Reaction kinetic analysis of the 3-hydroxypropionate/4-hydroxybutyrate CO2 fixation cycle in extremely thermoacidophilic archaea

Abstract

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle fixes CO₂ in extremely thermoacidophilic archaea and holds promise for metabolic engineering because of its thermostability and potentially rapid pathway kinetics. A reaction kinetics model was developed to examine the biological and biotechnological attributes of the 3HP/4HB cycle as it operates in Metallosphaera sedula, based on previous information as well as on kinetic parameters determined here for recombinant versions of five of the cycle enzymes (malonyl-CoA/succinyl-CoA reductase, 3-hydroxypropionyl-CoA synthetase, 3-hydroxypropionyl-CoA dehydratase, acryloyl-CoA reductase, and succinic semialdehyde reductase). The model correctly predicted previously observed features of the cycle: the 35-65% split of carbon flux through the acetyl-CoA and succinate branches, the high abundance and relative ratio of acetyl-CoA/propionyl-CoA carboxylase (ACC) and MCR, and the significance of ACC and hydroxybutyryl-CoA synthetase (HBCS) as regulated control points for the cycle. The model was then used to assess metabolic engineering strategies for incorporating CO₂ into chemical intermediates and products of biotechnological importance: acetyl-CoA, succinate, and 3-hydroxypropionate.

Keywords: 3-hydroxypropionate; 4-hydroxybutyrate; CO(2) fixation; Metallosphaera sedula.

Figure 1. Reactions of the 3-hydroxypropionate/4-hydroxybutyrate carbon fixation cycle

Reactions are color-coded by rate law. Enzyme abbreviations: ACC, acetyl-CoA/propionyl-CoA carboxylase; MCR, malonyl-CoA/succinyl-CoA reductase; MSR, malonic semialdehyde reductase; HPCS, 3-hydroxypropionyl-CoA synthetase; HBCS, 4-hydroxybutyryl-CoA synthetase; HPCD, 3-hydroxypropionyl-CoA dehydratase; ACR, acryloyl-CoA reductase; MCE, methylmalonyl-CoA epimerase; MCM, methylmalonyl-CoA mutase; SSR, succinic semialdehyde reductase; HBCD, 4-hydroxybutyryl-CoA dehydratase; CCH/HBCD, bifunctional crotonoyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase; AACT, acetoacetyl-CoA β-ketothiolase; SSADH, succinic semialdehyde dehydrogenase; IPPASE, inorganic pyrophosphatase; BM1, biomass production reaction #1; BM2, biomass production reaction #2. For a full list of rate law equations, see Supplementary Table S2.

Figure 2. Protein interaction assay for HPCS and HPCD, ACR and SSR by Yeast Two Hybrid

(A) Genomic context of HPCS (Msed_1456) and SSR (Msed_2001); ACR (Msed_1426) and SSR (Msed_1424). (B) Yeast two hybrid analyses of HPCS and HPCD, ACR and SSR. For HPCS and HPCD assay, Vector AD and BK, AD and BK-Msed_2001, AD-Msed_1456 and BK were applied as negative control, a commercial positive control was included. For ACR and SSR, Vector AD and BK, AD and BK-Msed_1424, AD-Msed_1426 and BK were applied as negative control.

Figure 3. Purification, and quaternary structure analysis of HPCS and HPCD

(A) HPCD was expressed in E. coli, purified by IMAC, and separated by size exclusion chromatography (Superdex 75). (B) Molecular assembly of HPCD as homooctomer. (C) HPCS and HPCD co-expressed in E. coli, purified by IMAC, and viewed on SDS-PAGE. (D) HPCS and HPCD separated by size exclusion chromatography (Superdex 75). (E) SDS-PAGE of two peaks eluted from size exclusion chromatography. (F) (HPCS)₄(HPCD)₈ determined by size exclusion chromatography.

Figure 4. Purification and quaternary structure analysis of ACR and SSR

(A) ACR and SSR were co-expressed in E. coli and recombinant enzymes were purified by IMAC. Quaternary structure was assayed by Superdex 75. (B) The two eluted peaks from (A) were collected and analyzed by SDS-PAGE. (C) NanoLC-MS/MS of the two peaks collected in (A). (D) SSR was expressed in E. coli and recombinant SSR purified by IMAC was analyzed by Superdex 75. (F) The collected peak of recombinant SSR in Superdex 75 was analyzed by SDS-PAGE.

