Engineering the Erythromycin-Producing Strain Saccharopolyspora erythraea HOE107 for the Heterologous Production of Polyketide Antibiotics.

Jin Lü,Yemin Wang,Zixin Deng,Weijun He,Qingshan Long,Lu Chen,Xiuhua Pang,Kai Liu,Meifeng Tao,Jiali Hong,Zhilong Zhao

doi:10.3389/fmicb.2020.593217

Jin Lü, Yemin Wang + Show 9 more

Open Access

https://doi.org/10.3389/fmicb.2020.593217

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Abstract

Bacteria of the genus Saccharopolyspora produce important polyketide antibiotics, including erythromycin A (Sac. erythraea) and spinosad (Sac. spinosa). We herein report the development of an industrial erythromycin-producing strain, Sac. erythraea HOE107, into a host for the heterologous expression of polyketide biosynthetic gene clusters (BGCs) from other Saccharopolyspora species and related actinomycetes. To facilitate the integration of natural product BGCs and auxiliary genes beneficial for the production of natural products, the erythromycin polyketide synthase (ery) genes were replaced with two bacterial attB genomic integration sites associated with bacteriophages ϕC31 and ϕBT1. We also established a highly efficient conjugation protocol for the introduction of large bacterial artificial chromosome (BAC) clones into Sac. erythraea strains. Based on this optimized protocol, an arrayed BAC library was effectively transferred into Sac. erythraea. The large spinosad gene cluster from Sac. spinosa and the actinorhodin gene cluster from Streptomyces coelicolor were successfully expressed in the ery deletion mutant. Deletion of the endogenous giant polyketide synthase genes pkeA1-pkeA4, the product of which is not known, and the flaviolin gene cluster (rpp) from the bacterium increased the heterologous production of spinosad and actinorhodin. Furthermore, integration of pJTU6728 carrying additional beneficial genes dramatically improved the yield of actinorhodin in the engineered Sac. erythraea strains. Our study demonstrated that the engineered Sac. erythraea strains SLQ185, LJ161, and LJ162 are good hosts for the expression of heterologous antibiotics and should aid in expression-based genome-mining approaches for the discovery of new and cryptic antibiotics from Streptomyces and rare actinomycetes.

Highlights

IntroductionBioactive natural products isolated through actinomycete fermentation processes are important sources of therapeutics and agrochemicals, including antibacterials (e.g., erythromycin A, tylosin, and vancomycin); antifungals (e.g., amphotericin B); immunosuppressants (e.g., FK-506 and rapamycin); anticancer agents (e.g., doxorubicin and epoxomicin); anthelmintics (e.g., avermectin); and insecticides (e.g., spinosad) (Challis, 2014; Pham et al, 2019)
Bioactive natural products isolated through actinomycete fermentation processes are important sources of therapeutics and agrochemicals, including antibacterials; antifungals; immunosuppressants (e.g., FK-506 and rapamycin); anticancer agents; anthelmintics; and insecticides (Challis, 2014; Pham et al, 2019)
The genotype of SLQ185 was verified by PCR, using primer pairs eryV1-F/R to confirm that the spectinomycin resistance gene aadA was successfully inserted at gene cluster ery (Figures 1A,B); using primer pairs eryV2-F/R to confirm deletion of the partial gene cluster ery; and using primer pairs eryV3-F/R to confirm the loss of the apramycin resistance gene, indicating that the plasmid pHLQ3 was eliminated (Figure 1B)

Summary

Introduction

Bioactive natural products isolated through actinomycete fermentation processes are important sources of therapeutics and agrochemicals, including antibacterials (e.g., erythromycin A, tylosin, and vancomycin); antifungals (e.g., amphotericin B); immunosuppressants (e.g., FK-506 and rapamycin); anticancer agents (e.g., doxorubicin and epoxomicin); anthelmintics (e.g., avermectin); and insecticides (e.g., spinosad) (Challis, 2014; Pham et al, 2019). Most of these compounds are isolated from the most dominant actinomycete genus, Streptomyces. Few heterologous hosts are derived from rare actinomycetes, mainly due to the lack of efficient genetic manipulation systems that would enable the substantial strain engineering required for removing internal competitive biosynthetic pathways, such as polyketide BGCs (Pfeifer and Khosla, 2001)

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