Petersen D J, Welch R W, Walter K A, Mermelstein L D, Papoutsakis E T, Rudolph F B, Bennett G N
Rice University, Department of Biochemistry and Cell Biology, Houston, Texas 77251.
Ann N Y Acad Sci. 1991 Dec 27;646:94-8. doi: 10.1111/j.1749-6632.1991.tb18567.x.
The acetone-butanol fermentation of C. acetobutylicum is characterized by the unique shift from acid to solvent production. The mechanism of the solventogenic switch involves the induction of several enzymes, including NADH-dependent butanol dehydrogenase (BDH) at the onset of solventogenesis. This enzyme is responsible for the final conversion of butyraldehyde to butanol, and is distinct from the NADPH-dependent alcohol dehydrogenase (ADH) also present in the organism. To characterize the genetic control of this gene, we have cloned and expressed it in E. coli. A lambda EMBL3 phage library of C. acetobutylicum DNA was screened via plaque hybridization using a [32P]-radiolabeled, 32-fold degenerate, 62-mer oligonucleotide probe. The probe was designed by reverse translation of the NH2-terminal amino acid sequence of purified BDH II. Southern blot experiments indicate that the phage insert was of clostridial origin and had no homology with the previously cloned NADPH-dependent ADH. Subcloning of DNA from purified positive plaques has localized the gene to a 3.5-kb EcoRI fragment from which the enzyme is well expressed. The sequence of the 25 NH2-terminal amino acids for the cloned enzyme purified from E. coli was determined and found to be identical to that for the clostridial NADH-dependent BDH II. Maxicell analysis of [35S]-radiolabeled plasmid-encoded proteins identified a species encoded by the clostridial insert with the expected Mr of 42 kD.
丙酮丁醇梭菌的丙酮-丁醇发酵的特点是从产酸到产溶剂的独特转变。溶剂生成转换的机制涉及几种酶的诱导，包括在溶剂生成开始时诱导的依赖于NADH的丁醇脱氢酶（BDH）。这种酶负责将丁醛最终转化为丁醇，并且与该生物体中也存在的依赖于NADPH的醇脱氢酶（ADH）不同。为了表征该基因的遗传控制，我们已将其克隆并在大肠杆菌中表达。使用[32P]放射性标记的、32倍简并的62聚体寡核苷酸探针通过噬菌斑杂交筛选丙酮丁醇梭菌DNA的λEMBL3噬菌体文库。该探针是通过对纯化的BDH II的NH2末端氨基酸序列进行反向翻译设计的。Southern印迹实验表明，噬菌体插入片段起源于梭菌，与先前克隆的依赖于NADPH的ADH没有同源性。从纯化的阳性噬菌斑中进行DNA亚克隆已将该基因定位到一个3.5 kb的EcoRI片段，该酶从该片段中得到良好表达。测定了从大肠杆菌中纯化的克隆酶的25个NH2末端氨基酸的序列，发现其与梭菌依赖于NADH的BDH II的序列相同。对[35S]放射性标记的质粒编码蛋白进行的最大细胞分析鉴定出一种由梭菌插入片段编码的、预期分子量为42 kD的蛋白。
