Fed-Batch Fermentation for Spinosad Production in an Improved Reactor

Abstract

As a kind of aerobic bacteria, Saccharopolyspora spinosa exhibits a high demand for oxygen. In the fermentation process, the methods of increasing ventilation and improving agitation speed are usually adopted to achieve higher values of dissolved oxygen. These methods decrease the efficiency of spinosad biosynthesis. In this study, an improved reactor was designed to solve these problems. The exhaust gas reflux device, impellers, and baffles were improved. Furthermore, we established the kinetic models for the cell growth, substrate consumption and spinosad generation in batch fermentation process. The simulation results were in good agreement with the experimental data. Spinosad production reached 583.86 mg/L after employing the suitable feeding strategy by fed-batch fermentation in the improved reactor, whereas it was only 157.01 mg/L before optimization. The method described can provide insight to strengthen spinosad production and can be extended to the culturing process of filamentous aerobic bacteria.

Keywords

Spinosad / Bioreactors / Fed-batch fermentation / Saccharopolyspora spinosa

References

[1] Hertlein MB, Mavrotas C, Jousseaume C, et al. A review of spinosad as a natural product for larval mosquito control. J Am Mosq Control Assoc, 2010, 26(1): 67-87.

[2] Williams T, Valle J, Vinuela E. Is the naturally derived insecticide Spinosad^® compatible with insect natural enemies?. Biocontrol Sci Tech, 2003, 13(5): 459-475.

[3] Dripps JE, Boucher RE, Chloridis A, et al. Lopez O, Fernandez-Bolanos JG, et al. Green Trends in Insect Control. The spinosad insecticides, 2011, London: Royal Society of Chemistry 163-212.

[4] Bai Y, Zhou PP, Fan P, et al. Four-stage dissolved oxygen strategy based on multi-scale analysis for improving spinosad yield by Saccharopolyspora spinosa ATCC49460. Microb Biotechnol, 2015, 8(3): 561-568.

[5] Liang Y, Lu WY, Wen JP (2009) Improvement of Saccharopolyspora spinosa and the kinetic analysis for spinosad production. Appl Biochem Biotechno l52(3):440–448

[6] Silva-Santisteban BOY, Maugeri F. Agitation, aeration and shear stress as key factors in inulinase production by Kluyveromyces marxianus. Enzyme Microb Technol, 2005, 36(5/6): 717-724.

[7] Xia X, Lin SJ, Xia XX, et al. Significance of agitation-induced shear stress on mycelium morphology and lavendamycin production by engineered Streptomyces flocculus. Appl Microbiol Biotechnol, 2014, 98(10): 4399-4407.

[8] Jin ZH, Xu B, Lin SZ, et al. Enhanced production of spinosad in Saccharopolyspora spinosa by genome shuffling. Appl Biochem Biotechnol, 2009, 159(3): 655-663.

[9] Jha AK, Pokhrel AR, Chaudhary AK, et al. Metabolic engineering of rational screened Saccharopolyspora spinosa for the Enhancement of Spinosyns A and D Production. Mol Cells, 2014, 37(10): 727-733.

[10] Tang Y, Xia LQ, Ding XZ, et al. Duplication of partial spinosyn biosynthetic gene cluster in Saccharopolyspora spinosa enhances spinosyn production. FEMS Microbiol Lett, 2011, 325(1): 22-29.

[11] Waldron C, Matsushima P, Rosteck PR, et al. Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa. Chem Biol, 2001, 8(5): 487-499.

[12] Wang C, Zhang XL, Chen Z, et al. Strain construction for enhanced production of spinosad via intergeneric protoplast fusion. Can J Microbiol, 2009, 55(9): 1070-1075.

[13] Luo YS, Ding XZ, Xia LQ, et al. Comparative proteomic analysis of Saccharopolyspora spinosa SP06081 and PR2 strains reveals the differentially expressed proteins correlated with the increase of spinosad yield. Proteome Sci, 2011, 9: 40-51.

[14] Wang XY, Zhang CB, Wang ML, et al. Genome-scale metabolic network reconstruction of Saccharopolyspora spinosa for Spinosad Production improvement. Microb Cell Fact, 2014, 13: 41-47.

[15] Zhao FL, Xue CY, Wang ML, et al. A comparative metabolomics analysis of Saccharopolyspora spinosa WT, WH124, and LU104 revealed metabolic mechanisms correlated with increases in spinosad yield. Biosci Biotechnol Biochem, 2013, 77(8): 1661-1668.

[16] Jin ZH, Cheng X, Cen PL. Effects of glucose and phosphate on spinosad fermentation by Saccharopolyspora spinosa. Chinese J Chem Eng, 2006, 14(4): 542-546.

[17] Yanez KP, Martin MT, Bernal JL, et al. Determination of spinosad at trace levels in bee pollen and beeswax with solid-liquid extraction and LC-ESI-MS. J Sep Sci, 2014, 37(3): 204-210.

[18] Jin ZH, Wu JP, Zhang Y, et al. Improvement of spinosad producing Saccharopolyspora spinosa by rational screening. J Zhejiang Univ Sci A, 2006, 7(2): 366-370.

[19] Sanchez S, Chavez A, Forero A, et al. Carbon source regulation of antibiotic production. J Antibiotics, 2010, 63(8): 442-459.

[20] Wang LP, Ridgway D, Gu TY, et al. Bioprocessing strategies to improve heterologous protein production in filamentous fungal fermentations. Biotechnol Adv, 2005, 23(2): 115-129.

[21] Li SY, Srivastava R, Suib SL, et al. Performance of batch, fed-batch, and continuous A-B-E fermentation with pH-control. Bioresour Technol, 2011, 102(5): 4241-4250.

[22] Kim HJ, Ruszczycky MW, Choi SH, et al. Enzyme-catalysed [4 + 2] cycloaddition is a key step in the biosynthesis of spinosyn A. Nature, 2011, 473(7345): 109-112.

[23] Xue CY, Duan YJ, Zhao FL, et al. Stepwise increase of spinosad production in Saccharopolyspora spinosa by metabolic engineering. BiochemEng, 2013, J72: 90-95.

[24] Puertas JM, Betton JM. Engineering an efficient secretion of leech carboxypeptidase inhibitor in Escherichia coli. Microb Cell Fact, 2009, 8: 57

[25] Miller GL. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem, 1959, 31(3): 426-428.

[26] Liu H, Zheng ZM, Wang P, et al. Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum. Appl Microbiol Biotechnol, 2013, 97(8): 3363-3372.

[27] Park EY, Koizumi K, Higashiyama K. Analysis of morphological relationship between micro- and macromorphology of mortierella species using a flow-through chamber coupled with image analysis. J Eukaryotic Microbiol, 2006, 53(3): 199-203.

[28] Gong GL, Huang YY, Liu LL, et al. Enhanced production of epothilone by immobilized Sorangium cellulosum in porous ceramics. J Microbiol Biotechnol, 2015, 25(10): 1653-1659.