Supplementary Material for: Heterologous Expression of Spinosyn Biosynthetic Gene Cluster in Streptomyces Species Is Dependent on the Expression of Rhamnose Biosynthesis Genes

Abstract

<p>Spinosyns are a group of macrolide insecticides produced by <i>Saccharopolyspora spinosa</i>. Although <i>S. spinosa</i> can be used for industrial-scale production of spinosyns, this might suffer from several limitations, mainly related to its long growth cycle, low fermentation biomass, and inefficient utilization of starch. It is crucial to generate a robust strain for further spinosyn production and the development of spinosyn derivatives. A BAC vector, containing the whole biosynthetic gene cluster for spinosyn (74 kb) and the elements required for conjugal transfer and site-specific integration, was introduced into different <i>Streptomyces</i> hosts in order to obtain heterologous spinosyn-producing strains. The exconjugants of different <i>Streptomyces</i> strains did not show spinosyn production unless the rhamnose biosynthesis genes from <i>S. spinosa</i> genomic DNA were present and expressed under the control of a strong constitutive <i>ermE</i>*<i>p</i> promoter. Using this heterologous expression system resulted in yields of 1 μg/mL and 1.5 μg/mL spinosyns in <i>Streptomyces coelicolor</i> and <i>Streptomyces lividans</i>, respectively. This report demonstrates spinosyn production in 2 <i>Streptomyces</i> strains and stresses the essential role of rhamnose in this process. This work also provides a potential alternative route for producing spinosyn analogs by means of genetic manipulation in the heterologous hosts.</p