National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
Jiangsu Provincial Research Center for Bioactive Product Processing Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
ACS Synth Biol. 2023 Jun 16;12(6):1794-1803. doi: 10.1021/acssynbio.3c00067. Epub 2023 May 12.
The GRAS (generally recognized as safe) strain is well known for its antibacterial and probiotic functions. Furthermore, as 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 . However, the rare gene expression elements greatly obstruct the development of engineering . In this study, we screened and constructed a library of promoter-5'-UTR (PUTR) complexes in 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 P 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 P 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 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 .
GRAS（通常被认为是安全的）菌株以其抗菌和益生菌功能而闻名。此外，由于具有出色的高温和耐盐性，因此它是生产食品酶、食品添加剂和药物的理想宿主。在这方面，通过代谢工程来提高这些产品的产量是可取且可行的。然而，稀有基因表达元件极大地阻碍了工程的发展。在这项研究中，我们筛选并构建了用于在 DY15 中调节转录和翻译水平基因表达的启动子-5'UTR（PUTR）复合物文库。在对数后期，90 个筛选出的天然 PUTR 的 mRNA 和蛋白质表达水平范围分别为 P 启动子的 0.059-2010%和 0.77-245%。此外，几种 PUTR 在高温、盐和乙醇胁迫下表现出很好的表达稳定性。我们分析了 PUTR 的结构，并获得了启动子和 5'UTR 的保守区。基于识别出的 PUTR 核心区域，我们构建了一组具有更高和更稳定蛋白质表达水平的组合 PUTR。最强的组合 PUTR 在蛋白质表达水平上是 P 启动子的 853%。最后，获得的 PUTR 被应用于优化转氨酶的表达水平并提高 DY15 中苯乳酸（PLA）的产量。达到的产率为 950.6mg/L，比野生型菌株高 79.2%。这些结果表明，具有梯度强度的获得的 PUTR 具有精确调节基因表达以实现 中的各种目标的巨大潜力。
