Genome-wide high-throughput signal peptide screening via plasmid pUC256E improves protease secretion in

Background: Proteases catalyze the hydrolysis of peptide bonds of proteins, thereby improving dietary protein digestibility, nutrient availability, as well as flavor and texture of fermented food and feed products. The lactobacilli Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) and Pediococcus acidilactici are widely used in food and feed fermentations due to their broad metabolic capabilities and safe use. However, extracellular protease activity in these two species is low. Here, we optimized protease expression and secretion in L. plantarum and P. acidilactici via a genetic engineering strategy. Results: To this end, we first developed a versatile and stable plasmid, pUC256E, which can propagate in both L. plantarum and P. acidilactici. We then confirmed expression and secretion of protease PepG1 as a functional enzyme in both strains with the aid of the previously described L. plantarum-derived signal peptide LP_0373. To further increase secretion of PepG1, we carried out a genome-wide experimental screening of signal peptide functionality. A total of 155 predicted signal peptides originating from L. plantarum and 110 predicted signal peptides from P. acidilactici were expressed and screened for extracellular proteolytic activity in the two different strains, respectively. We identified 12 L. plantarum signal peptides and eight P. acidilactici signal peptides that resulted in improved yield of secreted PepG1. No significant correlation was found between signal peptide sequence properties and its performance with PepG1. Conclusion: The vector developed here provides a powerful tool for rapid experimental screening of signal peptides in both L. plantarum and P. acidilactici. Moreover, the set of novel signal peptides identified was widely distributed across strains of the same species and even across some closely related species. This indicates their potential applicability also for the secretion of other proteins of interest in other L. plantarum or P. acidilactici host strains. Our findings © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Open Access *Correspondence: oleg.latypov@sg.wilmar-intl.com; sandra. kittelmann@sg.wilmar-intl.com 1 Wilmar International Limited, WIL@NUS Corporate Laboratory, Centre for Translational Medicine, National University of Singapore, Singapore, Singapore Full list of author information is available at the end of the article Page 2 of 16 Chen et al. BMC Genomics (2022) 23:48 Background The lactobacilli (or family Lactobacillaceae until 2020) are a highly diverse group of lactic acid-producing bacteria. Species within this group were formerly classified into only three genera, Lactobacillus, Paralactobacillus, and Pediococcus, and were only recently re-classified into 26 different genera, including the genera Lactiplantibacillus (formerly Lactobacillus) and Pediococcus [1]. They can be found in many ecological niches, such as on living and decaying plant material, as well as in naturally fermented meat, vegetables, milk and silages [2, 3]. Colonization of the digestive tract of mammalian hosts by members of the lactobacilli is also frequently observed [4, 5]. Some species of lactobacilli are “generally recognized as safe”, and these are some of the economically most important species as they are routinely used in a variety of industrial food and feed fermentations [6]. Many beneficial effects for human and animal health have been attributed to these species, some of which are supported by a large body of scientific literature, e.g., elimination of pathogens through lactic acid and bacteriocin production [7, 8], production of beneficial metabolites and vitamins [9], reduction of cholesterol [10], antioxidant activity [11], as well as a broad range of other health promoting and disease preventing effects [12, 13]. Moreover, fermented food and feed are generally characterized by an enhanced texture, flavor, aroma and nutritional value, due to the abundance and diversity of secreted metabolites (e.g., organic acids, ketones, and aldehydes) and enzymes (e.g., amylases, esterases, glucosidases, lipases, and proteases) [14, 15]. Proteases have been intensively studied in lactobacilli [16–19]. Proteases catalyze the hydrolysis of peptide bonds of proteins that are present in complex food and feed matrices. This process results in the release of peptides and free amino acids essential for cell growth. Hence, protease activity is particularly important to those species auxotrophic for amino acids, which often occur in milk fermentations [17]. Proteolytic activity improves dietary protein digestibility and nutrient utilization by increasing the relative amount of small peptides [20]. Moreover, proteases break down allergenic proteins and trypsin inhibitors, e.g., in soybean-derived substrates, which results in improved acceptance and higher uptake especially by monogastric animals [21]. In addition, proteases contribute to flavor and texture of fermented products [18]. For these reasons, investigations into the diversity and activity of native proteolytic enzymes in lactic acid bacteria has been a focal point of research for several decades [18]. However, most species harbor cell envelope-associated proteinase, and its attachment to the cell wall limits the amount of protease produced [17, 18]. Lactiplantibacillus plantarum and Pediococcus acidilactici are two of the industrially most important species in food and feed fermentation [2]. Several studies have explored the possibility of improving enzyme activity via genetic engineering using these two species as models [22]. One of the most critical parameters to determine if secretion of a desired target protein will be successful or not is the capacity of the signal peptide used to transport the protein into the extracellular space [23]. So far, engineered secretion in L. plantarum and P. acidilactici has mostly been achieved via heterologous signal peptides, e.g., sslipA of Bacillus subtilis [24], M6 of Streptococcus pyogenes [25] and Usp45 of Lactococcus lactis [26]. Only a limited number of studies have focused on the identification of homologous signal peptides in L. plantarum [27], and, to the best of our knowledge, none are available for P. acidilactici yet. Native signal peptides, however, have been shown to lead to similar or higher secretion than constructs with heterologous signal peptides [28]. It is conceivable that native signal peptides are best recognized by the native secretory machinery of the host. One key problem in selecting suitable signal peptides is the difficulty in predicting their efficiency based on primary sequence information alone. In this study, we carried out a genome-wide analysis of signal peptides from L. plantarum and P. acidilactici. Predicted native signal peptides were then assessed in L. plantarum or P. acidilactici host strain for their capacity in directing secretion of heterologous protease PepG 1[29]. Several novel native signal peptides were identified that resulted in recombinant strains with improved protease secretion. Use of these strains may increase extracellular protein degradation and peptide content in food and feed matrices.