Additional file 1 of Alcohol dehydrogenases AdhE and AdhB with broad substrate ranges are important enzymes for organic acid reduction in Thermoanaerobacter sp. strain X514
Creators: name: Hitschler, Lisa
name: Nissen, Laura Sofie
name: Kuntz, Michelle
name: Basen, Mirko
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: Pharmacology
subject: Biotechnology
subject: 39999 Chemical Sciences not elsewhere classified
subject: FOS: Chemical sciences
subject: Ecology
subject: 69999 Biological Sciences not elsewhere classified
subject: 110309 Infectious Diseases
subject: FOS: Health sciences

description: Additional file 1: Fig. S1. Time dependence of natural DNA uptake by Thermoanaerobacter sp. strain X514. Subsamples were incubated for one hour at 65 °C with 1 µg ml–1 plasmid pIKM1, carrying a thermostable kanamycin resistance cassette, and then embedded in complex agar medium, and incubated in a N2/CO2 (80/20 v/v) -atmosphere for 5 days. Filled circles, OD600; filled or open triangles, biological replicates of transformants per colony forming unit (CFU). Fig. S2. Affinity of the native AdhE (top chart) and the recombinant AdhE-His (bottom chart) for acetaldehyde, in the direction of ethanol formation. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers. To the cuvettes, 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE and 4 µM resazurin, 0.5 mM NADH and 5.8 µg AdhE µg or 17.3 AdhE-His was added, and assays were started by the addition of acetaldehyde. Fig. S3. Affinity of the native AdhE for NADH, in the direction of ethanol formation. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers. To the cuvettes, 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE and 4 µM resazurin, varying amounts of NADH, 5.8 µg AdhE and 10 mM acetaldehyde was added. Fig. S4. Affinity of the native AdhE for isobutyraldehyde, in the direction of isobutanol formation. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers under a N2 atmosphere. The assay mixture contained 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE, 4 µM resazurin, 5.8 µg AdhE and 0.5 mM NADH. Fig. S5. Thermal stability of AdhE. The recombinant enzyme was incubated for up to 120 min at 65 °C (squares), 70 °C (triangles), 75 °C (diamonds) or 80 °C (crosses) before NADH-dependent acetaldehyde reduction was recorded at 65 °C. Fig. S6. Affinity of the native AdhB for NADPH. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed with rubber stoppers, under a N2 atmosphere. The assay mixture contained 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE, 4 µM resazurin, 3.2 µg AdhB, 10 mM acetaldehyde and varying concentrations of NADPH. Fig. S7. Affinity of the native AdhB AdhB (top chart) and the recombinant AdhB-His (bottom chart) for acetaldehyde. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed with rubber stoppers, under a N2 atmosphere. The assay mixture contained 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE, 4 µM resazurin, 3.2 µg AdhB or 15 µg AdhB-His, 0.5 mM NADPH and varying concentrations of acetaldehyde. Fig. S8. Thermal stability of AdhB-His. The recombinant enzyme was incubated for up to 180 min at 65 °C (squares), 80 °C (triangles), 85 °C (inverted triangles) or 90 °C (diamonds) before NADPH-dependent acetaldehyde reduction was recorded at 65 °C. Fig. S9. Affinity of the recombinant Adh0564 for NADPH, in the direction of ethanol formation. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers. To the cuvettes, 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE and 4 µM resazurin, varying concentrations of NADPH and 9 µg Adh0564 was added, and assays were started by the addition of acetaldehyde. Fig. S10. Affinity of the recombinant Adh0564 for acetaldehyde, in the direction of ethanol formation. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers. To the cuvettes, 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE and 4 µM resazurin, 0.5 mM NADH and 9 µg Adh0564 was added, and assays were started by the addition of acetaldehyde. Fig. S11. Affinity of the recombinant Adh0564 for isobutyraldehyde, in the direction of isobutanol formation. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers under a N2 atmosphere. The assay mixture contained 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE, 4 µM resazurin, 16.4 µg Adh0564 and 0.5 mM NADH. Fig. S12. Affinity of the recombinant AdhA for NADPH, in the direction of ethanol formation. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers. To the cuvettes, 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE and 4 µM resazurin, varying amounts of NADH, 19.8 µg AdhA and 10 mM acetaldehyde was added. Fig. S13. Affinity of the recombinant AdhA for acetaldehyde, in the direction of ethanol formation. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers. To the cuvettes, 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE and 4 µM resazurin, 0.5 mM NADH and 18.5 µg AdhA was added, and assays were started by the addition of acetaldehyde. Fig. S14. Affinity of the recombinant AdhA for isobutyraldehyde, in the direction of isobutanol formation. The assays were performed at 65 °C in cuvettes (Glasgerätebau Ochs, Bovenden-Lenglern, Germany) sealed by rubber stoppers under a N2 atmosphere. The assay mixture contained 1 mL 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE, 4 µM resazurin, 50 µg AdhA and 0.5 mM NADH. Fig. S15. Thermal stability of Adh0564-His. The recombinant enzyme was incubated for up to 120 min at 65 °C (squares), 70 °C (triangles), 80 °C (inverted triangles), 85 °C (diamonds) or 90 °C (filled circles) before NADPH-dependent acetaldehyde reduction was recorded at 65 °C. Fig. S16. Thermal stability of His-AdhA. The recombinant enzyme was incubated for up to 20 min at 65 °C (squares) or 65 °C (triangles) before NADPH-dependent acetaldehyde reduction was recorded at 65 °C. Table S1. Co-purification of the major NADH-dependent aldehyde dehydrogenase AdhE of Thermoanaerobacter sp. Strain X514 with the major NADH-dependent ADH (Table 1). NADH-dependent ALDH activity was measured as in 50 mM Tris buffer (pH 7.5) supplemented with 2 mM DTE and 4 µM resazurin as coenzyme A—(0.2 mM) and NAD+ (2 mM)—dependent oxidation of acetaldehyde (2 mM) at 340 nm 65 °C. One unit represents one µmol of acetaldehyde oxidized per minute. Table S2. Oligonucleotides developed and used in this study.