Toxicants improve glycerol production in the fermentation of undetoxified hydrolysate by Candida glycerinogenes.

OBJECTIVES: Toxicants inhibit microbial fermentation and reduce product titres. This work investigated the glycerol production characteristics of Candida glycerinogenes in highly toxic unwashed undetoxified hydrolysate and provided new ideas for high glycerol production from hydrolysates. RESULTS: The unwashed hydrolysate contains higher concentrations of toxicants, such as furfural, acetic acid, phenols and NaCl than the washed alkali-treated bagasse hydrolysate. C. glycerinogenes fermented unwashed undetoxified hydrolysate yielded 36.1 g/L glycerol, 15.8% higher than the washed hydrolysate, suggesting that the toxicants stimulated glycerol synthesis. qRT-PCR analysis showed that toxicants of unwashed undetoxified hydrolysates greatly up-regulated the transcript levels of the genes GPD1, HXT4 and MSN4 et al. Overexpressing the above genes increased glycerol production by 27.9% to 46.1 g/L. And it was further increased by 8.8% to 50.1 g/L in a 5 L bioreactor. CONCLUSIONS: This result proves that toxicants in lignocellulosic hydrolysates can increase the titre of microbial glycerol production.

Spatiotemporal Regulation and Transport Engineering for Sustainable Production of Geraniol in Candida glycerinogenes.

Geraniol is an attractive natural monoterpene with significant industrial and commercial value in the fields of pharmaceuticals, condiments, cosmetics, and bioenergy. The biosynthesis of monoterpenes suffers from the availability of key intermediates and enzyme-to-substrate accessibility. Here, we addressed these challenges in Candida glycerinogenes by a plasma membrane-anchoring strategy and achieved sustainable biosynthesis of geraniol using bagasse hydrolysate as substrate. On this basis, a remarkable 2.4-fold improvement in geraniol titer was achieved by combining spatial and temporal modulation strategies. In addition, enhanced geraniol transport and modulation of membrane lipid-associated metabolism effectively promoted the exocytosis of toxic monoterpenes, significantly improved the resistance of the engineered strain to monoterpenes and improved the growth of the strains, resulting in geraniol yield up to 1207.4 mg L-1 at shake flask level. Finally, 1835.2 mg L-1 geraniol was obtained in a 5 L bioreactor using undetoxified bagasse hydrolysate. Overall, our study has provided valuable insights into the plasma membrane engineering of C. glycerinogenes for the sustainable and green production of valuable compounds.

Gene Editing of Candida glycerinogenes by Designed Toxin-Antitoxin Cassette.

Candida glycerinogenes is an industrial yeast with excellent multistress resistance. However, due to the diploid genome and the lack of meiosis and screening markers, its molecular genetic operation is limited. Here, a gene editing system using the toxin-antitoxin pair relBE from the type II toxin-antitoxin system in Escherichia coli as a screening marker was constructed. The RelBE complex can specifically and effectively regulate cell growth and arrest through a conditionally controlled toxin RelE switch, thereby achieving the selection of positive recombinants. The constructed editing system achieved precise gene deletion, replacement, insertion, and gene episomal expression in C. glycerinogenes. Compared with the traditional amino acid deficiency complementation editing system, this editing system produced higher biomass and the gene deletion efficiency was increased by 3.5 times. Using this system, the production of 2-phenylethanol by C. glycerinogenes was increased by 11.5-13.5% through metabolic engineering and tolerance engineering strategies. These results suggest that the stable gene editing system based on toxin-antitoxin pairs can be used for gene editing of C. glycerinogenes to modify metabolic pathways and promote industrial applications. Therefore, the constructed gene editing system is expected to provide a promising strategy for polyploid industrial microorganisms lacking gene manipulation methods.

Glycerol Production from Undetoxified Lignocellulose Hydrolysate by a Multiresistant Engineered Candida glycerinogenes.

Glycerol is an important platform compound with multidisciplinary applications, and glycerol production using low-cost sugar cane bagasse hydrolysate is promising. Candida glycerinogenes, an industrial yeast strain known for its high glycerol production capability, has been found to thrive in bagasse hydrolysate obtained through a simple treatment without detoxification. The engineered C. glycerinogenes exhibited significant resistance to furfural, acetic acid, and 3,4-dimethylbenzaldehyde within undetoxified hydrolysates. To further enhance glycerol production, genetic modifications were made to Candida glycerinogenes to enhance the utilization of xylose. Fermentation of undetoxified bagasse hydrolysate by CgS45 resulted in a glycerol titer of 40.3 g/L and a yield of 40.4%. This process required only 1 kg of bagasse to produce 93.5 g of glycerol. This is the first report of glycerol production using lignocellulose, which presents a new way for environmentally friendly industrial production of glycerol.

