Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Life Sciences and Health Engineering, Jiangnan University, Wuxi 214122, P.R. China.
National Engineering Research Center of Cereal Fermentation and Food Biomanufacturing, School of Biotechnology, Jiangnan University, Wuxi 214122, P.R. China.
ACS Synth Biol. 2024 Sep 20;13(9):2887-2898. doi: 10.1021/acssynbio.4c00280. Epub 2024 Sep 12.
Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins. Therefore, it is pivotal to strengthen the expression of exogenous genes at the genome level. Here, we systematically optimized the integrated expression system of from 3 aspects. First, the integration site with the highest expression activity was screened out of 18 sites in the ORI region of the BL21 (DE3) genome. Second, we characterized 16 endogenous promoters in and combined them with the T7 promoter. A constitutive promoter, Plpp-T7, exhibited significantly higher expression strength than the T7 promoter, achieving a 3.3-fold increase in expression levels. Finally, to further enhance the T7 expression system, we proceeded with overexpression of T7 RNA polymerase at the chassis cell level. The resulting constitutive efficient integrated expression system (CEIES_Ecoli) showed a 2-fold increase in GFP expression compared to the pET3b recombinant plasmid. Therefore, CEIES_Ecoli was applied to the integrated expression of nitrilase and hyaluronidase, achieving stable and efficient enzyme expression, with enzyme activities of 22.87 and 12,195 U·mL, respectively, comparable to plasmid levels. Overall, CEIES_Ecoli provides a stable and efficient method of gene expression without the need for antibiotics or inducers, making it a robust tool for synthetic biology, enzyme engineering, and related applications.
外源基因的整合表达是工业发酵相关菌株中首选的方法，因为质粒介导的微生物发酵存在一些缺点，包括额外的生长负担、遗传不稳定性和抗生素污染。合成生物学和基因组编辑技术的进步使得基因整合变得更加方便。整合表达在生物制造领域得到了广泛应用，并有望成为表达重组蛋白的主要方法。因此，在外源基因水平上加强表达至关重要。在这里，我们从 3 个方面系统地优化了 的整合表达系统。首先，从 BL21 (DE3)基因组的 ori 区的 18 个位点中筛选出具有最高表达活性的整合位点 。其次，我们对 中的 16 个内源性启动子进行了表征，并将其与 T7 启动子结合。组成型启动子 Plpp-T7 表现出明显更高的表达强度，比 T7 启动子高 3.3 倍。最后，为了进一步增强 T7 表达系统，我们在底盘细胞水平上过量表达 T7 RNA 聚合酶。所得的组成型高效整合表达系统（CEIES_Ecoli）与 pET3b 重组质粒相比，GFP 表达水平提高了 2 倍。因此，CEIES_Ecoli 被应用于腈水解酶和透明质酸酶的整合表达，实现了稳定高效的酶表达，酶活分别达到 22.87 和 12195 U·mL，与质粒水平相当。总的来说，CEIES_Ecoli 提供了一种稳定高效的基因表达方法，无需抗生素或诱导剂，是合成生物学、酶工程和相关应用的有力工具。
