Transcriptomics Analysis of Formic Acid Stress Response in Saccharomyces Cerevisiae

Formic acid is a representative small molecule acid cell inhibitor in lignocellulosic hydrolysate, which can inhibit the growth of yeast cells in the process of alcohol fermentation. However, the mechanism of formic acid cytotoxicity remains largely unknown. This study aimed to study the cytotoxicity of formic acid stress to Saccharomyces cerevisiae. We evaluated the effects of formic acid on growth metabolism and cell morphology of yeast cells, and comprehensively and systematically analyzed the molecular mechanism of formic acid stress tolerance through transcriptome technology. The results showed that when the concentration of formic acid was 1.8 g/L, the growth of yeast cells was signicantly inhibited, the cell surface was wrinkled, and the adhesion between cells was observed, and the cell wall and cell membrane of yeast were destroyed by changing the structure of proteins and carbohydrates, resulting in cell damage. Transcriptome sequencing results showed that formic acid stress inhibited protein biosynthesis, induced oxidative stress, resulted in autophagy, impaired intracellular ATP production and increased consumption, and then impaired normal physiological and metabolic functions of cells. Yeast cells provide sucient ATP by accelerating glucose metabolism, enhancing electron transport and ATP synthesis more energy to resist formic acid stress, and reduce the expression of genes related to energy metabolism such as intracellular amino acids to achieve an energy-saving strategy, In addition, it can also induce sexual reproduction and spore formation to improve cell tolerance to formic acid. This study initially revealed the molecular response mechanism of S. cerevisiae under formic acid stress, and provided a scientic basis for further research on methods to improve the tolerance of cell inhibitors in lignocellulose hydrolysate. Two glucose transporters in Saccharomyces cerevisiae are glucose sensors that generate a signal for induction of gene


Introduction
Lignocellulose is one of the most abundant clean and renewable resources in nature. Its hydrolysate can be fermented by Saccharomyces cerevisiae to produce cellulosic ethanol, which can effectively alleviate the urgent energy crisis and environmental pollution problems (Auesukaree et al. 2017; Mosier et al. 2005). However, toxic compounds are often introduced during the pretreatment and hydrolysis of lignocellulosic raw materials. According to the sources of these toxic compounds, they can be divided into three categories: weak acids ( on the performance of fermentation microorganisms. Speci cally,formic acid and acetic acid are common weak acid inhibitors in lignocellulosic hydrolysates, which are mainly produced by the degradation of 5-hydroxymethylfurfural (Larsson et al.1999), and a part of formic acid is produced by the degradation of furfural under acidic conditions (Zhang et al. 2012). Acetic acid is mainly produced in the process of deacetylation of hemicellulose, and its concentration is usually between 1-15 g/L (Wende et al. 1999). The concentration of formic acid is usually lower than that of acetic acid, but it is more toxic to S. cerevisiae than acetic acid (García-Aparicio et al. 2006; Martin et al. 2007). The concentration of other toxic and weak acid hydrolysates is rarely reported, and its concentration is even lower than that of formic acid. Although it is not easy to elucidate the inhibitory mechanism of weak acids, it has been suggested that the inhibitory effect of weak acids is due to uncoupling theory and intracellular anion accumulation (Palmqvist et al. 2000;Pampulha et al. 2000). The undissociated weak acid molecules can pass through the cell membrane and enter the cell, and dissociate into protons and acid radical ions in the cell, thereby causing intracellular acidi cation. This acidi cation may cause the intracellular pH to drop and inhibit cell growth and product formation, and thus affect cell metabolism (Lohmeier-Vogel et al. 1998; Guldfeldt et al. 1998). Intracellular anions believe that only the anion form of weak acids can accumulate in the cell, thus causing the toxicity of weak acids (Helle et al.2004; Russel et al. 1992) and resulting in the degradation of vacuoles (Suzuki et al. 2012).
In recent years, RNA sequencing, as a revolutionary tool for transcriptomics analysis through deep sequencing technology, has been successfully applied to S. cerevisiae (Nagalakshmi et al. 2008). Abbott et al. (2009) research found that overexpression of CTT1 encoding cytoplasmic catalase increased the speci c growth rate of yeast under acetic acid treatment at pH 3. The Mira study found that multiple genes in yeast cells are involved in the toxic effects of acetic acid. These genes are mainly involved in transcription, cell wall integrity, intracellular pH homeostasis, carbohydrate metabolism, and cell absorption of nutrients (Mira et al. 2010). However, as far as we know, the current tolerance mechanism of S. cerevisiae in response to formic acid stress needs to be further elucidated at the transcriptome level. In this study, in order to explore the molecular toxicity mechanism of formic acid on S. cerevisiae, and combined with scanning electron microscope (SEM) and Fourier transform infrared spectroscopy (FTIR) analysis. Transcriptome-based high-throughput sequencing technology was used to study the formic acid stress response and tolerance mechanism of S. cerevisiae, in order to initially reveal the molecular response mechanism of S. cerevisiae under formic acid stress at the molecular level, and provide a theoretical basis for further improving the tolerance of S. cerevisiae lignocellulose hydrolysate inhibitors.

