NDLI: Antisense RNA Downregulation of Coenzyme A Transferase Combined with Alcohol-Aldehyde Dehydrogenase Overexpression Leads to Predominantly Alcohologenic Clostridium acetobutylicum Fermentations

Content Provider	PubMed Central
Author	Tummala, Seshu B. Junne, Stefan G. Papoutsakis, Eleftherios T.
Copyright Year	2003
Abstract	Plasmid pAADB1 for the overexpression of the alcohol-aldehyde dehydrogenase (aad) gene and downregulation of the coenzyme A transferase (CoAT) using antisense RNA (asRNA) against ctfB (the second CoAT gene on the polycistronic aad-ctfA-ctfB message) was used in order to increase the butanol/acetone ratio of Clostridium acetobutylicum ATCC 824 fermentations. Acetone and butanol levels were drastically reduced in 824(pCTFB1AS) (expresses only an asRNA against ctfB) compared to 824(pSOS95del) (plasmid control). Compared to strain 824(pCTFB1AS), 824(pAADB1) fermentations exhibited two profound differences. First, butanol levels were ca. 2.8-fold higher in 824(pAADB1) and restored back to plasmid control levels, thus supporting the hypothesis that asRNA downregulation of ctfB leads to degradation of the whole aad-ctfA-ctfB transcript. Second, ethanol titers in 824(pAADB1) were ca. 23-fold higher and the highest (ca. 200 mM) ever reported in C. acetobutylicum. Western blot analysis confirmed that CoAT was downregulated in 824(pAADB1) at nearly the same levels as in strain 824(pCTFB1AS). Butyrate depletion in 824(pAADB1) fermentations suggested that butyryl-CoA was limiting butanol production in 824(pAADB1). This was confirmed by exogenously adding butyric acid to 824(pAADB1) fermentations to increase the butanol/ethanol ratio. DNA microarray analysis showed that aad overexpression profoundly affects the large-scale transcriptional program of the cells. Several classes of genes were differentially expressed [strain 824(pAADB1) versus strain 824(pCTFB1AS)], including genes of the stress response, sporulation, and chemotaxis. The expression patterns of the CoAT genes (ctfA and ctfB) and aad were consistent with the overexpression of aad and asRNA downregulation of ctfB.
Related Links	http://dx.doi.org/10.1128/JB.185.12.3644-3653.2003
Starting Page	3644
File Format	PDF
ISSN	10985530
e-ISSN	10985530
Journal	Journal of Bacteriology
Issue Number	12
Volume Number	185
Language	English
Publisher	American Society for Microbiology
Publisher Date	2003-06-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Microbiology

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Molecular Characterization and Transcriptional Analysis of adhE2, the Gene Encoding the NADH-Dependent Aldehyde/Alcohol Dehydrogenase Responsible for Butanol Production in Alcohologenic Cultures of Clostridium acetobutylicum ATCC 824

Molecular characterization of an aldehyde/alcohol dehydrogenase gene from Clostridium acetobutylicum ATCC 824.

Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum

Design of Antisense RNA Constructs for Downregulation of the Acetone Formation Pathway of Clostridium acetobutylicum

Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum.

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The alcohol dehydrogenase of Clostridium acetobutylicum.

Molecular cloning of an alcohol (butanol) dehydrogenase gene cluster from Clostridium acetobutylicum ATCC 824.

Cloning, sequencing, and expression of clustered genes encoding beta-hydroxybutyryl-coenzyme A (CoA) dehydrogenase, crotonase, and butyryl-CoA dehydrogenase from Clostridium acetobutylicum ATCC 824.

Antisense RNA Downregulation of Coenzyme A Transferase Combined with Alcohol-Aldehyde Dehydrogenase Overexpression Leads to Predominantly Alcohologenic Clostridium acetobutylicum Fermentations

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