Regulatory mechanisms and cell membrane properties of Candida glycerinogenes differ under 2-phenylethanol addition or fermentation conditions.

The biosynthesis of 2-phenylethanol (2-PE) at high yields and titers is often limited by its toxicity. In this study, we describe the molecular mechanisms of 2-PE tolerance in the multi-stress tolerant industrial yeast, Candida glycerinogenes. They were different under 2-PE addition or fermentation conditions. After extracellular addition of 2-PE, C. glycerinogenes cells became rounder and bigger, which reduced specific surface area. However, during 2-PE fermentation C. glycerinogenes cells were smaller, which increased specific surface area. Other differences in the tolerance mechanisms were studied by analyzing the composition and molecular parameters of the cell membrane. Extracellular 2-PE stress resulted in down-regulation of transcriptional expression of unsaturated fatty acid synthesis genes. This raised the proportion of saturated fatty acids in the cell membrane, which increased rigidity of the cell membrane and reduced 2-PE entry to the cell. However, intracellular 2-PE stress resulted in up-regulation of transcriptional expression of unsaturated fatty acid synthesis genes, and increased the proportion of unsaturated fatty acids in the cell membrane; this in turn enhanced flexibility of the cell membrane which accelerated efflux of 2-PE. These contrasting mechanisms are mediated by transcriptional factors Hog1 and Swi5. Under 2-PE addition, C. glycerinogenes activated Hog1 and repressed Swi5 to upregulate erg5 and erg4 expression, which increased cell membrane rigidity and resisted 2-PE import. During 2-PE fermentation, C. glycerinogenes activated Hog1 and repressed Swi5 to upregulate 2-PE transporter proteins cdr1 and Acyl-CoA desaturase 1 ole1 to increase 2-PE export, thus reducing 2-PE intracellular toxicity. The results provide new insights into 2-PE tolerance mechanisms at the cell membrane level and suggest a novel strategy to improve 2-PE production by engineering anti-stress genes.

Effect and application of proline metabolism-related gene CgMCUR1 on stress tolerance of Candida glycerinogenes and Saccharomyces cerevisiae.

AIMS: To investigate the effect of CgMCUR1 on the phenotype of Candida glycerinogenes and Saccharomyces cerevisiae. METHODS AND RESULTS: Inhibition of CgMCUR1 expression reduced acetate, H2O2, and high temperature tolerance of C. glycerinogenes. Expression of CgMCUR1 resulted in better acetic acid, H2O2, and high temperature tolerance in recombinant S. cerevisiae. Meanwhile, CgMCUR1 was able to enhance intracellular proline accumulation. The qRT-PCR analysis revealed that overexpression of CgMCUR1 affected proline metabolism in recombinant S. cerevisiae. The overexpression strain also showed reduced levels of cellular lipid peroxidation and an altered ratio of saturated fatty acid (SFA) to unsaturated fatty acid (UFA) in the cell membrane. The ethanol production of recombinant S. cerevisiae at high temperature was 30.9 g l-1, obtaining an increase of 12%, and the conversion rate was increased by 12%. In the undetoxified cellulose hydrolysate, the ethanol yield was 14.7 g l-1 at 30 h with an improvement of 18.5%, and the conversion rate was increased by 15.3%. CONCLUSIONS: Overexpression of CgMCUR1 rendered the acetic acid, H2O2, and high temperature tolerant of recombinant S. cerevisiae and enhanced the ethanol fermentation performance of recombinant S. cerevisiae under high temperature stress and in undetoxified cellulose hydrolysate by improving intracellular proline accumulation and by changing cellular physiological metabolism.

Metabolic Engineering of Candida glycerinogenes for Sustainable Production of Geraniol.