Materials And Methods
Strain culture The S. cerevisiae GGSF16 strain used in this study was isolated and preserved by the Microbiology Laboratory of Guangxi University of Science and Technology. The strain was stored on YPD medium plates (yeast extract 10 g/L, peptone 20 g/L, glucose 20 g /L, agar powder 20 g/L). Activate and culture for 14 hours in a shaker at 30°C and 150 r/min. Inoculate the activated GGSF16 yeast cells in a fresh YPD medium at 30°C with an initial yeast cell number of 4.2×10 7 CFU/mL. After culturing for 6 hours, report to the experimental group. Add an appropriate amount of formic acid to the culture medium to make the nal concentration 1.2 g/L, 1.5 g/L, 1.8 g/L, 2.1 g/L, and use the bacterial solution without formic acid as the control group. Then cultivated for 24 hours and monitored at 600 nm (OD 600 ) with an ultraviolet spectrophotometer UV2000. Three biological replicates were performed. Then we select 1.8 g/L formic acid for subsequent experiments according to the growth state of S. cerevisiae GGSF16. To repeat the above culture steps, the group with 1.8 g/L formic acid as treatment group (for1, for2, for3) and the nontreated group (ck1, ck2, ck3) as the control group. and the yeast cells were collected for RNA extraction after continuous culture for 2 hours.

SEM analysis
After treatment with formic acid, yeast cells were centrifuged at 8000 r/min 4 ℃ for 10 min to collect, washed with PBS buffer for 3 times, and the bacteria were xed in 2.5% glutaraldehyde, xed overnight at 4℃, centrifuged at 8000 r/min 4 ℃ for 10 min to collect the bacteria. 20%, 50%, 70%, 80% and 100% ethanol were washed successively, each concentration was treated for 10min, and the bacteria were collected by centrifugation at 8000 r/min at 4 ℃ for 10min. Finally, the yeast cells were dried, and after spraying gold, the yeast cells were observed and photographed using the FehnPhenom scanning electron microscope.

FTIR analysis
Collect the yeast cells by centrifugation, wash the cells 3 times with PBS, and freeze-dry. This experiment uses the potassium bromide tableting method (Rogowski et al. 2018), and the freeze-dried yeast cells and potassium bromide are in a mass ratio of 1:20 Mix and grind into a uniform powder with an agate mortar.
The scanning conditions were set as follows: the spectral range was 400cm -1~4 000cm -1 , the resolution was 4cm -1 , potassium bromide was used as a blank control, and each sample was repeated 3 times under the same conditions.

RNA extraction, library construction and sequencing
Samples containing yeast cells were centrifuged at 12000×g at 4 °C for 2 min. After turbid supernatant was removed by decantation, yeast cells were recovered from the pellets and stored at -80 ℃. Total RNA was extracted using the yeast RNA extraction kit. The extracted total RNA sample is subject to a simple quality test before it can be used for subsequent transcriptome analysis, including the detection of RNA integrity, concentration, and protein contamination to check whether they are quali ed. After the total RNA detection was quali ed, the mRNA was enriched with magnetic beads with Oligo (dT) and fragments were added with an appropriate amount of interrupting reagent. The cDNA library was constructed using the mRNA as template and was sequenced using the BGISEQ sequencing platform at last. The library was tested and sequenced were completed by Shenzhen BGI Technology Services Co., Ltd.

Reads mapping, annotation and analysis
Quality control was carried out on Raw Reads obtained by sequencing to obtain high-quality Clead Reads. First, the Reads containing joints and the Reads with high and low quality of unknown base N content should be removed to obtain Clead Reads, which were used as data for subsequent experimental studies. Sequence alignment analysis was performed between Clead reads and the speci ed reference genome using HISAT, and differential expression analysis was performed between the two sample groups using DESeq2 algorithm. Screen differentially expressed genes (DEGs) by qvalue<0.05 and Fold Change≥2. The GO and KEGG pathway enrichment analysis of DEGs were performed using David (https://david.ncifcrf.gov/) and Kobas (http ://kobas.cbi.pku.edu.cn/) database.

