Downregulation of ammonium uptake improves the growth and tolerance of Kluyveromyces marxianus at high temperature

Abstract The growth and tolerance of Kluyveromyces marxianus at high temperatures decreased significantly in the synthetic medium (SM), which is commonly used in industrial fermentations. After 100 days of adaptive laboratory evolution, a strain named KM234 exhibited excellent tolerance at a high temperature, without loss of its growth ability at a moderate temperature. Transcriptomic analysis revealed that the KM234 strain decreased the expression of the ammonium (NH4 +) transporter gene MEP3 and increased the synthesis of the amino acid carbon backbone, which may contribute greatly to the high‐temperature growth phenotype. High NH4 + content in SM significantly increased the reactive oxygen species (ROS) production at high temperatures and thus caused toxicity to yeast cells. Replacing NH4 + with organic nitrogen sources or increasing the concentration of potassium ions (K+) in the medium restored the growth of the wild‐type K. marxianus at a high temperature in SM. We also showed that the NH4 + toxicity mitigated by K+ might closely depend on the KIN1 gene. Our results provide a practical solution to industrial fermentation under high‐temperature conditions.


| INTRODUCTION
The growth ability of yeast at high temperatures is a critical phenotype for the industrial fermentation of ethanol, food products, and other useful metabolites. Especially, ethanol fermentation at high temperatures, for example within a 40°C-50°C range, is expected to achieve simultaneous saccharification and fermentation, which can effectively reduce the cooling costs and the risk of contamination (Limtong et al., 2007).
However, heat tolerance is a polygenic phenotype, and the current research is far from completely understanding it (Huang et al., 2018).
Heat shock proteins (HSPs) and chaperone proteins have been documented to be essential for yeast survival at high temperatures (Lertwattanasakul et al., 2015). In some cases, accumulating trehalose (Matsumoto et al., 2018) and decreasing lipid peroxidation (Mejía-Barajas et al., 2017) are well-known practical ways to improve the hightemperature tolerance of yeast. Although high-temperature tolerance is a property of industrial preference for yeast, previous studies on it were commonly carried out in yeast extract peptone dextrose medium (YPD), MicrobiologyOpen. 2022;11:e1290. www.MicrobiologyOpen.com not in synthetic medium (SM) that is mostly used in industrial applications.
As far as we know, only Caspeta et al. (2014) carried out the study in SM with an unnatural thermotolerance of Saccharomyces cerevisiae as the starting strain.
In YPD medium, peptone and yeast extract are the organic nitrogen sources. Due to the high cost, these nitrogen sources are rarely used in industrial production. As an alternative, ammonium salts, such as ammonium sulfate, ammonium chloride, and ammonium phosphate, are preferable nitrogen sources for large-scale fermentation production, which are inorganic and cheaper nitrogen sources conducive to reducing the production cost (Marini et al., 2006).
Kluyveromyces marxianus is a natural thermotolerant strain that can grow at 45°C or above (Fu et al., 2019;Matsumoto et al., 2018), which makes it a valuable tool for exploring the heat tolerance genes (Htg + ) and elucidating the heat tolerance mechanisms (Lertwattanasakul et al., 2015).
It is also generally recognized as a safe (GRAS) yeast that has proven to be a promising eukaryotic microbe for industrial applications (Leonel et al., 2021), such as the production of single-cell oil, fatty acids (Karim et al., 2020), lignocellulolytic enzymes , and β-mannanase (Pan et al., 2011), as well as efficient preparations of virus-like particles vaccines (Yang et al., 2021;Duan et al., 2019). However, to our knowledge, the thermotolerance of K. marxianus in SM has not been well studied yet.
The growth of K. marxianus decreased significantly at high temperatures in the industrial media using ammonium sulfate as the nitrogen source, which impedes its applications in protein expression and ethanol fermentation. Therefore, to improve its thermotolerance we adaptively evolved K. marxianus in SM at temperatures higher than 43°C. After 100 days of adaptive laboratory evolution (ALE), a mutant strain KM234 with significantly improved tolerance at 45°C was obtained. Meanwhile, it also showed no obvious change in growth at 30°C. To study the mechanism underlying the adaptive response to inorganic sources at high temperatures, the transcriptome was sequenced, and the result revealed that, in the KM234 strain, transcription of the genes that were involved in the carbon metabolism, multiple amino acid carbon skeleton synthesis pathways, NH 4 + uptake, and transportation were significantly changed in response to high temperature. In addition, the substitution of NH 4 + with glutamine or asparagine and competition of NH 4 + transport with K + in SM demonstrated that a high concentration of NH 4 + decreased the thermotolerance and growth of K. marxianus at high temperatures, which is probably due to the large production of reactive oxygen species (ROS). A high ratio of K + /NH + can alleviate the NH 4 + toxicity to K. marxianus at high temperatures, and this phenotype may be closely associated with the existence of the KIN1 gene.

