skip to main content

DOE PAGESDOE PAGES

Title: Ancillary contributions of heterologous biotin protein ligase and carbonic anhydrase for CO 2 incorporation into 3-hydroxypropionate by metabolically engineered Pyrococcus furiosus

Acetyl-Coenzyme A carboxylase (ACC), malonyl-CoA reductase (MCR), and malonic semialdehyde reductase (MRS) convert HCO 3 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 CO 2. 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 CO 2-sparged bioreactor reached titers of approximately 40 mg/L 3HP. Strains in which either the CA or BPLmore » 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,« less
Authors:
 [1] ;  [1] ;  [2] ;  [1] ;  [1] ;  [1] ;  [1] ;  [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: 113; Journal Issue: 12; Journal ID: ISSN 0006-3592
Publisher:
Wiley
Research Org:
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; biotin protein ligase; carboxylase; carbon dioxide; Pyrococcus furiosus; 3-hydroxypropionate; Metallosphaera sedula
OSTI Identifier:
1422399