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Background   Pichia pastoris  ( Komagataella phaffii ) is an important platform for heterologous protein production due to its growth to high cell density and outstanding secretory capabilities. Recent developments in synthetic biology have extended the toolbox for genetic engineering of  P. pastoris  to improve production strains. Yet, overloading the folding and secretion capacity of the cell by over-expression of recombinant proteins is still an issue and rational design of strains is critical to achieve cost-effective industrial manufacture. Several enzymes are commercially produced in  P. pastoris , with phytases being one of the biggest on the global market. Phytases are ubiquitously used as a dietary supplement for swine and poultry to increase digestibility of phytic acid, the main form of phosphorous storage in grains.    Results  Potential bottlenecks for expression of  E. coli  AppA phytase in  P. pastoris  were explored by applying bidirectional promoters (BDPs) to express AppA together with folding chaperones, disulfide bond isomerases, trafficking proteins and a cytosolic redox metabolism protein. Additionally, transcriptional studies were used to provide insights into the expression profile of BDPs. A flavoprotein encoded by  ERV2  that has not been characterised in  P. pastoris  was used to improve the expression of the phytase, indicating its role as an alternative pathway to  ERO1 . Subsequent AppA production increased by 2.90-fold compared to the expression from the state of the  AOX1  promoter.    Discussion  The microbial production of important industrial enzymes in recombinant systems can be improved by applying newly available molecular tools. Overall, the work presented here on the optimisation of phytase production in  P. pastoris  contributes to the improved understanding of recombinant protein folding and secretion in this important yeast microbial production host.

Synergistic optimisation of expression, folding, and secretion improves E. coli AppA phytase production in Pichia pastoris