| Yeast strains and experimental evolution
The K. marxianus strain FIM-1 used in this study has been described previously (Wu et al., 2020). Laboratory cultures of K. marxianus strains were carried out in flasks shaking at 220 rpm, and cell densities were determined by measuring the optical density (OD) at 600 nm (OD 600

| Gene deletions in K. marxianus
The KIN1 deletion in K. marxianus was performed by homologous recombination with the aid of a CRISPR plasmid according to the method described previously (Liu et al., 2018). Briefly, gRNA was inserted into Sap I site of pUKD-N122-AUC, obtaining the plasmid pUKD-N122-AUC/Kin1Del. Upstream and downstream homologous fragments of the KIN1 gene were amplified using the primer pairs UhfKIN1F/UhfFKIN1R and DhfKIN1F/UhfKIN1R from the genome of K. marxianus, respectively. The two amplified fragments were fused by PCR using the primers UhfKIN1F and UhfKIN1R, and then the amplified product was co-transformed with the CRISPR plasmid into K. marxianus according to the method by Antunes and de Souza Junior (2000). Transformants were selected on hygromycin plates (20 g/L poly-peptone, 20 g/L glucose, 10 g/L yeast extraction, 200 μg/mL hygromycin, and 20 g/L agar), and verified with the primers KIN1DvF and KIN1DvR. Primers and gRNA described above were listed in Table 3. 3 | RESULTS

| Growth phenotype of K. marxianus and ALE at high temperatures
A distinct difference between the complete medium and minimal medium for yeast growth is the form of nitrogen source. The effect of media on the wild-type (WT) K. marxianus growth at high temperatures was first evaluated using the common laboratory medium YPD and SM defined for the industrial fermentation of K. marxianus. As shown in Figure 1a, compared to 30°C, the cell density OD 600 of the WT strain incubated at 45°C for 24 h in YPD decreased by 19.89%, while it decreased by 54.8% in SM medium under the same culture conditions. These results demonstrated that the thermotolerance of K. marxianus significantly decreased in the SM medium. This phenotype seems to be a general physiological feature for K. marxianus after testing different K. marxianus strains including ATCC26548 and NBRC1777.
Accordingly, ALE of the WT strain was performed at 45°C in SM.
Among 135 clones derived from the 100-day ALE, a mutant strain, named KM234, exhibited high growth ability than the WT strain at high temperatures. This was further verified by the growth tests at gradient temperatures. As shown in Figure 1b, the growth of the KM234 strain was not significantly different from the WT strain when cultured at 30°C in SM, but at 45°C, its cell density OD 600 was about twice that of the WT strain. In particular, at 47°C, the cell density OD 600 of the KM234 strain was 4.2 times that of the WT strain. The spot assay likewise showed a significant difference in growth between the WT strain and the KM234 strain in the YPD medium at high temperatures. As shown in Figure 1c, the KM234 strain still grew well at 48°C, whereas the WT strain could hardly AI ET AL.
| 3 of 13 grow at temperatures over 44°C. These results demonstrate that the KM234 strain has significantly improved its high-temperature tolerance without obvious loss of growth ability at a moderate temperature.
Metabolites accumulated intracellularly and extracellularly may have a great effect on yeast thermotolerance. In yeast, ethanol and glycerol are two major metabolites of the overflow metabolism that occurred when the glucose uptake rate exceeds a threshold rate (Vemuri et al., 2007). Thus, the glucose, ethanol, and glycerol concentrations in media were detected at an interval of 3 h during culturing of both strains in SM at 30°C and 45°C. As shown in Figure 1d-f, there were no significant differences between the two strains in glucose consumption rates, as well as ethanol production, when grown at 30°C in SM. Growing at 45°C, the KM234 strain had a higher glucose consumption rate and produced lower than 0.8 g/L ethanol during the period of 6-8 h. In the case of the WT strain, however, >2 g/L glucose had been consumed, but no ethanol was produced, which is probably due to its poor growth at this temperature. Unlike glucose and ethanol, which are generally considered pro-aging carbon sources, glycerol contributes to protecting yeast against environmental stress and promoting life span extension (Wei et al., 2009). Compared to the WT strain, the KM234 F I G U R E 1 Growth phenotypes of the wild-type (WT) Kluyveromyces marxianus and the evolved strain KM234. (a) The cell densities optical densities at 600 nm (OD 600 ) of the WT strain cultured at 30°C and 45°C for 24 h in peptone dextrose medium (YPD) and synthetic medium (SM), respectively. (b) Growth of the WT and KM234 strains at different temperatures in SM. Both strains were grown at 30°C, 45°C, 46°C, 47°C, and 48°C in SM flasks shaking at 220 rpm for 24 h. (c) The spot assay of the WT and KM234 strains on YPD plates at different temperatures. Cell cultures grown in YPD liquid medium overnight were adjusted to an OD 600 of 1.0, and then serially diluted with sterilized water. From each dilution, 3 μL aliquots were spotted on YPD plates. The spotted plates were incubated at the indicated temperatures for 16-24 h before imaging. Determinations of the time courses of cell densities (d) and glucose consumption rates (e), and concentrations of ethanol (f) and glycerol (g) for the WT and KM234 strains grown at 30°C and 45°C in SM. Values are means with standard deviations (n = 3) and the p-values were calculated by the t-test. *p < .05; **p < .01; ***p < .001; ****p < .0001.
strain produced a higher amount of glycerol during its rapid growth period at either 30°C or 45°C. It seems that the increased glycerol production by the KM234 stain contributes to protecting it against high-temperature stress (Fu et al., 2019). This assumption is further supported by the subsequent RNA-Seq analysis.
3.2 | Global changes in the genomes and transcriptomes between the WT and KM234 strains  (Table 2), which also are upstream pathways for the synthesis of amino acid carbon skeletons (Figure 3).
The GUT1, GUT2, FPS1, and GPD2 genes involved in glycerol uptake and metabolism were upregulated in the KM234 strain when grown at 45°C. By contrast, the RHR2 gene, which encodes a glycerol-3phosphatase responsible for the terminal step in glycerol biosynthesis from glycerol-3-phosphate (Fan et al., 2005), was downregulated.
This was consistent with the above result that the KM234 strain accumulated glycerol during log-phase growth, and metabolized it as the carbon resource after glucose was depleted (Figure 1e)

