Improved metabolite production by the rewiring of transcriptional regulatory networks

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Rui Rodrigues, Travis S. Bayer

Centre for Synthetic Biology and Innovation, United Kingdom

A novel method for the development of industrial strains targeted at the production of chemical compounds is presented. This method consists on the rewiring of transcriptional regulatory networks through node addition to explore novel regulatory landscape architectures. A library of transcriptional regulatory components was created to randomly combine promoters and ORFs and installed over existing regulatory networks. New strains that are more adapted to the stress imposed by a heterologous pathway are isolated, resulting in improved phenotypes. The modified networks of improved strains are examined at the transcriptional level in order to better understand how the rewiring events produced the desired phenotype. This strategy was implemented in a Pichia pastoris strain producing the red pigment lycopene through the expression of the P. ananatis crtB, crtI and crtE genes. This pathway allows for simple screening procedures to be used, is a precursor to a number of medically and industrially relevant carotenoids and is itself relevant in the food, cosmetics and nutritional supplement markets. 67 promoters and 43 open reading frames of were combined using Gibson assembly to generate a library consisting of 2881 novel nodes. This library was then screened for increased production of lycopene after genomic integration. Validation of targets’ production levels and copy-number normalisation is ongoing and will be followed by scale up to confirm validity of the method for industrial application. Further use of this method across species and metabolic product classes should inform future rational engineering of strains and expand our knowledge of regulatory network evolution.