Next Article in Journal
Screening of Hanseniaspora Strains for the Production of Enzymes with Potential Interest for Winemaking
Next Article in Special Issue
Characterizing the Phenotypic Responses of Escherichia coli to Multiple 4-Carbon Alcohols with Raman Spectroscopy
Previous Article in Journal
Development of Novel Textile Bioreactor for Anaerobic Utilization of Flocculating Yeast for Ethanol Production
Previous Article in Special Issue
Activity of Lactobacillus brevis Alcohol Dehydrogenase on Primary and Secondary Alcohol Biofuel Precursors
Article Menu

Export Article

Open AccessArticle
Fermentation 2015, 1(1), 113-126; doi:10.3390/fermentation1010113

Antisense-RNA-Mediated Gene Downregulation in Clostridium pasteurianum

1
Department of Chemical Engineering, University of Waterloo, Waterloo, ON N2L 3G1, Canada
2
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)
View Full-Text   |   Download PDF [761 KB, uploaded 9 December 2015]   |  

Abstract

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
Figures

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).

Scifeed alert for new publications

Never miss any articles matching your research from any publisher
  • Get alerts for new papers matching your research
  • Find out the new papers from selected authors
  • Updated daily for 49'000+ journals and 6000+ publishers
  • Define your Scifeed now

SciFeed Share & Cite This Article

MDPI and ACS Style

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.

Show more citation formats Show less citations formats

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Fermentation EISSN 2311-5637 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top