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Title: Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis in Clostridium acetobutylicum

Abstract

ABSTRACT The recently revivedClostridium acetobutylicum-based acetone-butanol-ethanol (ABE) fermentation is widely celebrated and studied for its impact on industrial biotechnology.C. acetobutylicumhas been studied and engineered extensively, yet critical areas of the molecular basis for how solvent formation is regulated remain unresolved. The core solventogenic genes (adhE1/aad,ctfA,ctfB, andadc) are carried on the sol locus of the pSOL1 megaplasmid, whose loss leads to asporogenous, “degenerate” cells. The sol locus includes a noncoding small RNA (sRNA), SolB, whose role is presumed to be critical for solventogenesis but has eluded resolution. In the present study, SolB overexpression downregulated the sol-locus genes at the transcript level, resulting in attenuated protein expression and a solvent-deficient phenotype, thus suggesting that SolB affects expression of all sol-locus transcripts and seemingly validating its hypothesized role as a repressor. However, deletion ofsolBresulted in a total loss of acetone production and severe attenuation of butanol formation, with complex effects on sol-locus genes and proteins: it had a small impact onadcmRNA or its corresponding protein (acetoacetate decarboxylase) expression level, somewhat reducedadhE1andctfA-ctfBmRNA levels, and abolished thectfA-ctfB-encoded coenzyme A transferase (CoAT) activity. Computational predictions support a model whereby SolB expressed at low levels enables the stabilization and translation of sol-locus transcripts to facilitate tuning ofmore » the production of various solvents depending on the prevailing culture conditions. A key predicted SolB target is the ribosome binding site (RBS) of thectfAtranscript, and this was verified by expressing variants of thectfA-ctfBgenes to demonstrate the importance of SolB for acetone formation. IMPORTANCESmall noncoding RNAs regulate many important metabolic and developmental programs in prokaryotes, but their role in anaerobes has been explored minimally. Regulation of solvent formation in the important industrial organismC. acetobutylicumremains incompletely understood. While the genes for solvent formation and their promoters are known, the means by which this organism tunes the ratios of key solvents, notably the butanol/acetone ratio to balance its electron resources, remains unknown. Significantly, the roles of several coding and noncoding genes in the sol locus in tuning the solvent formation ratios have not been explored. Here we show that the small RNA SolB fine-tunes the expression of solvents, with acetone formation being a key target, by regulating the translation of the acetone formation rate-limiting enzyme, the coenzyme A transferase (CoAT). It is notable that SolB expressed at very low levels enables CoAT translation, while at high, nonphysiological expression levels, it leads to degradation of the corresponding transcript.« less

Authors:
; ORCiD logo; ; ORCiD logo;
Publication Date:
Research Org.:
Univ. of Delaware, Newark, DE (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1541704
DOE Contract Number:  
SC0007092
Resource Type:
Journal Article
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 84; Journal Issue: 14; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
Biotechnology & Applied Microbiology; Microbiology

Citation Formats

Jones, Alexander J., Fast, Alan G., Clupper, Michael, Papoutsakis, Eleftherios T., and Kelly, Robert M. Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis in Clostridium acetobutylicum. United States: N. p., 2018. Web. doi:10.1128/aem.00597-18.
Jones, Alexander J., Fast, Alan G., Clupper, Michael, Papoutsakis, Eleftherios T., & Kelly, Robert M. Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis in Clostridium acetobutylicum. United States. doi:10.1128/aem.00597-18.
Jones, Alexander J., Fast, Alan G., Clupper, Michael, Papoutsakis, Eleftherios T., and Kelly, Robert M. Fri . "Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis in Clostridium acetobutylicum". United States. doi:10.1128/aem.00597-18.
@article{osti_1541704,
title = {Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis in Clostridium acetobutylicum},
author = {Jones, Alexander J. and Fast, Alan G. and Clupper, Michael and Papoutsakis, Eleftherios T. and Kelly, Robert M.},
abstractNote = {ABSTRACT The recently revivedClostridium acetobutylicum-based acetone-butanol-ethanol (ABE) fermentation is widely celebrated and studied for its impact on industrial biotechnology.C. acetobutylicumhas been studied and engineered extensively, yet critical areas of the molecular basis for how solvent formation is regulated remain unresolved. The core solventogenic genes (adhE1/aad,ctfA,ctfB, andadc) are carried on the sol locus of the pSOL1 megaplasmid, whose loss leads to asporogenous, “degenerate” cells. The sol locus includes a noncoding small RNA (sRNA), SolB, whose role is presumed to be critical for solventogenesis but has eluded resolution. In the present study, SolB overexpression downregulated the sol-locus genes at the transcript level, resulting in attenuated protein expression and a solvent-deficient phenotype, thus suggesting that SolB affects expression of all sol-locus transcripts and seemingly validating its hypothesized role as a repressor. However, deletion ofsolBresulted in a total loss of acetone production and severe attenuation of butanol formation, with complex effects on sol-locus genes and proteins: it had a small impact onadcmRNA or its corresponding protein (acetoacetate decarboxylase) expression level, somewhat reducedadhE1andctfA-ctfBmRNA levels, and abolished thectfA-ctfB-encoded coenzyme A transferase (CoAT) activity. Computational predictions support a model whereby SolB expressed at low levels enables the stabilization and translation of sol-locus transcripts to facilitate tuning of the production of various solvents depending on the prevailing culture conditions. A key predicted SolB target is the ribosome binding site (RBS) of thectfAtranscript, and this was verified by expressing variants of thectfA-ctfBgenes to demonstrate the importance of SolB for acetone formation. IMPORTANCESmall noncoding RNAs regulate many important metabolic and developmental programs in prokaryotes, but their role in anaerobes has been explored minimally. Regulation of solvent formation in the important industrial organismC. acetobutylicumremains incompletely understood. While the genes for solvent formation and their promoters are known, the means by which this organism tunes the ratios of key solvents, notably the butanol/acetone ratio to balance its electron resources, remains unknown. Significantly, the roles of several coding and noncoding genes in the sol locus in tuning the solvent formation ratios have not been explored. Here we show that the small RNA SolB fine-tunes the expression of solvents, with acetone formation being a key target, by regulating the translation of the acetone formation rate-limiting enzyme, the coenzyme A transferase (CoAT). It is notable that SolB expressed at very low levels enables CoAT translation, while at high, nonphysiological expression levels, it leads to degradation of the corresponding transcript.},
doi = {10.1128/aem.00597-18},
journal = {Applied and Environmental Microbiology},
issn = {0099-2240},
number = 14,
volume = 84,
place = {United States},
year = {2018},
month = {5}
}