Transcriptomic analysis of thermotolerant yeast Kluyveromyces marxianus in multiple inhibitors tolerance

Abstract

During pretreatment of lignocellulosic biomass, toxic compounds were released and inhibited the growth and fermentation of microorganisms. Here the global transcriptional response of K. marxianus to multiple inhibitors including acetic acid, phenols, furfural and HMF, at 42 °C, was studied, via RNA-seq technology. Genes involved in the glycolysis pathway, fatty acid metabolism, ergosterol metabolism and vitamin B6 and B1 metabolic process were enriched in the down-regulated gene set, while genes involved in TCA cycle, respiratory chain, ROS detoxification and transporter coding genes were enriched in the up-regulated gene set in response to the multiple inhibitors stress. Further real time-PCR results with three single inhibitor stress conditions showed that different transporters responded quite differently to different inhibitor stress. Coenzyme assay results showed that the level of NAD⁺ was increased and both NADH/NAD⁺ and NADPH/NADP⁺ ratio decreased. Furthermore, genes involved with transcription factors related to carbohydrate metabolism, sulfur amino acids metabolism, lipid metabolism or those directly involved in the transcriptional process were significantly regulated. Though belonging to different GO categories or KEGG pathway, many differentially expressed genes were enriched in maintaining the redox balance, NAD(P)⁺/NAD(P)H homeostasis or NAD⁺ synthesis, energy production, and iron transportation or metabolism. These results suggest that engineering these aspects represents a possible strategy to develop more robust strains for industrial fermentation from cellulosic biomass.

Fig. 1. Response of cell growth to multiple inhibitors at 42 °C. Values shown are mean with SD (n = 3).

Fig. 2. Schematic representation of central carbon metabolism in K. marxianus in glycolysis, the pentose phosphate pathway (PPP) and TCA pathway, in response to multiple inhibitors challenges. The fold change (FC) of transcriptional levels with RNA-seq analysis compared with that under no challenge condition was presented by log₂ FC. Red indicates up-regulated and log₂ FC ≥ 1, green for down-regulated and log₂ FC ≤ −1, black indicates no significant change (−1 < log₂ FC < 1). Further details are given in Table 2.

Fig. 3. Ratio of intracellular NAD(P)H/NAD(P)⁺ with or without mixed inhibitors. The error bars represent the standard deviation calculated from triplicate experiments.

Fig. 4. RT-PCR results of various transporters with individual inhibitor stress condition in K. marxianus.