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

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

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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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.

Original languageEnglish
Article number18301
JournalScientific Reports
Volume11
Issue number1
Number of pages14
ISSN2045-2322
DOIs
Publication statusPublished - Dec 2021

Bibliographical note

Funding Information:
The authors would like to thank Alexandra Hoffmeyer for help with sequencing, Andreas Klitgaard and Yulia Radko for the help with analyzing LCMS data, Kai Blin for the help with bioinformatic analysis, Yaojun Tong for his help with CRISPR-Cas9 related experiments, Xinglin Jiang for the help with cloning experiments. The work of the authors is funded by grants of the Novo Nordisk Foundation, Denmark [NNF20CC0035580, NNF16OC0021746].

Publisher Copyright:
© 2021, The Author(s).

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