Refolding of a thermostable glyceraldehyde dehydrogenase for application in synthetic cascade biomanufacturing

Abstract

The production of chemicals from renewable resources is gaining importance in the light of limited fossil resources. One promising alternative to widespread fermentation based methods used here is Synthetic Cascade Biomanufacturing, the application of minimized biocatalytic reaction cascades in cell free processes. One recent example is the development of the phosphorylation independent conversion of glucose to ethanol and isobutanol using only 6 and 8 enzymes, respectively. A key enzyme for this pathway is aldehyde dehydrogenase from Thermoplasma acidophilum, which catalyzes the highly substrate specific oxidation of d-glyceraldehyde to d-glycerate. In this work the enzyme was recombinantly expressed in Escherichia coli. Using matrix-assisted refolding of inclusion bodies the yield of enzyme production was enhanced 43-fold and thus for the first time the enzyme was provided in substantial amounts. Characterization of structural stability verified correct refolding of the protein. The stability of the enzyme was determined by guanidinium chloride as well as isobutanol induced denaturation to be ca. -8 kJ/mol both at 25°C and 40°C. The aldehyde dehydrogenase is active at high temperatures and in the presence of small amounts of organic solvents. In contrast to previous publications, the enzyme was found to accept NAD(+) as cofactor making it suitable for application in the artificial glycolysis.

Figure 1. Function of glyceraldehyde dehydrogenase (AlDH) in our synthetic reaction cascade.

Glucose is degraded enzymatically to pyruvate and glyceraldehyde. AlDH catalyzes the oxidation of d-glyceraldehyde to d-glycerate, which is then dehydrated to pyruvate. Ethanol and isobutanol are synthesized by further reaction steps. AlDH must not catalyze isobutyraldehyde or acetaldehyde oxidation since the irreversibly formed carboxylates are unwanted side products, thus lowering the overall yield. Important reactions are shown in detail (continuous arrows), while some reaction steps are summarized (dashed arrows).

Figure 2. A) solubility of Thermoplasma acidophilum AlDH (TaAlDH) and B) inclusion body purification.

After recombinant expression of TaAlDH in E. coli, soluble (S) and insoluble fraction (P) were analyzed by SDS-PAGE. TaAlDH inclusion bodies from fermentation were purified with washing buffer in 2 steps (W1, W2) from insoluble fraction (P). Protein marker (M) indicates size of TaAlDH (arrow).

Figure 3. Size exclusion chromatography of TaAlDH.

Samples contained soluble TaAlDH (blue) and refolded TaAlDH (green).

Figure 4. CD spectra of TaAlDH.

A) Far-UV CD spectrum of soluble TaAlDH (blue) and refolded TaAlDH (green) with standard deviation (black). B) Near-UV CD of soluble TaAlDH (blue), refolded TaAlDH (green) and unfolded TaAlDH (red).

Figure 5. Michaelis-Menten kinetics of refolded TaAlDH with cofactors A) NAD⁺ and B) NADP⁺.

Reaction rates were determined in 100 mM HEPES pH 6.2 at 50°C with 5 mM d-glyceraldehyde and various concentrations of NAD⁺ or NADP⁺, respectively.

Figure 6. TaAlDH activity in the presence of different organic solvents.

Refolded TaAlDH was incubated at 50°C for 30 min in 100 mM HEPES pH 7 containing various concentrations of ethanol (green), isobutanol (blue) or n-butanol (red). Remaining enzyme activity was tested at 50°C in respective incubation buffers.

Figure 7. Time course of TaAlDH deactivation by organic solvent and its reactivation.

Deactivation of refolded TaAlDH was measured after incubation for indicated time at 50°C in 3% v/v isobutanol (red) or 0.3% v/v isobutanol (blue). Furthermore, samples incubated in 3% v/v isobutanol were also measured after 10-fold dilution to 0.3% v/v isobutanol (green) to test reactivation within 2 min.

Figure 8. Fluorescence analysis of unfolding and refolding of TaAlDH in the presence of A) GdmCl (25°C) and B) isobutanol (40°C).

The fluorescence emissions of TaAlDH at λ = 330 nm were monitored upon excitation at λ_max = 280 nm. Data were collected for protein unfolding (red symbols) and refolding (green symbols) at indicated concentrations of GdmCl or isobutanol. The transition curve for protein unfolding is presented as the best fit using nonlinear regression (black curve).