Effect of a Novel Alpha/Beta Hydrolase Domain Protein on Tolerance of K. marxianus to Lignocellulosic Biomass Derived Inhibitors.

Dan Wu,Dongmei Wang,Jiong Hong

doi:10.3389/fbioe.2020.00844

Abstract

The multiple inhibitors tolerance of microorganism is important in bioconversion of lignocellulosic biomass which is a promising renewable and sustainable source for biofuels and other chemicals. The disruption of an unknown α/β hydrolase, which was termed KmYME and located in mitochondria in this study, resulted in the yeast more susceptible to lignocellulose-derived inhibitors, particularly to acetic acid, furfural and 5-HMF. The KmYME disrupted strain lost more mitochondrial membrane potential, showed increased plasma membrane permeability, severer redox ratio imbalance, and increased ROS accumulation, compared with those of the non-disrupted strain in the presence of the same inhibitors. The intracellular concentration of ATP, NAD and NADP in the KmYME disrupted strain was decreased. However, disruption of KmYME did not result in a significant change of gene expression at the transcriptional level. The KmYME possessed esterase/thioesterase activity which was necessary for the resistance to inhibitors. In addition, KmYME was also required for the resistance to other stresses including ethanol, temperature, and osmotic pressure. Disruption of two possible homologous genes in S. cerevisiae also reduced its tolerance to inhibitors.

Highlights

Lignocellulosic biomass is deemed as a promising resource for renewable biofuels and other chemicals due to its low cost, large-scale availability, and non-competition with food production (Kamm and Kamm, 2004; Yinbo et al, 2006; Somerville et al, 2010)
This gene was named KmYME and its expression in YHJ010 cells (Table 2) in the presence of each single kind of inhibitor (2.5 g/L acetic acid, 1.5 g/L furfural + 1.5 g/L 5-HMF, or 1.0 g/L phenols (4-hydroxybenzaldehyde, syringaldehyde, catechol, and vanillin), respectively) at 42◦C was determined by quantitative real-time PCR (Figure 1)
The 215 unique differentially expressed genes (DEGs) in YWD001-I vs. YWD001-C pairwise comparison were too decentralized by KEGG or GO enrichment analysis, so we focused on the total DEGs in the YWD001-I vs. YWD001-C group, regardless of the comparison or not with those in the YWD005-I vs. YWD005-C group, especially those DEGs related to the mitochondrial respiratory chain, coenzyme-dependent proteins, NAD+ biosynthesis, reactive oxygen species (ROS) reduction, and fatty acid biosynthesis and degradation

Summary

Introduction

Lignocellulosic biomass is deemed as a promising resource for renewable biofuels and other chemicals due to its low cost, large-scale availability, and non-competition with food production (Kamm and Kamm, 2004; Yinbo et al, 2006; Somerville et al, 2010). GRAPHICAL ABSTRACT | Possible tolerance mechanisms to inhibitors of KmYME in K. marxianus. Degradation, including weak acids, furan derivatives and phenolic compounds These compounds have different toxic mechanisms and synergistic effects that inhibit microbial cell metabolism and fermentation (Palmqvist and Hahn-Hägerdal, 2000; Almeida et al, 2007). Determining the mechanism for tolerance to these inhibitors is important in the construction of a robust strain for industrial fermentation. Kluyveromyces marxianus is a ‘generally regarded as safe’ (GRAS) microorganism that has attracted increasing attention in bioethanol fermentation of lignocellulosic biomass due to its thermo-tolerance, high growth rate, and broad substrate spectrum (Zhang et al, 2015). Industrial fermentation at elevated temperature reduces cooling cost and risk of contamination (Banat and Marchant, 1995). The tolerance of K. marxianus to multiple inhibitors is not very strong and the knowledge of K. marxianus tolerance to multiple inhibitors was scarce in previous studies (Oliva et al, 2003; Oliva et al, 2004)

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Discussion

Conclusion