title: Establishing a high yielding streptomyces-based cell-free protein synthesis system.
abstract: Cell-free protein synthesis (CFPS) has emerged as a powerful platform for applied biotechnology and synthetic biology, with a range of applications in synthesizing proteins, evolving proteins, and prototyping genetic circuits. To expand the current CFPS repertoire, we report here the development and optimization of a Streptomyces-based CFPS system for the expression of GC-rich genes. By developing a streamlined crude extract preparation protocol and optimizing reaction conditions, we were able to achieve active enhanced green fluorescent protein (EGFP) yields of greater than 50 μg/mL with batch reactions lasting up to 3 h. By adopting a semi-continuous reaction format, the EGFP yield could be increased to 282 ± 8 μg/mL and the reaction time was extended to 48 h. Notably, our extract preparation procedures were robust to multiple Streptomyces lividans and Streptomyces coelicolor strains, although expression yields varied. We show that our optimized Streptomyces lividans system provides benefits when compared to an Escherichia coli-based CFPS system for increasing percent soluble protein expression for four Streptomyces-originated high GC-content genes that are involved in biosynthesis of the nonribosomal peptides tambromycin and valinomycin. Looking forward, we believe that our Streptomyces-based CFPS system will contribute significantly towards efforts to express complex natural product gene clusters (e.g., nonribosomal peptides and polyketides), providing a new avenue for obtaining and studying natural product biosynthesis pathways. Biotechnol. Bioeng. 2017;114: 1343-1353. © 2017 Wiley Periodicals, Inc.
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208.
authors.forename: Jian
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208.|Masters in Biotechnology Program, Northwestern University, Evanston, Illinois.
authors.forename: He
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208.
authors.forename: Yong-Chan
authors.initials: YC
authors.lastname: Kwon
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois 60208.|Masters in Biotechnology Program, Northwestern University, Evanston, Illinois.|Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois.|Member, Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois.|Simpson Querrey Institute, Northwestern University, Chicago, Illinois.|Center for Synthetic Biology, Northwestern University, Evanston, Illinois.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Jewett
mesh_terms: D058045:Artificial Cells / Q000378:metabolism; D053898:Biosynthetic Pathways / Q000235:genetics*; D024861:Genetic Enhancement / Q000379:methods*; D014176:Protein Biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000378:metabolism; D013302:Streptomyces / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins
keywords: Streptomyces; cell-free protein synthesis; high GC-content genes; in vitro transcription and translation; natural products; synthetic biology