Faculty of Pharmacy and Food Science Technology, Department of Biology, Healthcare and the Environment, Microbiology Section, Universitat de Barcelona, Avinguda Diagonal 643, 08028 Barcelona, Spain.
Int J Mol Sci. 2024 Jun 25;25(13):6957. doi: 10.3390/ijms25136957.
Achieving commercially significant yields of recombinant proteins in requires the optimization of its protein production pathway, including transcription, translation, folding, and secretion. Therefore, in this study, our aim was to maximize the secretion of a reporter α-amylase by overcoming potential bottlenecks within the secretion process one by one, using a clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) system. The strength of single and tandem promoters was evaluated by measuring the relative α-amylase activity of AmyQ integrated into the chromosome. Once a suitable promoter was selected, the expression levels of were upregulated through the iterative integration of up to six gene copies, thus boosting the α-amylase activity 20.9-fold in comparison with the strain harboring a single gene copy. Next, α-amylase secretion was further improved to a 26.4-fold increase through the overexpression of the extracellular chaperone PrsA and the signal peptide peptidase SppA. When the final expression strain was cultivated in a 3 L fermentor for 90 h, the AmyQ production was enhanced 57.9-fold. The proposed strategy allows for the development of robust marker-free plasmid-less super-secreting strains with industrial relevance.
在 中实现具有商业意义的重组蛋白产量需要优化其蛋白生产途径，包括转录、翻译、折叠和分泌。因此，在本研究中，我们的目的是使用成簇规律间隔短回文重复序列 - Cas9 (CRISPR-Cas9) 系统，通过逐个克服分泌过程中的潜在瓶颈，最大限度地分泌报告α-淀粉酶。通过测量整合到 染色体上的 AmyQ 的相对α-淀粉酶活性来评估单个和串联启动子的强度。选择合适的启动子后，通过迭代整合多达六个基因拷贝来上调 的表达水平，从而使α-淀粉酶活性与携带单个 基因拷贝的菌株相比提高了 20.9 倍。接下来，通过过表达细胞外分子伴侣 PrsA 和信号肽肽酶 SppA，进一步将α-淀粉酶分泌提高了 26.4 倍。当最终的表达菌株在 3 L 发酵罐中培养 90 h 时，AmyQ 的产量提高了 57.9 倍。所提出的策略允许开发具有工业相关性的稳健无标记无质粒超分泌 菌株。
