Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production
Construction and characterization of ack deleted mutant of Clostridium tyrobutyricum for enhanced butyric acid and hydrogen production
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DOI：
10.1021/bp060082g
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发表时间：
2006-10-06
影响因子：
2.9
通讯作者：
Yang, Shang-Tian
中科院分区：
文献类型：
--
作者：
Liu, Xiaoguang;Zhu, Ying;Yang, Shang-Tian
酪丁酸梭菌从葡萄糖和木糖中产生丁酸、乙酸、H-2 和 CO2 作为其主要发酵产物。为了提高丁酸和氢气的产量，采用整合诱变来创建具有失活ack基因的代谢工程突变体，该基因编码与乙酸盐形成途径相关的乙酸激酶（AK）。构建了含有乙酸激酶基因（ack）片段的非复制质粒，并通过电穿孔将其引入酪丁酸梭菌中。将质粒整合到染色体上的同源区域应使目标 ack 基因失活并产生 ack 删除的突变体 PAK-Em。酶活性测定表明，PAK-Em 中的 AK 活性降低了约 50%；与此同时，磷酸转乙酰酶 (PTA) 和氢化酶活性分别增加了约 40%。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示，突变体中32 kDa分子量相似的蛋白表达明显减少。与野生型相比，突变体在 pH 6.0 和 37 C 下生长更慢，比生长速率较低，为 0.14 h(-1)（野生型为 0.21 h-1），这可能是由于 PTA-AK 途径部分受损所致。然而，突变体在最终浓度为 41.7 g/L（vs 19.98 g/L）时产生的丁酸盐多了 23.5%（0.42 g/g 葡萄糖 vs 0.34 g/g 葡萄糖），这是由于其较高的丁酸盐耐受性（如使用培养基中不同初始丁酸盐浓度的生长动力学研究所示）。与野生型相比，突变体从葡萄糖中产生的氢多了 50%（0.024 g/g）。 PAK-Em 在纤维床生物反应器 (FBB) 中的固定化细胞发酵进一步提高了最终丁酸浓度 (50.1 g/L) 和丁酸盐产量 (0.45 g/g 葡萄糖)。此外，在pH 5.0以木糖为底物的FBB发酵中，突变体仅产生丁酸，而野生型产生大量的乙酸盐（0.43 g/g木糖）和乳酸（0.61 g/g木糖）和少量的丁酸（0.05 g/g木糖），这表明突变体中ack缺失引起了显着的代谢途径转变。
Clostridium tyrobutyricum produces butyrate, acetate, H-2, and CO2 as its main fermentation products from glucose and xylose. To improve butyric acid and hydrogen production, integrational mutagenesis was used to create a metabolically engineered mutant with inactivated ack gene, encoding acetate kinase (AK) associated with the acetate formation pathway. A non-replicative plasmid containing the acetate kinase gene (ack) fragment was constructed and introduced into C. tyrobutyricum by electroporation. Integration of the plasmid into the homologous region on the chromosome should inactivate the target ack gene and produce ack-deleted mutant, PAK-Em. Enzyme activity assays showed that the AK activity in PAK-Em decreased by similar to 50%; meanwhile, phosphotransacetylase (PTA) and hydrogenase activities each increased by similar to 40%. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the expression of protein with similar to 32 kDa molecular mass was reduced significantly in the mutant. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37 C, with a lower specific growth rate of 0.14 h(-1) (vs 0.21 h-1 for the wild type), likely due to the partially impaired PTA-AK pathway. However, the mutant produced 23.5% more butyrate (0.42 vs 0.34 g/g glucose) at a higher final concentration of 41.7 g/L (vs 19.98 g/L) as a result of its higher butyrate tolerance as indicated in the growth kinetics study using various intial concentrations of butyrate in the media. The mutant also produced 50% more hydrogen (0.024 g/g) from glucose than the wild type. Immobilized- cell fermentation of PAK-Em in a fibrous-bed bioreactor (FBB) further increased the final butyric acid concentration (50.1 g/L) and the butyrate yield (0.45 g/g glucose). Furthermore, in the FBB fermentation at pH 5.0 with xylose as the substrate, only butyric acid was produced by the mutant, whereas the wild type produced large amounts of acetate (0.43 g/g xylose) and lactate (0.61 g/g xylose) and little butyrate (0.05 g/g xylose), indicating a dramatic metabolic pathway shift caused by the ack deletion in the mutant.