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 production of 3HP in M. sedula.

CO2-dependent carbon isotope fractionation in Archaea, Part I: Modeling the 3HP/4HB pathway

“…Relatively fast catalytic rates and high K M values are typical of scenarios in which the substrate is not limiting (e.g., elevated CO 2 levels at pH 2-3). Catalytic properties of the -CA of M. sedula, either purified from overexpression in Escherichia coli or engineered into P. furiosus (Lian et al, 2016), agree with those observed for -CA of Methanobacterium thermoautotrophicum (k cat = 1.7 10 4 s -1 , K M = 2.9 mM; and are characteristic of high CO 2 conditions. The CAs in Archaea are similar in overall efficiency to the CAs of Bacteria and Eukarya (Smith and Ferry, 2000).…”

“…Carbon fixation in the 3HP/4HB cycle is performed by a single, promiscuous enzyme that acts both as an acetyl-CoA carboxylase and a propionyl-CoA carboxylase (ACC; Menendez et al, 1999;Hügler et al, 2003). For M. sedula, activity of this enzyme has been studied in vivo, in vitro, and through heterologous expression in Pyrococcus furiosus (Hügler et al, 2003;Estelmann et al, 2011;Lian et al, 2016;, collectively determining the ACC properties shown in Table 1. The kinetic properties of ACC in N. maritimus have proven difficult to characterize in vitro due to apparent instability of the complex (Könneke et al, 2014), so the values in Table 1 are derived from in vivo carbon fixation rate data and total cellular carbon content (Könneke et al, 2014;Hurley et al, 2016).…”

“…Nitrosopumilus spp or other marine strains. Studies on the catalytic activity of the  and -CAs in M. sedula indicate that only the -CA is active, while the -CA is inactive or serves another function (Auernik and Kelly, 2010;Lian et al, 2016). Regardless, N. maritimus contains neither option.…”

“…Here, C e (extracellular CO 2 concentration) is known from culture conditions, and the K M1 and k cat1 values of CA in M. sedula are conservatively based on the slowest CAs of Archaea (Smith and Ferry, 2000) and on expression of the -CA of M. sedula in P. furiosus (Lian et al, 2016). Values of K M1 and k cat1 for the HYD process in N. maritimus are modeled to achieve flux and isotope balance at steady state.…”

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

“…Figure 5 shows that recombinant versions of HPCS, HPCD, ACC (Lian et al, 2016), MCE, MCM, MCR and SSR do indeed catalyze the formation of intermediates the second sub-pathway of the cycle, involved in the conversion of 3HP to 4HB. This information, in conjunction with previous studies (Estelmann et al, 2011; Hawkins et al, 2014; Hawkins et al, 2015; Hawkins et al, 2013; Keller et al, 2013; Lian et al, 2016), forms the basis for the development of a mathematical model describing the 3HP/4HB cycle.…”