High Level of Spinosad Production in the Heterologous Host Saccharopolyspora erythraea

Abstract

Spinosad, a highly effective insecticide, has an excellent environmental and mammalian toxicological profile. Global market demand for spinosad is huge and growing. However, after much effort, there has been almost no improvement in the spinosad yield from the original producer, Saccharopolyspora spinosa. Here, we report the heterologous expression of spinosad using Saccharopolyspora erythraea as a host. The native erythromycin polyketide synthase (PKS) genes in S. erythraea were replaced by the assembled spinosad gene cluster through iterative recombination. The production of spinosad could be detected in the recombinant strains containing the whole biosynthesis gene cluster. Both metabolic engineering and UV mutagenesis were applied to further improve the yield of spinosad. The final strain, AT-ES04PS-3007, which could produce spinosad with a titer of 830 mg/liter, has significant potential in industrial applications. IMPORTANCEThis work provides an innovative and promising way to improve the industrial production of spinosad. At the same time, it also describes a successful method of heterologous expression for target metabolites of interest by replacing large gene clusters.

Heterologous Expression of Spinosyn Biosynthetic Gene Cluster in <b><i>Streptomyces</i></b> Species Is Dependent on the Expression of Rhamnose Biosynthesis Genes

“…Since overexpression of spn cluster genes and precursor synthesis in S. spinosa have indeed been demonstrated to improve spinosyn production in recent years [Jha et al, 2014;Tang et al, 2011], enhancing the positive regulator of secondary metabolites in the hosts will likely be an efficient strategy 196 to boost spinosyn production. Traditional mutagenesis combined with a metabolic engineering method is effective for further improving the yield of spinosyn [Huang et al, 2016]. The successful production of spinosyn proved that BAC-spn -152-rha contains all the necessary elements for the biosynthesis of spinosyn.…”

“…The known sequence of the spinosyn biosynthetic gene cluster, together with the completion of whole-genome sequencing of S. spinosa , greatly facilitates the process of heterologous expression and further engineering in heterologous hosts. Recently, successful heterologous expression of spinosad was reported in Saccharopolyspora erythreae [Huang et al, 2016]. Many other actinomycetes hosts have been used in combinatorial biosynthesis and gene cluster expression studies, in particular Streptomyces spp., some of which are easy to manipulate genetically.…”

Strategies for Enhancing the Yield of the Potent Insecticide Spinosad in Actinomycetes

“…Based on this information, the spinosyn gene cluster was heterologously expressed by replacing the native erythromycin PKS genes in Sa. erythraea ATCC 40137 and the high‐yield erythromycin‐producing strain HS067 . Four cosmids containing fragments in the same orientation and spanning the whole spinosad gene cluster were modified separately and sequentially transformed into two Sa.…”

“…5) The final strain was selected for ultraviolet mutagenesis 90-fold, to 830 mg L À1 Huang et al [49] A BAC vector containing the whole spinosyn biosynthetic gene cluster and the rhamnose biosynthesis genes under the control of PermE Ã was introduced into S. coelicolor and S. lividans To 1.5 mg L À1 Zhao et al [50] 1) Heterologous expression of the whole spinosyn biosynthetic gene cluster in S. albus J1074 and S. lividans TK24 using BAC plasmid. 2) Transcriptomics, proteomics, and metabolomics were performed to identify rate-limiting steps and evaluate heterologous hosts.…”

“…erythraea ATCC 40137 and the high-yield erythromycin-producing strain HS067. [49] Four cosmids containing fragments in the same orientation and spanning the whole spinosad gene cluster were modified separately and sequentially transformed into two Sa. erythraea hosts for homologous recombination.…”