Signal peptides (SPs) are N‐terminus sequences on the nascent polypeptide for protein export or localization delivery, which are essential for maintaining cell function. SPs are also employed as a key element for industrial production of secreted recombinant proteins. Yet, detailed information and rules about SPs and their cellular interactions are still not well understood. Here, systematic bioinformatics analysis and secretion capacity measurement of genome‐wide SPs from the model organism Saccharomyces cerevisiae is performed. Several key features of SPs, including region properties, consensus motifs, evolutionary relationships, codon bias, e.g., are successfully revealed. Diverse cell metabolism can be trigged by using different SPs for heterologous protein secretion. Influences on SPs with different properties by chaperones can cause different secretory efficiencies. Protein secretion by the SP NCW2 in SEC72 deletion strain is 10 times than the control. These findings provide insights into the properties and functions of SPs and contribute to both fundamental research and industrial application.

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In the originally published article, Table S8, Supporting Information, contained an error. Some primer sequences did not match their names in primer lists due to re-sorting in the table. The corrected Table S8 Supporting Information file was published on February 24, 2023, after initial article online publication. The authors state that the correction does not affect the discussion or conclusions and that they sincerely apologize for the unintentional errors.