Characterization and engineering of Streptomyces griseofuscus DSM 40191 as a potential host for heterologous expression of biosynthetic gene clusters

Tetiana Gren,Christopher M Whitford,Eftychia E Kontou,Omkar S Mohite,Julie B Nielsen,Sang Yup Lee,Tue S Jørgensen,Tilmann Weber

doi:10.1038/s41598-021-97571-2

Abstract

Streptomyces griseofuscus DSM 40191 is a fast growing Streptomyces strain that remains largely underexplored as a heterologous host. Here, we report the genome mining of S. griseofuscus, followed by the detailed exploration of its phenotype, including the production of native secondary metabolites and ability to utilise carbon, nitrogen, sulphur and phosphorus sources. Furthermore, several routes for genetic engineering of S. griseofuscus were explored, including use of GusA-based vectors, CRISPR-Cas9 and CRISPR-cBEST-mediated knockouts. Two out of the three native plasmids were cured using CRISPR-Cas9 technology, leading to the generation of strain S. griseofuscus DEL1. DEL1 was further modified by the full deletion of a pentamycin BGC and an unknown NRPS BGC, leading to the generation of strain DEL2, lacking approx. 500 kbp of the genome, which corresponds to a 5.19% genome reduction. DEL2 can be characterized by faster growth and inability to produce three main native metabolites: lankacidin, lankamycin, pentamycin and their derivatives. To test the ability of DEL2 to heterologously produce secondary metabolites, the actinorhodin BGC was used. We were able to observe a formation of a blue halo, indicating a potential production of actinorhodin by both DEL2 and a wild type.

Highlights

The vast majority of clinically used antibiotics, antifungals, anticancer and immunosuppressive drugs are derived from natural products[1] originating from soil-inhabiting Actinobacteria[2]
We found that 3918 genes were shared across all three genomes, whereas S. griseofuscus shared additional 937 and 522 genes with S. coelicolor and S. venezuelae respectively (Fig. 2A, Supplementary Data 1)
We found that the number of genes involved in subsystems such as membrane transport, signalling and cellular processes were significantly lower in S. griseofuscus than in others

Summary

Introduction

The vast majority of clinically used antibiotics, antifungals, anticancer and immunosuppressive drugs are derived from natural products[1] originating from soil-inhabiting Actinobacteria[2]. Heterologous hosts are typically well-characterized strains, that possess a plurality of needed characteristics, e.g. fast and disperse growth, amenability to genetic engineering, high yields of produced secondary m etabolites[11,13,14]. The use of such chassis strains is highly advantageous, when the native producer strain can not be genetically engineered or is slow g rowing[13]. Even though several publications have stipulated multiple positive characteristics of S. griseofuscus, e.g. fast growth, ease of transformation and genetic manipulation, it was never methodically studied regarding its qualities as a potential platform for the expression of BGCs. In this paper, we describe a comprehensive genotypic and phenotypic characterization of S. griseofuscus DSM40191 as a potential heterologous production host. All of the generated knockouts were tested regarding their growth and production of metabolites

Methods

Results

Discussion

Conclusion