Bioprocessing analysis of Pyrococcus furiosus strains engineered for CO₂-based 3-hydroxypropionate production

Journal Article · Thu Jun 11 00:00:00 EDT 2015 · Biotechnology and Bioengineering

DOI:https://doi.org/10.1002/bit.25584· OSTI ID:1342894

Hawkins, Aaron B. ^[1]; Lian, Hong ^[1]; Zeldes, Benjamin M. ^[1]; Loder, Andrew J. ^[1]; Lipscomb, Gina L. ^[2]; Schut, Gerrit J. ^[2]; Keller, Matthew W. ^[2]; Adams, Michael W. W. ^[2]; Kelly, Robert M. ^[1]

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1. North Carolina State Univ., Raleigh, NC (United States)
2. Univ. of Georgia, Athens, GA (United States)

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 CO₂ 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 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.

• Research Organization: Univ. of Georgia, Athens, GA (United States); North Carolina State University, Raleigh, NC (United States)
• Sponsoring Organization: USDOE Advanced Research Projects Agency - Energy (ARPA-E)
• Grant/Contract Number: AR0000081
• OSTI ID: 1342894
• Journal Information: Biotechnology and Bioengineering, Vol. 112, Issue 8; ISSN 0006-3592
• Publisher: Wiley
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
3-hydroxypropionate
CO2 fixation
Metallosphaera sedula
Pyrococcus furiosus