Study On The Strain Improvement, Culture Condition Optimization And Fermentation Kinetics For Mildiomycin Production

Mildiomycin is a kind of nucleotide antibiotics produced by fermentation with Streptoverticillium rimofaciens. It is able to strongly inhibit or even kill various fungus, and is especially effective to prevent powdery mildew diseases in many kinds of plants with remarkably low toxicity in mammals and fishes. Mildiomycen has been commercially produced and applied as a new generation of pesticides and have not studied in China currently. The aim of this work is to increase the productivity of mildiomycin fermentation. After thorough and particularity study in strain improvement by rational screening, protoplast fusion and genome shuffling, medium composition and culture condition optimization as well as fermentation kinetics, the mildiomycin productivity is increased greatly. This work lays a sound foundation for the industrialization of mildiomycin fermentation.The following major progresses were achieved in this work:A bioassay method for quantitative determination of mildiomycin was developed using a strain of Rhodotorula rubra AS 2.166 as the indicator organism and the potato dextrose agar at pH 7.0 as the test medium. Based on this technique, a simple, low-cost and timesaving agar-plug method was constructed for preliminary screening of mutants. This method was successfully applied in the high throughput screening procedure.According to the biosynthesis path of mildiomycin, a high-yielding strain MIL29 of mildiomycin was obtained by rational breeding of the original wild type strain MIL01 with the traditional induced mutation. Mildiomycin productivity of MIL29 was 1010 mg/L, which was 2.6 times higher than that of M1L01.The optimum conditions for preparing and regenerating MIL29 protoplast were determined. Under the optimum conditions, both the protoplast concentration and the regeneration rate of protoplast were raised by about 1.6 times. The effect of poly(ethylene glycol) with various molecular weights at various concentrations on the fusion frequency of protoplasts was evaluated. The results show that 40% of poly(ethylene glycol) with molecular weight of 6000 was the optimum fusion agent and the optimum fusion time was 5 minutes. A high-producing strain MIL32, with which mildiomycin productivity was increased by about 9% as compared with M1L29, was obtained.Through two runs of multi-parent protoplast fusion based on the theory of whole genome shuffling, a high-yielding strain MIL89 was obtained, with which mildiomycin productivity reached 1950 mg/L, which was about 77% higher than that with MIL32.Several precursors which could enhance mildiomycin biosynthesis were found. Among them, the combination of 80 mmol/L N,N-dimethyl acetamide and 150 mmol/L choline chloride could accelerate the consumption of sugar and ammonium nitrogen and enhance mildiomycin biosynthesis, also the production of mildiomycin was advanced. The mildiomycin productivity was increased by at least 12%.The effect of the production medium composition and the fermentation conditions on the biosynthesis of mildiomycin was investigated with the Plackett-Burman design. Three most important factors, corn starch, choline chloride and N,N-dimethyl acetamide, for mildiomycin biosynthesis were selected from total of 10 factors. The statistical analysis by response surface methodology also shows that choline chloride and N,N-dimethyl acetamide exhibited a positive cooperative effect on mildiomycin biosynthesis. Mildiomycin productivity under the optimum fermentation conditions was 2607 mg/L in shake flask culture, which was 13% higher than that under the original conditions.The characteristics of the batch, semi-fed-batch and fed-batch fermentation in a 10-liter bioreactor were investigated. In the late phase of the batch fermentation, mildiomycin biosynthesis would be repressed due to the exhaustion of nutrition substances. Semi-fed-batch culture could improve the substrate supply and increase mildiomycin productivity by about 38%. Fed-batch fermentation could prevent the dramatic transient phenomenon in fermentation environment aroused by semi-fed-batch culture; therefore, the mildiomycin productivity was further increased by about 16% to 2011 mg/L.A kinetics model of mycelial growth comprised of a segregated morphological model, a population growth model and a macroscopical pellets model was established. The proposed model could well simulate the mycelial growth, substrate consumption and mildiomycin biosynthesis during the batch fermentation. Based on some idealized assumption, a relatively simple model was derived and demonstrated practical.