Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824
Cloning, sequencing, and expression of genes encoding phosphotransacetylase and acetate kinase from Clostridium acetobutylicum ATCC 824
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DOI：
10.1128/aem.62.8.2758-2766.1996
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发表时间：
1996-08-01
影响因子：
4.4
通讯作者：
Rudolph, FB
中科院分区：
文献类型：
--
作者：
Boynton, ZL;Bennett, GN;Rudolph, FB
在丙酮丁醇梭菌发酵中，磷酸转乙酰酶（PTA）和乙酸激酶（AK）催化乙酰辅酶A转化为乙酸。产乙酸步骤是产酸发酵阶段的重要元素，并产生用于梭菌细胞生长的ATP。对编码PTA和AK的基因pta和ack进行了克隆和序列测定。对大肠杆菌和C. acetobutylicum窝藏的亚克隆，和AK和PTA活性都被证明是升高的。DNA序列分析表明，pta和ack基因在梭菌染色体上相邻，pta位于上游。pta基因编码333个氨基酸残基的蛋白质，计算分子量为36.2 kDa，ack编码401个氨基酸残基的多肽，分子量为44.3 kDa。引物延伸分析确定了一个单一的转录起始位点位于70 bp的pta基因的起始密码子上游，这表明这些串联基因的操纵子安排。ack和pta基因在C.丙酮丁醇的结果表明乙酸酯与其它主要产物的最终比率更高，并且存在更大比例的二碳衍生产物对四碳衍生产物。
The enzymes phosphotransacetylase (PTA) and acetate kinase (AK) catalyze the conversion of acetyl coenzyme A to acetate in the fermentation of Clostridium acetobutylicum. The acetate-producing step is an important element in the acidogenic fermentation stage and generates ATP for clostridial cell growth. The genes pta and ack, encoding PTA and AK, respectively, were cloned and sequenced. Enzyme activity assays were performed on cell extracts from Escherichia coli and C. acetobutylicum harboring the subclone, and both AK and PTA activities were shown to be elevated. DNA sequence analysis showed that the pta and ack genes are adjacent in the clostridial chromosome, with pta upstream. The pta gene encodes a protein of 333 amino acid residues with a calculated molecular mass of 36.2 kDa, and ack encodes a polypeptide of 401 residues with a molecular mass of 44.3 kDa. Primer extension analysis identified a single transcriptional start site located 70 bp upstream of the start codon for the pta gene, suggesting an operon arrangement for these tandem genes. The results from overexpression of ack and pta in C. acetobutylicum showed that the final ratios of acetate to other major products were higher and that there was a greater proportion of two- versus four-carbon-derived products.