Fumarate Production by Torulopsis glabrata: Engineering Heterologous Fumarase Expression and Improving Acid Tolerance

Abstract

Fumarate is a well-known biomass building block compound. However, the poor catalytic efficiency of fumarase is one of the major factors preventing its widespread production. To address this issue, we selected residues 159HPND162 of fumarase from Rhizopus oryzae as targets for site-directed mutagenesis based on molecular docking analysis. Twelve mutants were generated and characterized in detail. Kinetic studies showed that the Km values of the P160A, P160T, P160H, N161E, and D162W mutants were decreased, whereas Km values of H159Y, H159V, H159S, N161R, N161F, D162K, and D162M mutants were increased. In addition, all mutants displayed decreased catalytic efficiency except for the P160A mutant, whose kcat/Km was increased by 33.2%. Moreover, by overexpressing the P160A mutant, the engineered strain T.G-PMS-P160A was able to produce 5.2 g/L fumarate. To further enhance fumarate production, the acid tolerance of T.G-PMS-P160A was improved by deleting ade12, a component of the purine nucleotide cycle, and the resulting strain T.G(△ade12)-PMS-P160A produced 9.2 g/L fumarate. The strategy generated in this study opens up new avenues for pathway optimization and efficient production of natural products.

Fig 1. Major metabolic pathways leading to fumarate formation in T. glabrata.

Boldface arrows indicate variants for fumarate synthesis implemented in strains featured in this study. RoPYC: pyruvate carboxylase from R. oryzae; RoMDH: malate dehydrogenase from R. oryzae; RoFUM: fumarase from R. oryzae; SpMAE1: C₄-dicarboxylic acid transporter from Schizosaccharomyces pombe; ade12: adenylosuccinate synthase; ade13: adenylosuccinate lyase; amd1: AMP deaminase.

Fig 2. Structural model of RoFUM constructed by the Swiss Model server.

The positions of the residues that are critical for substrate binding in RoFUM are shown as “sticks”. (A) Overview, (B) binding site B, (C) binding site A, and (D) the residues in binding site A.

Fig 3. SDS-PAGE analysis of wild-type RoFUM and mutant proteins.

Fig 4. Effect of temperature and pH on RoFUM and its mutants.

(A) The effect of temperature on the activity of RoFUM and its mutants was measured at pH 7.3 with temperatures ranging from 15–35°C. (B) The effect of pH on RoFUM (quadrangle, black), H159Y (circle, red), H159V (upper triangle), H159S (reverse triangle), P160T (quadrangle, green), P160H (left triangle), P160A (right triangle), N161E (sexangle), N161F (star), N161R (pentacle), D162K (circle, blue), D162M (vertical bar), D162W (cross). All values presented in graphs are the means of three replications.

Fig 5. Effect of mutations on fumarate production.

Fig 6. Effect of mutations on fermentation parameters.

(A) Glucose consumption, (B) cell growth, (C) malate production, (D) fumarate production. ■T.G-PMS, ●T.G-PMS-RoFUM, ▼T.G-PMS-P160A. All values presented in graphs are the means of three replications.

Fig 7. Effect of gene deletions on acid tolerance and fumarate production.

(A) Growth assays under various pH values. Logarithmic-phase cells of each T. glabrata strain were adjusted to 2×10⁷ cells/mL, and then 5 μL of serial 10-fold dilutions were spotted onto the corresponding fermentation medium. Pictures were taken after 4 days of growth at 30°C. (B) Concentrations of fumarate obtained in shake flask cultivation of the different strains. All values presented in graphs are the means of three replications.