Expanding the bio-production palette: how to speed up the construction and optimization of novel pathways

In the last couple of years, metabolic engineering is undergoing a transition in which the challenge of microbial synthesis of more complex molecules is tackled by merging metabolic engineering with other disciplines such as systems biology, synthetic biology and protein engineering. As the synthetic pathway of these complex molecules involves multiple, heavily controlled and poorly expressed reactions steps, fine tuning of the pathway is mandatory. To find the optimum flux through the pathway of the multivariate, non-lineair solution space, a combinatorial / semi-rational approach is being developed. The solution space is explored by introducing promotor, ribosome binding site or protein libraries, or combinations hereof. Optimal pathways can be traced back using a high throughput screen. A novel method, called Single Stranded Assembly (SSA), allows for a fast and reliable introduction of different libraries like e.g. promoter, RBS and protein libraries. This SSA method was optimized and validated for different oligonucleotide concentrations and setups. Furthermore, this developed system was applied for the introduction of multiple libraries at once: promoter U+2013 RBS as well as two promoter libraries in front of two different reporter genes. In view of the current evolutions in the field of metabolic engineering and systems biology, which increasingly focus on the biotechnological production of highly complex secondary molecules and the concomitant need to independently fine tune the expression of the multiple genes involved in the pathway, due to, e.g., metabolic burden and the accumulation of toxic intermediates, the SSA method described here is a valuable addition to the state of the art tools since it can be used successfully for the modulation of transcription, translation and enzyme activity in order to make an optimal production strain.