Isobutanol tolerance and production of Saccharomyces cerevisiae can be improved by engineering its TATA-binding protein Spt15

Low isobutanol tolerance of Saccharomyces cerevisiae limits its application in isobutanol fermentation. Here, we used global transcription machinery engineering to screen mutants with higher isobutanol tolerance and elevated isobutanol titres. TATA-binding protein Spt15 was used as the target of global transcription machinery engineering for improvement of such complex phenotypes. A random mutagenesis library of S. cerevisiae TATA-binding protein Spt15 was constructed and subjected to screening under isobutanol stress. A mutant strain (denoted as spt15-3) with improved isobutanol tolerance was identified. There were three mutations of Spt15 in strain spt15-3, including deletion of A at position −132 nt upstream of initiation codon, insertion of G at position −65 nt upstream of initiation codon and a synonymous mutation at position 315 nt (T → C) downstream of initiation codon. We then metabolically engineered isobutanol synthesis in strains harbouring plasmids YCplac22 containing these Spt15 mutations. Delta integration was used to overexpress ILV3 gene, and 2μ plasmids carrying PGK1p-ILV2 and PGK1p-ARO10 were used to overexpress ILV2 and ARO10 genes. After 24-h micro-aerobic fermentation, Engi-3 produced 0·556 g l⁻¹ isobutanol, which was 404% and 25·3% greater than isobutanol produced by control Engi-1 and engineered Engi-2, respectively. After 28 h, Engi-4 produced 0·459 g l⁻¹ isobutanol, which was 315% and 3·2% greater than isobutanol produced Engi-1 and Engi-2, respectively. RNA-Seq-based transcriptome analysis shows that mutations of Spt15 in strain spt15-3 increased the expression of SPT15. Meanwhile, compared with strain Engi-3, the spt15-3 mutation downregulated the expression of genes involved in the TCA cycle and glyoxylic acid cycle, but increased the expression of genes related to cell stability. This work demonstrates that isobutanol tolerance and production of S. cerevisiae can be improved by engineering its TATA-binding protein Spt15. This study clarified the molecular mechanisms regulating isobutanol production and tolerance in S. cerevisiae.