P12: Novel gene co-expression strategies enabled by synthetic biology

The co-expression of multiple genes is a common problem in heterologous protein production and metabolic engineering (e.g. production of dimeric proteins, multi subunit enzymes, antibodies, or entire pathways). Common strategies rely either on using multiple expression vectors with different selection markers or providing multiple expression cassettes on the same vector. These efforts are limited by the decreased transformation efficiency of large plasmids and genetic instability when using repeatedly identical promoters. Furthermore, co-expression of two genes or a pathway require transcriptional fine-tuning that is hardly achievable with conventional vectors and in specific ratios that are hard to predict.

Exemplified by studies using Pichia pastoris, we have developed an innovative co-expression strategy based on synthetic bidirectional promoters combined with screening which now becomes available for protein expression and metabolite production in different hosts. New native histone promoters identified in the genome of Pichia pastoris enable gene expression in two directions with half of the size of a GAP promoter and higher yield. A library of new synthetic bidirectional promoters showed varying promoter strength and regulations in both directions from very low to higher strength than the strongest known Pichia promoters as exemplified on fluorescent reporter proteins and enzymes (lipase and redox enzymes).

In addition the new promoters allow new feeding and induction strategies with and without methanol as inductor.

Acknowledgements: This project was supported by the European Union in the framework of the FP7 KBBE project BIONEXGEN and by the Austrian Comet programme managed by FFG