Fast and combinatorial construction and optimization of synthetic pathways

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Pieter Coussement, Jo Maertens, Joeri Beauprez, Wim Soetaert and Marjan De Mey

University of Ghent, Belgium

The advancements in the field of metabolic engineering and synthetic biology have allowed the rapid de novo construction of multi-enzyme heterologous pathways. However, in order to obtain an optimal flux through a pathway, the various regulatory elements (e.g. promoters, ribosome binding sites) need to be optimized. For example, the massive over-expression of a gene may result in metabolic burden, due to the withdrawal of NADH, ATP, and amino acids from the central metabolism required for the synthesis of the corresponding protein and the concurrent depletion of intermediates required for biomass synthesis or may lead to the accumulation of toxic intermediates due to an unbalanced pathway. The current lack of in-depth knowledge on the various regulatory control levels renders combinatorial approaches popular for pathway optimization. In this context, methods to rapidly and efficiently create variability are crucial. To generate this variability, the promoter and the ribosome binding sites are typically randomized to modulate gene expression and to create gene expression libraries. In this research Gibson assembly was used to introduce promoter and RBS variability into a construct. The biggest advantages are the speed of assembly and the standardized procedure which allow for high-throughput assembly lines. Several libraries were tested: promoter and RBS libraries, but also combinations of both. Furthermore, this developed system was applied for the introduction of multiple libraries at once. Multiple genes could be differentially expressed using a promoter, RBS or promoter-RBS combination library. The research was successfully used for the fast and scarless introduction of various promoter and RBS libraries. This methodology was validated using several fluorescent proteins, and can thus be applied for the combinatorial building of new and synthetic pathways.