KEVIN ENRICK ALVES DE ABREU1,2, Ana Cristina Araújo Collaço3, Denise Maria Guimaraes Freire3,4, Gilberto Barbosa Domont1,2,4, Erika Cristina Gonçalves Aguieiras3,5, Fábio César Sousa Nogueira1,2,4
Introduction: Diutina rugosa is a yeast species of significant biotechnological interest. Its ability to secrete extracellular proteins—such as lipases—makes it especially relevant for industrial applications, including the production of biodiesel and biolubricants. However, studies indicate that the variation carbon source used in submerged cultures can alter the profile of proteins secreted into the extracellular medium, particularly affecting the enzymatic composition of the secretome.
Objective: This study aims to compare the secretome of D. rugosa cultured with three different carbon sources to elucidate potential differences in protein abundance under distinct cultivation conditions.
Methodology: Cultures were performed in simple batch mode (200mL, erlenmeyer flask) and fed-batch mode (1000mL, bioreactor), with cultivation times of 24 and 72 hours, respectively. Protein quantification was performed using the Qubit Protein Assay method. Six protein enrichment methods were evaluate: Solvent precipitation methods (1–Methanol, Chloroform, and Water; 2–Acetone; 3–Acetone and TCA; 4–Acetone, TCA, and DTT; 5–Acetone, TCA, and β-Mercaptoethanol) and an ultrafiltration method (6–Amicon Ultra-0.5 [10 kDa cutoff]). Proteins were digested in solution with DTT reduction (10mM), IAA alkylation (40mM), and trypsin digestion (1:50) for 18h. Peptides were desalted (manual R2 column), dried in a speedvac, resuspended in 0.1% formic acid, and analyzed by LC-MS/MS on a Vanquish Neo system coupled to an Orbitrap Exploris™ 480. Data were processed using Proteome Discoverer v2.5 and analyzed in Perseus v2.1.3.0.
Results and Discussion: Both cultures performed in erlenmeyer flasks and in the bioreactor using oleic acid as a carbon source showed very similar growth curves. However, a difference in hydrolase activity of the secretome was observed between the two conditions: 323.0±46.4 U/L in erlenmeyer flasks and 213.1±37.6 U/L in the bioreactor. This variation may be attributed to operational differences such as agitation, aeration, and handling, which affect oxygenation and substrate dispersion in the medium. Method optimization was carried out using samples of Diutina rugosa cultivated for 72 hours in the bioreactor with oleic acid. Among the protein enrichment methods tested, methods 1 and 6 showed the highest yields (50.0±1.8% and 68.6±2.8%, respectively). SDS-PAGE (12.5%) analysis did not reveal significant differences between these two methods, leading to the selection of ultrafiltration as the standard. Different amounts of tryptic peptides derived from secretome proteins enriched by method 6 (200ng, 500ng, and 800ng) were injected into the Orbitrap system, and the chromatograms obtained showed reproducibility across profiles. Data analysis is still being carried out, but 21 proteins have already been identified in the samples.
Conclusion: In conclusion, yeast cultivation in erlenmeyer flasks can effectively replicate the initial cultivation conditions of a bioreactor, and further comparison of the protein profiles under both conditions is feasible. Ultrafiltration demonstrated the best yield for secretome enrichment (~68.6%), making it the chosen method for the pre-treatment of all samples. Based on the results obtained, a standardized methodology for sample preparation was defined. The next steps will involve continued analysis of the acquired spectra, as well as preparation of new cultures for sample collection to further advance the study.
Agradecimentos: The authors would like to thank CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the financial support provided for this research.