A High-Yield Streptomyces Transcription-Translation Toolkit for Synthetic Biology and Natural Product Applications

Overview

Journal J Vis Exp

Specialties Biology
General Medicine

Date 2021 Sep 27

PMID 34570109

Citations 2

Authors

Ming Toh

Kameshwari Chengan

Tanith Hanson

Paul S Freemont

Simon J Moore

Affiliations

Soon will be listed here.

Abstract

Streptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 μL) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 μM) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.

Citing Articles

cell-free systems for natural product discovery and engineering.

Moore S, Lai H, Li J, Freemont P Nat Prod Rep. 2022; 40(2):228-236.

PMID: 36341536 PMC: 9945932. DOI: 10.1039/d2np00057a.

A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems.

Nagappa L, Sato W, Alam F, Chengan K, Smales C, von der Haar T Front Bioeng Biotechnol. 2022; 10:992708.

PMID: 36185432 PMC: 9524191. DOI: 10.3389/fbioe.2022.992708.

References

Cai Q, Hanson J, Steiner A, Tran C, Masikat M, Chen R . A simplified and robust protocol for immunoglobulin expression in Escherichia coli cell-free protein synthesis systems. Biotechnol Prog. 2015; 31(3):823-31. PMC: 5029582. DOI: 10.1002/btpr.2082. View

Hunter D, Bhumkar A, Giles N, Sierecki E, Gambin Y . Unexpected instabilities explain batch-to-batch variability in cell-free protein expression systems. Biotechnol Bioeng. 2018; 115(8):1904-1914. DOI: 10.1002/bit.26604. View

Navarro-Munoz J, Selem-Mojica N, Mullowney M, Kautsar S, Tryon J, Parkinson E . A computational framework to explore large-scale biosynthetic diversity. Nat Chem Biol. 2019; 16(1):60-68. PMC: 6917865. DOI: 10.1038/s41589-019-0400-9. View

Song J, Yoo Y, Lim S, Cha S, Kim J, Roe J . Complete genome sequence of Streptomyces venezuelae ATCC 15439, a promising cell factory for production of secondary metabolites. J Biotechnol. 2016; 219:57-8. DOI: 10.1016/j.jbiotec.2015.12.028. View

Gregorio N, Levine M, Oza J . A User's Guide to Cell-Free Protein Synthesis. Methods Protoc. 2019; 2(1). PMC: 6481089. DOI: 10.3390/mps2010024. View

Moore S, Lai H, Chee S, Toh M, Coode S, Chengan K . A Cell-Free Toolkit for Synthetic Biology. ACS Synth Biol. 2021; 10(2):402-411. PMC: 7901020. DOI: 10.1021/acssynbio.0c00581. View

Geertz M, Shore D, Maerkl S . Massively parallel measurements of molecular interaction kinetics on a microfluidic platform. Proc Natl Acad Sci U S A. 2012; 109(41):16540-5. PMC: 3478601. DOI: 10.1073/pnas.1206011109. View

Kim W, Lee N, Hwang S, Lee Y, Kim J, Cho S . Comparative Genomics Determines Strain-Dependent Secondary Metabolite Production in Strains. Biomolecules. 2020; 10(6). PMC: 7357120. DOI: 10.3390/biom10060864. View

McManus J, Emanuel P, Murray R, Lux M . A method for cost-effective and rapid characterization of engineered T7-based transcription factors by cell-free protein synthesis reveals insights into the regulation of T7 RNA polymerase-driven expression. Arch Biochem Biophys. 2019; 674:108045. DOI: 10.1016/j.abb.2019.07.010. View

10.

Karim A, Heggestad J, Crowe S, Jewett M . Controlling cell-free metabolism through physiochemical perturbations. Metab Eng. 2017; 45:86-94. DOI: 10.1016/j.ymben.2017.11.005. View

11.

Medema M, Fischbach M . Computational approaches to natural product discovery. Nat Chem Biol. 2015; 11(9):639-48. PMC: 5024737. DOI: 10.1038/nchembio.1884. View

12.

Zimmerman E, Heibeck T, Gill A, Li X, Murray C, Madlansacay M . Production of site-specific antibody-drug conjugates using optimized non-natural amino acids in a cell-free expression system. Bioconjug Chem. 2014; 25(2):351-61. DOI: 10.1021/bc400490z. View

13.

Moore S, MacDonald J, Wienecke S, Ishwarbhai A, Tsipa A, Aw R . Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria. Proc Natl Acad Sci U S A. 2018; 115(19):E4340-E4349. PMC: 5948957. DOI: 10.1073/pnas.1715806115. View

14.

Hoff G, Bertrand C, Piotrowski E, Thibessard A, Leblond P . Genome plasticity is governed by double strand break DNA repair in Streptomyces. Sci Rep. 2018; 8(1):5272. PMC: 5869714. DOI: 10.1038/s41598-018-23622-w. View

15.

Hoskisson P, Seipke R . Cryptic or Silent? The Known Unknowns, Unknown Knowns, and Unknown Unknowns of Secondary Metabolism. mBio. 2020; 11(5). PMC: 7587438. DOI: 10.1128/mBio.02642-20. View

16.

Garenne D, Thompson S, Brisson A, Khakimzhan A, Noireaux V . The all-E. coliTXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform. Synth Biol (Oxf). 2021; 6(1):ysab017. PMC: 8546610. DOI: 10.1093/synbio/ysab017. View

17.

Jakeman D, Bandi S, Graham C, Reid T, Wentzell J, Douglas S . Antimicrobial activities of jadomycin B and structurally related analogues. Antimicrob Agents Chemother. 2008; 53(3):1245-7. PMC: 2650534. DOI: 10.1128/AAC.00801-08. View

18.

Caschera F, Noireaux V . Synthesis of 2.3 mg/ml of protein with an all Escherichia coli cell-free transcription-translation system. Biochimie. 2013; 99:162-8. DOI: 10.1016/j.biochi.2013.11.025. View

19.

Shin J, Noireaux V . Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70. J Biol Eng. 2010; 4:8. PMC: 3161345. DOI: 10.1186/1754-1611-4-8. View

20.

Kodani S, Sato K, Hemmi H, Ohnish-Kameyama M . Isolation and structural determination of a new hydrophobic peptide venepeptide from Streptomyces venezuelae. J Antibiot (Tokyo). 2014; 67(12):839-42. DOI: 10.1038/ja.2014.81. View