Quantitative real-time PCR analysis
Totally, twelve representative genes were selected to evaluate the validity of RNA-seq by real-time PCR. The Revertaid First Strand cDNA Synthesis Kit from Thermo Fisher Scienti c was used to remove genomic DNA and reverse transcribed RNA to synthesize cDNA. qRT-PCR was performed using Fluorecent Quantitative PCR Detection system Line Gene 4800 (FQD-48A, HANGZHOU BIOR TECHNOLOGY CO., LTD). The PCR reaction conditions were as follows: pre-denaturation at 95 ℃ for 2 min, denaturation at 95 ℃ for 15 s, annealing at 55 ℃ for 15 s, and extension at 72 ℃ for 1min, with a total of 40 cycles. All reactions were performed in triplicate, The relative expression level of target genes was measured with the 2 -△△CT method and ACT1 was used as the reference gene.

Results
The effects of different concentrations of formic acid on the growth of S. cerevisiae GGSF16 cells The cell growth of GGSF16 under different concentrations of formic acid stress (Fig. 1). The growth inhibition of formic acid on S. cerevisiae increased with the increase of formic acid mass concentration. We can see that there was little effect on the growth of GGSF16 when exposed to 1.2 g/L formic acid. the growth of GGSF16 was immediately inhibited under the stress of adding 1.5 g/L formic acid, but as time does on, the inhibition gradually weakened, and the nal growth rate was slightly lower than that of the formic acid-free treatment group. When the mass concentration of formic acid was increased from 1.5 g/L to 1.8 g/L, the growth of yeast cells was signi cantly inhibited, and the stabilization time was prolonged compared with the formic acid-free treatment group. When exposed to 2.1 g/L formic acid stress group, the GGSF16 cells hardly grew. In order to obtain valuable transcriptome data, the GGSF16 cells exposed to 1.8 g/L formic acid stress was selected as the experimental group in this study.
The effects of formic acid on the morphology of yeast cells The morphology of yeast cells in the control and formic acid treatment groups was signi cantly different (Fig. 2). In the formic acid treatment group (Fig. 2B), the surface of yeast cells was obviously deformed, the surface of cell membrane became rough, and the edge of cell wall collapsed. While in the control group ( Fig. 2A), the cell surface was smooth and full, and the appearance was oval or round, indicating that formic acid had obvious damage to the cell wall and cell membrane of S. cerevisiae, causing the cells lose the protection of cell wall, causing the normal physiological metabolism of cells to be blocked, and it was di cult for nutrients to enter and exit the cells, leading to cell damage and death.

FTIR analysis
FTIR was a structure analysis technology based on the vibration of functional groups and polar bonds in compounds. It was characterized by simple operation, fast speed and high sensitivity. In recent years, it has been widely used in the structure analysis of macromolecular compounds and the analysis of the secondary structure of proteins. and it is a powerful tool for molecular structure information (Yu et al.

2019
; Bosch et al. 2006). Studies have shown that yeast cell wall is mainly composed of β-glucan, mannoglycoprotein, a small amount of chitin and lipids, which plays an important role in maintaining cell morphology and inter-cell recognition (Galichet et al. 2001). These substances can provide a large number of active groups, and the stretching, bending and deformation vibrations generated by these groups can generate obvious absorption peaks in the infrared spectrum. In the spectrogram of yeast, the most important band to characterize protein is the amide band, and the amide I band at 1650 cm -1 is formed by the stretching vibration of C=O and the bending vibration of N-H. which indicates that the secondary structure of protein in yeast is dominated by α helix ( suggesting that formic acid stress may cause protein denaturation in yeast cells (Fig. 3). In addition, the peak of the amide band (1243.3/1242.4 cm -1 ) produced red shift, which may be caused by the phospholipid bilayer damaged by formic acid treatment, and the permeability of the cell membrane was changed and some nucleic acids and proteins were released. The peak sites were signi cantly increased at 3307.7 cm -1 , 3002.6 cm -1 and 2928.6 cm -1 , indicating that formic acid altered the structure of chitin in the cell wall and damaged the lipid of yeast cell membrane. The peak at 914.2 cm -1 was enhanced, indicating that formic acid changed the polysaccharide hydroxyl skeleton on the surface of S. cerevisiae, which in turn changed the structure of the yeast cell wall. Therefore, it is speculated that formic acid damages the yeast cell wall and membrane by changing the structure of proteins, lipids and chitin, leading to cell death.

Transcriptome data analysis
The original data was processed through a series of data processing to lter impurities to get ltered data. The average clean read of each sample reaches 6.41 Gb, and the average Q20 of the two groups were 97.27% and 97.26%, and the Q30 averages are 89.73% and 89.53%. High-quality reads were compared with the designated reference genome using Hisat software, and the results showed that more than 95.91% and 96.31% reads of the two groups covered the reference genome, and the quality of the original sequencing was quali ed and could be used for subsequent data analysis.