| The dual effects of ammonium affecting the yeast growth at high temperatures
To further verify the connection between NH 4 + and hightemperature growth, we tested the effect of the (NH 4 ) 2 SO 4 contents in the medium on yeast growth. As shown in Figure 4a, increasing the (NH 4 ) 2 SO 4 concentration in SM enhanced the growth of both the WT and KM234 strains if it was not higher than 5 g/L. However, when the (NH 4 ) 2 SO 4 content was increased to 10 g/L, twice the normal As ammonium sulfate is the sole source of nitrogen in SM, insufficient NH 4 + may cause poor growth of yeast, which interferes with its behavior in affecting the thermotolerance of yeast. Nevertheless, the NH 4 + toxicity can be alleviated by replacing ammonium sulfate with organic nitrogen asparagine (Hess et al., 2006). Given this, we replaced ammonium sulfate in SM with 5 g/L glutamine (Gln) or asparagine (Asn). As a result, substitution with Gln or Asn in SM substantially rescued the growth defect of the WT strain at 45°C but had no apparent effect on the growth of the KM234 strain at high temperatures ( Figure 4b). This result was in agreement with our previous speculation that the KM234 strain had evolved an adaptive mechanism to avoid uptake of excess NH 4 + at high temperatures.
In yeast, NH 4 + uptake competes with K + transportation, and increasing K + concentration can inhibit NH 4 + uptake and mitigate the toxicity of high NH 4 + concentration (Hess et al., 2006). This competition may help to explain how the KM234 strain enhanced its thermotolerance.
Therefore, we varied the K + content in SM and tested the growth phenotypes of the WT and KM234 strains. At 30°C, increasing the KH 2 PO 4 concentrations from 1.5 to 12 g/L slightly decreased the growth of the WT strain, but has no apparent effect on that of the KM234 strain ( Figure 4c)

| Ammonium affects the high-temperature growth of yeast by producing ROS
Heat usually stimulates yeast to produce more harmful reactive oxygen species (ROS) that can affect its growth at high temperatures (Mejía-Barajas et al., 2017). Excess NH 4 + also induces the production of ROS in yeast (Yang et al., 2020). To investigate whether the loss of growth ability at high temperatures in SM was ascribed to the high level of ROS, ROS in both strains under different temperatures were determined by a fluorescent probe (Yang et al., 2020). As shown in

