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.
Highlights
Clostridium pasteurianum is a strictly anaerobic, Gram-positive, spore-forming bacterium that has received growing biotechnological attention for its capacity to ferment low-value waste feedstocks, crude glycerol, to commodity chemicals and biofuels [1,2,3]
Cell growth was quantified spectrophotometrically by measuring culture optical density at 600 nm (OD600). β-Galactosidase activity was assayed as described [35] at 60 °C by measuring optical density at 420 nm (OD420) following development of yellow color resulting from cleavage of ortho-nitrophenyl-β-galactoside (ONPG) to orthonitrophenol
We show that plasmid-based heterologous gene overexpression and asRNA-mediated gene downregulation are effective tools for altering gene expression in C. pasteurianum
Summary
Clostridium pasteurianum is a strictly anaerobic, Gram-positive, spore-forming bacterium that has received growing biotechnological attention for its capacity to ferment low-value waste feedstocks, crude glycerol, to commodity chemicals and biofuels [1,2,3]. Cheap crude glycerol is currently available in abundance as a result of the tremendous growth experienced by global biodiesel and bioethanol industries in recent years [8,9]. In 2007, global biodiesel production capacity from a total of 119 countries was estimated at 51 billion liters [11], leading to a plummet in the price of crude glycerol to approximately $0.05/lb [8]. C. pasteurianum has become an attractive prospective industrial host for the production of butanol from abundant and renewable waste glycerol [3]. Few genetic tools have been developed for metabolic engineering of this important bacterium
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
