Hormônio de crescimento recombinante de tambaqui (Colossoma macropomum): otimização da expressão em frascos agitados e produção em biorreator

Abstract

The heterologous expression of recombinant growth hormone from tambaqui (rtGH), Colossoma macropomum, in the methylotrophic yeast Pichia pastoris points to the prospect of generating an innovative biotechnological product for Brazilian fish farming, especially for the cultivation of tambaqui. In this perspective, this research aimed to evaluate the potential of rtGH expression in yeast P. pastoris, in shaken flasks and in bioreactor. Initially, a selection of the transforming clones of P. pastoris with the capacity to produce the best rtGH levels was carried out, taking into account the phenotype. In order to improve the level of rtGH expression in the shaken flasks system, the statistical tools of factorial experimental planning were used. First, a screening design based on the Plackett and Burman design was used to select the most significant parameters related to the production of rtGH and biomass: yeast extract, peptone, urea, phosphate buffer, biotin, methanol, inoculum and pH. Subsequently, the selected parameters, methanol and peptone, were evaluated in DCCR and analyzed using the response surface methodology. Finally, a fermentation was carried out in batch fed by pulses of methanol and the kinetic profile of the determined rtGH production. The results indicated clone 32b, Mut+ phenotype, as the most productive. Methanol was identified as the most important parameter for rtGH production, followed by peptone, biotin and inoculum. In relation to biomass formation, methanol, yeast extract and phosphate buffer were the most important parameters. According to the obtained model, the concentrations of 1.55% methanol and 2.8% peptone were considered optimal for rtGH production. The model was validated with increased rtGH production from 0.3 mg / L to 2.4 mg / L in the optimized condition. Bioreactor fermentation conducted in batch pulsed methanol resulted in a rtGH production level of 250 mg / L, approximately 100 times greater than that obtained in shaken flasks. This research pioneered the development of optimization strategies for rtGH production in P. pastoris using shaken flasks and bioreactor.