F I G U R E 4
The specific toxicity of ammonium as a nitrogen source at high temperatures. (a) Influence of ammonium in synthetic medium (SM) on the growth ability of Kluyveromyces marxianus at 30°C and 45°C. The cell densities of the wild type (WT) and the KM234 strains were recorded for 24 h in SM containing 0-10 g/L (NH 4 ) 2 SO 4 . (b) The cell densities of the WT and the KM234 strains grown in SM at 30°C and 45°C for 24 h, in which (NH 4 ) 2 SO 4 was replaced by the same concentration (g/L) of glutamine or asparagine. (c) Effects of K + concentrations on the growth abilities of the WT and KM234 at different temperatures. Cell densities of the WT and the KM234 strains grown at 30°C and 45°C were recorded for 24 h in SM containing 1.5-12 g/L KH 2 PO 4 . However, for the WT-KIN1Δ and KM234 ΔKIN1 strains, only 12 g/L of KH 2 PO 4 was assayed. *p < .05; **p < .01; ***p < .001; ****p < .0001; ns, no significant difference. (d) Effects of the KIN1 deletion on the growth of the WT, the KM234, the WT-KIN1Δ, and KM234-KIN1Δ strains at 45°C. Values were means ± SE from four independent replicates. *p < .05; ****p < .0001; ns, no significant difference.
accumulate a lower level of ROS than the WT strain when grown at a high temperature. To validate this, we quantified the ROS contents in cells for both strains. As shown in Figure 5b, there was no significant difference in the ROS contents between the two strains grown at 30°C. Although the ROS contents were substantially increased in both strains at 45°C, the WT strain accumulated a significantly higher ROS content than did the KM234 strain when grown in SM both at 30°C and 45°C. If grown in SM contained high K + content the result was the opposite. In addition, ROS contents in both strains were significantly decreased when grown at 45°C in the presence of 9 g/L KH 2 PO 4 . These results suggest that excess NH 4 + induces the production of ROS in SM. In addition to ROS from hightemperature stress, yeast needs to cope with more ROS than organic nitrogen source alone, which may exceed the handling capacity of yeast and leads to the loss of growth ability at high temperatures.
GSH is one of the major free radical scavengers that maintains the intracellular redox balance. As ROS burst is usually accompanied by an increase in GSH production, we measured the intracellular GSH contents in both strains grown at 45°C. As shown in Figure 5c, except that the GSH content in the KM234 strain was significantly higher than in the WT strain at 12 h, there was no significant difference in the intracellular GSH between the two strains. These results suggest that the GSH synthesis pathway in the KM234 strain has not been changed after ALE.

