Abstract
The bacteria of the genus Streptomyces are important producers of a large number of biologically active natural products. Examination of their genomes has revealed great biosynthetic potential for the production of new products, but many of them are silent under laboratory conditions. One of the promising avenues for harnessing this biosynthetic potential is the refactoring and heterologous expression of relevant biosynthetic gene clusters (BGCs) in suitable optimized chassis strains. Although several Streptomyces strains have been used for this purpose, the efficacy is relatively low, and some BGCs have not been expressed. In this study, we optimized our long-term genetically studied Streptomyces lavendulae subsp. lavendulae CCM 3239 strain as a potential host for heterologous expression along with its stable large linear plasmid pSA3239 as a vector system. Two reporter genes, mCherry and gusA under the control of ermEp* promoter, were successfully integrated into pSA3239. The activity of GUS reporter was four-fold higher in pSA3239 than in a single site in S. lavendulae subsp. lavendulae CCM 3239 chromosome, consistent with a higher copy number of pSA3239 (4 copies per chromosome). In addition, the two Att/Int systems (based on PhiC31 and pSAM2) were able to integrate into the corresponding individual attB sites in the chromosome. The BGC for actinorhodin was successfully integrated into pSA3239. However, the resulting strain produced very low amounts of actinorhodin. Its level increased dramatically after integration of the actII-ORF4 gene for the positive regulator under the control of the kasOp* promoter into this strain using the PhiC31 phage integration system.
Key points
• New Streptomyces chassis for heterologous expression of genes and BGCs
• Optimized strategy for insertion of heterologous genes into linear plasmid pSA3239
• Efficient heterologous production of actinorhodin after induction of its regulator
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Acknowledgements
We are grateful to Bertold Gust (John Innes Centre, Norwich, UK) for kindly providing all the plasmids and strains used in the PCR-targeting system and the plasmid pSET152; to Andriy Luzhetskyy (Universitat des Saarlandes, Saarbrucken, Germany) for the plasmids pGUS, pGUS-ermE*, and pCLY10; and to Justin Nodwell (McMaster University, Ontario, Canada) for the plasmid pMU1s.
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This work was supported by the Slovak Research and Development Agency under contract No. APVV-19–0009 and by VEGA grant 2/0026/20 from the Slovak Academy of Sciences.
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JK conceived and designed the research. RN, DH, DC, RK, BR, BS, RJ, and LF conducted experiments. RN and DH analyzed data. RN and JK wrote the manuscript. All authors read and approved the manuscript.
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Novakova, R., Homerova, D., Csolleiova, D. et al. A stable vector for efficient production of heterologous proteins and secondary metabolites in streptomycetes. Appl Microbiol Biotechnol 106, 7285–7299 (2022). https://doi.org/10.1007/s00253-022-12187-4
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DOI: https://doi.org/10.1007/s00253-022-12187-4