title: Cloning and Expression of Metagenomic DNA in Streptomyces lividans and Subsequent Fermentation for Optimized Production.
abstract: The choice of an expression system for the metagenomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the gram-negative bacterium Escherichia coli as a host for metagenomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40 % of the enzymatic activities may be readily recovered by random cloning in E. coli. To recover some of the remaining 60 %, alternative cloning hosts such as Streptomyces spp. have been used. Streptomycetes are high-GC gram-positive bacteria belonging to the Actinomycetales and they have been studied extensively for more than 15 years as an alternative expression system. They are extremely well suited for the expression of DNA from other actinomycetes and genomes of high GC content. Furthermore, due to its high innate, extracellular secretion capacity, Streptomyces can be a better system than E. coli for the production of many extracellular proteins. In this article an overview is given about the materials and methods for growth and successful expression and secretion of heterologous proteins from diverse origin using Streptomyces lividans has a host. More in detail, an overview is given about the protocols of transformation, type of plasmids used and of vectors useful for integration of DNA into the host chromosome, and accompanying cloning strategies. In addition, various control elements for gene expression including synthetic promoters are discussed, and methods to compare their strength are described. Integration of the gene of interest under the control of the promoter of choice into S. lividans chromosome via homologous recombination using pAMR4-based system is explained. Finally a basic protocol for benchtop bioreactor experiments which can form the start in the production process optimization and upscaling is provided.
authors.affiliation: Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), University of Saarland, Saarbrücken, Germany.
authors.forename: Yuriy
authors.initials: Y
authors.lastname: Rebets
authors.affiliation: Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Kormanec
authors.affiliation: Actinobacteria Metabolic Engineering Group, Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), University of Saarland, Saarbrücken, Germany.|Department of Pharmaceutical Biotechnology, University of Saarland, Saarbrücken, Germany.
authors.forename: Andriy
authors.initials: A
authors.lastname: Luzhetskyy
authors.affiliation: Department of Chemical Engineering, KU Leuven (University of Leuven), Leuven, Belgium.
authors.forename: Kristel
authors.initials: K
authors.lastname: Bernaerts
authors.affiliation: Lab. Molecular Bacteriology, Department Microbiology and Immunology, Rega Institute, KU Leuven (University of Leuven), Box 1037, Herestraat 49, B-3000, Leuven, Belgium. jozef.anne@rega.kuleuven.be.
authors.forename: Jozef
authors.initials: J
authors.lastname: Anné
mesh_terms: D003001:Cloning, Molecular; D005285:Fermentation; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D054892:Metagenome; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D048170:Streptomyces lividans / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins
keywords: Actinomycetes; Cloning; Cloning vectors; Expression; Fermentation; Integrative vectors; Plasmids; Protein secretion; Streptomyces