RECOMBINANT ENZYME EXPRESSION IN Pichia pastoris: A GATEWAY FOR POLYPHENOLS’ RECOVERY FROM PLANT BIOMASS

PIAZZA, DANA M; GENTILE, JULIA; MEINI, MARÍA-ROCÍO; ROMANINI, DIANA

Buenos Aires

Congreso; XVIII Congreso Argentino de Microbiología General, SAMIGE 2023; 2023

In recent years, increasing health awareness and the desire for natural products sparked a growing demand for natural additives that can replace synthetic ones in various industries. Agro-industrial byproducts represent a promising source of high-value bioactive compounds, such as polyphenols, which hold great potential as natural additives and functional ingredients. Due to the increasing demand, highly efficient and sustainable polyphenols production processes must be developed to meet the market’s requirements. In this way, we are currently working on the recombinant production and optimization of fungal feruloyl esterase (FAE) and tannase enzymes using Pichia pastoris. We aim to selectively produce hydroxycinnamic acids and gallic acid from different agro-industrial byproducts.The coding sequences, without their signal peptides, of FAE (type A FAE from Aspergillus niger, AnFaeA; and type B FAE from Myceliophthora thermophila, MtFae1a) and tannase enzymes (from A. oryzae, AoTan) were optimized according to the codon usage bias of P. pastoris, synthesized, and expressed in P. pastoris X-33. Expression was carried out in YPM medium (YP, 100 mM sodium phosphate buffer pH 6.0, and 0.5% v/v methanol) in a 24-well plate (30ºC, 200 rpm). Methanol was added every 24 h to maintain a 0.5% v/v methanol concentration. After 72 h of induction, the cultures were centrifuged, and the supernatants were used for enzymatic activity assays and SDS-PAGE analysis. SDS-PAGE analysis showed that there were almost no other proteins except for the recombinant enzymes (AnFaeA, MtFae1a, and AoTan) in the cultured supernatants. The molecular weights obtained were: 40.0 kDa for AnFaeA; 36.0 kDa for MtFae1a; two bands of 37 and 39 kDa for AoTan. In all cases the values were larger than expected, probably due to glycosylation. The maximum activities were obtained after 72 h of induction: 9.5 U/mL for AnFaeA, 2.2 U/mL for MtFae1a, and 0.75 U/mL for AoTan. The specific activities (U/mg total proteins) were: 120.4 for AnFaeA, 47.6 for MtFae1a and 17.9 for AoTan. Lastly, the enzymes’ supernatants were combined with other hydrolytic enzymes to test their capability of releasing polyphenols from plant biomass. The extracts obtained showed higher polyphenols concentrations when compared to the respective control extractions.In summary, the synthetic genes of two fungal FAEs and a tannase enzyme were optimized according to the codon usage bias of P. pastoris and were successfully expressed and secreted by this organism. The supernatants from cultures obtained at 48 h and 72 h after induction exhibited high purity in SDS-PAGE analysis. This would simplify their industrial application as complex purification procedures become unnecessary. Moreover, the three enzymes showed considerable activity towards the substrates tested. This study also provided an insight in the potential application of FAE and tannase enzymes in the recovery of valuable polyphenols.