title: One step DNA assembly for combinatorial metabolic engineering.
abstract: The rapid and efficient assembly of multi-step metabolic pathways for generating microbial strains with desirable phenotypes is a critical procedure for metabolic engineering, and remains a significant challenge in synthetic biology. Although several DNA assembly methods have been developed and applied for metabolic pathway engineering, many of them are limited by their suitability for combinatorial pathway assembly. The introduction of transcriptional (promoters), translational (ribosome binding site (RBS)) and enzyme (mutant genes) variability to modulate pathway expression levels is essential for generating balanced metabolic pathways and maximizing the productivity of a strain. We report a novel, highly reliable and rapid single strand assembly (SSA) method for pathway engineering. The method was successfully optimized and applied to create constructs containing promoter, RBS and/or mutant enzyme libraries. To demonstrate its efficiency and reliability, the method was applied to fine-tune multi-gene pathways. Two promoter libraries were simultaneously introduced in front of two target genes, enabling orthogonal expression as demonstrated by principal component analysis. This shows that SSA will increase our ability to tune multi-gene pathways at all control levels for the biotechnological production of complex metabolites, achievable through the combinatorial modulation of transcription, translation and enzyme activity.
authors.affiliation: Centre of Expertise - Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Electronic address: pietera.coussement@ugent.be.
authors.forename: Pieter
authors.initials: P
authors.lastname: Coussement
authors.affiliation: Centre of Expertise - Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Electronic address: jo.maertens@ugent.be.
authors.forename: Jo
authors.initials: J
authors.lastname: Maertens
authors.affiliation: Centre of Expertise - Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Electronic address: joeri.beauprez@ugent.be.
authors.forename: Joeri
authors.initials: J
authors.lastname: Beauprez
authors.affiliation: Centre of Expertise - Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Electronic address: wouter.vanbellegem@ugent.be.
authors.forename: Wouter
authors.initials: W
authors.lastname: Van Bellegem
authors.affiliation: Centre of Expertise - Industrial Biotechnology and Biocatalysis, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Electronic address: marjan.demey@ugent.be.
authors.forename: Marjan
authors.initials: M
authors.lastname: De Mey
mesh_terms: D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*
chemical_list: D004269:DNA, Bacterial
keywords: Metabolic engineering; Pathway optimization; Promoter library; Protein library; RBS library