Effect of Different Signal Peptides on the Expression of Glucoamylase from Aspergillus awamori in the Filamentous Fungus Penicillium verruculosum

Filamentous fungi are widely used in biotechnological processes because they secrete significant amounts of protein, use inexpensive nutrient media, and are predictably scalable in technological processes. <i>Penicillium verruculosum</i> B1-537 (now renamed <i>Talaromyces verruculosus</i>) produces large amounts of secreted protein (up to 70 g/L) and is used for large-scale enzyme production. Although <i>P. verruculosum</i> has an excellent protein expression system under the control of a strong <i>cbh1</i> promoter, some heterologous enzymes such as <i>Aspergillus awamori</i> glucoamylase (aaGlaA) are still produced in insufficient quantities (15-20% of the total secreted protein), and this limits the application of enzyme preparations derived from <i>P. verruculosum</i> strains in the alcohol industry for the enzymatic treatment of grain starch together with α-amylase. One of the well-known approaches to addressing this is signal peptide replacement to increase protein expression. Therefore, the aim of this study was to investigate the effectiveness of signal peptide replacement. Various signal peptides (SPs), which were previously used for other well-expressed heterologous proteins, such as xylanases, β-glucosidases, and others, were analyzed to determine their effect on aaGlaA secretion. Five plasmids containing signal peptide sequences fused to the <i>glaA</i> gene were constructed and used to transform <i>P. verruculosum</i>. The resulting strains were cultured and screened for protein content and glucoamylase activity. Copy number analysis was performed on the most productive strains. The best one was an SP of homologous glucoamylase in <i>P. verruculosum</i> (pvGlaA). The use of this particular SP increased the secretion of heterologous aaGlaA by 2.5 times when cultivating recombinant strains on cellulose-containing fermentation media for <i>P. verruculosum</i>. Thus, SP replacement is a useful way to increase expression levels in the <i>P. verruculosum</i> expression system. Application of this method in <i>P. verruculosum</i> could address some productivity issues and enable the large-scale production of other industrial and food enzymes.