Integration-coupled activation of promoterless combinatorial pathway libraries inavoids burden during DNA assembly
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
Abstract Combinatorial DNA design and assembly is an efficient and pragmatic way to obtain high-performing metabolic pathway designs quickly. However, implementation may require organism-specific technical barriers to be overcome. Firstly, suitable expression control parts such as promoters and ribosome-binding sites (RBSs), which provide a suitable range of expression levels, need to be identified or developed. Secondly, these need to be assembled into pathway-encoding combinatorial libraries of sufficient size, quality, and diversity. For organisms with transformation frequencies too low to allow direct transformation of library assembly reactions, such as many, assembly and amplification is typically carried out usingHowever, if constructs are deleterious (or ‘burdensome’) to, which is often the case when usinggenetic parts, poor libraries may be obtained. Here we develop a new approach called integration-coupled activation of promoterless sequences to overcome this barrier and therefore enable combinatorial assembly in. Libraries were designed and assembled as promoterless synthetic operons, preventing expression during DNA assembly, and expression was only activated later, when constructs were integrated into the host genome downstream of a promoter. Variation of expression levels was achieved using a range of context-resistant RBS sequences. This approach was used to produce alibrary with combinatorial expression variants of an introduced hexanol pathway. This proof of
Abstract
Abstract Combinatorial DNA design and assembly is an efficient and pragmatic way to obtain high-performing metabolic pathway designs quickly. However, implementation may require organism-specific technical barriers to be overcome. Firstly, suitable expression control parts such as promoters and ribosome-binding sites (RBSs), which provide a suitable range of expression levels, need to be identified or developed. Secondly, these need to be assembled into pathway-encoding combinatorial libraries of sufficient size, quality, and diversity. For organisms with transformation frequencies too low to allow direct transformation of library assembly reactions, such as many, assembly and amplification is typically carried out usingHowever, if constructs are deleterious (or ‘burdensome’) to, which is often the case when usinggenetic parts, poor libraries may be obtained. Here we develop a new approach called integration-coupled activation of promoterless sequences to overcome this barrier and therefore enable combinatorial assembly in. Libraries were designed and assembled as promoterless synthetic operons, preventing expression during DNA assembly, and expression was only activated later, when constructs were integrated into the host genome downstream of a promoter. Variation of expression levels was achieved using a range of context-resistant RBS sequences. This approach was used to produce alibrary with combinatorial expression variants of an introduced hexanol pathway. This proof of concept provides a generally-applicable approach to implement combinatorial metabolic pathway-encoding libraries in, circumventing the excessive strength ofexpression control parts in, and is applicable to other organisms. Graphical Abstract Graphical Abstract http://www.w3.org/1999/xlink float portrait ysag008ga1.webp float ga1 portrait graphical