Banerjee Areen, Leang Ching, Ueki Toshiyuki, Nevin Kelly P, Lovley Derek R
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, USA.
Appl Environ Microbiol. 2014 Apr;80(8):2410-6. doi: 10.1128/AEM.03666-13. Epub 2014 Feb 7.
The development of tools for genetic manipulation of Clostridium ljungdahlii has increased its attractiveness as a chassis for autotrophic production of organic commodities and biofuels from syngas and microbial electrosynthesis and established it as a model organism for the study of the basic physiology of acetogenesis. In an attempt to expand the genetic toolbox for C. ljungdahlii, the possibility of adapting a lactose-inducible system for gene expression, previously reported for Clostridium perfringens, was investigated. The plasmid pAH2, originally developed for C. perfringens with a gusA reporter gene, functioned as an effective lactose-inducible system in C. ljungdahlii. Lactose induction of C. ljungdahlii containing pB1, in which the gene for the aldehyde/alcohol dehydrogenase AdhE1 was downstream of the lactose-inducible promoter, increased expression of adhE1 30-fold over the wild-type level, increasing ethanol production 1.5-fold, with a corresponding decrease in acetate production. Lactose-inducible expression of adhE1 in a strain in which adhE1 and the adhE1 homolog adhE2 had been deleted from the chromosome restored ethanol production to levels comparable to those in the wild-type strain. Inducing expression of adhE2 similarly failed to restore ethanol production, suggesting that adhE1 is the homolog responsible for ethanol production. Lactose-inducible expression of the four heterologous genes necessary to convert acetyl coenzyme A (acetyl-CoA) to acetone diverted ca. 60% of carbon flow to acetone production during growth on fructose, and 25% of carbon flow went to acetone when carbon monoxide was the electron donor. These studies demonstrate that the lactose-inducible system described here will be useful for redirecting carbon and electron flow for the biosynthesis of products more valuable than acetate. Furthermore, this tool should aid in optimizing microbial electrosynthesis and for basic studies on the physiology of acetogenesis.
用于李氏梭菌基因操作的工具的发展，增加了其作为从合成气和微生物电合成中自养生产有机商品和生物燃料的底盘的吸引力，并使其成为产乙酸基本生理学研究的模式生物。为了扩展李氏梭菌的基因工具箱，研究了采用先前报道的用于产气荚膜梭菌的乳糖诱导系统进行基因表达的可能性。最初为产气荚膜梭菌开发的带有gusA报告基因的质粒pAH2，在李氏梭菌中作为有效的乳糖诱导系统发挥作用。含有pB1的李氏梭菌经乳糖诱导，其中醛/醇脱氢酶AdhE1的基因位于乳糖诱导型启动子的下游，使得adhE1的表达比野生型水平增加了30倍，乙醇产量增加了1.5倍，同时乙酸产量相应降低。在adhE1和adhE1同源物adhE2已从染色体上缺失的菌株中，adhE1的乳糖诱导型表达将乙醇产量恢复到与野生型菌株相当的水平。类似地，诱导adhE2的表达未能恢复乙醇产量，这表明adhE1是负责乙醇生产的同源物。将乙酰辅酶A（acetyl-CoA）转化为丙酮所需的四个异源基因的乳糖诱导型表达，在以果糖为生长底物时，约60%的碳流转向丙酮生产，而当一氧化碳作为电子供体时，25%的碳流用于丙酮生产。这些研究表明，本文所述的乳糖诱导系统将有助于重新引导碳和电子流，用于生物合成比乙酸更有价值的产品。此外，该工具应有助于优化微生物电合成以及用于产乙酸生理学的基础研究。