Screening of differentially expressed genes
In order to study the differentially expressed genes before and after the addition of formic acid, differential expression identi ed was conducted for all genes obtained after sequencing between the two groups. With Fold Change>2 and qvalue<0.05 as the identi ed conditions, A total of 1504 DEGs were identi ed, of which 797 were up-regulated genes and 707 were down-regulated genes (Fig. 4). Each dot in the gure represents a gene, The red ones are up-regulated gene, the green ones are down-regulated gene, and blue ones are that is not signi cantly different.
GO enrichment analysis GO enrichment analysis can provide three types of descriptions for gene products, namely Biological Process (BP), Cellular Component (CC) and Molecular Function (Molecular Function, MF). Through the GO enrichment analysis of differential genes, The function of genes can be studied (Fig. 5). Through GO enrichment, we found that 8 of 25 signi cantly enriched pathways were related to membrane transport function, including fructose transmembrane transport, mannose transmembrane transport, glucose transmembrane transport, fructose transmembrane transporter activity, transporter activity. These DEGs changed differently under formic acid stress, suggesting that formic acid affected the function of cell membrane and membrane transporter. In addition, most DEGs are enriched in ribosome metabolic pathways, indicating that formic acid has a certain impact on ribosomes. It can be seen from the scanning electron microscope that the surface morphology of the yeast cells is greatly changed after formic acid treatment, indicating that the cell membrane and cell wall are damaged, so that the cell loses the protection of the cell wall, causing the normal physiological metabolism of the cell to be blocked, and it is di cult for substances to enter and exit the cell, resulting in poor cell growth or death. The infrared analysis results show that formic acid destroys yeast cell walls and cell membranes by changing the structure of proteins, polysaccharides, lipids and chitin, which is consistent with the changes in cell morphology observed in the electron microscope. GO enrichment analysis showed that under formic acid stress, yeast cells deal with the damage of formic acid to the cell membrane and cell wall by regulating the expression of genes related to membrane transport.

KEGG pathway analysis
The KEGG enrichment analysis of DEGs between the control group and the formic acid treatment group is shown in Fig. 6. It is important to note that there are 115 DEGs are enriched in the ribosome pathways, and 27 DEGs are enriched in the ribosomal biogenesis of eukaryotes, indicating that ribosomes play an important role under the formic acid stress, and found that most genes related to body biosynthesis were down-regulated, while genes related to mitochondrial ribosome biosynthesis were up-regulated.It was also found that 26 DEGs were enriched in glycolysis/gluconeogenesis, 20 DEGs were enriched in starch and sucrose metabolism, 13 DEGs were enriched in TCA cycle, 12 DEGs were enriched in galactose metabolism, 10 DEGs were enriched in fructose and mannose metabolism, 38 DEGs were enriched in meiotic yeast, 40 DEGs were enriched in MAPK signaling pathway, It was speculated that S. cerevisiae accelerates glucose metabolism through glucose transmembrane transport, thus synthesizing a large amount of ATP, providing su cient energy for resisting formic acid stress, which is consistent with the GO enrichment analysis results. In addition, it is possible to enter sexual reproduction and spore formation to improve offspring adaptation to the environment, thereby enhancing formic acid tolerance. Interestingly, we also found that the differential genes involved in cysteine and methionine metabolism and purine metabolism are also signi cantly expressed, indicating that amino acid metabolism and purine metabolism also play a role in formic acid stress.

RNA-seq expression validation by quantitative real-time PCR
In order to quantitatively determine the reliability of the transcriptome results, we detected the expression of 12 candidate DEGs by RT-qPCR. These candidate genes include 10 up-regulated genes and 2 downregulated genes (Fig.7). The RT-qPCR results showed that, after the addition of formic acid, the expression of HXK2 and ENO2, which were involved in glycolysis pathway, IMA3 involved in galactose metabolism and SOR1 and DSF1 involved in fructose and mannose metabolism, HSP30 involved in protein folding, GND2 is involved in pentose phosphate pathway, ATP14, ATP19 and COX6, which were involved in ATP biosynthesis were up-regulated, while PRS3 and ADE5,7 were involved in purine metabolism were all down-regulated. A high consistency was displayed between the RNA-seq and RT-qPCR data, proving the validity of RNA-seq data for genes with distinct transcript abundance.