Geraniol is a class of natural products that are widely used in the aroma industry due to their unique aroma. Here, to achieve the synthesis of geraniol and alleviate the intense competition from the yeast ergosterol pathway, a transcription factor-mediated ergosterol feedback system was developed in this study to autonomously regulate ergosterol metabolism and redirect carbon flux to geraniol synthesis. In addition, the modification of ergosterol-responsive promoters, the optimization of transcription factor expression intensity, and stepwise metabolic engineering resulted in a geraniol titer of 531.7 mg L-1. For sustainable production of geraniol, we constructed a xylose assimilation pathway in Candida glycerinogenes (C. glycerinogenes). Then, the xylose metabolic capacity was ameliorated and the growth of the engineered strain was rescued by activating the pentose phosphate (PP) pathway. Finally, we obtained 1091.6, 862.4, and 921.8 mg L-1 of geraniol in a 5 L bioreactor by using pure glucose, simulated wheat straw hydrolysates, and simulated sugarcane bagasse hydrolysates, with yields of 47.5, 57.9, and 59.1 mg g-1 DCW, respectively. Our study demonstrated that C. glycerinogenes has the potential to produce geraniol from lignocellulosic biomass, providing a powerful tool for the sustainable synthesis of other valuable monoterpenes.

Balancing Pyruvate Node Based on a Dual-Layered Dynamic Regulation System to Improve the Biosynthesis of Caffeic Acid in Candida glycerinogenes.

Caffeic acid is a phenolic acid compound widely applied in the food and pharmaceutical fields. Currently, one of the reasons for the low yield of caffeic acid biosynthesis is that the carbon flow enters mainly into the TCA cycle via pyruvate, which leads to low concentrations of erythrose 4-phosphate (E4P) and phosphoenolpyruvate (PEP), the precursors of caffeic acid synthesis. Here, we developed a growth-coupled dual-layered dynamic regulation system. This system controls intracellular pyruvate supply in real time by responding to intracellular pyruvate and p-coumaric acid concentrations, autonomously coordinates pathway gene expression, and redirects carbon metabolism to balance cell growth and caffeic acid synthesis. Finally, our constructed engineered strain based on the dual-layered dynamic regulation system achieved a caffeic acid titer of 559.7 mg/L in a 5 L bioreactor. Thus, this study demonstrated the efficiency and potential of this system in boosting the yield of aromatic compounds.

Candida glycerinogenes-Promoted α-Pinene and Squalene Co-production Strategy Based on α-Pinene Stress.

α-Pinene is a naturally occurring monoterpene, which is widely used in fragrances, cosmetics, and foods. Due to the high cellular toxicity of α-pinene, this work considered the application of Candida glycerinogenes, an effective industrial strain with high resistance, in α-pinene synthesis. It was found that α-pinene-induced stress resulted in an intracellular accumulation of reactive oxygen species with an increased formation of squalene as a cytoprotective compound. As squalene is a downstream product in the mevalonate (MVA) pathway for α-pinene synthesis, a strategy based on the promotion of α-pinene and squalene co-production under α-pinene stress is proposed. By introducing the α-pinene synthesis pathway and enhancing the MVA pathway, the production of both α-pinene and squalene is increased. We have demonstrated that intracellular synthesis of α-pinene is effective in promoting squalene synthesis. The generation of intercellular reactive oxygen that accompanies α-pinene synthesis promotes squalene synthesis with a resultant cellular protection and upregulation of MVA pathway genes that facilitate α-pinene production. In addition, we have overexpressed phosphatase and introduced NPP as a substrate to synthesize α-pinene, where co-dependent fermentation yielded 208 mg/L squalene and 12.8 mg/L α-pinene. This work establishes a viable strategy to promote terpene-co-dependent fermentation based on stress.

Construction of a novel plasmid for an industrial yeast Candida glycerinogenes by dual-autonomously replicating sequence strategy.

Due to the lack of available episomal plasmid, the improvement of many industrial strains, especially exogenous gene expression, is severely restricted. The failure of autonomous replication or low copy number of episomal plasmids is the main reason for the failure of many episomal plasmids construction. In this paper, Candida glycerinogenes, an industrial strain lacking episomal plasmids, was employed as the topic. A series of GFP-based plasmids containing autonomously replicating sequence (ARS) from different strain sources were constructed and analyzed for performance, and it was found that only the panARS from Kluyveromyces lactis compared with other nine low capacity ARSs proved to have the best performance and could be used to construct episomal plasmid. Further, the dual-ARS strategy was used to optimize the episomal plasmid, and the results indicated that only the dual-ARS plasmid +PPARS2 with double different ARSs, not the dual-ARS plasmid +panARS with double same ARSs, showed an improvement in all properties, with an increase in transformation efficiency of about 36% and a synchronous trend of fluorescence intensity and copy number, both by about 40%. In addition, constructed episomal plasmids were used to express the exogenous gene CrGES, and the fact that geraniol was found proved the versatility of the plasmids. The successful construction of episomal plasmids will also substantially facilitate genetic engineering research and industrial use of C. glycerinogenes in the future, as well as providing a feasible approach to create episomal plasmids for industrial strains.

