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PMID: 10049845 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum.

Applied and environmental microbiology ·Vol. 65 ·No. 3 ·1999-03-00 ·Pages 936-45

Desai RP, Papoutsakis ET

Abstract

We examined the effectiveness of antisense RNA (as RNA) strategies for metabolic engineering of Clostridium acetobutylicum. Strain ATCC 824(pRD4) was developed to produce a 102-nucleotide asRNA with 87% complementarity to the butyrate kinase (BK) gene. Strain ATCC 824(pRD4) exhibited 85 to 90% lower BK and acetate kinase specific activities than the control strain. Strain ATCC 824(pRD4) also exhibited 45 to 50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase specific activities than the control strain. This strain exhibited earlier induction of solventogenesis, which resulted in 50 and 35% higher final concentrations of acetone and butanol, respectively, than the concentrations in the control. Strain ATCC 824(pRD1) was developed to putatively produce a 698-nucleotide asRNA with 96% complementarity to the PTB gene. Strain ATCC 824(pRD1) exhibited 70 and 80% lower PTB and BK activities, respectively, than the control exhibited. It also exhibited 300% higher levels of a lactate dehydrogenase activity than the control exhibited. The growth yields of ATCC 824(pRD1) were 28% less than the growth yields of the control. While the levels of acids were not affected in ATCC 824(pRD1) fermentations, the acetone and butanol concentrations were 96 and 75% lower, respectively, than the concentrations in the control fermentations. The lower level of solvent production by ATCC 824(pRD1) was compensated for by approximately 100-fold higher levels of lactate production. The lack of any significant impact on butyrate formation fluxes by the lower PTB and BK levels suggests that butyrate formation fluxes are not controlled by the levels of the butyrate formation enzymes.

MeSH Terms
Acetone/metabolism Base Sequence Bioreactors Butanols/metabolism Butyrates/metabolism Clostridium/enzymology,genetics,growth & development Ethanol/metabolism Fermentation Gene Expression Regulation, Bacterial Gene Expression Regulation, Enzymologic Genetic Engineering Molecular Sequence Data Phosphate Acetyltransferase/genetics,metabolism Phosphotransferases (Carboxyl Group Acceptor)/genetics,metabolism Plasmids/genetics RNA, Antisense/genetics RNA, Bacterial/genetics
Chemicals
Butanols Butyrates RNA, Antisense RNA, Bacterial Acetone Ethanol Phosphate Acetyltransferase Phosphotransferases (Carboxyl Group Acceptor) butyrate kinase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Desai R P
Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208, USA.
Papoutsakis E T
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Article Info
Journal
Applied and environmental microbiology
Abbr.
Appl Environ Microbiol
ISSN
0099-2240
DOI
10.1128/AEM.65.3.936-945.1999
Published
1999-03-00
Pages
936-45
Language
English
Region
United States
NLM ID
7605801
PMCID
PMC91126
Subset
IM
Grants
NIGMS NIH HHS · T32 GM008449 · United States
NIGMS NIH HHS · GM 08449 · United States
External Links
PubMed source DOI: 10.1128/AEM.65.3.936-945.1999 PMC Full Text View journal details
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