Abstract
The ability to program cellular behaviour is a major goal of synthetic biology, with applications in health, agriculture and chemicals production. Despite efforts to build ‘orthogonal’ systems, interactions between engineered genetic circuits and the endogenous regulatory network of a host cell can have a significant impact on desired functionality. We have developed a strategy to rewire the endogenous cellular regulatory network of yeast to enhance compatibility with synthetic protein and metabolite production. We found that introducing novel connections in the cellular regulatory network enabled us to increase the production of heterologous proteins and metabolites. This strategy is demonstrated in yeast strains that show significantly enhanced heterologous protein expression and higher titers of terpenoid production. Specifically, we found that the addition of transcriptional regulation between free radical induced signalling and nitrogen regulation provided robust improvement of protein production. Assessment of rewired networks revealed the importance of key topological features such as high betweenness centrality. The generation of rewired transcriptional networks, selection for specific phenotypes, and analysis of resulting library members is a powerful tool for engineering cellular behavior and may enable improved integration of heterologous protein and metabolite pathways.
Highlights
The production of proteins and metabolites using engineered microbial strains is an area of significant interest for many industries, including therapeutics, biomass processing, food and beverage, agriculture and materials [1,2,3,4,5]
We constructed a library consisting of promoters and coding sequence (CDS) that regulate cellular functions known to be important in protein overproduction, such as the unfolded protein response, heat shock response, carbon and nitrogen metabolism, and oxidative stress (Figure 1C)
This library was constructed in tandem with a cassette for highlevel expression of a heterologous reporter: green fluorescent protein (GFP) under the control of the methanol inducible promoter AOX1 (Figure 1B)
Summary
The production of proteins and metabolites using engineered microbial strains is an area of significant interest for many industries, including therapeutics, biomass processing, food and beverage, agriculture and materials [1,2,3,4,5]. Expression of proteins and metabolic pathways results in a highly unnatural cellular state that invokes a number of cellular stress responses, reducing the quality and quantity of desired products [6,7]. Strategies to engineer regulatory networks such that they are ‘tailor made’ for heterologous protein and metabolite production are of significant interest. Many approaches have focused on fine-tuning the expression of the heterologous protein or pathway, while relatively few have addressed manipulation of the endogenous regulatory and metabolic network that synthetic pathways are embedded in
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