Tummala Seshu B, Junne Stefan G, Papoutsakis Eleftherios T
Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208, USA.
J Bacteriol. 2003 Jun;185(12):3644-53. doi: 10.1128/JB.185.12.3644-3653.2003.
Plasmid pAADB1 for the overexpression of the alcohol-aldehyde dehydrogenase (aad) gene and downregulation of the coenzyme A transferase (CoAT) using antisense RNA (asRNA) against ctfB (the second CoAT gene on the polycistronic aad-ctfA-ctfB message) was used in order to increase the butanol/acetone ratio of Clostridium acetobutylicum ATCC 824 fermentations. Acetone and butanol levels were drastically reduced in 824(pCTFB1AS) (expresses only an asRNA against ctfB) compared to 824(pSOS95del) (plasmid control). Compared to strain 824(pCTFB1AS), 824(pAADB1) fermentations exhibited two profound differences. First, butanol levels were ca. 2.8-fold higher in 824(pAADB1) and restored back to plasmid control levels, thus supporting the hypothesis that asRNA downregulation of ctfB leads to degradation of the whole aad-ctfA-ctfB transcript. Second, ethanol titers in 824(pAADB1) were ca. 23-fold higher and the highest (ca. 200 mM) ever reported in C. acetobutylicum. Western blot analysis confirmed that CoAT was downregulated in 824(pAADB1) at nearly the same levels as in strain 824(pCTFB1AS). Butyrate depletion in 824(pAADB1) fermentations suggested that butyryl-CoA was limiting butanol production in 824(pAADB1). This was confirmed by exogenously adding butyric acid to 824(pAADB1) fermentations to increase the butanol/ethanol ratio. DNA microarray analysis showed that aad overexpression profoundly affects the large-scale transcriptional program of the cells. Several classes of genes were differentially expressed [strain 824(pAADB1) versus strain 824(pCTFB1AS)], including genes of the stress response, sporulation, and chemotaxis. The expression patterns of the CoAT genes (ctfA and ctfB) and aad were consistent with the overexpression of aad and asRNA downregulation of ctfB.
为了提高丙酮丁醇梭菌ATCC 824发酵产物中丁醇/丙酮的比例，使用了质粒pAADB1，该质粒可通过针对ctfB（多顺反子aad - ctfA - ctfB转录本上的第二个辅酶A转移酶基因）的反义RNA（asRNA）来实现醇醛脱氢酶（aad）基因的过表达以及辅酶A转移酶（CoAT）的下调。与824(pSOS95del)（质粒对照）相比，824(pCTFB1AS)（仅表达针对ctfB的asRNA）中的丙酮和丁醇水平大幅降低。与菌株824(pCTFB1AS)相比，824(pAADB1)发酵表现出两个显著差异。首先，824(pAADB1)中的丁醇水平约高2.8倍，并恢复到质粒对照水平，从而支持了ctfB的asRNA下调导致整个aad - ctfA - ctfB转录本降解的假说。其次，824(pAADB1)中的乙醇滴度约高23倍，是丙酮丁醇梭菌中报道的最高水平（约200 mM）。蛋白质印迹分析证实，824(pAADB1)中CoAT的下调水平与菌株824(pCTFB1AS)几乎相同。824(pAADB1)发酵中丁酸的消耗表明丁酰辅酶A限制了824(pAADB1)中丁醇的产生。通过向824(pAADB1)发酵中外源添加丁酸以提高丁醇/乙醇比例，这一点得到了证实。DNA微阵列分析表明，aad的过表达深刻影响了细胞的大规模转录程序。几类基因存在差异表达[824(pAADB1)菌株与824(pCTFB1AS)菌株相比]，包括应激反应、芽孢形成和趋化性相关基因。CoAT基因（ctfA和ctfB）和aad的表达模式与aad的过表达以及ctfB的asRNA下调一致。
