Materials and reagents
The tested thermotolerant yeast strain was Pichia kudriavzevii (ZG-3) preserved at Henan Liquor Style Technology Research Center, Henan University of Animal Husbandry and Economy.
Glucose, agaragar (Tianjin Kemiou Chemical Reagent Co. Ltd.); peptone, trehalose (Beijing Abxing Biotech Co. Ltd.); yeast powder (UK Oxoid); KH2PO4 (Tianjin De'en Chemical Reagent Co. Ltd.); NaCl (Xilong Chemical Co. Ltd.); Trizol kits (US Invitrogen); CaCl2, CCl3COOH, (NH4)2SO4 (Zhengzhou, Paini Chemical Reagent Plant); MgSO4, sodium citrate (Sinopharm Chemical Reagent Co., Ltd); double distilled water (Shanghai Sangon Biotech Co. Ltd.); anthrone (Beijing Solarbio Co. Ltd.) were used here.
Liquid culture medium (pH 4.5): 20.0 g of glucose, 20.0 g of peptone, and 10.0 g of yeast powder were dissolved in 1000.0 mL of water, and sterilized at 121 ºC for 15 min.
Solid culture medium YPD: medium (natural pH): 10 g of yeast powder, 20 g of tryptone, and 20 g of anhydrous glucose were dissolved in 1000 mL of water, and sterilized at 115 ºC for 15 min.
Fermentation medium: 20 g of peptone, 10 g of yeast extract powder, 100 g of glucose, 1 g of MgSO4, 1 g of (NH4)2SO4, and 1 g of KH2PO4 were diluted by distilled water to 1000 mL (natural pH) and sterilized at 121 °C for 20 min.
A DRP-9052 thermostatic oscillation incubator (Shanghai Senxin Laboratory Instrument Co. Ltd.), an SW-CJ-2FD double two-sided purification workbench and a Sigma high-speed freezing centrifuge (Suzhou Purification Equipment Co. Ltd.), An LDZX-50FBS high-pressure steam sterilization pot (Shanghai Shenan Medical Instrument), a BCD-216SDN refrigerator (Qingdao Haier Co. Ltd.), an A590 ultraviolet spectrophotometer (Shanghai Aoyi Instrument Co. Ltd.), and a CX31 biological microscope (Guangzhou Olympus Co. Ltd.) were used here.
Phenotypes of the thermotolerant yeast strain
Activation of the thermotolerant yeast strain
The strain was taken out from the refrigerator at -80 ºC, and recovered from the preservation state. It was inoculated on a superclean bench. The inoculated culture medium was put into a thermostatic incubator at 28 ºC, until milk white colonies appeared. Then the colonies with representative characteristics of yeast strain colonies were selected, and lineated twice. Only the colonies microscopically examined to be pure were used in subsequent experiments[22-23].
Macromorphology of the thermotolerant yeast strain
From the activated strain, single colonies were selected and lineated. After cultivation in a yeast incubator at 28 ºC for 48 h, colonial morphology was observed. Then after cultivation in a thermostatic shaking table at 28 ºC for 24 h, the strain was thermalactivated at 43 ºC for 24 h. The strain was put back to the thermostatic shaking table at 28 ºC for 24 h of recovering. The above steps were repeated, during which the strain was photographed to record the morphological changes before and after thermalactivation.
Micromorphology of the thermotolerant yeast strain
The strain was dyed by cultivation with methylene blue at 28 or 43 ºC and then microscopically observed.
Basic research on the thermotolerant yeast strain
Effects of medium components on thermotolerant ability of the yeast strain
Effects of peptone and yeast extractum on thermotolerant ability of the yeast strain
The thermotolerant yeast strain treated at 43 ºC was inoculated at the 5% amount into 10, 15, 20, 25, 30 g/L YEPD liquid media, which were cultured at 43 ºC and 150 r/min for 48 h. Then the absorbance of each bacterial solution at λ=600 nm was measured. With the same method, the thermotolerant ability at different concentrations of yeast powders was detected.
Effects of inorganic salt ions on thermotolerant ability of the yeast strain
The thermotolerant yeast strain treated at 43 ºC was inoculated at the 5% amount into 0.5, 1, 1.5, 2, 2.5, 3 mmol/L CaCl2 YEPD liquid media, which were cultured at 43 ºC and 150 r/min for 48 h. Then the absorbance of each bacterial solution at λ=600 nm was measured. A system without adding inorganic salt ions was set as the control. With the same method, the effects of ZnSO4, MgSO4, NaCl, KH2PO4 at different concentrations on the thermotolerant ability were detected.
Association between strain cell structure and thermotolerance of yeast
Association between trehalose concentration and thermotolerant resistance of yeast
Fucose concentrations were measured by anthrone colorimetry. Li Yanling et al. compared three sulfuric acid - anthrone colorimetric methods, and found sulfuric acid - anthrone dilute sulphuric acid method was the most effective and economical. The specific conditions were: 85% H2SO4, 1.0 mL of trehalose solution, 3.0 mL of anthrone sulphuric acid reagent, boiling coloration 10 min, and absorbance at 625 nm.
