Abstract
A program of mutation and screening, with stepwise reverse engineering or “decoding” of the improved strain, is a way to better understand the genetics and physiology of the strain improvement process. As more is learned about the genetics of strain improvement, it is hoped that more fundamental principles will emerge about the types of mutations and genetic manipulations that reliably lead to higher producing strains. This will accelerate the construction of higher producing strains by metabolic engineering in the future. In this chapter, a detailed tagged mutagenesis approach is described using in vitro transposon mutagenesis which allowed the successful identification of key genes involved in macrolide (erythromycin) antibiotic biosynthesis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Queener SW, Lively DH (1986) Screening and selection for strain improvement, pp. 155-169. In: Demain AL and Solomon NA (eds) Manual of Industrial Microbiology and Biotechnology. American Society for Microbiology, Washington, DC.
Vinci VA, Byng, G (1999) Strain Improvement by Non-recombinant Methods, p. 103-113. In: Demain AL and Davies JE (eds), Manual of Industrial Microbiology and Biotechnology, 2nd ed. ASM Press, Washington, DC.
Goryshin IY, Reznikoff WS (1998) Tn5 in vitro transposition. J Biol Chem 273, 7367–7374.
Kirby JR (2007) In vivo Mutagenesis using EZ-Tn5. Methods in Enzymology 421, 17–21.
Rothberg JM, Leamon JH (2008) The development and impact of 454 sequencing. Nat Biotechnol 26, 1117–1124.
Alper H, Miyaoku K, Stephanopoulos G (2005) Construction of lycopene-overproducing E. coli strains by combining systematic and combinatorial gene knockout targets. Nat Biotechnol 23, 612–616.
Gehring AM, Wang ST, Kearns DB, Storer NY, Losick R (2004) Novel genes that influence development in Streptomyces coelicolor. J Bacteriol 186, 3570–3577.
Reeves AR, Cernota WH, Brikun IA, Wesley RK, Weber JM (2004) Engineering precursor flow for increased erythromycin production in Aeromicrobium erythreum. Metab Eng 6, 300–312.
Tannler S, Zamboni N, Kiraly C, Aymerich S, Sauer U (2008) Screening of Bacillus subtilis transposon mutants with altered riboflavin production. Metab Eng 10, 216–226.
Trötschel C, Kandirali S, Diaz-Achirica P, Meinhardt A, Morbach S, Krämer R, Burkovski A (2003) GltS, the sodium-coupled L-glutamate uptake system of Corynebacterium I: identification of the corresponding gene and impact on L-glutamate production. Appl Microbiol Biotechnol 60, 738–742.
Ikeda M, Ohnishi J, Hayashi M, Mitsuhashi S (2006) A genome-based approach to create a minimally mutated Corynebacterium glutamicum strain for efficient L-lysine production. J Ind Microbiol Biotechnol 33, 610–615.
Ikeda M, Mitsuhashi S, Tanaka K, Hayashi M (2009) Re-engineering of an L-Arginine and L-Citrulline Producer of Corynebacterium glutamicum. Appl Environ Microbiol 75, 1635–1641.
Reeves AR, Brikun IA, Cernota WH, Leach BI, Gonzalez MC, Weber JM (2006) Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea. J Ind Microbiol Biotechnol 7, 600–609.
Reeves AR, Brikun IA, Cernota WH, Leach BI, Gonzalez MC, Weber JM (2007) Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production. Metab Eng 9, 293–303.
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Hopwood DA, Bibb MJ, Chater KF, Kieser T, Bruton CJ, Kieser HM, Lydiate DJ, Smith CP, Ward JM, Schrempf H (1985) Genetic Manipulation of Streptomyces, A laboratory manual. Norwich, UK: John Innes Foundation.
Roberts AN, Barnett L, Brenner S (1987) Transformation of Arthrobacter and studies on the transcription of the Arthrobacter ermA gene in Streptomyces lividans and E. coli. Biochem J 243, 431–436.
Oh SH, Chater KF (1997) Denaturation of circular or linear DNA facilitates targeted integrative transformation of Streptomyces coelicolor A3(2): possible relevance to other organisms. J Bacteriol 129, 122–127.
Reeves AR, Seshadri R, Brikun IA, Cernota WH, Gonzalez MC, Weber JM (2008) Knockout of the erythromycin biosynthetic cluster gene, eryBI, blocks isoflavone glucoside bioconversion during erythromycin fermentations in Aeromicrobium erythreum but not in Saccharopolyspora erythraea. Appl Env Microbiol 74, 7383–7390.
Kanfer I, Skinner MF, Walker RB (1998). Analysis of macrolide antibiotics. J Chromato 812, 255–286.
Acknowledgments
This work was supported by The National Institutes of Health, Small Business Innovation Research (SBIR) awards R44GM58943 and R44GM063278.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Reeves, A.R., Weber, J.M. (2012). Metabolic Engineering of Antibiotic-Producing Actinomycetes Using In Vitro Transposon Mutagenesis. In: Cheng, Q. (eds) Microbial Metabolic Engineering. Methods in Molecular Biology, vol 834. Springer, New York, NY. https://doi.org/10.1007/978-1-61779-483-4_11
Download citation
DOI: https://doi.org/10.1007/978-1-61779-483-4_11
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-61779-482-7
Online ISBN: 978-1-61779-483-4
eBook Packages: Springer Protocols