Strain construction for enhanced production of spinosad via intergeneric protoplast fusion.(Report)

Introduction

Saccharopolyspora spinosa was isolated and identified in 1986 from soil in a defunct sugar-mill rum still in the Virgin Islands (Mertz and Yao 1990). Spinosyns produced by this organism belong to a novel class of insect-control agents (Kirst et al. 1992) that have a unique structure, spectrum of activity, and mode of action (Salgado et al. 1998). Spinosad, a mixture of spinosyn A and spinosyn D, is marketed by Dow AgroSciences for the control of agricultural pests. It exhibits activity of varying degrees against a broad spectrum of insect pests (Thompson et al. 1995) without apparent damage to the environment. In the fermentation process, we found that S. spinosa can use glucose but not starch as a carbon source. Its capacity for spinosad production is relatively low. By limiting the glucose concentration in the fermentation, one can obtain more efficient spinosad production. To obtain a strain suitable for industrial production, one of the aims is to develop a strain that can use soluble starch as the sole carbon source during fermentation. Unfortunately, the regulatory mechanism of spinosad production in S. spinosa is unclear. Genetic approaches to improve secondary-metabolite production in insecticide-producer strains were hampered by restriction barriers. To circumvent these problems, we used an inactive intergeneric protoplast fusion approach to construct a starch-utilizing strain that overproduced spinosad.

Protoplast fusion is a common method in microorganism breeding, and it can overcome some difficulties in the construction of genetically engineered strains. In Streptomyces coelicolor, protoplast fusion has led to the discovery of new intermediates and hybrid antibiotics with beneficial properties (Baltz 1999). This technique has also led to the largescale enhancement of the yield of metabolites (Hamlyn et al. 1981; Rubinder et al. 2000). Hopwood and Wright (1978) reported high recombination frequencies after protoplast fusion. Furthermore, they obtained a 2- to 10-fold increase in recombination frequency when the parental protoplasts were inactivated to a survival rate of 0.1% by UV light. Inactivation of the parental protoplasts also improved the efficiency of the selection of recombinants.

Avermectin, a marcolide produced by Streptomyces avermitilis, has potent anthelmintic and insecticidal activities (Burg et al. 1979). It is a biological pesticide that has the largest application in the world. Streptomyces avermilitis can use a variety of macromolecular materials in fermentation, including low-cost corn starch and yeast meal. Both spinosad and avermectin are polyketides that are synthesized by type I polyketide synthase (PKS) from the common precursors.

In this study, an intergeneric protoplast fusion between S. spinosa ATCC 49460 and S. avermilitis UA-G, an avermectin high producer, was performed to breed for starch-utilizing recombinants with high yields of spinosad. Two stable recombinants (F17 and F70) were obtained with enhanced spinosad production. Strain F17 was able to use soluble starch as the sole carbon source in fermentation and is suitable for further strain improvement and industrial applications.

Materials and methods

Microbial strains

Strain ATCC 49460 of S. spinosa was a low producer of spinosad obtained from the American Type Culture Collection (ATCC). Strain UA-G of S. avermilitis (Chen 2004), a high producer of avermectin with green spores, …