Ribosome
The ribosome is an important organelle in eukaryotic cells, which is mainly involved in protein biosynthesis in cells. It consists of a 40S subunit, a 60S subunit and 4 types of RNA (Horsey et al. 2004). RNA-Seq analysis showed that under formic acid stress, our research found that most of the DEGs related to ribosomal and ribosomal biogenesis in eukaryotic pathways were signi cantly down-regulated ( Table  1). The expression of genes related to mitochondrial ribosomal proteins is up-regulated, which means that even if formic acid inhibits the biosynthesis of yeast cytoplasmic ribosomes, cells can still use mitochondrial ribosomes to synthesize other proteins to resist the stress of formic acid. Ribosomal protein is an important component of ribosomes. Studies have found that ribosomal protein is not only involved in protein synthesis, but also involved in cell differentiation, cell development regulation, DNA repair and other processes. It plays a crucial role in the growth and development of eukaryotes (Tedesco et al. 2014). In yeast cells, ribosome biosynthesis is highly conserved. It starts from the precursor rRNA in the cytoplasm and ends with the synthesis of mature 40S and 60S ribosomal subunits. In addition, the ribosome synthesizes cells through ribosomes. The required protein can consume about 2/3 of the energy. Li et al. (2010) showed that the expression of genes related to ribosome synthesis was downregulated under acetic acid stress, indicating that formic acid also had an inhibitory effect on ribosome synthesis in yeast cells. In addition, we also found that the expression levels of differentially expressed genes involved in ribosomal biosynthesis in eukaryotes are also down-regulated. UTP14, UTP13, UTP21, and UTP5 are components of complexes that have the activity of processing precursor rRNA and participate in the process. The synthesis of 18S rRNA in the nucleolus and the assembly process of small ribosomal subunits (Dragon et al. 2002), the down-regulation of its expression indicates that formic acid stress may inhibit the biosynthesis of the yeast cell polypeptide chain. Transcriptome analysis shows that formic acid signi cantly reduces the expression of genes involved in protein and RNA synthesis in yeast cells, indicating that formic acid affects the biosynthesis of yeast cells. Cells allocate the energy they produce reasonably and synthesize materials needed to relieve stress, To reduce the synthesis of some ribosomes and degrade non-essential substances, thereby improving the resistance to formic acid. The endoplasmic reticulum is an important organelle in yeast cells and has many general functions, including folding and transport of synthetic proteins. It is the center of various chaperone proteins and enzymes in eukaryotes. Chemical stress will destroy the conformation of the protein, leading to the unfolding and aggregation of the protein (Goldberg et al. 2003). Small HSPs act as chaperones to help fold or refold newborn or denatured proteins and enzymes to maintain functional conformation (Burnie et al. 2006). Interestingly, we found that the expression levels of genes involved in protein folding and degradation were signi cantly up-regulated under formic acid stress (Table 2) (Table 2). ATG9 encodes a transmembrane protein that participates in the formation of autophagy vesicles and cytoplasmic-cytoplasmic (Cvt) pathways. ATG7 and ATG13 encode autophagyrelated proteins, which participate in the formation of vesicles in the process of autophagy, and regulate the binding of Atg12p, Atg5p, Atg8p and phosphatidylethanolamine. ATG2 is involved in autophagy repair. The peripheral membrane protein encoded by ATG2 is involved in the Cvt pathway and the entry of autophagic vesicles. It is a necessary condition for the formation of isolated cytoplasmic vesicles. Vps34p and vps15p jointly encode the autophagy of the phosphatidylinositol 3-kinase complex, which is a necessary condition for the formation of autophagy vesicles. Studies have shown that autophagy is an important way for cells to get rid of oxidative stress and is the second level of self-protection against environmental oxidative stress (Jain et al. 2010). In the absence of autophagy, environmental stress induces the accumulation of intracellular reactive oxygen species (Tal et al. 2009). Interestingly, the expression levels of autophagy related genes were signi cantly increased under formic acid stress, suggesting that autophagy plays an important role in the response of S. cerevisiae to formic acid stress. Therefore, it is speculated that formic acid stress induces oxidative stress through the accumulation of reactive oxygen species in yeast cells, which induces autophagy in yeast cells, and then regulates protein degradation and autophagy genes, making S. cerevisiae resistant to formic acid stress. By improving the activity of molecular chaperones, degrading misfolded proteins, reducing misfolded stress, providing raw materials for the synthesis of new proteins, and helping cells to restore normal physiological and metabolic functions. Mitochondria, as an energy center, drive a large number of energy-needed processes in cells, and the expression of differential genes involved in this process are up-regulated (Table 3). COX17, COX10, COX8 and COX6 are not only related to mitochondrial respiratory chain complexes III, IV, mitochondrial electron transport and oxidative phosphorylation, but also encode cytochrome oxidase (COX). which catalyzes electron transfer from cytochrome C to molecular oxygen and catalyzes proton pump across the mitochondrial inner membrane to produce large amounts of ATP. Interestingly, the expression of energyrelated genes ATP19, ATP14, ATP20, ATP4 and PPA2 increased signi cantly, which played an important role in ATP synthesis. Our study also found that the expression level of PMA2 a gene regulating plasma membrane H + -ATPase activity, was also signi cantly up-regulated, which could pump protons out of cells and regulate cytoplasmic pH and plasma membrane potential. Yeast cells will choose to enhance the expression of genes related to electron transport and ATP synthesis, Expression provides su cient energy for H + and acid radical ion transport, maintain the stability of intracellular pH, and thus improve the tolerance of yeast cells to formic acid.