Shang Lei  used a pipette to accurately suck 0, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0 mL of a standard trehalose solution into 7 test tubes, which were all diluted with distilled water to 2.0 mL and then added with 4.0 mL of a newly-prepared sulfuric acid - anthrone reagent. The first tube served as a blank. Then the absorbance at 625 nm was detected. A standard curve was plotted with the absorbance as Y-axis and trehalose concentration as X-axis.
The yeast strain was cultured at 28 or 43 ºC for 24 h. Then the fermentation culture solutions (each 5 mL) were centrifuged at 2000 rpm/min for 5 min, washed with deionized water that was placed in an ice-bath in advance, and centrifuged again. Then 4 mL of a 0.5 mol/L CCl3COOH solution was added. In the resulting solutions, when only trehalose existed and after oscillation to be uniform, the solutions were put into an ice water mixture with ice cubes for extraction. Oscillation was operated every 15 min. After 1.5 h, the solutions were centrifuged at 950 rpm/min for 5 min. Then the supernate (1 mL) was collected and added with 3.0 mL of the sulfuric acid - anthrone reagent, followed by measurement of optical density (OD).
Association between cell membrane permeability and thermotolerance of yeast
The thermotolerant yeast strain treated at 43 ºC was inoculated by a 10% amount into the YEPD medium for cultivation. Samples were collected every 1 h. With the yeast strain treated at 28 ºC and other similar conditions as the control, extracellular nucleotide concentration of the strain was measured.
In brief, each sample was washed with double distilled water until the absorbance at λ=260 nm of the supernatant was nearly 0. Then the cleaned strain was removed to an alcohol buffer solution (pH 5.0 and containing 0.1 mol/L sodium citrate), which was put into an oscillation incubator for culture. Samples were collected every 12 h. Then absorbance values of each sample at λ=260 nm and 280 nm were measured until the two absorbance values both reached the corresponding average values. The extracellular nucleotide concentration of the strain upon sampling was Ce (μg·mL-1)=[(OD260×11.87 - OD280×10.40)×9]/100.
Transcriptomics research on thermotolerant yeast
ZG-3 was activated and then cultured at 28 ºC for 3 d. An appropriate amount of each sample was collected, in which the count of fresh yeast was no less than 6×105 cells. After raising to 37 ºC, cultivation was continued for 24 h. Then an appropriate amount of the sample was collected. The temperature was raised to 43 ºC, and the final sampling was collected. The three times of sampling were named Sce28, Sce37, and Sce43 respectively. Then the strain was washed with 5 mL of sterile water 1-3 times, and then removed to a 1.5 mL or 2.0 mL centrifuge tube, which was centrifuged at 4 ºC for 10 min (1500 r·min-1). The supernatant was removed, and the sediment strain was reserved, which was immediately deepfrozen by liquid nitrogen. After that, each of the three samplings was sent to RNA extraction.
Total RNA extraction
Transcriptome sequencing was conducted in Guangzhou Genedenovo. Specifically, the qualified samples were sent to total RNA extraction using Trizol kits. RNA quality was assessed on Agilent 2100 Bioanalvzer, and examined by RNase-free agarose gel electrophoresis.
Library construction and RNA-Seq
After extraction of total RNA, eukaryotic mRNA was enriched using Oligo(dT) beads, and rRNA was removed using a Ribo-ZeroM magnetic kit to enrich prokaryotic mRNA. Then the enriched mRNA fragments were cut using a fragment buffer solution into short fragments, and reverse-transcribed using random primers into cDNA. Two-chain cDNA was synthesized using DNA polymerase I, RNase H. dNTP, and a buffer. Then the cDNA fragments were purified using a QiaQuick PCR extraction kit, followed by terminal restoration, A base addition, and connection to an Illumina sequencing adapter. The connected products were screened by agarose gel electrophoresis, amplified by PCR, and sequenced using Illumina Novaseq6000 (Gene Denovo Biotechnology).
Filtering and processing of original sequencing data
The reads from the sequencing machine included the original data of low-quality bases, which will interfere the subsequent assembly and analysis. Hence, to acquire high-quality clean data, we filtered the data on Fastp 0.18.0. The parameters were set as follows: 1) the reads of inset, 2) reads containing > 10% unknown nucleotides (N) and 3) low-quality reads with > 50% low-quality (Q20) bases were removed.
On the comparison tool Bowtie2 2.2.8, the reads were mapped to a ribosome RNA (rRNA) database. The rRNA-mapped data were deleted, and the remaining clean data were used for subsequent assembly and gene abundance computation. Then after comparison with the reference genome, an index of reference genome was established. The clean reads at the paired ends were mapped on HISAT 2.2.4 onto the reference genome. Based on the reference genome sequence, the mapped reads were spliced on StringTie, and compared with the original genome annotations. The raw counts were statistically analyzed on DESeq2, and at the condition of P<0.05 & |log2FC|≥2, the differentially expressed genes (DEGs) between the test group and the blank group were screened out.