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.

Advanced Insights into Catalytic and Structural Features of the Zinc‐Dependent Alcohol Dehydrogenase from Thauera aromatica

“…TkADH showed slightly higher residual activities in methanol, ethanol, isopropanol, acetone, and DMSO, but only after a relatively short incubation over 4 h in 20 % (v/v) solvent. [25] The glyceraldehyde dehydrogenase from Thermoplasma acidophilum showed a residual activity of about 40 % after only 30 min incubation in also 20 % (v/v) ethanol, [26] while ThaADH hardly lost any activity within the same time.…”

Cell‐free metabolic engineering: Biomanufacturing beyond the cell

“…“Heat” purification was also utilized during efforts to engineer a thermotolerant aldehyde dehydrogenase for glucose‐to‐ethanol CFME [49]. Here, reaction velocity and NAD + affinity were improved via protein refolding [84] and evolution by random mutagenesis [85]. Shorter pathways have also leveraged “heat” purification in conjunction with ammonium sulfate precipitation, a technique where the salt concentration is increased until proteins begin sequentially precipitating [86].…”

Optimization of a reduced enzymatic reaction cascade for the production of L-alanine

“…Regarding the economy of our process, D-glyceraldehyde needs to undergo further conversion in order to eliminate the byproduct and double the theoretical L-alanine yield. The oxidation of the aldehyde was previously done by a mutated aldehyde dehydrogenase (ALDH) from Thermoplasma acidophilum 38 . With the original enzyme being strictly NADP dependent, a directed evolution mutagenesis resulted in some activity for NAD 39 .…”