Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus

Lian, Hong; Zeldes, Benjamin M.; Lipscomb, Gina L.; Hawkins, Aaron B.; Han, Yejun; Loder, Andrew J.; Nishiyama, Declan; Adams, Michael W. W.; Kelly, Robert M.

doi:10.1002/bit.26033

Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO₂ incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus

Journal Article · Sat Jun 18 00:00:00 EDT 2016 · Biotechnology and Bioengineering

DOI:https://doi.org/10.1002/bit.26033· OSTI ID:1422399

Lian, Hong ^[1]; Zeldes, Benjamin M. ^[2]; Lipscomb, Gina L. ^[3]; Hawkins, Aaron B. ^[2]; Han, Yejun ^[2]; Loder, Andrew J. ^[2]; Nishiyama, Declan ^[2]; Adams, Michael W. W. ^[3]; Kelly, Robert M. ^[2]

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

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO₃^– and acetyl-CoA into 3-hydroxypropionate (3HP) in the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle resident in the extremely thermoacidophilic archaeon Metallosphaera sedula. These three enzymes, when introduced into the hyperthermophilic archaeon Pyrococcus furiosus, enable production of 3HP from maltose and CO₂. Sub-optimal function of ACC was hypothesized to be limiting for production of 3HP, so accessory enzymes carbonic anhydrase (CA) and biotin protein ligase (BPL) from M. sedula were produced recombinantly in Escherichia coli to assess their function. P. furiosus lacks a native, functional CA, while the M. sedula CA (Msed_0390) has a specific activity comparable to other microbial versions of this enzyme. M. sedula BPL (Msed_2010) was shown to biotinylate the β-subunit (biotin carboxyl carrier protein) of the ACC in vitro. Since the native BPLs in E. coli and P. furiosus may not adequately biotinylate the M. sedula ACC, the carboxylase was produced in P. furiosus by co-expression with the M. sedula BPL. The baseline production strain, containing only the ACC, MCR, and MSR, grown in a CO₂-sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPL accessory enzyme from M. sedula was added to the pathway resulted in improved titers, 120 or 370 mg/L, respectively. The addition of both M. sedula CA and BPL, however, yielded intermediate titers of 3HP (240 mg/L), indicating that the effects of CA and BPL on the engineered 3HP pathway were not additive, possible reasons for which are discussed. Here, while further efforts to improve 3HP production by regulating gene dosage,

View Accepted Manuscript (DOE)

Research Organization:: North Carolina State Univ., Raleigh, NC (United States)

Sponsoring Organization:: USDOE Advanced Research Projects Agency - Energy (ARPA-E)

Grant/Contract Number:: AR0000081

OSTI ID:: 1422399

Journal Information:: Biotechnology and Bioengineering, Journal Name: Biotechnology and Bioengineering Journal Issue: 12 Vol. 113; ISSN 0006-3592

Publisher:: WileyCopyright Statement

Country of Publication:: United States

Language:: English

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Related Subjects

3-hydroxypropionate
59 BASIC BIOLOGICAL SCIENCES
Metallosphaera sedula
Pyrococcus furiosus
biotin protein ligase
carbon dioxide
carboxylase