A new source of resistance to 2-furaldehyde from Scheffersomyces (Pichia) stipitis for sustainable lignocellulose-to-biofuel conversion

Authors: WANG, XU - Sichuan Agricultural University; Liu, Zonglin; MA, MENGGEN - Sichuan Agricultural University; ZHANG, XIAOPING - Sichuan Agricultural University

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/28/2016
Publication Date: 6/6/2017
Citation: Wang, X., Liu, Z.L., Ma, M., Zhang, X. 2017. A new source of resistance to 2-furaldehyde from Scheffersomyces (Pichia) stipitis for sustainable lignocellulose-to-biofuel conversion. Applied Microbiology and Biotechnology. 101:4981–4993. doi: 10.1007/s00253-017-8208-6.

Interpretive Summary: Aldehyde inhibitory compounds derived from lignocellulosic biomass pretreatment have been identified as a major class of toxic chemicals that interfere with microbial growth and subsequent fermentation for advanced biofuels production. Overcoming aldehyde inhibition is one of significant challenges for low-cost advanced biofuels including cellulosic ethanol production. This study investigated functions of five uncharacterized genes from naturally occurred xylose utilizing yeast Scheffersomyces stipitis (S. stipitis) against fermentation inhibitor furfural. This research quantitatively analyzed gene expression response to furfural challenges; cloned five genes; confirmed the gene function by heterologous expression; isolated partially purified proteins; and provided direct enzyme assay evidence to characterize functions of SsAAD1, SsAAD2, SsAAD3, SsAAD4, and SsAAD5. Among which, SsAAD2, SsAAD3, and SsAAD4 were found to be closely related to each other and serve as core aldehyde reduction function genes. New knowledge obtained from this research not only contributes to gene annotation of S. stipitis, but also aids broad applications of more tolerant microbial strain development for advanced biofuels production.

Technical Abstract: Aldehyde inhibitory compounds derived from lignocellulosic biomass pretreatment have been identified as a major class of toxic chemicals that interfere with microbial growth and subsequent fermentation for advanced biofuel production. Development of robust next-generation biocatalyst is a key for a low-cost biofuel production industry. Scheffersomyces (Pichia) stipitis is a naturally occurring C-5 sugar utilization yeast; however, little is known about the genetic background underlying its potential tolerance to biomass conversion inhibitors. We investigated and identified five uncharacterized putative aryl-alcohol dehydrogenase genes (SsAADs) from this yeast as a new source of resistance against biomass fermentation inhibitor 2-furaldehyde furfural) by gene expression, gene cloning, and direct enzyme assay analysis using partially purified proteins. All five proteins from S. stipitis showed furfural reduction using cofactor NADH. An optimum active temperature was observed at 40°C for SsAad1p; 30°C for SsAad3p, SsAad4p, and SsAad5p; and 20°C for SsAad2p. SsAad2p, SsAad3p, and SsAad4p showed tolerance to a wide range of pH from 4.5 to 8, but SsAad1p and SsAad5p were sensitive to pH changes beyond 7. Genes SsAAD2, SsAAD3, and SsAAD4 displayed significantly enhanced higher levels of expression in response to the challenge of furfural. Their encoding proteins also showed higher levels of specific activity toward furfural and were suggested as core functional enzymes contributing aldehyde resistance in S. stipitis.