Interestingly, genes involved in starch and sucrose metabolism, such as GSY1, GSY2, GLG1, GLG2, GPH1, PGM2, genes involved in fructose and mannose metabolism, such as PFK27, SOR1, DSF1, MAN2, DAK2, genes involved in galactose metabolism, such as IMA1, IMA2, IMA3, GAL7, GAL2, MPH3, were signi cantly up-regulated under formic acid stress (Table 3). GSY1 and GSY2 encode glycogen synthase, which catalyzes glycogen synthesis. When yeast cells are undernourished by environmental stress, they synthesize glycogen to provide energy metabolism and adjust the energy demand for biosynthesis. GPH1 encodes glycogen phosphorylase, which can form glucose-1-phosphate catalyzed by glycogen and further convert glucose-6-phosphate catalyzed by glucose phosphate mutant enzyme. This is a key step in hexose metabolism. The up-regulation of the gene expression accelerates the conversion of glucose-1phosphate to glucose-6-phosphate. SOR1, DSF1 and MAN2 encode sorbitol dehydrogenase and mannitol dehydrogenase, respectively, providing fructose-6-phosphate for EMP pathway, thereby accelerating ATP generation. IMA1, IMA2, IMA3, and GAL7 are involved in the decomposition of maltose, and GAL2, MPH3, MPH2, and MAL31 are involved in the transport of galactose and maltose. These genes related to energy metabolism were signi cantly up-regulated in the response of S. cerevisiae to formic acid stress. It is speculated that when yeast cells are subjected to formic acid stress, certain energy is stored to resist damage, which indicates that energy metabolism is very important in the response to formic acid stress in yeast cells. The growth and development of yeast cells, including the synthesis of nucleic acids and proteins, the delivery of bio lms, and transport functions, all require energy consumption. What is interesting is that we found that the overall expression of genes involved in the sugar metabolism pathway is up-regulated (Table 4). HXK1 and HXK2 encode hexokinase, which catalyzes glucose-phosphorus to glucose-6phosphate, The expression level was signi cantly up-regulated under formic acid stress, indicating that the utilization of glucose by yeast was accelerated. TDH1 is a gene that regulates glyceraldehyde-3phosphate dehydrogenase activity, and its upregulation accelerates the conversion of glyceraldehyde-2phosphate to pyruvic acid. PGK1 and GPM1 encode 3-phosphoglycerate kinase and phosphoglycerate mutase, respectively. which are mainly involved in the conversion of 1,3-diphosphoglyceric acid to 2phosphoglyceric acid in glycolysis, and produce a small amount of ATP. ENO2 and ENO1 encode enolase, which play a role in the main metabolic pathways of glycolysis and gluconeogenesis (Kornblatt et al. 2013). PYK2 encodes pyruvate kinase, which is an important rate-limiting enzyme in the glycolysis pathway, and the of its activity helps to accelerate the smooth progress of EMP (Mitsui et al. 2020). PDC6, which regulates the activity of pyruvate kinase, is down-regulated, which slows down the conversion of pyruvate to acetaldehyde. In addition, the expression of alcohol dehydrogenase gene ADH3 was up-regulated, but the expression of the enzyme gene ADH2, which is the reverse reaction of ethanol production of acetaldehyde, was down-regulated. Studies have shown that knocking out ADH2 gene in S. cerevisiae strain As2.4 can increase ethanol production by 52% (Ye et al. 2016). ALD4, which is related to aldehyde dehydrogenase, is down-regulated and the conversion of acetaldehyde is reduced. This may be due to the fact that yeast gradually adapts to environmental pressure by regulating its metabolic activity and avoiding the decline of activity, thus affecting the fermentation environment. PCK1 encodes phosphoenolpyruvate carboxykinase, which catalyzes the conversion of oxalylacetic acid to allylpyruvate. It is a reverse reaction of glycolysis pathway, and the down-regulation of its transcription level promotes the metabolism of EMP pathway.
In yeast, glucose transport is mainly accomplished by the transporter encoded by the HXT gene, a member of the Major facilitator superfamily (MFS) (Özcan et al. 1999). In this study, we found that the expression of glucose transporters encoded by HXT1, HXT2, HXT3, HXT4, HXT6, HXT7 and HXT15 were signi cantly up-regulated (Table 4). HXTI and HXT3 encode low a nity glucose transporters, HXT2, HXT4, HXT6 and HXT7 all encoded glucose transporters with high a nity. Among them, HXTI was induced under high glucose concentration, while the expressions of HXT6 and HXT7 were inhibited under high glucose concentration. HXT2 and HXT4 were induced under low glucose concentration (Özcan et al. 1996). In addition, SNF3 does not directly encode sugar transporters, but acts as an extracellular plasma membrane glucose sensor, and at the same time assists in the induction of expression of HXT1 and HXT3 (Özcan et al. 1999). It is suggested that yeast cells respond to formic acid stress mainly by regulating the expression of glucose transporters, thus improving the tolerance of formic acid. As a special way of cell proliferation, meiosis also has a certain impact on cell proliferation. Compared with the control group, the expression of genes involved in meiosis in formic acid-treated yeast cells was all up-regulated (Table 5). RIM15 is responsible for activating the activity of meiotic genes. RAD53, DDC1 and MRC1 control the S-phase and G1/G2 DNA damage checkpoints. SWM1 and APC2 are essential genes that promote the activity of the complex/loop body (APC/C) in late meiosis. MCM7, MCM4, and MCM2 encode proteins for DNA replication, which are part of the MCM2-7 hexamer helicase complex, participate in the starting point of DNA replication in G1, and can promote the melting and extension of Sphase DNA. ESP1 is an isolating enzyme that cuts the meiosis-adhesin subunit Rec8p along the chromosome arm during meiosis I and the centromeric site during meiosis II, and its expression is inhibited by Pds1p. The subunit synthesis of lectin complex (encoded by SMC4) increased, and the lectin recombines chromosomes during mitosis and meiosis. Therefore, yeast cells can respond to formic acid stress through sexual reproduction, because sexual reproduction can improve the adaptability of offspring to the environment through gene recombination.
