Adaptive laboratory evolution to obtain furfural tolerant Saccharomyces cerevisiae for bioethanol production and the underlying mechanism

Lan Yao; Youpiao Jia; Qingyan Zhang; Xueyun Zheng; Haitao Yang; Jun Dai; Xiong Chen

doi:10.3389/fmicb.2023.1333777

Adaptive laboratory evolution to obtain furfural tolerant Saccharomyces cerevisiae for bioethanol production and the underlying mechanism

Front Microbiol. 2024 Jan 4:14:1333777. doi: 10.3389/fmicb.2023.1333777. eCollection 2023.

Authors

Lan Yao^#¹, Youpiao Jia^#¹, Qingyan Zhang¹, Xueyun Zheng¹, Haitao Yang², Jun Dai¹, Xiong Chen¹

Affiliations

¹ Key Laboratory of Fermentation Engineering (Ministry of Education), Cooperative Innovation Center of Industrial Fermentation (Ministry of Education and Hubei Province), College of Bioengineering, Hubei University of Technology, Wuhan, China.
² Hubei Provincial Key Laboratory of Green Materials for Light Industry, Hubei University of Technology, Wuhan, China.

^# Contributed equally.

Abstract

Introduction: Furfural, a main inhibitor produced during pretreatment of lignocellulose, has shown inhibitory effects on S. cerevisiae.

Method: In the present study, new strains named 12-1 with enhanced resistance to furfural were obtained through adaptive laboratory evolution, which exhibited a shortened lag phase by 36 h, and an increased ethanol conversion rate by 6.67% under 4 g/L furfural.

Results and discussion: To further explore the mechanism of enhanced furfural tolerance, ADR1_1802 mutant was constructed by CRISPR/Cas9 technology, based on whole genome re-sequencing data. The results indicated that the time when ADR1_1802 begin to grow was shortened by 20 h compared with reference strain (S. cerevisiae CEN.PK113-5D) when furfural was 4 g/L. Additionally, the transcription levels of GRE2 and ADH6 in ADR1_ 1802 mutant were increased by 53.69 and 44.95%, respectively, according to real-time fluorescence quantitative PCR analysis. These findings suggest that the enhanced furfural tolerance of mutant is due to accelerated furfural degradation. Importance: Renewable carbon worldwide is vital to achieve "zero carbon" target. Bioethanol obtained from biomass is one of them. To make bioethanol price competitive to fossil fuel, higher ethanol yield is necessary, therefore, monosaccharide produced during biomass pretreatment should be effectively converted to ethanol by Saccharomyces cerevisiae. However, inhibitors formed by glucose or xylose oxidation could make ethanol yield lower. Thus, inhibitor tolerant Saccharomyces cerevisiae is important to this process. As one of the main component of pretreatment hydrolysate, furfural shows obvious impact on growth and ethanol production of Saccharomyces cerevisiae. To get furfural tolerant Saccharomyces cerevisiae and find the underlying mechanism, adaptive laboratory evolution and CRISPR/Cas9 technology were applied in the present study.

Keywords: CRISPR/Cas9; Saccharomyces cerevisiae; adaptive laboratory evolution; bioethanol; furfural tolerance.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the National Natural Science Foundation of China (Nos. 21978074 and 31871789) and Key Project of Hubei Provincial Department of Education (Nos. D20211404 and T2022011).