William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, USA.
Metab Eng. 2011 Jul;13(4):373-82. doi: 10.1016/j.ymben.2011.04.002. Epub 2011 Apr 22.
Clostridium tyrobutyricum ATCC 25755, a butyric acid producing bacterium, has been engineered to overexpress aldehyde/alcohol dehydrogenase 2 (adhE2, Genebank no. AF321779) from Clostridium acetobutylicum ATCC 824, which converts butyryl-CoA to butanol, under the control of native thiolase (thl) promoter. Butanol titer of 1.1g/L was obtained in C. tyrobutyricum overexpressing adhE2. The effects of inactivating acetate kinase (ack) and phosphotransbutyrylase (ptb) genes in the host on butanol production were then studied. A high C4/C2 product ratio of 10.6 (mol/mol) was obtained in ack knockout mutant, whereas a low C4/C2 product ratio of 1.4 (mol/mol) was obtained in ptb knockout mutant, confirming that ack and ptb genes play important roles in controlling metabolic flux distribution in C. tyrobutyricum. The highest butanol titer of 10.0g/L and butanol yield of 27.0% (w/w, 66% of theoretical yield) were achieved from glucose in the ack knockout mutant overexpressing adhE2. When a more reduced substrate mannitol was used, the butanol titer reached 16.0 g/L with 30.6% (w/w) yield (75% theoretical yield). Moreover, C. tyrobutyricum showed good butanol tolerance, with >80% and ∼60% relative growth rate at 1.0% and 1.5% (v/v) butanol. These results suggest that C. tyrobutyricum is a promising heterologous host for n-butanol production from renewable biomass.
丙酮丁醇梭菌（Clostridium tyrobutyricum）ATCC 25755 是一种产丁酸细菌，通过过表达来自丙酮丁醇梭菌（Clostridium acetobutylicum）ATCC 824 的醛/醇脱氢酶 2（adhE2，Genebank 编号 AF321779），利用内源性硫解酶（thl）启动子将丁酰辅酶 A 转化为丁醇。在过表达 adhE2 的 C. tyrobutyricum 中，丁醇产量达到 1.1g/L。然后研究了宿主中乙酰激酶（ack）和磷酸转丁酰酶（ptb）基因失活对丁醇生产的影响。在 ack 敲除突变体中获得了高的 C4/C2 产物比 10.6（摩尔/摩尔），而在 ptb 敲除突变体中获得了低的 C4/C2 产物比 1.4（摩尔/摩尔），证实了 ack 和 ptb 基因在控制 C. tyrobutyricum 代谢通量分布方面起着重要作用。在过表达 adhE2 的 ack 敲除突变体中，从葡萄糖中获得了最高的丁醇产量 10.0g/L 和丁醇产率 27.0%（w/w，理论产率的 66%）。当使用更还原的底物甘露醇时，丁醇产量达到 16.0g/L，产率为 30.6%（w/w）（理论产率的 75%）。此外，C. tyrobutyricum 表现出良好的丁醇耐受性，在 1.0%和 1.5%（v/v）丁醇下相对生长率分别为>80%和∼60%。这些结果表明，C. tyrobutyricum 是一种很有前途的从可再生生物质生产正丁醇的异源宿主。
