title: Streptomyces venezuelae TX-TL - a next generation cell-free synthetic biology tool.
abstract: Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the tetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, the authors demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway.
authors.affiliation: Centre for Synthetic Biology and Innovation, South Kensington Campus, London, UK.|Department of Medicine, South Kensington Campus, London, UK.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Moore
authors.affiliation: Centre for Synthetic Biology and Innovation, South Kensington Campus, London, UK.|Department of Medicine, South Kensington Campus, London, UK.
authors.forename: Hung-En
authors.initials: HE
authors.lastname: Lai
authors.affiliation: Department of Life Science, South Kensington Campus, London, UK.
authors.forename: Hannah
authors.initials: H
authors.lastname: Needham
authors.affiliation: Centre for Synthetic Biology and Innovation, South Kensington Campus, London, UK.|Department of Life Science, South Kensington Campus, London, UK.
authors.forename: Karen M
authors.initials: KM
authors.lastname: Polizzi
authors.affiliation: Centre for Synthetic Biology and Innovation, South Kensington Campus, London, UK.|Department of Medicine, South Kensington Campus, London, UK.
authors.forename: Paul S
authors.initials: PS
authors.lastname: Freemont
mesh_terms: D039903:Actinobacteria / Q000235:genetics; D002474:Cell-Free System; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D010118:Oxytetracycline / Q000378:metabolism; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D058615:Synthetic Biology; D014158:Transcription, Genetic
chemical_list: D010118:Oxytetracycline
keywords: Cell-free; Gene expression; Streptomyces; Synthetic biology; Tetracycline