Response mechanisms of xylitol-producing Saccharomyces cerevisiae strains to acetic acid and furfural and the role of SPI1, CTT1, and CLB1 in tolerance.

BACKGROUND

The efficient bioconversion of lignocellulosic hydrolysates to xylitol is hampered by inhibitors such as acetic acid and furfural. This study investigated the response mechanisms of two engineered xylitol-producing Saccharomyces cerevisiae strains, TX and CXAU, to acetic acid and furfural during fermentation in pretreated straw slurry through comparative transcriptomics.

RESULTS

TX showed greater acetic acid tolerance, while CXAU was more resistant to furfural. Transcriptomic analysis under acetic acid stress indicated that TX downregulated genes associated with respiration and acetate accumulation but upregulated those involved in NADPH generation, proton efflux, and protective amino acid synthesis. CXAU exhibited more limited transcriptional changes, primarily downregulating genes for acetate, ammonium, and polyamines uptake while upregulating genes related to sporulation and protein clearance. Under furfural stress, both strains showed transcriptional patterns indicative of repressed acetate accumulation and enhanced furfural detoxification, proton efflux, ribosomal biogenesis, and protective amino acid synthesis. TX further downregulated genes involved in central carbon metabolism, NADPH production, and mitochondrial function, whereas CXAU downregulated genes for glycolysis, membrane protein synthesis, and nitrogen uptake but upregulated genes supporting NADPH supply and filamentous growth. SPI1 and CTT1 were downregulated under both stresses, while CLB1 was upregulated under furfural. Overexpression of SPI1 or CTT1 enhanced tolerance to both inhibitors in both strains, and CLB1 overexpression improved furfural tolerance. CXAU-SPI1 achieved the highest xylitol titer (42.56 ± 1.12 g/L) under acetic acid stress, and TX-SPI1 performed best (41.00 ± 0.70 g/L) under furfural stress.

CONCLUSIONS

This study reveals distinct transcriptional responses to inhibitors in xylitol-producing strains and identifies SPI1, CTT1, and CLB1 as promising engineering targets for improving strain robustness.