Tuning Geraniol Biosynthesis via a Novel Decane-Responsive Promoter in Candida glycerinogenes.

Geraniol is a rose-scented monoterpene with significant commercial and industrial value in medicine, condiments, cosmetics, and bioenergy. Here, we first targeted geraniol as a reporter metabolite and explored the suitability and potential of Candida glycerinogenes as a heterologous host for monoterpenoid production. Subsequently, dual-pathway engineering was employed to improve the production of geraniol with a geraniol titer of 858.4 mg/L. We then applied a synthetic hybrid promoter approach to develop a decane-responsive hybrid promoter based on the native promoter PGAP derived from C. glycerinogenes itself. The hybrid promoter was able to be induced by n-decane with 3.6 times higher transcriptional intensity than the natural promoter PGAP. In particular, the hybrid promoter effectively reduces the conflict between cell growth and product formation in the production of geraniol. Ultimately, 1194.6 mg/L geraniol was obtained at the shake flask level. The strong and tunable decane-responsive hybrid promoter developed in this study provides an important tool for fine regulation of toxic terpenoid production in cells.

Production of Caffeic Acid with Co-fermentation of Xylose and Glucose by Multi-modular Engineering in Candida glycerinogenes.

Caffeic acid (CA), a natural phenolic compound, has important medicinal value and market potential. In this study, we report a metabolic engineering strategy for the biosynthesis of CA in Candida glycerinogenes using xylose and glucose. The availability of precursors was increased by optimization of the shikimate (SA) pathway and the aromatic amino acid pathway. Subsequently, the carbon flux into the SA pathway was maximized by introducing a xylose metabolic pathway and optimizing the xylose assimilation pathway. Eventually, a high yielding strain CG19 was obtained, which reached a yield of 4.61 mg/g CA from mixed sugar, which was 1.2-fold higher than that of glucose. The CA titer in the 5 L bioreactor reached 431.45 mg/L with a yield of 8.63 mg/g of mixed sugar. These promising results demonstrate the great advantages of mixed sugar over glucose for high-yield production of CA. This is the first report to produce CA in C. glycerinogenes with xylose and glucose as carbon sources, which developed a promising strategy for the efficient production of high-value aromatic compounds.

Identification of key residues for efficient glucose transport by the hexose transporter CgHxt4 in high sugar fermentation yeast Candida glycerinogenes.

Efficient hexose transporters are essential for the development of industrial yeast strains with high fermentation performance. We previously identified a hexose transporter, CgHxt4, with excellent sugar uptake performance at ultra-high glucose concentrations (200 g/L) in the high sugar fermenting yeast C. glycerinogenes. To understand the working mechanism of this transporter, we constructed 87 mutants and examined their glucose uptake performance. The results revealed that five residues (N321, N322, F325, G426, and P427) are essential for the efficient glucose transport of CgHxt4. Subsequently, we focused our analysis on the roles of N321 and P427. Specifically, N321 and P427 are likely to play a role in glucose coordination and conformational flexibility, respectively. Our results help to expand the application potential of this transporter and provide insights into the working mechanism of yeast hexose transporter. KEY POINTS: • Five residues, transmembrane segments 7 and 10, were found to be essential for CgHxt4. • N321 and P427 are likely to play a role in glucose coordination and conformational flexibility, respectively. • Chimeric CgHxt5.4TM7 significantly enhanced the performance of CgHxt5.

Genetic engineering of an industrial yeast Candida glycerinogenes for efficient production of 2-phenylethanol.

