Collections of characterized promoters of different strengths are key resources for synthetic biology, but are not well established for many important organisms, including industrially relevant Clostridium spp. When generating promoters, reporter constructs are used to measure expression, but classical fluorescent reporter proteins are oxygen-dependent and hence inactive in anaerobic bacteria like Clostridium. We directly compared oxygen-independent reporters of different types in Clostridium acetobutylicum and found that glucuronidase (GusA) from E. coli performed best. Using GusA, a library of synthetic promoters was first generated by a typical approach entailing complete randomization of a constitutive thiolase gene promoter (P_thl) except for the consensus −35 and −10 elements. In each synthetic promoter, the chance of each degenerate position matching P_thl was 25%. Surprisingly, none of the tested synthetic promoters from this library were functional in C. acetobutylicum, even though they functioned as expected in E. coli. Next, instead of complete randomization, we specified lower promoter mutation rates using oligonucleotide primers synthesized using custom mixtures of nucleotides. Using these primers, two promoter libraries were constructed in which the chance of each degenerate position matching P_thl was 79% or 58%, instead of 25% as before. Synthetic promoters from these “stringent” libraries functioned well in C. acetobutylicum, covering a wide range of strengths. The promoters functioned similarly in the distantly related species Clostridium sporogenes, and allowed predictable metabolic engineering of C. acetobutylicum for acetoin production. Besides generating the desired promoters and demonstrating their useful properties, this work indicates an unexpected “stringency” of promoter sequences in Clostridium, not reported previously.

中文翻译：

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调谐启动子库突变率揭示和规避梭菌中合成启动子序列的严格性。

具有不同强度的特征性启动子的集合是合成生物学的关键资源，但对于许多重要的生物（包括与工业有关的梭状芽孢杆菌），并没有得到很好的建立。当产生启动子时，报道分子构建体用于测量表达，但是经典的荧光报道蛋白是氧依赖性的，因此在厌氧细菌如梭状芽胞杆菌中是无活性的。我们直接比较了丙酮丁醇梭菌中不同类型的非氧依赖性报道分子，发现大肠杆菌中的葡糖醛酸糖苷酶（GusA）表现最佳。使用GusA，首先通过典型的方法将组成型硫解酶基因启动子（P_thl），除了共识-35和-10元素。在每个合成启动子中，每个简并位置匹配P _thl的机会为25％。出人意料的是，尽管该文库在大肠杆菌中具有预期的功能，但该文库中测试的合成启动子均未在丙酮丁醇梭菌中起作用。接下来，代替完全随机化，我们使用使用核苷酸的定制混合物合成的寡核苷酸引物，指定了较低的启动子突变率。使用这些引物，构建了两个启动子文库，其中每个简并位置匹配的P _thl的机会为79％或58％，而不是以前的25％。这些“严格”文库的合成启动子在丙酮丁醇梭菌（C. acetobutylicum），具有广泛的优势。这些启动子在远缘的产孢梭状芽胞杆菌中具有相似的功能，并允许可预测的丙酮丁醇梭菌代谢工程化，以生产丙酮酸。除了产生所需的启动子并证明其有用的特性外，这项工作还表明了梭状芽孢杆菌中启动子序列的意外“严格性”，这是以前未曾报道过的。