Cyclin is a type of protein that expresses, accumulates, and decomposes in a cell cycle-speci c or phased manner, and they together with cyclin-dependent kinases affect the operation of cell cycle. The cell cycle is mainly divided into the early stage of DNA synthesis (G1 stage), the DNA synthesis stage (S stage), the late DNA synthesis stage (G2 stage) and the cell division stage (M stage). Most cell cycle-related genes are up-regulated (Table 5), such as CLB1, CLB2, CLB6, CLN1, CLN2, CLN3, SPS4, PCL1, DUN1, MBP1. PCL1 encodes the G1/S phase-speci c cyclic protein involved in cell cycle regulation, interacts with the cyclin-dependent kinase Pho85p, and participates in the regulation of polarized growth and morphogenesis and progression during cell growth. Its up-regulated expression means Promote the transition from G1 phase to S phase. The up-regulated expression of cyclin CLN1, CLN2 and CLN3 genes means that they activate Cdc28p kinase to promote the transition from G1 to S phase. MBP1 is involved in the regulation of cell cycle from G1 to S phase. In addition, CLB6 can activate Cdc28p to promote the initiation of DNA synthesis, and plays a role in the formation of mitotic spindles together with CIb3p and CIb4p. CLB1 and CLB2 (cyclin) can activate Cdc28p to promote the transformation from G2 to M phase (Hadwiger et al. 1989;Wittenberg et al. 1990). CLB1 is involved in meiosis, but CLB2 is only involved in mitosis (Grandin et al. 1993). It is worth noting that the up-regulated expression of DUN1, as a cell cycle checkpoint protein gene, and Dun1p, as a signal transducer for cell cycle arrest and transcription response to damaged or unreplicated DNA, Its up-regulated expression often means DNA damage. In addition, the gene SPS4 regulating spore formation was up-regulated, and the yeast showed meiosis under nitrogen stress, forming four haploid ascospores (Hill et al. 2007). Studies have shown that meiosis, spore formation and pseudohypha growth are the responses of S. cerevisiae to nitrogen starvation (Schrder et al. 2000). Therefore, we believe that formic acid stress may cause nitrogen starvation, and yeast cells can improve the adaptability of offspring to the environment through sexual reproduction and spore formation, thereby improving the tolerance of formic acid.  (Chen et al. 2007). In our study, the expressions of MF(ALPHA)1, MF(ALPHA)2, STE3, STE2, and SST2 involved in the pheromone signaling pathway were all up-regulated (Table 6). MF(ALPHA)1 and MF(ALPHA)2, as mating pheromone α factors, are composed of a cells and interact with mating type a cells, inducing cell cycle arrest and other mating reactions. STE3 and STE2, as receptors of α factor pheromone, cascade with MAP kinase, and are required for transcription in α cells and mating with α cells. The ligand-bound receptor is endocytosed and recycled to the plasma membrane. As GTPase activator protein of Gpa1p, SST2 can regulate the desensitization of α-factor pheromone, and it also necessary to prevent receptor-independent signal transduction in mating pathway. Bnilp is the key to the formation of linear actin laments and participates in the cell processes that need to polarize actin clusters, such as budding and mitotic spindle orientation (Matheos et al. 2004;Yu et al. 2008 FKS3 is involved in the assembly of spore wall proteins required for occulation and bio lm formation. The up-regulation of these genes indicates that formic acid has adverse effects on the growth of S. cerevisiae. The tolerance of S. cerevisiae to formic acid may be improved by regulating the signal transduction pathway and lamentous or invasive growth pathway of pheromone. the differential genes related to cell growth and division and protein synthesis are signi cantly inhibited under external environmental stress, which was precisely to reduce energy loss and achieve selfprotection. It can be seen that the reduction of amino acid metabolism in yeast cells may be to reduce energy requirements and rationally allocate the energy produced by themselves to make them survive the stress of formic acid. Studies have shown that yeasts are faced with multiple environmental pressures such as high ethanol, high acid and insu cient nutrients, and nitrogen source is the most common factor limiting the growth of yeasts (Bely et al. 1990). SER3 encodes 3-phosphoglycerate dehydrogenase, which catalyzes the synthesis of serine and glycine. Up-regulation of its expression accelerates the synthesis of these two amino acids. CHA1 encodes L-serine deaminase, which catalyzes the degradation of L-serine and threonine. requiring the use of serine or threonine as the only nitrogen source, indicating that maintaining the balance of carbon and nitrogen metabolism in yeast cells plays an important role under formic acid stress. Glutathione is an important antioxidant substance, which can reduce active oxygen free radicals to generate oxidized glutathione to exert its antioxidant function and protect important organelles in the cell from damage (Mailloux et al. 2013). In this study, it was found that genes involved in the glutathione metabolism pathway were up-regulated (Table 7). GSH2 encodes glutathione synthetase, which catalyzes the synthesis of glutathione from γ-glutamylcysteine and glycine. GPX2 encodes glutathione peroxidase, which protects cells from phospholipid hydroperoxides and non-phospholipid peroxides during oxidative stress (Izawa et al. 1995;Grant et al. 1998). Since glutathione is responsible for the redox state in cells and acts as a protective agent against reactive oxygen species (Morano et al. 2012), these results indicate that formic acid stress can disturb the redox balance of yeast. GND1 and GND2 encode glucose-6 phosphate dehydrogenase, which catalyzes the production of ribulose-5-phosphate from phosphogluconate, produces a large amount of NADPH in the pentose phosphate pathway, provides reducing power for various synthesis reactions of cells, and maintains The level of redox in the cell. At the same time provide synthetic DNA, RNA, tryptophan, tyrosine and other biological synthesis of the premise substances 4-phosphoerythritol and 5-phosphoribose. It is because of the increased activity of glucose 6phosphate dehydrogenase that yeast cells have more raw materials to synthesize cells, thus improving the tolerance of GGSF16 to formic acid.

