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Title: Expression of benzoyl-CoA metabolism genes in the lignocellulolytic host Caldicellulosiruptor bescii

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 inmore » 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.Keywords« less

Authors:
ORCiD logo [1];  [2]; ORCiD logo [3]; ORCiD logo [4]; ORCiD logo [2]; ORCiD logo [5]; ORCiD logo [2]; ORCiD logo [2];  [6]
  1. Univ. of Tennessee, Knoxville, TN (United States); Univ. of California, Berkeley, CA (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Becton Dickinson Diagnostics, Glencoe, MD (United States)
  5. Univ. of Tennessee, Knoxville, TN (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); LanzaTech, Skokie, IL (United States)
  6. Delaware State Univ., Dover, DE (United States)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1542243
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
AMB Express
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2191-0855
Publisher:
Springer
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; 59 BASIC BIOLOGICAL SCIENCES; Caldicellulosiruptor bescii; Aromatic; Benzoyl-CoA; Heterologous expression

Citation Formats

Sander, Kyle, Yeary, Meredith, Mahan, Kristina M., Whitham, Jason, Giannone, Richard J., Brown, Steven D., Rodriguez, Jr., Miguel, Graham, David E., and Hankoua, Bertrand B. Expression of benzoyl-CoA metabolism genes in the lignocellulolytic host Caldicellulosiruptor bescii. United States: N. p., 2019. Web. doi:10.1186/s13568-019-0783-8.
Sander, Kyle, Yeary, Meredith, Mahan, Kristina M., Whitham, Jason, Giannone, Richard J., Brown, Steven D., Rodriguez, Jr., Miguel, Graham, David E., & Hankoua, Bertrand B. Expression of benzoyl-CoA metabolism genes in the lignocellulolytic host Caldicellulosiruptor bescii. United States. doi:10.1186/s13568-019-0783-8.
Sander, Kyle, Yeary, Meredith, Mahan, Kristina M., Whitham, Jason, Giannone, Richard J., Brown, Steven D., Rodriguez, Jr., Miguel, Graham, David E., and Hankoua, Bertrand B. Sat . "Expression of benzoyl-CoA metabolism genes in the lignocellulolytic host Caldicellulosiruptor bescii". United States. doi:10.1186/s13568-019-0783-8. https://www.osti.gov/servlets/purl/1542243.
@article{osti_1542243,
title = {Expression of benzoyl-CoA metabolism genes in the lignocellulolytic host Caldicellulosiruptor bescii},
author = {Sander, Kyle and Yeary, Meredith and Mahan, Kristina M. and Whitham, Jason and Giannone, Richard J. and Brown, Steven D. and Rodriguez, Jr., Miguel and Graham, David E. and Hankoua, Bertrand B.},
abstractNote = {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.Keywords},
doi = {10.1186/s13568-019-0783-8},
journal = {AMB Express},
number = 1,
volume = 9,
place = {United States},
year = {2019},
month = {5}
}

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