The secretome of Pichia pastoris in fed‐batch cultivations is largely independent of the carbon source but changes quantitatively over cultivation time

The quantitative changes of the secretome of recombinant Pichia pastoris (Komagataella phaffii) CBS7435 over the time‐course of methanol‐ or glucose‐limited fed‐batch cultures were investigated by LC‐ESI‐MS/MS to define the carbon source‐specific secretomes under controlled bioreactor conditions. In both set‐ups, no indication for elevated cell lysis was found. The quantitative data revealed that intact and viable P. pastoris cells secrete only a low number of endogenous proteins (in total 51), even during high cell density cultivation. Interestingly, no marked differences in the functional composition of the P. pastoris secretome between methanol‐ and glucose‐grown cultures were observed with only few proteins being specifically affected by the carbon source. The ‘core secretome’ of 22 proteins present in all analysed carbon sources (glycerol, glucose and methanol) consists mainly of cell wall proteins. The quantitative analysis additionally revealed that most secretome proteins were already present after the batch phase, and depletion rather than accumulation occurred during the fed‐batch processes. Among the changes over cultivation time, the depletion of both the extracellularly detected chaperones and the only two identified proteases (Pep4 and Yps1‐1) during the methanol‐ or glucose‐feed phase appear as most prominent.