Research On The Relationship Between Val-A Production And Melanin Secretion From Streptomyces Hygroscopicus5008

Vallidamycin A (Val-A) is a safe and effective anti-fungal antibiotic. Streptomyces hygroscopicus5008is a commonly used atinomycetes strain in industrial fermentation of Val-A for40years. In the process of Val-A fermentation, we found that there was a novel melanin produced by S. hygroscopicus5008, and the production of the melanin was increasing when fermentation went on. In this study, we worked on the relationship between the producing of pigment and Val-A for correlation studies, starting with the relationship between the absorbance values of the fermentation broth and the production of Val-A, suggesting the correlation between melanin and Val-A producing in molecular level by qRT-PCR. And the melanin in the fermentation broth was extracted for the research of physicochemical properties and stability of the pigment. Then we established a Val-A rapid detection method on the basis of studies above. Meanwhile, we detected and conjectured the structure of the melanin using FT-IR infrared spectroscopy methods. If we could extract the melanin of the fermentation broth in the process of industrial Val-A fermentation, we could make the best use of fermentation product and provided a theoretical basis for the development and utilization of an novel melanin.The main research results are as follows:1. The relationship between Val-A production and pigment secretionIn this experiment, we found that the Val-A synthesis and pigment secretion in different fermentation conditions, such as adding exogenous stimulus, changing fermentation culture medium and temperature, had a stable changing trend. In the preponderant environment, both synthetic quantity of Val-A and pigment were increased. In a disadvantage environment, both synthetic quantity of Val-A and pigment were reduced. This suggests that the Val-A synthesis and pigment secretion have a stable correlationship.2. The relationship between Val-A production and pigment secretion in gene levelThe transcriptional level of three operons (valABC, valKLMN, valG) which represent eight structural genes for Val-A biosynthesis, pigment encoding gene (SHJG4405) and two putative regulator genes (afsR, ugp) in different fermentation conditions were monitored during fermentation. The results suggest that when the fermentation was conducted at a disadvantage environment, such as low yield medium and low temperature situation, the expression level of these genes were significantly down-regulated. Likewise, when the fermentation was conducted at a preponderant^environment, such as adding exogenous stimulant and changing the initial pH of the fermentation medium, the expression levels of these genes were significantly up-regulated. Although the transcriptional level changes in different growth periods, it shows collaborative related to the Val-A production overall.3. Characterization and stability of the pigmentThe pigment was extracted from the fermentation supernatant of Streptomyces hygroscopicus5008. Its stability to sunlight, ultraviolet light, temperature, pH valve, hydrogen peroxide (H2O2) as an oxidant, vitamin C as a deoxidizer, sucrose, glucose, sodium benzoate and metal ions was evaluated. Results showed that the pigment exhibited strong tolerance to sunlight and radiation and it had good heat stability and certain oxidation and reducing resistance. pH exerted a large effect on the pigment stability. It would precipitate and became dark claret-red in strong acid condition and it was stable in neutral and alkaline solution. Ca+, Mn2+and Fe3+affected the pigment much more than Mg2+, K+and Na+, while Al3+could protect its color. The pigment was stable to food additives such as glucose, sucrose, dicarbonate and sodium benzoate, while citric acid could make the pigment precipitate.4. The presumably structural information of pigmentAccording to the infrared spectrogram of the pigment extracted from Streptomyces hygroscopicus5008comparing with other natural melanin, this pigment has the common characteristic with other melanin pigment, i.e., the elementary structure is indole ring, but melanin came from different sources exerted certain structure different, the infrared absorption peaks were not exactly the same. Thus, we speculate this pigment is a compound of eumelanin and phaeomelanin.5. Construction of a fast detecting method of Val-AWe associated the Val-A production with spectral absorption value of supernatant, confirmed the fitting equation is y=19.410x4-51.750x3+39.262x2+8.604x-0.643, in which x is referred to spectral absorption value of supernatant and y referred to Val-A concentration. This fitting formula shows a high degree of accuracy with a Adj R-square reached to0.9928,which means it has broad application prospects in the future industrial development.6. A method to increase the production of Val-A by quorum sensingIn this study, quorum sensing was used as a method to improving the production of Val-A in Streptomyces hygroscopicus5008. We added fermentation supernatant containing quorum sensing signal molecules into the fermentation broth to stimulate the cell response and promote the expression of related genes, and making the secretion of certain secondary metabolites in advance. This experiment determined the optimum fermentation supernatant adding time was12h after fermentation began, the optimum concentration of added fermentation supernatant was0.5%. After96h fermentation, the absolute amount of accumulated Val-A increased from12.47g/L to16.39g/L, which inferred that without increasing the energy consumption, this method could achieve higher Val-A production and reduced production costs. Meanwhile, after96h fermentation, it reached the highest yield of Val-A, which shortened the fermentation period from120h, and achieved a greater productivity.