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

Abstract

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 thermallymore » diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.« less

Authors:

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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+ Show Author Affiliations

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

Publication Date:

Thu Jun 11 00:00:00 EDT 2015

Research Org.:

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

Sponsoring Org.:

USDOE Advanced Research Projects Agency - Energy (ARPA-E)

OSTI Identifier:

1342894

Grant/Contract Number:  

AR0000081

Resource Type:

Accepted Manuscript

Journal Name:

Biotechnology and Bioengineering

Additional Journal Information:

Journal Volume: 112; Journal Issue: 8; Journal ID: ISSN 0006-3592

Publisher:

Wiley

Country of Publication:

United States

Language:

English

Subject:

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