Microbial cell factories offer an economic approach for synthesizing "natural'" aromatic flavor compounds. During their fermentation process, the inefficient synthesis pathway and product cytotoxicity are the major barriers to the high-level production. This study combined metabolic engineering and tolerance engineering strategies to maximize the valuable rose-smell 2-phenylethanol (2-PE) production in Candida glycerinogenes, a GRAS diploid industrial yeast. Firstly, 2-PE metabolic networks involved in Ehrlich pathway were stepwise rewired using metabolic engineering, including the following: (1) overexpressing L-phenylalanine permease Aap9 enhanced precursor uptake; (2) overexpressing enzymes (aminotransferase Aro9 and decarboxylase Aro10) of Ehrlich pathway increased catalytic efficiency; and (3) disrupting the formation of by-product phenylacetate catalyzed by Ald2 and Ald3 maximized the metabolic flux toward 2-PE. Then, tolerance engineering was applied by overexpression of a stress-inducible gene SLC1 in the metabolically engineered strain to further enhance 2-PE production. Combining these two approaches finally resulted in 5.0 g/L 2-PE in shake flasks, with productivity reaching 0.21 g/L/h, which were increased by 38.9% and 177% compared with those of the non-engineered strain, respectively. The 2-PE yield of this engineered strain was 0.71 g/g L-phenylalanine, corresponding to 95.9% of theoretical yield. This study provides a reference to efficiently engineering of microbial cell factories for other valuable aromatic compounds. KEY POINTS: • Metabolic engineering improved 2-PE biosynthesis. • Tolerance engineering alleviated product inhibition, contributing to 2-PE production. • The best strain produced 5.0 g/L 2-PE with 0.959 mol/mol yield and high productivity.

Transcription factor Hap5 induces gsh2 expression to enhance 2-phenylethanol tolerance and production in an industrial yeast Candida glycerinogenes.

2-Phenylethanol (2-PE) is an important flavor compound but also impairs cell growth severely, which in turn blocks its bioproduction. However, the molecular mechanism of 2-PE tolerance is unclear. In this study, a superb 2-PE stress-tolerant and producing yeast, Candida glycerinogenes, was selected to uncover the underlying mechanism of 2-PE tolerance. We discovered that Hap5 is an essential regulator to 2-PE resistance, and its induction by 2-PE stress occurs at the post-transcriptional level, rather than at the transcriptional level. Under 2-PE stress, Hap5 is activated and imported into the nucleus rapidly. Then, the nuclear Hap5 binds to the glutathione synthetase (gsh2) promoter via CCAAT box, to induce the expression of gsh2 gene. The increased gsh2 expression contributes to enhanced cellular glutathione content, and consequently alleviates ROS accumulation, lipid peroxidation, and cell membrane damage caused by 2-PE toxicity. Specifically, increasing the expression of gsh2 is effective in improving not just 2-PE tolerance (33.7% higher biomass under 29 mM 2-PE), but also 2-PE production (16.2% higher). This study extends our knowledge of 2-PE tolerance mechanism and also provides a promising strategy to improve 2-PE production.

Selection and application of novel high temperature inducible promoters in the tolerant yeast Candida glycerinogenes.

High temperature fermentation can substantially reduce the contaminations and condensation costs. Candida glycerinogenes was more resistant to high temperature than Saccharomyces cerevisiae. Quantitative real-time PCR results showed that 7 of 33 candidates served as potential high temperature inducible promoters in C. glycerinogenes. Fluorescence analysis indicated that PCgcwp1 showed the highest activity at 42°C. PCgaac and PCgpot1 showed medium-high expression, while PCghsp12, PCgatp1, PCgino1 and PCgscl were modest or weaker expression. Above seven promoters were further used to control the expression of xylose reductase gene from Neurospora crassa in C. glycerinogenes. Compared with 30°C, the xylitol yields of the seven recombinant strains were all improved at 42°C, and PCgcwp1 showed the highest xylitol production which was increased by 204% and reached 15.4 g/L. These results showed a potential to high temperature fermentation on the stress tolerant C. glycerinogenes and some of novel high temperature inducible promoters.

Establishment of a transient CRISPR-Cas9 genome editing system in Candida glycerinogenes for co-production of ethanol and xylonic acid.

