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 and generic co-expression strategy based on synthetic bidirectional promoters combined with screening for protein expression and metabolite production in different hosts. New short native histone promoters identified in the Pichia genome enable more efficient gene expression than the GAP promoter but in two directions. 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 GFP & RFP and enzymes (CalB and redox enzymes).
In addition the new synthetic bidirectional promoters allow new feeding and induction strategies and consecutive induction processes with and without methanol as inductor.
(supported by the FP7 KBBE project BIONEXGEN and by the Austrian Comet programme)