Abstract
Spinosyns, a secondary metabolite from the fermentation of Saccharopolyspora spinosa, exhibits evident insecticidal activity. The most active components of the spinosyns family are spinosyns A and D, which are macrocyclic lactone antibiotics. Spinosad is a defined combination of the two principal fermentation factors, spinosyns A and D. Spinosad is used on grain storage, vegetable and fruit crops, ornamentals, and turf for pest control because it is toxic to many insects, but relatively nontoxic to mammals. In this study, we combined drug resistance screening and genome shuffling to achieve rapid improvement of spinosad yield of S. spinosa. The starting mutant population was generated by UV irradiation of S. spinosa ATCC 49460 protoplasts, which were then screened for erythromycin or neomycin resistance. Two mutant strains, Ery-13 (erythromycin resistant) and Neo-127 (neomycin resistant), were selected according to their spinosad yield. The highest titers of Ery-13 and Neo-127 strain reached 188 μg/ml and 165 μg/ml, respectively, which are 3.7-fold and 3.3-fold higher than that of the parental strain ATCC 49460. After four rounds of genome shuffling, an improved recombinant EN4-33 with both erythromycin and neomycin resistance was obtained. The highest spinosad yield of the recombinant EN4-33 reached 332 μg/ml, which is 6.6-fold higher than that of ATCC 49460. Results demonstrated that combining genome shuffling with antibiotics resistance screening is an effective approach for the molecular breeding of high-producing strains.
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Acknowledgments
This research was supported by the Key Project Fund of the Shanghai Municipal Committee of Agriculture (No. 2011-1-8), International Scientific and Technological Cooperation (11230705900, 13440701700), Agriculture Science Technology Achievement Transformation Fund (133919N1300), National Natural Science Foundation (31071486), and Key Project Fund of Shanghai Min hang Science and Technology Committee (2012MH059). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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Fig. S1
Spinosad structure (GIF 641 kb)
Fig. S2
Growth trend of spinosad production by genome shuffling and drug resistance. Ery-13, Ery-70, Ery-150: strains that have erythromycin resistance in drug resistance screening. Neo-25, Neo-42, Neo-127: strains that have neomycin resistance in drug resistance screening. Ery-13 and Neo-127 strains were used as the starting strains for genome shuffling. EN1: strains generated from the first round of genome shuffling. EN2: strains generated from the second round of genome shuffling. EN3: strains generated from the third round of genome shuffling. EN4: strains generated from the fourth round of genome shuffling. The best isolate after four rounds of genome shuffling (EN4-33) exhibited a spinosad yield of 332 μg/ml. Bar represents mean spinosad yield with less than 15 % standard deviation (GIF 517 kb)
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Wang, H., Xue, W., He, YM. et al. Improvement of the ability to produce spinosad in Saccharopolyspora spinosa through the acquisition of drug resistance and genome shuffling. Ann Microbiol 65, 771–777 (2015). https://doi.org/10.1007/s13213-014-0917-9
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DOI: https://doi.org/10.1007/s13213-014-0917-9