Abstract
With the emergence of synthetic biology and the vast knowledge about individual biocatalytic reactions, the challenge nowadays is to implement whole natural or synthetic pathways into microorganisms. For this purpose balanced enzyme activities throughout the pathway need to be achieved in addition to simple functional gene expression to avoid bottlenecks and to obtain high titers of the desired product. As the optimization of pathways in a specific biological context is often hard to achieve by rational design, combinatorial approaches have been developed to address this issue. Here, current strategies and proof of concepts for combinatorial pathway assembly in yeasts are reviewed. By exploiting its ability to join multiple DNA fragments in a very efficient and easy manner, the yeast Saccharomyces cerevisiae does not only constitute an attractive host for heterologous pathway expression, but also for assembling pathways by recombination in vivo .
Highlights
Metabolic engineering and synthetic biology provide powerful tools to modify existing metabolic pathways and to extend them with new functions [1,2]
In the current review we explore how the efficient homologous recombination machinery of S. cerevisiae can be exploited for combinatorial pathway assembly
Combinatorial pathway assembly is a powerful tool to generate and identify pathways with improved flux that allow the production of valuable molecules in recombinant microorganisms
Summary
Metabolic engineering and synthetic biology provide powerful tools to modify existing metabolic pathways and to extend them with new functions [1,2]. A broad variety of DNA assembly technologies has been developed that enable the generation of large pathways from small DNA fragments [4]. There are methods that harness sequence homology to assemble parts Examples for these overlap-directed methods are Gibson cloning [7] and in vivo recombination in yeast [8,9]. The aim is to maximize the flux through the pathway without the accumulation of intermediates or side products in order to obtain the desired product in industrial relevant amounts with suitable rates and yields In this context, combinatorial approaches for pathway optimization are of interest to tackle this issue. In the current review we explore how the efficient homologous recombination machinery of S. cerevisiae can be exploited for combinatorial pathway assembly
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