| DISCUSSION
Nitrogen provides an essential element for all forms of life. It is also a structural component for the synthesis of basic macromolecules such as nucleic acids, proteins, and other types of molecules, for example, alkaloids and thioglucosides (Landi et al., 2019). The main organic nitrogen sources in nature are urea and allantoin, amino acids, short peptides, and proteins (Lea et al., 2001). Inorganic nitrogen sources include inorganic compounds such as ammonium (NH 4 + ), nitrates (NO 3 − ), nitrites (NO 2 − ), and nitrogen gas. Nitrogen metabolism in a cell is precisely regulated and insufficient supply will lead to poor growth (Zhang et al., 2018), but excess ammonium can bring toxicity (Vidotto et al., 1993). For example, in plants, nitrogen fertilizers are rapidly converted to NH 4 + by urease, which provides nitrogen nutrients for their growth. However, excess NH 4 + leads to the accumulation of large amounts of ROS that can affect plant growth (Yang et al., 2020). Similarly, high levels of NH 4 + in human blood cause hyperammonemia (Santos et al., 2012). So, NH 4 + is a preferred source of inorganic nitrogen for yeast, and also acts as a "negative factor" implicated in the ROS production in the senescent yeast cells, as well as the regulation of the chronological lifespan (CLS) (Santos et al., 2015).
Although excess NH 4 + has been proved to be cytotoxic, little is known about the effect of NH 4 + on the thermotolerance and growth ability of yeast at high temperatures. This study demonstrates for the first time the toxicity of ammonium to K. marxianus at high temperatures when using inorganic nitrogen as the sole nitrogen source. Uptake of excess NH4 + promotes the production of ROS and adversely impacts the growth ability of K. marxianus.
How does NH 4 + affect the thermotolerance of yeast? Probably, this is mainly due to the ROS production promoted by both heat and NH 4 + when incubated at high temperatures in SM. If the total amount of intracellular ROS exceeds the antioxidant defense system, the yeast cell is in a status of oxidative stress that may drastically reduce its growth ability. At high temperatures, ROS is rapidly generated by the protons leaked from the electron transport chain (ETC) and caused damage to proteins, lipids, and DNA (Zorov et al., 2014). To respond the oxidative stress, K. marxianus need to increase the NAPDH output via upregulating the pentose phosphate pathway (PPP) and reduce the NADH output and consumption by downregulating the TCA cycle and ETC pathway when grown in YPD (Lertwattanasakul et al., 2015). In this study, however, the oxidative phosphorylation pathway that is closely related to ATP production, as well as the PPP pathway, were not significantly changed when grown in SM at high temperatures. Excessive intake of NH 4 + triggers ROS production (Yang et al., 2020), and removal of NH 4 + or replacement with amide amino acids can extend the CLS of yeast (Santos et al., 2015 (Hess et al., 2006;Shi et al., 2020). Therefore, to confirm our speculation, nitrogen resource substitution and competition assays with K + were conducted. Consistent with the previous study, replacing the nitrogen source with glutamine or asparagine restored the growth ability of the WT strain at high temperatures, and a similar result was obtained by increasing the K + concentration in SM.
However, nitrogen resource substitution and high concentration of upregulated (Graham, 2008). In the KM234 strain, glyoxylate and gluconeogenesis pathways were simultaneously upregulated during the logarithmic phase (8 h). However, as shown in Figure 1e, glucose was sufficient during this phase. Thus we supposed that upregulating gluconeogenesis in K. marxianus at high temperatures tended to produce more intermediates for other metabolic pathways, not to replenish carbon sources. This is because the glyoxylate pathway is a faster pathway for oxaloacetate production than the TCA cycle, which is conducive to synthesizing amino acids of the aspartate family, such as aspartic acid, asparagine, threonine, methionine, and lysine ( Figure 3). To assimilate inorganic nitrogen, yeast also needs to constantly synthesize carbon skeletons that are mainly from the intermediates of sugar metabolism (Maslanka & Zadrag-Tecza, 2020).
Therefore, it is reasonable that PCK1, a key enzyme for sugar gluconeogenesis, is the most significantly upregulated gene in the KM234 strain at high temperatures.
Yeast Kin1 is a serine/threonine kinase located on the cell plasma membrane surface (Lamb et al., 1991;Tibbetts et al., 1994). Current studies have not yet mentioned that it participates in NH 4 + uptake, K + transport, and high-temperature tolerance. In the KM234 strain, the KIN1 gene contains an SNP that an acidic amino acid glutamate is changed to nonpolar valine. Knockout of the KIN1 gene reduced the growth ability of K. marxianus at high temperatures. More importantly, the loss of the KIN1 gene has a higher impact on the growth of the WT strain than the KM234 strain, and the high ratio of K + /NH 4 + cannot rescue the growth defect of the WT-KIN1Δ strain. As described above, to cope with the hightemperature stress, the KM234 strain decreased its NH 4 + uptake by downregulation of NH 4 + transport genes and increasing the synthesis of the amino acid carbon backbone. In WT K. marxianus, ammonium toxicity can be mitigated by the replacement of ammonium sulfate with amide amino acids or increasing the K + content in SM, while the latter approach relies on the gene KIN1, which may act similarly to CIPK23 in Arabidopsis (Shi et al., 2020). However, the underlying mechanism for the regulation of the NH 4 + transport genes including MEP2 and MEP3 by KIN1 needs to be investigated in a further study.

| CONCLUSIONS
Ammonium regulates the growth of K. marxianus in the SM, and uptake of excess NH4 + promotes the production of ROS and decreases the growth ability of K. marxianus at high temperatures.
A high ratio of K + /NH 4 + rescues the growth defect of the WT strain at high temperatures, and the serine/threonine kinase Kin1 may participate in this regulation. An increase in K + content can alleviate the ammonium toxicity and significantly increase the hightemperature growth ability of K. marxianus in the SM. Our result provides a practical way for K. marxianus in industrial fermentations at high temperatures.