Breeding And Fermentation Optimization Of Avilamycin High-producing Strains

Avilamycin is a feed antibiotic with excellent growth promoting and metabolic regulation functions produced by Streptomyces viridochromogenes,which has been widely used in livestock or poultry breeding.At present,the low production capacity of avilamycin production strains,immature fermentation process,and high production costs restrict its industrial production and application in China.Multiple mutagenesis breeding and rational screening of avilamycin wild strains were carried out to obtain high-quality and high-yielding mutant strains in this research.The fermentation process was optimized by various strategies to construct a more efficient and reasonable fermentation system,which might provide some theoretical reference for the breeding and fermentation of avilamycin.The main results of this research are listed as follows:（1）Ultraviolet（UV）,Atmospheric and Room Temperature Plasma（ARTP）,and the combinational mutagenesis of UV and ARTP was respectively performed on Streptomyces viridochromogenes S219 in this research with Ca Cl₂as a resistance screening pressure to obtain avilamycin high-yielding mutant strains.After parameter optimization,the optimal mutagenesis time of UV,ARTP,UV+ARTP was 180 s,70 s and 70 s,respectively,and the corresponding lethality of the three modes was 86.05%,94.17%and 92.59%,respectively.The highest positive mutation rate of the three modes was 8.75%,10.00%and 9.58%,respectively.Three mutant strains with significant production improvement and better genetic stability were obtained by multiple rounds of mutagenesis screening at 25g/L Ca Cl₂as the screening pressure,named Z-6,A-9 and F-23,whose avilamycin production was 29.31,36.84 and 45.73 mg/L,respectively.Compared with the wild strain S219（18.56 mg/L）,their avilamycin yield increased by 57.92%,98.49%and 146.39%,respectively.（2）The morphological comparison of mycelium development of the high-yielding mutant strains were further conducted,and their fermentation performance were analyzed and compared from various parameters such as avilamycin yield,dry cell weight,reducing sugars,total sugars,and amino nitrogen consumption during the fermentation process.Morphological observation showed that the mutant strains Z-6,A-9 and F-23 had smaller colonies,weaker pigment-producing ability,more nodular elliptic spores,faster growth rate and stronger bacteriostatic effect.The maximum DCW of the mutant strains Z-6,A-9 and F-23 was 14.21,15.48 and 18.99 mg/m L,respectively,which was 10.24%,20.09%and47.32%higher than the wild strain（12.89 mg/m L）.Compared with the wild strains,the utilization rate of total sugars by mutant strains Z-6,A-9 and F-23 increased by 2.14%,10.11%and 15.82%,and the utilization rate of reducing sugars was increased by 9.16%,15.14%and 16.28%,respectively.The utilization rate of nitrogen source increased by 9.55%,14.08%and 18.02%,respectively,and the synthesis period of avilamycin was longer than that of wild strain by 36 h,48 h and 48 h,respectively.The cell growth,substrate consumption,precursor supply and antibiotic synthesis ability of mutant strains Z-6,A-9 and F-23 were all stronger than those of wild strains,which indicated that UV and ARTP mutagenesis technology combined with Ca Cl₂rational screening was an effective method of breeding avilamycin high-yielding mutant strains.（3）The seed medium and fermentation medium were optimized by single factor and orthogonal experiments,and the optimal medium formula was obtained.Moreover,the fermentation parameters were further optimized to determine the optimal fermentation basic process.The optimal formulation of seed medium was as follows（w/v）:Glucose 2%,Peptone 2%,Ca²⁺0.05%,Mg²⁺0.05%,Na Cl 0.2%,K₂HPO₄0.01%.Therefore,the yield of avilamycin was increased to 46.14 mg/L by optimizing the seed medium.The optimal combination of fermentation medium was（w/v）:Soluble starch 1.5%,Dextrin 0.5%,Glucose 1%,Corn syrup 0.2%,Soybean meal powder 0.6%,Soy flour 0.4%,Peptone 0.6%,Ca Cl₂0.2%,Mg SO₄7H₂O 0.1%,K₂HPO₄0.1%,Fe SO₄7H₂O 0.01%,KNO₃0.1%.The best fermentation conditions of mutant strain F-23 were as follows:initial p H of fermentation medium 7.4,inoculum volume 6%（w/v）,seed age 30 h,fermentation time 216 h,fermentation temperature 28℃,rotation speed 200 r/min,liquid volume 35 m L/250 m L.Finally,the avilamycin yield of the mutant strain F-23 has been increased to 53.25 mg/L under the optimal fermentation conditions,which was 16.44%higher than before.These results indicated that optimization of the initial p H of fermentation medium,seed quality,inoculation conditions,shaking fermentation conditions and other basic parameters was an effective measure to improve the production of avilamycin mutant strain F-23.（4）Different types of oxygen vectors were added to the fermentation system to improve the fermentation dissolved oxygen level.The results showed that adding 1.3%（v/v）n-dodecane as the oxygen vector at 24 h of fermentation had a significant effect on improving the fermentation of avilamycin.Compared with the control group,.the addition of n-dodecane（1%,v/v）increased the dry cell weight of the strain F-23 by 7.24%and the yield of avilamycin by 13.03%（52.58 mg/L）.After adding Tween-20 or N-hexane,the dry cell weight of the strain F-23 decreased by 11.53%and 17.60%,and the production of avilamycin decreased by 20.41%and 31.66%,respectively,compared with the control group,indicating that Tween-20 and N-hexane might inhibit the synthesis of avilamycin.In addition,N-decane and N-hexadecane had no significant effect on the growth of the strain F-23 and the synthesis of avilamycin.（5）By adding precursors such as L-valine,D-xylose and sodium acetate and optimizing the feeding mode of glucose,the biosynthesis of avilamycin was improved and the optimal fermentation feeding strategy was established.The optimal fermentation supplement strategy was as follows:adding 1.25%sodium acetate at 30 h,adding 0.25%L-valine and 1.00%D-xylose at 36 h,and adding 0.15%glucose at constant speed intermittently.Finally,the avilamycin yield of mutant strain F-23 was increased to 64.88 mg/L,which was 21.84%higher than before.