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Title: Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO 2-based 3-hydroxypropionate production

In this paper, metabolically engineered strains of the hyperthermophile Pyrococcus furiosus (T opt 95–100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO 2 using enzymes from the Metallosphaera sedula (T opt 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas–liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO 2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. Lastly, the results here support themore » prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.« less
Authors:
 [1] ;  [1] ;  [1] ;  [1] ;  [2] ;  [2] ;  [2] ;  [2] ;  [1]
  1. North Carolina State Univ., Raleigh, NC (United States)
  2. Univ. of Georgia, Athens, GA (United States)
Publication Date:
Grant/Contract Number:
AR0000081
Type:
Accepted Manuscript
Journal Name:
Biotechnology and Bioengineering
Additional Journal Information:
Journal Volume: 112; Journal Issue: 8; Journal ID: ISSN 0006-3592
Publisher:
Wiley
Research Org:
Univ. of Georgia, Athens, GA (United States); North Carolina State Univ., Raleigh, NC (United States)
Sponsoring Org:
USDOE Advanced Research Projects Agency - Energy (ARPA-E)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 3-hydroxypropionate; CO2 fixation; Metallosphaera sedula; Pyrococcus furiosus
OSTI Identifier:
1342894

Hawkins, Aaron B., Lian, Hong, Zeldes, Benjamin M., Loder, Andrew J., Lipscomb, Gina L., Schut, Gerrit J., Keller, Matthew W., Adams, Michael W. W., and Kelly, Robert M.. Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO2-based 3-hydroxypropionate production. United States: N. p., Web. doi:10.1002/bit.25584.
Hawkins, Aaron B., Lian, Hong, Zeldes, Benjamin M., Loder, Andrew J., Lipscomb, Gina L., Schut, Gerrit J., Keller, Matthew W., Adams, Michael W. W., & Kelly, Robert M.. Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO2-based 3-hydroxypropionate production. United States. doi:10.1002/bit.25584.
Hawkins, Aaron B., Lian, Hong, Zeldes, Benjamin M., Loder, Andrew J., Lipscomb, Gina L., Schut, Gerrit J., Keller, Matthew W., Adams, Michael W. W., and Kelly, Robert M.. 2015. "Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO2-based 3-hydroxypropionate production". United States. doi:10.1002/bit.25584. https://www.osti.gov/servlets/purl/1342894.
@article{osti_1342894,
title = {Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO2-based 3-hydroxypropionate production},
author = {Hawkins, Aaron B. and Lian, Hong and Zeldes, Benjamin M. and Loder, Andrew J. and Lipscomb, Gina L. and Schut, Gerrit J. and Keller, Matthew W. and Adams, Michael W. W. and Kelly, Robert M.},
abstractNote = {In this paper, metabolically engineered strains of the hyperthermophile Pyrococcus furiosus (Topt 95–100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (Topt 73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas–liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/h to 11 mg/L/h, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. Lastly, the results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.},
doi = {10.1002/bit.25584},
journal = {Biotechnology and Bioengineering},
number = 8,
volume = 112,
place = {United States},
year = {2015},
month = {6}
}