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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.

Metabolically engineered strains of the hyperthermophile Pyrococcus furiosus(Topt 95-100°C), designed to produce 3-hydroxypropionate (3HP) from maltose and CO2 using enzymes from the Metallosphaera sedula (Topt73°C) carbon fixation cycle, were examined with respect to the impact of heterologous gene expression on metabolic activity, fitness at optimal and sub-optimal temperatures, gas-liquid mass transfer in gas-intensive bioreactors, and potential bottlenecks arising from product formation. Transcriptomic comparisons of wild-type P. furiosus, a genetically-tractable, naturally-competent mutant (COM1), and COM1-based strains engineered for 3HP production revealed numerous differences after being shifted from 95°C to 72°C, where product formation catalyzed by the heterologously-produced M. sedula enzymes occurred. At 72°C, significantly higher levels of metabolic activity and a stress response were evident in 3HP-forming strains compared to the non-producing parent strain (COM1). Gas-liquid mass transfer limitations were apparent, given that 3HP titers and volumetric productivity in stirred bioreactors could be increased over 10-fold by increased agitation and higher CO2 sparging rates, from 18 mg/L to 276 mg/L and from 0.7 mg/L/hr to 11 mg/L/hr, respectively. 3HP formation triggered transcription of genes for protein stabilization and turnover, RNA degradation, and reactive oxygen species detoxification. The results here support the prospects of using thermally diverse sources of pathways and enzymes in metabolically engineered strains designed for product formation at sub-optimal growth temperatures.

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-hydroxyproprionate.

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