Multiplying the heterologous production of spinosad through tandem amplification of its biosynthetic gene cluster in Streptomyces coelicolor

Abstract

Summary Heterologous expression of the biosynthetic gene cluster (BGC) is important for studying the microbial natural products (NPs), especially for those kept in silent or poorly expressed in their original strains. Here, we cloned the spinosad BGC through the Cas9‐Assisted Targeting of Chromosome segments and amplified it to five copies through a ZouA‐dependent DNA amplification system in Streptomyces coelicolor M1146. The resulting strain produced 1253.9 ± 78.2 μg l −1 of spinosad, which was about 224‐fold compared with that of the parent strain carrying only one copy of the spinosad BGC. Moreover, we further increased spinosad to 1958.9 ± 73.5 μg l −1 by the dynamic regulation of intracellular triacylglycerol degradation. Our study indicates that tandem amplification of the targeted gene cluster is particularly suitable to enhance the heterologous production of valuable NPs with efficiency and simplicity.

Consequences of the deletion of the major specialized metabolite biosynthetic pathways of Streptomyces coelicolor on the metabolome and lipidome of this strain

“…The introduction of heterologous polyketide or peptidic biosynthetic pathways in these deleted strains may avoid or limit the establishment of metabolic features described in this paper and allow the efficient production of these heterologous polyketide and peptidic antibiotics. Indeed peptidic antibiotics such as congocidin, chloramphenicol, caprazamycin (Flinspach et al., 2010 ; Gomez‐Escribano & Bibb, 2011 ) as well as some small polyketide antibiotics such as spinosad, germicidin and flaviolin (Li et al., 2022 ; Thanapipatsiri et al., 2015 ) were reported to be efficiently produced in M1146 (ΔACT, ΔRED, ΔCPK, ΔCDA). However, to improve further the efficiency of these strains as chassis strains, genetic engineering strategies aimed at reducing the TAG content of these strains via inhibition of synthesis or activation of degradation of theses storage lipids, should be beneficial for enhanced production of heterologous polyketide and/or peptidic antibiotics as already reported by some authors (Li et al., 2022 ; Wang et al., 2020 ).…”

Improving the Yield and Quality of Daptomycin in Streptomyces roseosporus by Multilevel Metabolic Engineering

“…For example, the start and engineered 5-oxomilbemycin-producing strains, such as KF200, KF201, KF202, and KF203, harboring 1–4 copies of 5-oxomilbemycin BGCs by advanced Multiplex Site-specific Genome Engineering (aMSGE) system, respectively, can make a stepwise increase in 5-oxomilbemycin production (from 2,228, 4,415, 5,592 to 6,368 mg/l; Li et al, 2019 ). Previous studies also suggested that tandem amplification of BGCs could increase the copy number (4–12 copies), thereby enhancing the production of natural products, such as actinorhodin, bleomycin, and validamycin A ( Murakami et al, 2011 ; Zhou et al, 2014 , Li et al, 2021a , b ). These studies strongly imply that increasing the cluster copies can further enhance the production of target metabolites.…”

A tunable and reversible thermo-inducible bio-switch for streptomycetes

“…The ZouA ampli cation system consists of three genetic elements, a site-speci c relaxase ZouA, and two oriT-like recombination sites RsA and RsB. It has been used successfully for ampli cation of the act gene cluster in S. coelicolor MT1110 10 , bleomycin in Streptomyces verticillus 11 , and spinosad in S. coelicolor M1146 12 . To expand the utility of StrepT-switch, the regulatory system was used to drive the expression of zouA.…”