Orthogonal Gene Expression: Creating 3 Promoter Libraries Linked to Different Sigma Factors Which Are Orthogonal Between Eachother and Towards the Host

Technological advances in synthetic biology, systems biology, and metabolic engineering have boosted applications of industrial biotechnology for an increasing number of complex and high added-value molecules. In general, the transfer of multi-gene or poorly understood heterologous pathways into the production host leads to imbalances due to lack of adequate regulatory mechanisms. Hence, fine-tuning expression of synthesis pathways in specific conditions is mandatory together with the decoupling of host systems. 

Here we develop an orthogonal expression system in Escherichia coli with three heterologous sigma factors that function orthogonal between each other and towards the host organism. Furthermore, for each of these sigma factors, a promoter library will be created that retains this orthogonal feature.

First, several sigma factors originating from Bacillus subtilis were tested for their orthogonality in  E. coli on the level of promoter recognition, by using a red-fluorescent reporter system. Secondly, the potential of sigma factors, and mutants thereof, from B. subtilis  to work together with the E. coli core RNA polymerase was tested. This was accomplished by expressing these proteins together with their promoters; three distinct promoters for each factor. Subsequently, cross talk between the heterologous sigma factors and the promoters was evaluated by examining all combinations. Based on the results three heterologous sigma factors with one of their respective promoters were chosen for further work. For each of these factors a promoter library will be generated that remains orthogonal towards the host and towards the alternative sigma factors. The promoter libraries will be attained by randomising part of the promoter, one strategy will be to randomise regions of 5 nucleotides throughout the promoter while the second strategy will be randomisation of the complete linker sequence. Subsequently the promoter strengths will be measured through sequential rounds of fluorescent-activated cell sorting (FACS) to result in a set of orthogonal promoters for each sigma factor, covering a range of different strengths.

The use of these different orthogonal sigma factors combined with a range of promoter strengths for each factor offers the opportunity to fine-tune expression of different genes in heterologous pathways.