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Abstract:

The genus Streptomyces is well known for producing specialized metabolites that
exhibit wide spectrum of bioactivities, many of which are encoded by biosynthetic
gene clusters (BGCs) (Lee et al., 2020). However, a significant number of BGCs
remain cryptic in native strains, being necessary to develop methods to express
these yet undiscovered compounds (Rutledge et al., 2015). A reliable option is the
construction of a chassis for synthetic biology, for the reconstruction of synthetic
cellular behaviour and efficient implementation of biomanufacturing (Yan et al., 2024).
This project aims to develop a Streptomyces chassis strain by systematically deleting
endogenous BGCs using three different methods of CRISPR/Cas: Cas9, Cas3, and
cBEST; as well as evaluating each method for efficiency, precision, and potential off-
target effects (Whitford et al., 2023).
To fulfil this objective, it is proposed to begin with the bioinformatic identification of
BGCs within a new native Streptomyces strain, using tools such as AntiSMASH for
detailed annotation. The three CRISPR methods offer unique advantages, such as
double-strand breaks that facilitate homologous recombination, deletion of large BGC
regions, or a base-editing system that permits specific base modifications without
double-strand breaks (Tong et al., 2019). Each method is to be assessed for its
deletion efficiency, ease of use, and ability to maintain genomic stability. Following
each deletion, it is necessary to confirm the absence of target clusters. Finally, it is
necessary to characterize the metabolic and transcriptomic profiles to make sure that
essential functions remain intact.
This work aims to perform NRPS-encoding BGC deletion in a novel native
Streptomyces strain to establish a versatile and stable chassis strain for secondary
metabolite production of geographically co-isolated strains.
Acknowledgement:
This project is supported by the National Council of Science and Technology (Mexico) and the
DTU-alliance PHD between DTU and Tecnologico de Monterrey (DTU, Denmark).
References:
Lee, L. H., Goh, B. H., & Chan, K. G. (2020). Actinobacteria: Prolific producers of bioactive metabolites.
Frontiers in Microbiology, 11, 1612. Doi: 10.3389/fmicb.2020.01612
Rutledge, P. J., & Challis, G. L. (2015). Discovery of microbial natural products: Signalling and silent
gene clusters. Nature Reviews Microbiology, 13(8), 509-523. Doi: 10.1038/nrmicro3496
Yan, H., Li, S., & Wang, W. (2024). Reprogramming naturally evolved switches for Streptomyces
chassis development. Trends in Biotechnology. Doi: 10.1016/j.tibtech.2024.07.001
Whitford, C. M., Gockel, P., Faurdal, D., Gren, T., Sigrist, R., & Weber, T. (2023). CASCADE-Cas3
enables highly efficient genome engineering in Streptomyces species. BioRxiv, 2023-05. Doi:
10.1101/2023.05.09.539971
Tong, Y., Whitford, C. M., Robertsen, H. L., Blin, K., Jørgensen, T. S., Klitgaard, A. K., ... & Lee, S. Y.
(2019). Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST. Proceedings of
the National Academy of Sciences, 116(41), 20366-20375. Doi: 10.1073/pnas.1913493116