Journal of Zhejiang University SCIENCE A
(Monthly)

2006   Vol. 7   Suppl. II   p. 366-370

Improvement of spinosad producing Saccharopolyspora spinosa by rational screening

Jin Zhi-Hua^†1,2, Wu Jian-Ping², Zhang Yuan², Cheng Xiu², Yang Li-Rong², Cen Pei-Lin²

(¹Department of Biological and Pharmaceutical Engineering, Ningbo Institute of Technology, Zhejiang University, Ningbo 315100, China)
(²Institute of Bioengineering, School of Materials Science and Chemical Engineering, Zhejiang University, Hangzhou 310027, China)
^†E-mail: zhking@zju.edu.cn
Received Nov. 1, 2005 revision accepted May 29, 2006

Abstract: Spinosad (spinosyns A and D) is a mixture of secondary metabolites produced by Saccharopolyspora spinosa. It is used in agriculture as a potent insect control agent with exceptional safety to non-target organisms. Spinosyns are macrolides with a 21-carbon, tetracyclic lactone backbone to which the deoxysugars forosamine and tri-O-methylrhamnose are attached. According to the pathway and regulation of spinosad biosynthesis, a rational selection procedure with u.v. mutation was performed to obtain high spinosad producing strain. The spinosad resistant mutants, the rhamnose resistant mutants, the 2-deoxygen-D-glucose resistant mutants were selected, successively. Eventually, a strain S. spinosa 4~6 was obtained, with its production of spinosad reaching 268 mg/L, which is increased by 121% in comparison with that of the parent strain S. spinosa 1~5. The subculture experiments indicated that the hereditary character of high production of S. spinosa 4~6 is stable. The spinosad fermentation with S. spinosa 4~6 was scaled up in a 10 L fermentor, and a production of 458 mg/L was obtained, which was 71% higher than the production with shaking-flask fermentation.

Key words: Spinosad, Saccharopolyspora spinosa, Strain improvement, Rational screening
doi:10.1631/jzus.2006.AS0366             CLC number: Q815

References:

[1] Chen, J.M., Xu, L.T., 1991. Analysis of Antibiotic Industry. Chinese Press of Pharmacetical Science, Beijing, p.109- 139 (in Chinese).

[2] Jin, Z.H., Wang, M.R., Cen, P.L., 2002a. Production of teicoplanin by valine analogue-resistant mutant strains of Actinoplanes teichomyceticus. Appl. Microbiol. Biotechnol., 58(1):63-66.

[3] Jin, Z.H., Lin, J.P., Xu, Z.N., Cen, P.L., 2002b. Improvement of industry-applied rifamycin B-producing strain, Amycolatopsis mediterranei, by rational screening. J. Gen. Appl. Microbiol., 48(6):329-334.

[4] Jin, Z.H., Lei, Y.L., Lin, J.P., Cen, P.L., 2006. Improvement of pristinamycin-producing Streptomyces pristinaespiralis by rational screening. World J. Microbiol. Biotechnol., 22(2):129-134.

[5] Kirst, H.A., Michel, K.H., Martin, J.W., Creemer, L.C., Chio, E.H., Yao, R.C., Nakatsukasa, W.M., Boeck, L., Occolowitz, J.L., Paschal, J.W., et al., 1991. A83543A-D, unique fermentation-derived tetracyclic macrolides. Tetrahedron Lett., 32(37):4839-4842.

[6] Leblihi, A., Germain, P., Lefebvre, G., 1987. Phosphate repression of cephamycin and clavulanic acid production by Streptomyces clavuligerus. Appl. Microbiol. Biotechnol., 28:44-51.

[7] Madduri, K., Waldron, C., Matsushima, P., Broughton, M.C., Crawford, K., Merlo, D.J., Baltz, R.H., 2001a. Genes for the biosynthesis of spinosyns: Applications for yield improvement in Saccharopolyspora spinosa. J. Ind. Microbiol. Biotechnol., 27(6):399-402.

[8] Madduri, K., Waldron, C., Merlo, D.J., 2001b. Rhamnose biosynthesis pathway supplies precursors for primary and secondary metabolism in Saccharopolyspora spinosa. J. Bacteriol., 183(19):5632-5638.

[9] Mertz, F.P., Yao, R.C., 1990. Saccharopolyspora spinosa sp. nov. isolated from soil collected in a sugar mill rum still. Int. J. Syst. Bacteriol., 40:34-39.

[10] Sparks, T.C., Thompson, G.D., Kirst, H.A., Hertline, M.B., Larson, L.L., Worden, T.V., Thibault, S.T., 1998a. Biological activity of the spinosyns, new fermentation derived insect control agents, on tobacco budworm (Lepidoptera: Nocuidae) larvae. J. Econ. Entomol., 91:1277-1283.

[11] Sparks, T.C., Thompson, G.D., Kirst, H.A., Hertlein, M.B., Mynderse, J.S., Turner, J.R., Worden, T.V., 1998b. Fermentation-derived Insect Control Agents: the Spinosyns. In: Hall, F.R., Menn, J.J. (Eds.), Methods in Biotechnology, Biopesticides: Use and Delivery. Humana Press, Totowa, NJ, 5:171-188.

[12] Waldron, C., Madduri, K., Crawford, K., Merlo, D.J., Treadway, P., Broughton, M.C., Baltz, R.H., 2001a. A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa. Antonie Van Leeuwenhoek, 78(3-4):385-390.

[13] Waldron, C., Matsushima, P., Rosteck, P.R.Jr, Broughton, M.C., Turner, J., Madduri, K., Crawford, K.P., Merlo, D.J., Baltz, R.H., 2001b. Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa. Chem. Biol., 8(5):487-499.

[14] Zhang, Y., Jin, Z.H., Lin, J.P., Cen, P.L., 2003. Determination of spinosyns by HPLC. Pesticides, 42(10):27-28 (in Chinese).