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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