@article {3130,
	title = {Lactose-inducible system for metabolic engineering of Clostridium ljungdahlii.},
	journal = {Appl Environ Microbiol},
	volume = {80},
	year = {2014},
	month = {2014 Apr},
	pages = {2410-6},
	abstract = {<p>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.</p>},
	keywords = {Acetic Acid, Acetone, Acetyl Coenzyme A, Alcohol Dehydrogenase, Carbon, Clostridium, Ethanol, Fructose, Gene Expression, Gene Expression Regulation, Bacterial, Lactose, Metabolic Engineering, Metabolic Flux Analysis, Transcriptional Activation},
	issn = {1098-5336},
	doi = {10.1128/AEM.03666-13},
	author = {Banerjee, Areen and Leang, Ching and Ueki, Toshiyuki and Nevin, Kelly P and Lovley, Derek R}
}
@article {3150,
	title = {A genetic system for Clostridium ljungdahlii: a chassis for autotrophic production of biocommodities and a model homoacetogen.},
	journal = {Appl Environ Microbiol},
	volume = {79},
	year = {2013},
	month = {2013 Feb},
	pages = {1102-9},
	abstract = {<p>Methods for genetic manipulation of Clostridium ljungdahlii are of interest because of the potential for production of fuels and other biocommodities from carbon dioxide via microbial electrosynthesis or more traditional modes of autotrophy with hydrogen or carbon monoxide as the electron donor. Furthermore, acetogenesis plays an important role in the global carbon cycle. Gene deletion strategies required for physiological studies of C. ljungdahlii have not previously been demonstrated. An electroporation procedure for introducing plasmids was optimized, and four different replicative origins for plasmid propagation in C. ljungdahlii were identified. Chromosomal gene deletion via double-crossover homologous recombination with a suicide vector was demonstrated initially with deletion of the gene for FliA, a putative sigma factor involved in flagellar biogenesis and motility in C. ljungdahlii. Deletion of fliA yielded a strain that lacked flagella and was not motile. To evaluate the potential utility of gene deletions for functional genomic studies and to redirect carbon and electron flow, the genes for the putative bifunctional aldehyde/alcohol dehydrogenases, adhE1 and adhE2, were deleted individually or together. Deletion of adhE1, but not adhE2, diminished ethanol production with a corresponding carbon recovery in acetate. The double deletion mutant had a phenotype similar to that of the adhE1-deficient strain. Expression of adhE1 in trans partially restored the capacity for ethanol production. These results demonstrate the feasibility of genetic investigations of acetogen physiology and the potential for genetic manipulation of C. ljungdahlii to optimize autotrophic biocommodity production.</p>},
	keywords = {Clostridium, Electroporation, Gene Deletion, Genetic Complementation Test, Genetic Vectors, Genetics, Microbial, Metabolic Engineering, Molecular Biology, Plasmids, Transformation, Bacterial},
	issn = {1098-5336},
	doi = {10.1128/AEM.02891-12},
	author = {Leang, Ching and Ueki, Toshiyuki and Nevin, Kelly P and Lovley, Derek R}
}
