Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance
Metabolic engineering of the non-sporulating, non-solventogenic Clostridium acetobutylicum strain M5 to produce butanol without acetone demonstrate the robustness of the acid-formation pathways and the importance of the electron balance
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DOI：
10.1016/j.ymben.2008.07.005
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发表时间：
2008-11-01
影响因子：
8.4
通讯作者：
Papoutsakis, Eleftherios T.
中科院分区：
文献类型：
--
作者：
Sillers, Ryan;Chow, Alison;Papoutsakis, Eleftherios T.
在乙酰丁酸梭菌中，由aad基因编码的原醇/醛脱氢酶负责丁醇的形成。我们补充了不产孢子、不产溶剂的C. acetobutylicum M5菌株（该菌株失去了含有aad和丙酮形成基因的pSOL1巨质粒），并从磷酸转丁基化酶启动子中表达了aad，将丁醇产量恢复到野生型水平。由于不产生丙酮，没有酸（乙酸或丁酸）被重新吸收，导致高丁酸，尤其是乙酸水平。为了对抗乙酸的产生，我们检测了巯基酶的过表达，以减少乙酰辅酶库和增加丁基辅酶库。我们将巯基酶过表达与巯基酶过表达结合起来，旨在增强丁醇的形成。虽然限制了乙酸和乙醇的形成，但丁醇滴度没有提高。我们还使用改进的方案生成了M5的醋酸激酶（AK）和丁酸激酶（BK）敲除（KO）突变体，以增加抗生素抗性基因的表达。这些菌株的乙酸酯和丁酸酯的生成分别减少了60%以上。我们用aad过表达补充AKKO M5菌株，但不能成功转化BKKO M5菌株。与过表达的M5菌株相比，过表达的AKKO M5菌株产生的乙酸较少，但丁醇也较少。这些数据表明，pSOL1巨质粒的缺失使细胞抵抗两种酸形成途径的变化，尤其是丁酸形成途径的变化。我们认为，在没有丙酮和酸生产的情况下产生高丁醇生产者的困难是由于无法控制电子流，这似乎受到未知pSOL1基因的影响。(C) 2008爱思唯尔公司版权所有。
The primary alcohol/aldehyde dehydrogenase (coded by the aad gene) is responsible for butanol formation in Clostridium acetobutylicum. We complemented the non-sporulating, non-solvent-producing C. acetobutylicum M5 strain (which has lost the pSOL1 megaplasmid containing aad and the acetone-formation genes) with aad expressed from the phosphotransbutyrylase promoter and restored butanol production to wild type levels. Because no acetone was produced, no acids (acetate or butyrate) were re-assimilated leading to high butyrate but especially acetate levels. To counter acetate production, we examined thiolase overexpression in order reduce the acetyl-CoA pool and enhance the butyryl-CoA pool. We combined thiolase overexpression with aad overexpression aiming to also enhance butanol formation. While limiting the formation of acetate and ethanol, the butanol titers were not improved. We also generated acetate kinase (AK) and butyrate kinase (BK) knockout (KO) mutants of M5 using a modified protocol to increase the antibiotic-resistance gene expression. These strains exhibited greater than 60% reduction in acetate or butyrate formation, respectively. We complemented the AKKO M5 strain with aad overexpression, but could not successfully transform the BKKO M5 strain. The AKKO M5 strain overexpressing aad produced less acetate, but also less butanol compared to the M5 aad overexpression strain. These data suggest that loss of the pSOL1 megaplasmid renders cells resistant to changes in the two acid-formation pathways, and especially so for butyrate formation. We argue that the difficulty in generating high butanol producers without acetone and acid production is hindered by the inability to control the electron flow, which appears to be affected by unknown pSOL1 genes. (C) 2008 Elsevier Inc. All rights reserved.