Optimization of cultivation conditions to achieve high titers of recombinant trypsinogen with Pichia pastoris expression system cultured on different substrates and their mixtures

Pichia pastoris became a popular host for expression of recombinant proteins, due to the use of the strong and tightly regulated alcohol oxidase I promoter. Methanol has been used in most cultivation protocols for Pichia pastoris Mut
⁺
strains, to induce expression of heterologous protein and support cell growth. Limitations associated with methanol metabolism encouraged the research for mixed-substrate strategies, where part of methanol was replaced with other source of carbon and energy. Thus, mixtures of glycerol or glucose with methanol were tested as alternative carbon feed-stocks for production of recombinant porcine trypsinogen, cloned under control of AOX1 promoter of Pichia pastoris. Under carbon-limited condition both substrates were consumed simultaneously and mixed feeding strategy demonstrated to be successful. The protocol where a mixture of glucose and methanol was used, proved to be the most suitable to support biomass growth, achieving 63.1 g
CDW
.L
⁻¹
, and to reach the highest titer of trypsinogen, 14.2 mg
p
.g
CDW
⁻¹
.