Conclusions
In this study, transcriptomics was used to study the cytotoxicity of formic acid to S. cerevisiae GGSF16 and its molecular mechanism. Formic acid can obviously inhibit the growth of yeast cells, and cause cell damage and even death by changing the structure of proteins and carbohydrates on the cell wall and cell membrane. Notably, transcriptomics data show that formic acid stress can not only inhibit protein biosynthesis, but also induce oxidative stress, resulting in autophagy, impaired intracellular ATP production and increased consumption, leading to impaired cellular physiological and metabolic functions. GGSF16 provides su cient energy to resist formic acid stress by accelerating energy metabolism, enhancing electron transfer and ATP synthesis. It can also reduce the expression of some genes related to the biosynthesis of energy-consuming metabolic pathways such as intracellular amino acids, thereby achieving an energy-saving strategy, and It can rationally use its own energy to synthesize substances needed to cope with stress to protect cells. In addition, sexual reproduction and spore formation can improve the adaptability of offspring to the environment, promote the absorption of nutrients, and improve the tolerance of cells to formic acid. This review preliminarily revealed the molecular response mechanism of S. cerevisiae under formic acid stress, which provided a theoretical basis for the subsequent study on the tolerance of cell inhibitors in lignocellulose hydrolysates. The effect of formic acid on the growth of yeast cells