Random Mutagenesis of the Pm Promoter as a Powerful Strategy for Improvement of Recombinant-Gene Expression
Random Mutagenesis of the Pm Promoter as a Powerful Strategy for Improvement of Recombinant-Gene Expression
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DOI：
10.1128/aem.02315-08
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发表时间：
2009-04-01
影响因子：
4.4
通讯作者：
Valla, Svein
中科院分区：
文献类型：
--
作者：
Bakke, Ingrid;Berg, Laila;Valla, Svein
诱导型 Pm-xylS 启动子系统已被证明可用于在几种革兰氏阴性物种和大肠杆菌的高细胞密度培养中生产重组蛋白。在这项研究中，我们对 Pm 的 24 bp 区域（包括 -10 元件）进行随机诱变，从而在大肠杆菌中产生了大型突变体文库。通过使用 β-内酰胺酶作为报告基因，可以有效地识别低频出现的 Pm 突变体，该突变体显示出启动子活性显着增加（上突变体）。上突变体通常在整个诱变区域携带多个点突变，并结合转录起始位点周围的缺失。鉴定出β-内酰胺酶表达增加约 14 倍（相对于野生型 Pm）的突变体，且不损失诱导表型。这些突变体还强烈刺激了另外两个报告基因 luc（编码萤火虫荧光素酶）和 celB（编码磷酸葡萄糖变位酶）的表达，并被发现显着改善（两倍）先前优化的工艺，用于在高细胞密度条件下在大肠杆菌中高水平重组生产医学上重要的粒细胞巨噬细胞集落刺激因子。这些结果证明了使用启动子随机诱变在工业水平上改善蛋白质表达的潜力，并表明单个功能元件的靶向修饰不足以获得优化的启动子序列。
The inducible Pm-xylS promoter system has proven useful for production of recombinant proteins in several gram-negative species and in high-cell-density cultivations of Escherichia coli. In this study we subjected a 24-bp region of Pm (including the -10 element) to random mutagenesis, leading to large mutant libraries in E. coli. Low-frequency-occurring Pm mutants displaying strongly increased promoter activity (up-mutants) could be efficiently identified by using beta-lactamase as a reporter. The up-mutants typically carried multiple point mutations positioned throughout the mutagenized region, combined with deletions around the transcription start site. Mutants displaying up to about a 14-fold increase in beta-lactamase expression (relative to wild-type Pm) were identified without loss of the inducible phenotype. The mutants also strongly stimulated the expression of two other reporter genes, luc (encoding firefly luciferase) and celB (encoding phosphoglucomutase), and were found to significantly improve (twofold) a previously optimized process for high-level recombinant production of the medically important granulocyte-macrophage colony-stimulating factor in E. coli under high-cell-density conditions. These results demonstrate the potential of using random mutagenesis of promoters to improve protein expression at industrial levels and indicate that targeted modifications of individual functional elements are not sufficient to obtain optimized promoter sequences.