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

Abstract

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. While further efforts to improve 3HP production by regulating gene dosage, improving carbon flux and optimizing bioreactor operation are needed, these results illustrate the ancillary benefits of accessory enzymes for incorporating CO₂ into 3HP production in metabolically engineered P. furiosus, and hint at the important role that CA and BPL likely play in the native 3HP/4HB pathway in M. sedula. Biotechnol. Bioeng. 2016;113: 2652-2660. © 2016 Wiley Periodicals, Inc.

Keywords: 3-hydroxypropionate; Metallosphaera sedula; Pyrococcus furiosus; biotin protein ligase; carbon dioxide; carboxylase.

Figure 1

3-Hydroxypropionate formation and accessory enzymes. The first three enzymes of the 3HP/4HB cycle (sub-pathway 1 [SP1]) catalyze the formation of 3HP from acetyl-CoA and bicarbonate. Two accessory enzymes are potentially involved in carboxylase function—CA catalyzes the hydration of dissolved CO₂ gas to supply bicarbonate and BPL is required for covalently ligating biotin to the β-subunit of ACC. CA, carbonic anhydrase; BPL, biotin protein ligase; ACC, acetyl-CoA carboxylase; MCR, malonyl-CoA reductase; MSR, malonic semialdehyde reductase.

Figure 2

Amino acid sequence alignment of BCCP around the canonical lysine residue (K). The conserved—E-X-M-K-M-motif is indicated by triangles. Mutated site in the Sulfolobales is marked by arrow. Msed, M. sedula Msed0148, Stok, S. tokodaii ST0592, Pfu, P. furiosus PF0673, Eco, E. coli ECs4127.

Figure 3

In vitro biotinylation of recombinant Msed ACC-β with recombinant Msed BPL. Purified M. sedula ACC-β and BPL were combined with biotin cofactor and incubated for 30 min at 70°C. This “reaction mixture” was applied to a streptavidin column and washed. Bound fraction consisting of biotinylated ACC-β was released from the beads by heat denaturation.

Figure 4

Representative bioreactor runs of metabolically engineered P. furiosus strains. a) Cell counts, showing difference in growth rate before and after temperature shift from 95°C to 72°C. Time course of concentrations of b) 3HP, c) acetate, and d) maltose in supernatant. Vertical black line indicates temperature switch from 95°C to 72°C.