Rapid optimization of engineered metabolic pathways with serine integrase recombinational assembly (SIRA)

Merrick, C.A., Wardrope, C., Paget, J.E., Colloms, S.D. and Rosser, S.J. (2016) Rapid optimization of engineered metabolic pathways with serine integrase recombinational assembly (SIRA). In: O'Connor, S. E. (ed.) Synthetic Biology and Metabolic Engineering in Plants and Microbes Part A: Metabolism in Microbes. Series: Methods in enzymology (575). Elsevier, pp. 285-317. ISBN 9780128046166 (doi:10.1016/bs.mie.2016.02.009)

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Abstract

Metabolic pathway engineering in microbial hosts for heterologous biosynthesis of commodity compounds and fine chemicals offers a cheaper, greener, and more reliable method of production than does chemical synthesis. However, engineering metabolic pathways within a microbe is a complicated process: levels of gene expression, protein stability, enzyme activity, and metabolic flux must be balanced for high productivity without compromising host cell viability. A major rate-limiting step in engineering microbes for optimum biosynthesis of a target compound is DNA assembly, as current methods can be cumbersome and costly. Serine integrase recombinational assembly (SIRA) is a rapid DNA assembly method that utilizes serine integrases, and is particularly applicable to rapid optimization of engineered metabolic pathways. Using six pairs of orthogonal attP and attB sites with different central dinucleotide sequences that follow SIRA design principles, we have demonstrated that ΦC31 integrase can be used to (1) insert a single piece of DNA into a substrate plasmid; (2) assemble three, four, and five DNA parts encoding the enzymes for functional metabolic pathways in a one-pot reaction; (3) generate combinatorial libraries of metabolic pathway constructs with varied ribosome binding site strengths or gene orders in a one-pot reaction; and (4) replace and add DNA parts within a construct through targeted postassembly modification. We explain the mechanism of SIRA and the principles behind designing a SIRA reaction. We also provide protocols for making SIRA reaction components and practical methods for applying SIRA to rapid optimization of metabolic pathways.

Item Type:Book Sections
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Rosser, Professor Susan and Colloms, Dr Sean
Authors: Merrick, C.A., Wardrope, C., Paget, J.E., Colloms, S.D., and Rosser, S.J.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:Methods in Enzymology
Publisher:Elsevier
ISSN:0076-6879
ISSN (Online):1557-7988
ISBN:9780128046166
Published Online:23 March 2016

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
519113Sandpit: Synthetic integrons for continuous directed evolution of complex genetic ensemblesSusan RosserEngineering & Physical Sciences Research Council (EPSRC)EP/H019154/1RI MOLECULAR CELL & SYSTEMS BIOLOGY
519113Sandpit: Synthetic integrons for continuous directed evolution of complex genetic ensemblesSusan RosserEngineering & Physical Sciences Research Council (EPSRC)EP/H019154/1RI MOLECULAR CELL & SYSTEMS BIOLOGY
519114Sandpit: Synthetic integrons for continuous directed evolution of complex genetic ensemblesSusan RosserEngineering & Physical Sciences Research Council (EPSRC)EP/K034359/1RI MOLECULAR CELL & SYSTEMS BIOLOGY