verification experiment was tested.3. Construction, analysis and application of metabolic networks pathway of producing GA3Adopt as the resting cell culture technology, the mycelia of Gibberella fujikuroi were fluidized or immobilized in calcium alginate gel beads. The mycelium age was suit of 96 h to establish the GA3 metabolic pathways analysis in vivo. Based on the results, simplified the GA3 synthesis pathway, glucose consumption, GA3 synthesis, fatty acid C18 synthesis in the mycelium and red pigments (carotenoids) were measuremented, and the synthesis of GA3 metabolic pathways in vivo was constructed in Gibberella fujikuroi after fermentation 96 h. And the effect the mycelium metabolism in vivo on the changes in the external fermentation environment factors (pH、T、DO) were studied. The experimental results before, could be a good suit and mutual confirm compared to the optimization fermentation environment in the previous section. So we construct and provide a method of simple, fast optimization of hyphae anabolic pathway distribution. By Plackett-Burman design and response surface methodology, the optimal concentrations of the variables were determined as:oxalacetate 0.2086 g/L, calcium gluconate 0.0482 g/L, vitamin B20.04096 mg/L. Under such conditions, the GA3 production was increased to 2396 mg/L, which was 11.6% higher than the maximum value in the single factor tests. After the precursor promoters added in the fermentation system, the mycelial metabolism network pathways were analyzed. So the biochemical mechanism was clarified, GA3 synthesis metabolic networks would provide a reference for the implementation in the field of the terpene synthesis metabolites engineering.4. Fermentation process modeling and optimization feeding strategiesAccording to the nitrogen repression phenomena in GA3 fermentation process, the process was divided into two parts:the mycelium growth (0-60 h) and the GA3 synthetic production (60-192 h). For estimation of the optimal model parameters, a non-linear regression technique assisted by MATLAB program was used to minimize the deviation between the model predictions and batch experimental data. The model predictions the system of differential equations describing GA3 fermentation kinetics was solved by an integration program based on the Runge-Kutta method, and so on its scope of application was studied. Then using the model, the optimum initial concentration of carbon source, nitrogen source concentration and C/N were explored. On this basis, glucose feeding strategy was optimized by the genetic algorithm in the fed batch fermentation. Through feeding, GA3 fermentation titer was 2210 mg/L, increased 7.91% compared with the original fermentation process. In addition, we also try to research the structure modeling of mycelia average age. The dynamic structure model as the basic work would discover the relationship between the mycelial morphology analysis and production potential in the future.5. Resin adsorption coupling GA3 fermentationThe mechanism of end product inhibition exists in GA3 biosynthesis of Gibberella fujikuroi. Fermentation technology coupling with resin extraction in situ would decrease the product inhibition and enhance the efficiency of GA3 production. Thought the static adsorption/desorption performance screening, D-100 resin was selected as adsorbent through the method compared the adsorption capacity of the GA3 and elution efficiency. The facts about addition time and the amount of resin were studied by regression and response surface analysis. The experimental results showed that the optimal addition time and amount of resin were 70.75h and 2.02%, accordingly. The total gibberellin productivity and average specific production rate in the fermentation was 221.75 mg and 0.96 mgGA3’gBiomass-1·h-1, respectively. Using the coupling technology in the fermentation, the gibberellin production increased by 149.6% compared with controls without D-100 resin. Based on the results, we constructed GA3 magnetic molecularly imprinted core-shell microspheres (Fe3O4@SiO2 as core, AM-St-TRIM as shell) MMPs, the resin reuse and non-specific adsorption problems were solved. The results of the fermentation experiments in shake flask Indicated that:the MMIPs has good specific absorption capacity, the average GA3 titer in fermentation was 220.82±0.36 mg/100mL and the average GA3 production rate of 0.931±0.042 mg/(g·h).