Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Dec 31:52:kuaf019.
doi: 10.1093/jimb/kuaf019.

Engineering Streptomyces coelicolor for heterologous expression of the thiopeptide GE2270A-A cautionary tale

Francesco Del Carratore  1   2 , Erik K R Hanko  2 , Kamila Schmidt  2   3 , Oksana Bilyk  2 , Suhui Ye Huang  2   4 , Marianna Iorio  5 , Mercedes Pérez-Bonilla  6 , Rosario Pérez-Redondo  7   8 , Michelle Rudden  9 , Emmanuele Severi  9   10 , Arianna Tocchetti  5 , Margherita Sosio  5 , Emily J Johnson  11 , Timothy Kirkwood  2 , Dominic R Whittall  2 , Alkisti Manousaki  2 , Olga Genilloud  6 , Antonio Rodríguez-García  12   7 , Gavin H Thomas  8 , Stefano Donadio  5 , Rainer Breitling  2 , Eriko Takano  2
Affiliations

Engineering Streptomyces coelicolor for heterologous expression of the thiopeptide GE2270A-A cautionary tale

Francesco Del Carratore et al. J Ind Microbiol Biotechnol. .

Abstract

The thiopeptide GE2270A is a clinically relevant, ribosomally synthesised, and post-translationally modified peptide naturally produced by Planobispora rosea. Due to the genetically intractable nature of P. rosea, heterologous expression is considered a possible route to yield improvement. In this study, we focused on improving GE2270A production through heterologous expression of the biosynthetic gene cluster (BGC) in the model organism Streptomyces coelicolor M1146. A statistically significant yield improvement was obtained in the S. coelicolor system through the data-driven rational engineering of the BGC, including the introduction of additional copies of key biosynthetic and regulatory genes. However, despite our best efforts, the highest production level observed in the strains generated in this study is 12× lower than published titres achieved in the natural producer and 50× lower than published titres obtained using Nonomuraea ATCC 39727 as expression host. These results suggest that, while using the most genetically amenable strain as host can be the right choice when exploring different BGC designs, the choice of the most suitable host has a major effect on the achievable yield and should be carefully considered. The analysis of the multiomics data obtained in this study suggests an important role of PbtX in GE2270A biosynthesis and provides insights into the differences in production metabolic profiles between the different strains. One Sentence Summary: Data-driven rational engineering of Streptomyces coelicolor for heterologous production of the thiopeptide antibiotic GE2270A resulted in increased production but encountered unexpected challenges compared to production in the natural producer or the alternative host Nonomuraea ATCC 39727.

Keywords: Streptomyces coelicolor; GE2270A; Metabolic engineering; Synthetic biology; Thiopeptide.

PubMed Disclaimer

Conflict of interest statement

There are no conflicts of interest.

Figures

Graphical Abstract
Graphical Abstract
Fig. 1.
Fig. 1.
Summary of strains generated in this study. (A) Pedigree chart for the Streptomyces coelicolor strains used and generated in this study. Strains for which metabolomics, transcriptomics, and proteomics data were collected are denoted by asterisks. GE2270A titres are indicated for each strain (mean ± standard deviation). N.D.—not detected. Titres represent means of biological replicates (N = 2–9). (B) The strains outlined in A differ from each other only in the GE2270A BGC. Differences are illustrated here graphically. Promoters are indicated by arrows and protein functions are colour coded. Gene names are annotated in LW204, containing the unmodified version of the GE2270A cluster, including the hygromycin selection marker (Hyg) and the elongation factor TU (EF-Tu).
Fig. 2.
Fig. 2.
Gene expression and protein levels of the GE2270 cluster for a selection of strains considered in this study. (A) Gene expression levels of the genes from the GE2270 BGC measured in the natural producer P. rosea (left) and strain LW207 (right) at different time points. Gene expression levels are shown in transcripts per million (TPM). (B) Protein levels measured for the GE2270 BGC in the natural producer P. rosea and three S. coelicolor strains: LW207, LW296, and LW298.
Fig. 3.
Fig. 3.
Summary of the GE2270-associated peaks detected in an untargeted metabolomics experiment considering whole-broth extraction of the fermentation of six S. coelicolor strains (LW207, LW290, LW291, LW292, LW293, and LW294) and four Nonomuraea strains (Nono2F7, Nonocos3, Nono-Δ12, and Nono-Δ12pbtG1). The S. coelicolor strains were analysed at the University of Manchester, while the Nonomuraea strains were analysed both in MEDINA (M) and Naicons (N) labs. (A) Heatmap showing the relative abundance (normalised by row) of the GE2270-related peaks detected in positive and negative mode. The features without any annotation mass-to-charge ratio (m/z), predicted charge and retention time are shown. (B) Similarity network for the fragmentation spectra acquired for ten of the selected features related to GE2270A from the S. coelicolor strains. All features considered here show a significant similarity in the fragmentation spectra with GE2270A, hence providing additional evidence that these features are indeed related with the GE2270 cluster.
See this image and copyright information in PMC

References

    1. Abramson J., Adler J., Dunger J., Evans R., Green T., Pritzel A., Ronneberger O., Willmore L., Ballard A. J., Bambrick J., Bodenstein S. W., Evans D. A., Hung C.-C., O'Neill M., Reiman D., Tunyasuvunakool K., Wu Z., Žemgulytė A., Arvaniti E., Jumper J. M. (2024). Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature, 630(8016), 493–500. 10.1038/s41586-024-07487-w - DOI - PMC - PubMed
    1. Arnison P. G., Bibb M. J., Bierbaum G., Bowers A. A., Bugni T. S., Bulaj G., Camarero J. A., Campopiano D. J., Challis G. L., Clardy J., Cotter P. D., Craik D. J., Dawson M., Dittmann E., Donadio S., Dorrestein P. C., Entian K.-D., Fischbach M. A., Garavelli J. S., van der Donk W. A. (2013). Ribosomally synthesized and post-translationally modified peptide natural products: Overview and recommendations for a universal nomenclature. Natural Product Reports, 30(1), 108–160. 10.1039/C2NP20085F - DOI - PMC - PubMed
    1. Bai C., Zhang Y., Zhao X., Hu Y., Xiang S., Miao J., Lou C., Zhang L. (2015). Exploiting a precise design of universal synthetic modular regulatory elements to unlock the microbial natural products in Streptomyces. Proceedings of the National Academy of Sciences, 112(39), 12181–12186. 10.1073/pnas.1511027112 - DOI - PMC - PubMed
    1. Bennett P. M., Grinsted J., Richmond M. H. (1977). Transposition of TnA does not generate deletions. Molecular and General Genetics MGG, 154(2), 205–211. 10.1007/BF00330839 - DOI - PubMed
    1. Bibb M. J., Janssen G. R., Ward J. M. (1985). Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus. Gene, 38(1-3), 215–226. 10.1016/0378-1119(85)90220-3 - DOI - PubMed

MeSH terms