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Additional file 1 of Tryptophan plays an important role in yeast’s tolerance to isobutanol

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posted on 14.10.2021, 03:27 by Hsien-Lin Liu, Christine H.-T. Wang, En-Pei Isabel Chiang, Chieh-Chen Huang, Wen-Hsiung Li
Additional file 1: Figure S1. Growth assay of a tryptophan biosynthesis defective strain under a standard growth condition. The wild type and a tryptophan biosynthesis defective strain were tested in a standard medium with or without tryptophan addition (200 μg/ml). The growth rate shows no significantly difference between strains and between media with and without tryptophan addition. The data represent the mean ± SD (n = 3). Figure S2. Survival test of a tryptophan biosynthesis defective strain (Sc12) and a pentose phosphate pathway defective strain (Sc3). Both mutant strains Sc3 and Sc12 were sensitive to isbutanol, but could be recovered by adding tryptophan. Figure S3. Cell density assays of the wildtype and a tryptophan biosynthesis defective strain (Sc12) under different growth conditions. Yeasts were inoculated in 4ml medium with/without isobutanol and/or tryptophan. After 24 hours, the cell density was measured and RNA was harvested. The data represent the mean ± SD (n = 3). Notations: Trp, Tryptophan; IB, isobutanol. Figure S4. Checking the gene deletion and expression level of the mutant gene TRP5. a PCR was applied to verify the gene deletion from genome. The arrows represent the direction of 6 primers, which include A, B, C, D, kB, and kC. b The expression levels of the TRP5 gene under different growth conditions were estimated from NGS data. Figure S5. Expression level of gene BNA2. BAN2 is responsible for de novo biosynthesis of NAD+ from tryptophan via kynurenine pathway. It was highly expressed in WT and Sc12 under isobutanol. Figure S6. The recovery assay of wild type (WT) and tryptophan defective strain (Sc12) by supplementing of vitamin B1 (50 μg/ml) or tryptophan (200 μg/ml) under isobutanol (1%) stress. Under isobutanol stress, Sc12 can be only recovered by trptophan. Vitamin B1 did not help Sc12 to tolerate isobutanol. Figure S7. Expression level of gene GLN3. GLN3 plays a key role in yeast’s response to nitrogen starvation, including depletion of glutamine. It was upregulated when Sc12 was under isobutanol pressure; its expression level could be recovered by adding external tryptophan. Figure S8. Gene expression related to glutamine biosynthesis and transporter. The gene expression related to glutamine biosynthesis and transport was affected by adding tryptophan (Trp) and/or isobutanol (IB). GLN1 was upregluated in WT when isobutanol was added, and was upregulated in the mutant strain Sc12. DIP5 and GNP1 were up-regulated when isobutanol was added. Avt7 was up-regulated when WT and Sc12 were under isobutanol stress. Figure S9. Expression level of the ACC1 gene. ACC1 is the rate-limiting step for de novo fatty acid biosynthesis. It was up-regulated in WT under isobutanol stress. When the tryptophan pathway was defective, the expression of ACC1 was down-regulated under isobutanol stress but could be recovered by adding external tryptophan. Figure S10. Expression level of the TAT2 gene. TAT2 is a tryptophan amino acid transporter and was up-regulated when WT and Sc12 were under isobutanol stress. Table S1. Genes related to isobutanol tolerance. Table S2. Tophat mapping rate of NGS reads. Table S3. Gene names and numbers used in clustering analysis.

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