Screening and Constructing a Library of Promoter-5'-UTR Complexes with Gradient Strength in Pediococcus acidilactici

The GRAS (generally recognized as safe) strain Pediococcus acidilactici is well known for its antibacterial and probiotic functions. Furthermore, as P. acidilactici has excellent high temperature and salt resistance, it is an ideal host for the production of food enzymes, food additives, and pharmaceuticals. In this regard, it is desirable and feasible to enhance the production of these products through the metabolic engineering of P. acidilactici. However, the rare gene expression elements greatly obstruct the development of engineering P. acidilactici. In this study, we screened and constructed a library of promoter-5'-UTR (PUTR) complexes in P. acidilactici DY15 for regulating gene expression at the transcription and translation levels. In the post-log phase, the mRNA and protein expression level ranges of the 90 screened native PUTRs were 0.059-2010% and 0.77-245%, respectively, of the P₃₂ promoter. Besides, several PUTRs exhibited great expression stability under high temperature, salt, and ethanol stress. We analyzed the structure of PUTRs and obtained the conserved regions of the promoter and 5'-UTR. Based on the identified core regions of PUTRs, we constructed a panel of combinatorial PUTRs with higher and stable protein expression levels. The strongest combinatorial PUTR was 853% of the P₃₂ promoter in the protein expression level. Finally, the obtained PUTRs were applied to optimize the expression level of aminotransferase and improve the phenyllactic acid (PLA) production in P. acidilactici DY15. The achieved yield was 950.6 mg/L, which was 79.2% higher than the wild-type strain. These results indicated that the obtained PUTRs with gradient strength had great potential for precisely regulating gene expression to achieve various goals in P. acidilactici.