Production of recombinant Aedes albopictus akirin in Pichia pastoris using an aqueous two-phase semicontinuous fermentation process

The yeast, Pichia pastoris, has been successfully used as an efficient system to produce heterologous proteins. The secretion of the recombinant Aedes albopictus mosquito akirin (AKR) from P. pastoris using an extractive bioconversion in an aqueous two-phase system (ATPS) fed-batch fermentation process allowed the conduction of vaccination trials to assay its protective efficacy against different arthropod vector species. Herein, we report the development of a semicontinuous process for the extractive bioconversion in an ATPS coupled with a simple and easy to scale up process for polymer recycling as an effective alternative to improve production of recombinant A. albopictus AKR in P. pastoris. Six repetitive batches of the extractive bioconversion were conducted in a semicontinuous process by replacing 2/3 of the culture and recycling the top polymeric phase every 72 h. Overall process in a 5-L fermentor yielded a protein concentration in the bottom phase of 0.46 ± 0.13 mg mL−1 and 6 g of total purified protein in 490 h with a reduction of 25% of the operation time and 27% of the operation cost when compared to the fed-batch ATPS process. These results improve the production process for mosquito AKR and suggest the possibilities to develop similar processes for the expression of other recombinant proteins in P. pastoris.

► Akirin was produced in Pichia pastoris using an aqueous two-phase semicontinuous process.
► Effective recycling and recovery of PEG4000 was obtained.
► Higher protein production when compared to the process with fresh organic phase.
► Reduction of the operation time and production costs by 25% and 27%, respectively.