Heterologous Biosynthesis of Spinosad: An Omics-Guided Large Polyketide Synthase Gene Cluster Reconstitution in Streptomyces

Abstract

With the advent of the genomics era, heterologous gene expression has been used extensively as a means of accessing natural products (NPs) from environmental DNA samples. However, the heterologous production of NPs often has very low efficiency or is unable to produce targeted NPs. Moreover, due to the complicated transcriptional and metabolic regulation of NP biosynthesis in native producers, especially in the cases of genome mining, it is also difficult to rationally and systematically engineer synthetic pathways to improved NPs biosynthetic efficiency. In this study, various strategies ranging from heterologous production of a NP to subsequent application of omics-guided synthetic modules optimization for efficient biosynthesis of NPs with complex structure have been developed. Heterologous production of spinosyn in Streptomyces spp. has been demonstrated as an example of the application of these approaches. Combined with the targeted omics approach, several rate-limiting steps of spinosyn heterologous production in Streptomyces spp. have been revealed. Subsequent engineering work overcame three of selected rate-limiting steps, and the production of spinosad was increased step by step and finally reached 1460 μg/L, which is about 1000-fold higher than the original strain S. albus J1074 (C4I6-M). These results indicated that the omics platform developed in this work was a powerful tool for guiding the rational refactoring of heterologous biosynthetic pathway in Streptomyces host. Additionally, this work lays the foundation for further studies aimed at the more efficient production of spinosyn in a heterologous host. And the strategy developed in this study is expected to become readily adaptable to highly efficient heterologous production of other NPs with complex structure.

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

“…However, heterologous biosynthesis of target compounds is just the starting point as the production of NPs is very low. The subsequent rational and systematic engineering of target pathways remains challenging; therefore, an omics‐guided metabolic engineering approach was developed for heterologous biosynthesis of spinosyns in the Streptomyces hosts, S. albus J1074 and S. lividans TK24 . The intact spinosyn biosynthetic gene cluster was obtained from the bacterial artificial chromosome (BAC) library, which was constructed using the genomic DNA of Sa.…”

“…One notable advantage is that spinosad can be produced more rapidly by S. albus J1074 compared with that by the Saccharopolyspora host. Although spinosad production in S. albus J1074 is still very low, Tan et al provided a new strategy for future overproduction of NPs in heterologous Streptomyces hosts. Under the guidance of in vitro reconstitution and omics analysis, rational and systematic refactoring of biosynthetic pathways in actinomycetes can be achieved…”

Identification and Engineering of Post‐PKS Modification Bottlenecks for Ansamitocin P‐3 Titer Improvement in Actinosynnema pretiosum subsp. pretiosum ATCC 31280

“…It was reported that overexpression of the halogenase led to a significant enhancement in CTC yield . The production of another polyketide, spinosad, was improved about 500‐fold when the sugar (rhamnose and forosamine) biosynthetic modules and the methyltransferase SpnI were overexpressed . These findings prompted investigation on ansamitocin yield improvement by engineering the tailoring steps.…”

Streptomycetes: Surrogate hosts for the genetic manipulation of biosynthetic gene clusters and production of natural products

“…Omics-guided pathway reconstitution and host engineering have become a very effective strategy for optimized production of targeted products in heterologous hosts. A nearly 1,000-fold increase in the yield of the insecticide spinosyn (Figure 3) in S. albus is a recent example of this approach (Tan et al, 2017). Introduction of mutations with resistance to drugs such as rifampin and streptomycin has proven an efficient strategy to activate secondary metabolic potential (Ochi, 2017).…”