Yeasts are prominent expression systems for recombinant protein production due to the advantages they offer in basic research laboratories and the biotechnology industry. These systems enable the production of proteins in higher quantities and industrial conditions more efficiently and economically than natural sources. It also facilitates downstream processes, especially in intracellularly produced proteins, compared to production in its natural source [1], [2]. In this regard, the secretory signal sequences are important molecular elements that function in extracellular protein production. These short peptide sequences located in the N-terminal region of the synthesized proteins are involved in the translocation of proteins to the endoplasmic reticulum. Also, the successful selection of this component stands out as one of the effective approaches used to improve the secretion of heterologous proteins [3], [4].

Today, S. cerevisiae α-mating factor prepro-leader is still the most preferred option for the secretion of recombinant protein production in yeasts [5], [6]. However, it cannot be concluded that it may be the best secretion signal for all proteins. Besides, the native secretion sequence of the target protein can be used [7], [8]. Previous studies have also reported different alternative secretion signals providing higher efficiency for the secretion of different proteins compared to S. cerevisiae α-mating factor prepro-leader [9], [10]. Therefore, studies have been directed to the search for alternative peptides to expand the secretory signal repertoire. Some of them focused on modifying existing ones [11], [12], [13], [14], [15], [16] or characterized the alternative signal peptides isolated from different sources [17], [18], [19] and the others have established the synthetic sequences [20], [21], [22].

A methylotrophic yeast P. pastoris is one of the most popular yeast expression systems. Its ability to secrete high titers of recombinant proteins into the culture media is among the main advantages that make it attractive as a host expression system [23]. In this study, the potential of alternative secretory signal sequences (P. pastoris MF, Kluyveromyces lactis MF, Kluyveromyces marxianus MF, Hansenula polymorpha PIR, K. lactis PIR and K. marxianus PIR) was investigated by producing two different reporter proteins, xylanase (XylB) and α-amylase (AmyE) in P. pastoris.