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Fermentation 2015, 1(1), 113-126;

Antisense-RNA-Mediated Gene Downregulation in Clostridium pasteurianum

Department of Chemical Engineering, University of Waterloo, Waterloo, ON N2L 3G1, Canada
Algaeneers Inc., Hamilton, ON L8N 3Z5, Canada
Present address: Department of Biology and Centre for Structural and Functional Genomics, Concordia University, Montréal, QC H4B 1R6, Canada
Authors to whom correspondence should be addressed.
Academic Editor: George N. Bennett
Received: 22 September 2015 / Revised: 6 November 2015 / Accepted: 2 December 2015 / Published: 9 December 2015
(This article belongs to the Special Issue Metabolic Engineering)
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Clostridium pasteurianum is receiving growing attention for its unique metabolic properties, particularly its ability to convert waste glycerol and glycerol-rich byproducts into butanol, a prospective biofuel. Genetic tool development and whole genome sequencing have recently been investigated to advance the genetic tractability of this potential industrial host. Nevertheless, methodologies for tuning gene expression through plasmid-borne expression and chromosomal gene downregulation are still absent. Here we demonstrate plasmid-borne heterologous gene expression and gene knockdown using antisense RNA in C. pasteurianum. We first employed a common thermophilic β-galactosidase (lacZ) gene reporter system from Thermoanaerobacterium thermosulfurogenes to characterize two promoters involved in the central fermentative metabolism of C. pasteurianum. Due to a higher level of constitutive lacZ expression compared to the ferredoxin gene (fdx) promoter, the thiolase (thl) promoter was selected to drive expression of asRNA. Expression of a lacZ asRNA resulted in 52%–58% downregulation of β-galactosidase activity compared to the control strain throughout the duration of culture growth. Subsequent implementation of our asRNA approach for downregulation of the native hydrogenase I gene (hydA) in C. pasteurianum resulted in altered end product distribution, characterized by an increase in production of reduced metabolites, particularly butyrate (40% increase) and ethanol (25% increase). Knockdown of hydA was also accompanied by increased acetate formation and lower levels of 1,3-propanediol, signifying a dramatic shift in cellular metabolism in response to inhibition of the hydrogenase enzyme. The methodologies described herein for plasmid-based heterologous gene expression and antisense-RNA-mediated gene knockdown should promote rational metabolic engineering of C. pasteurianum for enhanced production of butanol as a prospective biofuel. View Full-Text
Keywords: antisense RNA; biofuels; Clostridium; gene reporter; genetic engineering; glycerol; gene knockdown; metabolic engineering antisense RNA; biofuels; Clostridium; gene reporter; genetic engineering; glycerol; gene knockdown; metabolic engineering

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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Pyne, M.E.; Moo-Young, M.; Chung, D.A.; Chou, C.P. Antisense-RNA-Mediated Gene Downregulation in Clostridium pasteurianum. Fermentation 2015, 1, 113-126.

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