Transcriptomic analysis of thermotolerant yeastKluyveromyces marxianusin 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.

Effect of a novel alpha/beta hydrolase domain protein on tolerance of K. marxianus to lignocellulosic biomass derived inhibitors

“…Considering the synergistic effects of these inhibitors, the construction of a strain with tolerance to multiple inhibitors has an increased practical application value. In our previous study, the significant up-regulation of KmYME was detected in the presence of multiple inhibitors [13]. Here, its upregulation was confirmed in the presence of multiple inhibitors or each single kind of inhibitor (Fig.…”

“…In our previous study, the expression of an uncharacterized α/β-hydrolase domain (ABHD)-containing gene (GenBank BAP69980, locus tag KMAR_10772) of K. marxianus was significantly up-regulated in the presence of inhibitors derived from the pretreatment of lignocellulosic biomass [13]. In this study, after the up-regulation of its expression was confirmed through real time PCR, this gene was disrupted or overexpressed to evaluate its effect on tolerance to inhibitors.…”

“…In our previous study, the gene KMAR_10772, encoding an uncharacterized ABHD-containing protein was significantly up-regulated when the cells were cultivated with multiple inhibitors (acetate acid, furfural, HMF, and phenols) [13]. In this study, this gene was named KmYME and its expression in YHJ010 cells (Table 1) 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 also determined by quantitative real-time PCR (qPCR) (Fig.…”

“…Total RNA of each sample was extracted and cDNA libraries were prepared as previously described [13]. The resulting cDNA library products were then shotgun sequenced (101 bp paired-end read) with the Illumina HiSeq 4000 instrument (Illumina, San Diego, CA, USA) using a customer sequencing service (Majorbio Co., Ltd, Shanghai, China).…”

“…Annotation and bioinformatics analysis were performed as previously reported [13]. Clean reads were mapped to the reference genomic sequence of K. marxianus NBRC1777 from GenBank with accession No.…”