Candida glycerinogenes, an industrial yeast with excellent multi-stress tolerance, has been applied to glycerol production for decades. However, its genetic manipulation was limited by the absence of meiosis, the diploid genome, and the lack of molecular tools. We described here the implementation of a transient CRISPR-Cas9 system for efficient genome editing in C. glycerinogenes. By targeting the counterselectable marker genes (TRP1, URA3), single and double gene knock-outs were achieved and the auxotroph obtained can be used as a background for targeting other gene (HOG1) at a mutation efficiency of 80%. Further, a xylonic acid producing C. glycerinogenes strain was constructed by knock-in of the xylose dehydrogenase gene, which produced up to 28 g/L ethanol and 9 g/L xylonic acid simultaneously from simulated lignocellulosic hydrolysate (contained 70 g/L glucose and 24 g/L xylose). These results indicated that the CRSIPR-Cas9 system developed here can facilitate the study of gene functions and metabolic pathways in C. glycerinogenes.

Identification and application of novel low pH-inducible promoters for lactic acid production in the tolerant yeast Candida glycerinogenes.

Bioproduction of organic acids under low pH condition without adding inducer and neutralizer is an economical craft to decrease downstream cost. Candida glycerinogenes had higher tolerances to low pH and lactic acid than Saccharomyces cerevisiae. QRT-PCR analysis showed that four of fifteen candidates functioned as potentially acid-inducible promoters in C. glycerinogenes. In particular, PCggmt1 showed the strongest induction ability at pH 2.5. Fluorescence analysis indicated that the induction ability of PCggmt1 gradually increased as the pH decreased. In addition, PCggmt1 had the binding sites of Msn2p/4p, Azf1p, and Nrg1p. PCggmt1 was further used to control the expression of lactate dehydrogenase gene from Rhizopus oryzae in C. glycerinogenes. Compared with pH 5.5, the specific activity of lactate dehydrogenase and lactic acid titer at pH 2.5 were increased by 229% and 218%, reaching 13.8 mU/mg and 12.3 g/L, respectively. These results presented here showed a potential to produce organic acid economically at low pH by the stress tolerant C. glycerinogenes and the novel low-pH inducible promoter PCggmt1.

Ethanol fermentation from non-detoxified lignocellulose hydrolysate by a multi-stress tolerant yeast Candida glycerinogenes mutant.

The aim of this work was to study ethanol fermentation properties of the robust mutant Candida glycerinogenes UG21 from non-detoxified lignocellulose hydrolysate. C. glycerinogenes UG21 with high tolerance to elevated temperature, acetic acid, and furfural was obtained and applied in lignocellulose-based ethanol production. C. glycerinogenes UG21 exhibited highly-efficient degradation ability to furfural. High levels of acetic acid and furfural inhibited cell growths and ethanol production of Saccharomyces cerevisiae ZWA46 and industrial Angel yeast but had a slight impact on biomass and ethanol titer of C. glycerinogenes UG21. Using non-detoxified sugarcane bagasse hydrolysate, C. glycerinogenes UG21 reached 1.24 g/L/h of ethanol productivity at 40 °C but ethanol production of S. cerevisiae ZWA46 and Angel yeast was inhibited. Further, C. glycerinogenes UG-21 exhibited 2.42-fold and 1.58-fold higher productivity than S. cerevisiae ZWA46 and Angel yeast under low-toxicity hydrolysate. Therefore, C. glycerinogenes UG-21 could be an excellent candidate for low-cost lignocelluloses ethanol production.

Gene expression profiles of Candida glycerinogenes under combined heat and high-glucose stresses.

Low cell tolerance is a basic issue in high-glucose fermentation under high temperature to economically obtain high product titer. Candida glycerinogenes, an industrial yeast, has excellent tolerance to the combined heat and high-glucose stress than Saccharomycescerevisiae. The potential mechanism responsible for the high tolerance was illustrated here. The transcription of the potential stress-responsive genes in two strains were varied under single stress (heat or high-glucose), especially the ribosome-related genes. Unlike S. cerevisiae, C. glycerinogenes up-regulated 17 genes, including most of the single stress responsive genes, and genes Avt1 and Pfk1 under the combined stress, indicating a more systematic stress-responsive system in C. glycerinogenes. Further down-regulating the 17 potential key responsive genes indicated that genes Dip5, Gpd1, Pfk1, Hxt4, Hxt6, and Ino4 are important for cell tolerance to the combined stress. Furthermore, most of the ribosomal function related genes, such as Mrt4, Nug1, Nop53, Rpa190, Rex4, and Nsr1, play important role in cell tolerance. Therefore, the wider responsive gene spectrum and the activated expression of ribosomal function related genes might be key and prerequisite factors for the excellent tolerance to the combined stress of C. glycerinogenes.