Abstract
Genes responsible for the anaerobic catabolism of benzoate in the thermophilic archaeon Ferroglobus placidus were expressed in the thermophilic lignocellulose-degrading bacterium Caldicellulosiruptor bescii, as a first step to engineering this bacterium to degrade this lignin metabolite. The benzoyl-CoA ligase gene was expressed individually, and in combination with benzoyl-CoA reductase and a putative benzoate transporter. This effort also assessed heterologous expression from a synthetically designed operon whereby each coding sequence was proceeded by a unique C. bescii ribosome binding site sequence. The F. placidicus benzoyl-CoA ligase gene was expressed in C. bescii to produce a full-length protein with catalytic activity. A synthetic 6-gene operon encoding three enzymes involved in benzoate degradation was also successfully expressed in C. bescii as determined by RNA analysis, though the protein products of only four of the genes were detected. The discord between the mRNA and protein measurements, especially considering the two genes lacking apparent protein abundance, suggests variable effectiveness of the ribosome binding site sequences utilized in this synthetic operon. The engineered strains did not degrade benzoate. Although the heterologously expressed gene encoding benzoyl-CoA ligase yielded a protein that was catalytically active in vitro, expression in C. bescii of six benzoate catabolism-related genes combined in a synthetic operon yielded mixed results. More effective expression and in vivo activity might be brought about by validating and using different ribosome binding sites and different promoters. Expressing additional pathway components may alleviate any pathway inhibition and enhance benzoyl-CoA reductase activity.
Original language | English |
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Article number | 59 |
Journal | AMB Express |
Volume | 9 |
Issue number | 1 |
DOIs | |
State | Published - Dec 1 2019 |
Funding
This work was supported by the BioEnergy Science Center (BESC), which is a U.S. Department of Energy (DOE) Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. This research was supported in part by appointments to the Higher Education Research Experiences Program and the ASTRO Programs at Oak Ridge National Laboratory, administered by ORAU through the DOE Oak Ridge Institute for Science and Education. This work was supported in part by the U.S. Department of Energy, Office of Science, and Office of Workforce Development for Teachers and Scientists (WDTS) under the Visiting Faculty Program (VFP) administered by the Oak Ridge Institute for Science and Education (ORISE). Oak Ridge National Laboratory is managed by UT-Battelle LLC for DOE under contract DE-AC05-00OR22725. The authors would like to acknowledge Dale Pelletier, Michael W.W. Adams, and Farris Poole for valuable discussions, assistance with analysis, and critical review of the manuscript. The authors would like to acknowledge Dawn Klingeman for valuable insight and assistance with RNA extraction, cDNA generation and qPCR quantification. The authors would like to also acknowledge Dr. Janet Westpheling for the kind gift of the C. bescii strain JWCB018 and the plasmid pJGW07 used in this study. This manuscript has been authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/ doe-public-access-plan).
Keywords
- Aromatic
- Benzoyl-CoA
- Caldicellulosiruptor bescii
- Heterologous expression