Engineering of Streptomyces lividans for heterologous expression of secondary metabolite gene clusters

Yousra Ahmed,Maksym Myronovskyi,Josef Zapp,Andriy Luzhetskyy,Marta Rodríguez Estévez,Yuriy Rebets

doi:10.1186/s12934-020-1277-8

Abstract

BackgroundHeterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds.ResultsIn this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains.ConclusionThe constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.

Highlights

Introduction of additionalattB sites into the chromosome of the S. lividans ΔYA9 strain Most of the gene cluster expression constructs are based on actinobacterial vectors integrating into the host chromosome
The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression
Transcriptome‐based identification of actively expressed secondary metabolite gene clusters The genome of S. lividans TK24 was sequenced, and twenty-five gene clusters potentially involved in the biosynthesis of secondary metabolites were identified (Additional file 1: Table S1) [24]

Summary

Introduction

AttB sites into the chromosome of the S. lividans ΔYA9 strain Most of the gene cluster expression constructs are based on actinobacterial vectors integrating into the host chromosome. The final constructs were sequentially introduced into the S. lividans ΔYA9 strain by conjugation, and secondary crossover strains were selected, resulting in markerless replacement of the targeted gene clusters by attB sites. This approach often caused difficulties in the identification of produced metabolites or interactions between expressed and endogenous pathways, resulting in aberrant product formation [8, 9] To overcome these complications, the first modified host strain, S. coelicolor CH999, deficient in the production of internal natural products was constructed [10]. These mutations were reported to enhance the production of secondary metabolites in actinobacteria due to increased

Methods

Results

Discussion

Conclusion