Figure 5. Conversion of 3-hydroxypropionate to 4-hydroxybutyrate by enzymes in the CO₂ fixation cycle

Stepwise confirmation of sub-cycle operation using HPLC: (A) Formation of propionyl-CoA from 3HP by HPCS, HDCD, and ACR – 1: Propionyl-CoA standard, 2: Reaction mixture, 3: Reaction mixture control (no enzymes); (B) Formation of succinyl-CoA from (S)-methylmalonyl-CoA by MCE and MCM – 1: Reaction mixture with both MCE and MCM, 2: Reaction mixture with MCM only, 3: Reaction mixture control (no enzymes); (C) Formation of 4-hydroxybutyrate from succinyl-CoA by MCR and SSR – 1: 4HB standard, 2: Reaction mixture, 3: reaction mixture control (no enzymes).

Figure 6. Optimized amounts of 3HP/4HB cycle enzymes and flux control coefficients

(A) Relative enzyme amounts, on a mass basis, for maximum biomass production as predicted by the model for the reactions summarized in Figure 1. (B) Flux control coefficients of each enzyme for biomass production from acetyl-CoA (r₂₁), from succinate (r₂₂), or total (r₂₁ + r₂₂). Only enzymes with flux control coefficients >0.01 are shown.

Figure 7. Effect of cofactor concentrations and parameters on 3HP/4HB cycle

(A) Effect of NADPH concentration on biomass production rates, relative to the biomass production rate predicted after optimization. The y-intercept represents the concentration used for optimization. (B) Effect of CoA concentration on biomass production rates. (C) Parameter response coefficients for biomass production rates. Only parameter response coefficients >0.01 are shown.

Figure 8. Thermodynamic landscape and kinetic utilization of 3HP/4HB cycle enzymes

(A) −ΔG’, the Gibbs energy of reaction at steady-state metabolite concentrations and pH 5.4, represents the thermodynamic driving force of each reaction. The dotted line represents a driving force of 10 kJ/mol, above which the forward reaction accounts for >99% of the total enzyme reaction rate. (B) The ratio of actual reaction rate to maximum reaction rate for the enzyme (r/V_max), representing the kinetic utilization of each enzyme.

Figure 9. Optimized enzyme amounts for metabolic engineering applications of 3HP/4HB cycle enzymes

Relative amounts predicted by model, on a mass basis, shown for each pathway derived from the 3HP/4HB cycle. The pathways are (A) acetyl-CoA autotrophic, (B) succinate autotrophic, (C) succinate heterotrophic (via acetyl-CoA), and (D) 3HP heterotrophic (via acetyl-CoA). Enzyme abbreviations: Acetyl-CoA/propionyl-CoA carboxylase (ACC), Malonyl-CoA/succinyl-CoA reductase (MCR), Malonic semialdehyde reductase (MSR), 3-Hydroxypropionyl-CoA synthetase (HPCS), 4-Hydroxybutyryl-CoA synthetase (HBCS), 3-Hydroxypropionyl-CoA dehydratase (HPCD), Acryloyl-CoA reductase (ACR), Methylmalonyl-CoA epimerase (MCE), Methylmalonyl-CoA mutase (MCM), Succinic semialdehyde reductase (SSR), 4-Hydroxybutyryl-CoA dehydratase (HBCD), Bifunctional crotonoyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (CCH/HBCD), Acetoacetyl-CoA β-ketothiolase (AACT), Succinic semialdehyde dehydrogenase (SSADH).

Figure 10. Parameter response coefficients for metabolic engineering applications of 3HP/4HB cycle at optimum enzyme ratios predicted by model

Only parameters with response coefficients >0.1 are shown.

Figure 11. Flux control coefficients for metabolic engineering applications of 3HP/4HB cycle at optimum enzyme ratios predicted by model

Only enzymes with FCCs >0.01 are shown.

Figure 12. Effect of (A) NADPH and (B) coenzyme A concentrations on specific carbon fixation rate for metabolic engineering applications of 3HP/4HB cycle at optimum enzyme ratios predicted by model

Dotted lines indicate the baseline NADPH and CoA concentrations at which enzymes ratios were optimized. *Sharp corners in curves for autotrophic succinate pathway represent points above which buildup of pathway intermediates prevents reaching steady-state. ^Sharp corner in heterotrophic succinate pathway represents the point below which buildup of pathway intermediates prevents reaching steady-state.