Exploring the Nonconserved Sequence Space of Synthetic Expression Modules in Bacillus subtilis.
Exploring the Nonconserved Sequence Space of Synthetic Expression Modules in Bacillus subtilis.
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DOI：
10.1021/acssynbio.8b00110
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发表时间：
2018-06
影响因子：
4.7
通讯作者：
Christopher Sauer;E. Ver Loren van Themaat;Léonie G M Boender;D. Groothuis;Rita Cruz;L. Hamoen;C. Harwood
中科院分区：
文献类型：
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作者：
Christopher Sauer;E. Ver Loren van Themaat;Léonie G M Boender;D. Groothuis;Rita Cruz;L. Hamoen;C. Harwood
提高蛋白质表达水平是酶商业化生产的关键一步。到目前为止，仅基于一致序列预测启动子活性和翻译启动效率的结果喜忧参半。在这里，我们通过设计和合成一个大型组合文库来应对这一挑战，该文库包括枯草芽孢杆菌的∼12000个独特的合成表达模块(SM)。利用GFP荧光作为基因表达的报告基因，我们获得了跨越5个数量级的动态表达范围，并且与已经很强的VEG表达模块相比，表达最大增加了13倍。对合成模块的分析表明，mRNA 5‘端的序列是导致表达水平差异的最重要因素，可能是通过防止形成影响翻译起始的强大二级mRNA结构。当GFP编码区被编码工业相关枯草杆菌木聚糖酶的xynA基因的编码区取代时，木聚糖酶的产量仅提高了3倍。此外，绿色荧光蛋白和木聚糖酶的表达水平之间的相关性很弱。这表明GFP和xynA构建体之间表达水平的差异是由于5‘端mRNA折叠的差异以及由此导致的翻译起始率的差异。我们的数据表明，使用大型扫描电子显微镜文库，结合高通量技术，是提高特定蛋白质产量的有效方法，但结果不一定适用于其他蛋白质。
Increasing protein expression levels is a key step in the commercial production of enzymes. Predicting promoter activity and translation initiation efficiency based solely on consensus sequences have so far met with mixed results. Here, we addressed this challenge using a "brute-force" approach by designing and synthesizing a large combinatorial library comprising ∼12 000 unique synthetic expression modules (SEMs) for Bacillus subtilis. Using GFP fluorescence as a reporter of gene expression, we obtained a dynamic expression range that spanned 5 orders of magnitude, as well as a maximal 13-fold increase in expression compared with that of the already strong veg expression module. Analyses of the synthetic modules indicated that sequences at the 5'-end of the mRNA were the most important contributing factor to the differences in expression levels, presumably by preventing formation of strong secondary mRNA structures that affect translation initiation. When the gfp coding region was replaced by the coding region of the xynA gene, encoding the industrially relevant B. subtilis xylanase enzyme, only a 3-fold improvement in xylanase production was observed. Moreover, the correlation between GFP and xylanase expression levels was weak. This suggests that the differences in expression levels between the gfp and xynA constructs were due to differences in 5'-end mRNA folding and consequential differences in the rates of translation initiation. Our data show that the use of large libraries of SEMs, in combination with high-throughput technologies, is a powerful approach to improve the production of a specific protein, but that the outcome cannot necessarily be extrapolated to other proteins.