Optimization Of Fermentation Conditions Of Collaborative Production Of Natamycin And Chitinase From Streptomyces Lydicus A01-chit33CT

Streptomyces spp and Trichoderma spp are both well-known antagonistic organisms. Streptomyces usually depends upon production of a range of antibiotics including natamycin against plant diseases, however, Trichoderma performs biocontrol of plant diseases mainly relying on massive production of cell wall degrading enzymes, e.g. chitinase which can degrade chitin component within cell wall of fungal pathogens and could be used for inhibiting a range of fungal pathogens growth. Therefore the two antagonistic organisms offer two different mechanisms against plant pathogens involving antibiosis and lysis of pathogen as well as mycoparasitism, respectively.For using the advantages of two biocontrol factors from Trichoderma and Streptomyces, in previous study we had transfered gene chit33 of Trichoderma into Streptomyces and obtained engineering strain Streptomyces A01-chit33 CT. Streptomyces A01-chit33 CT, on the one hand, can inhibit plant pathogen growth by production of natamycin, and on the other hand, enables destruction of plant pathogen cell wall structures since chitinase expressed by chit33 can hydrolyze the chitin component in fungal pathogen cell wall, thus the engineering strain can provide a coordination control effect of natamycin and chitinase against plant diseases. To optimize the synergistic action of both biocontrol factors as much as possible during fermentation process, firstly, we conducted some single-factor experiments to determine the primary fermentation conditions. The major components contained in basic medium were involved as follows: chitin powder 10g/L(after 4days), glucose 40g/L, soybean meal 30g/L,soya peptone 10g/L.The cultural conditions were set up as follows: initial pH 6.0, liquid medium volume 50/250 mL,inoculum volume 3%,temperature 28 ℃, rotation speed 180r/min. The highest production of natamycin could reach 1.5g/L and chitinase activity reach 990U/mL under the optimal fermentation conditions. Secondly, to increase yields of natamcyin, the fermentation conditions were further optimized by using Plackett-Burman design and response surface methodology(RSM). The optimal medium concentrations were as follows: 40.37g/L glucose, 28.12g/L soybean meal, 14.91g/L soya peptone, CaCO3 5g/L, Mg SO4﹒7H2O 0.5g/L,K2HPO4 0.5g/L. Under the optimal fermentation conditions, natamycin yield was enhanced by more than 0.5 times. Thirdly, we searched the optimal conditions for maximal production of natamycin and chitinase along the course of fermentation process either by adding chitin powder at different time points or by adding different concentrations of chitin powder. The optimal parameters of fermentation condition were achieved as follows: adding chitin powder 11g/L in fermentation medium at 4 day during fermentation process. Under the fermentation conditions, the productions of natamycin and chitinase reached 2.23g/L and 1049U/mL, respectively. Fourthly, to validate the model described above, the further amplified experiments were performed in a 7 liter fermentor and results showed that the predicted data obtained from flask are matched well to practical ones in the fermentor. The biocontrol efficiency of fermentation broths from Streptomyces A01-chit33 CT was also evaluated against Botrytis cinerea a tomato gray mold pathogen in vitro and in vivo. Results demonstrated that the fermentation broth product from optimized model showed significantly inhibition against pathogen growth and disease infection.