Furfural tolerance of mutant Saccharomyces cerevisiae selected via ionizing radiation combined with adaptive laboratory evolution

Abstract

Background: Lignocellulose is a renewable and sustainable resource used to produce second-generation biofuel ethanol to cope with the resource and energy crisis. Furfural is the most toxic inhibitor of Saccharomyces cerevisiae cells produced during lignocellulose treatment, and can reduce the ability of S. cerevisiae to utilize lignocellulose, resulting in low bioethanol yield. In this study, multiple rounds of progressive ionizing radiation was combined with adaptive laboratory evolution to improve the furfural tolerance of S. cerevisiae and increase the yield of ethanol.

Results: In this study, the strategy of multiple rounds of progressive X-ray radiation combined with adaptive laboratory evolution significantly improved the furfural tolerance of brewing yeast. After four rounds of experiments, four mutant strains resistant to high concentrations of furfural were obtained (SCF-R1, SCF-R2, SCF-R3, and SCF-R4), with furfural tolerance concentrations of 4.0, 4.2, 4.4, and 4.5 g/L, respectively. Among them, the mutant strain SCF-R4 obtained in the fourth round of radiation had a cellular malondialdehyde content of 49.11 nmol/mg after 3 h of furfural stress, a weakening trend in mitochondrial membrane potential collapse, a decrease in accumulated reactive oxygen species, and a cell death rate of 12.60%, showing better cell membrane integrity, stable mitochondrial function, and an improved ability to limit reactive oxygen species production compared to the other mutant strains and the wild-type strain. In a fermentation medium containing 3.5 g/L furfural, the growth lag phase of the SCF-R4 mutant strain was shortened, and its growth ability significantly improved. After 96 h of fermentation, the ethanol production of the mutant strain SCF-R4 was 1.86 times that of the wild-type, indicating that with an increase in the number of irradiation rounds, the furfural tolerance of the mutant strain SCF-R4 was effectively enhanced. In addition, through genome-transcriptome analysis, potential sites related to furfural detoxification were identified, including GAL7, MAE1, PDC6, HXT1, AUS1, and TPK3.

Conclusions: These results indicate that multiple rounds of progressive X-ray radiation combined with adaptive laboratory evolution is an effective mutagenic strategy for obtaining furfural-tolerant mutants and that it has the potential to tap genes related to the furfural detoxification mechanism.

Keywords: Saccharomyces cerevisiae; Evolution; Furfural; Second generation biofuel ethanol; Whole genome resequencing.

Fig. 1

Evaluation of the growth stability of the strains. WT: wild-type strain; SCF-R0: strain screened by adaptive laboratory evolution; SCF-R1: mutant strain obtained by the first round of radiation plus furfural domestication; SCF-R2: mutant strain obtained by the second round of radiation and furfural domestication; SCF-R3: mutant strain obtained by the third round of radiation plus furfural domestication; SCF-R4: mutant strain obtained by the fourth round of radiation plus furfural domestication. The results shown are the average of the three experiments

Fig. 2

Performance of the Saccharomyces cerevisiae mutant strains and WT strain tolerant to high-concentration furfural stress. A The furfural tolerance of the four mutant strains and WT strain was observed by plate dot; B growth curves of the mutant and wild-type strains in YPD medium containing 3.5 g/L furfural. The results shown are the average of the three experiments. WT, wild-type; YPD, yeast extract peptone dextrose

Fig. 3

Changes in the intracellular MDA and mitochondrial membrane potential of four mutant strains and the WT strain under furfural stress. a Intracellular MDA content of the four mutant strains and WT strain under furfural stress at 0 and 3 h (the results shown are the average of the three experiments); b and c changes in the intracellular MMP of four mutant strains and the WT strain under furfural stress at 0 and 3 h. MDA, malondialdehyde; WT, wild-type; MMP, mitochondrial membrane potential

Fig. 4

Analysis of changes in ROS in Saccharomyces cerevisiae cells under furfural stress via flow cytometry. a The growth ability of the four mutant strains and WT strain under 1.0 mmol/L hydrogen peroxide stress; b and c change in the ROS in the cells of the four mutant strains and WT strain under furfural stress at 0 and 3 h; d the proportion of dead cells in the four mutant strains and WT strain under furfural stress for 3 h. The results shown are the average of the three experiments. ROS, reactive oxygen species; WT, wild-type

Fig. 5

Analysis of the transcriptome data of Saccharomyces cerevisiae under furfural stress. a Volcanic map of differentially expressed genes; b DEG statistics in each pathway; c and d KEGG enrichment map of up-regulated and down-regulated genes under furfural stress. DEG, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes

Fig. 6

Analysis of mutant genome data. a Statistics of various mutation types; b conversion and transversion ratio of single base substitution; c chromosome distribution of four furfural-resistant mutants and genomic mutations; the change from one base sequence (a) to another base sequence (b) is represented by a–b; d statistics of mutation sites

Fig. 7

Verification and analysis of mutation sites related to improving the furfural tolerance of Saccharomyces cerevisiae. a Mutation sequence characteristics of mutation sites; b fluorescence quantitative PCR verification results of the mutation sites; c interaction network diagram of the mutation sites. The fluorescence quantitative PCR results are the average of three experiments. PCR, polymerase chain reaction

Fig. 8

Comparison of the ethanol production ability between four mutant strains tolerant to high furfural stress and the WT strain. a The ability of four mutant strains and the WT strain to utilize glucose at 3.5 g/L furfural; b the ethanol production by four mutant strains and the WT strain at 3.5 g/L furfural. The results shown are the average of three experiments. WT, wild-type

Fig. 9

Schematic diagram of multiple rounds of progressive X-ray radiation combined with ALE