Transcription Analysis of the Acid Tolerance Mechanism of <i>Pichia kudriavzevii</i> NBRC1279 and NBRC1664

Simultaneous saccharification and fermentation (SSF) has been investigated for the efficient production of ethanol because it has several advantages such as simplifying the manufacturing process, operating easily, and reducing energy input. Previously, using lignocellulosic biomass as source materials, we succeeded in producing ethanol by SSF with Pichia kudriavzevii NBRC1279 and NBRC1664. However, various acids that fermentation inhibitors are also produced by the hydrolysis of lignocellulosic biomass, and the extent to which these acids affect the growth and ethanol productivity of the two strains has not yet been investigated. In this study, to better understand the acid tolerance mechanism of the two strains, a spot assay, growth experiment, and transcriptome analysis were carried out using Saccharomyces cerevisiae BY4742 as a control. When the three strains were cultured in SCD medium containing 15 mM formic acid, 35 mM sulfuric acid, 60 mM hydrochloric acid, 100 mM acetic acid, or 550 mM lactic acid, only P. kudriavzevii NBRC1664 could grow well under all conditions, and it showed the fastest growth rates. The transcriptome analysis showed that “MAPK signaling pathway-yeast” was significantly enriched in P. kudriavzevii NBRC1664 cultured with 60 mM hydrochloric acid, and most genes involved in the high osmolarity glycerol (HOG) pathway were up-regulated. Therefore, the up-regulation of the HOG pathway may be important for adapting to acid stress in P. kudriavzevii. Moreover, the log2-transformed fold change value in the expression level of Gpd1 was 1.3-fold higher in P. kudriavzevii NBRC1664 than in P. kudriavzevii NBRC1279, indicating that high Gpd1 expression may be accountable for the higher acid tolerance of P. kudriavzevii NBRC1664. The transcriptome analysis performed in this study provides preliminary knowledge of the molecular mechanism of acid stress tolerance in P. kudriavzevii. Our data may be useful for future studies on methods to improve the tolerance of P. kudriavzevii to acids produced from lignocellulose hydrolysis

Fermentation of xylose causes inefficient metabolic state due to carbon/energy starvation and reduced glycolytic flux in recombinant industrial Saccharomyces cerevisiae.

In the present study, comprehensive, quantitative metabolome analysis was carried out on the recombinant glucose/xylose-cofermenting S. cerevisiae strain MA-R4 during fermentation with different carbon sources, including glucose, xylose, or glucose/xylose mixtures. Capillary electrophoresis time-of-flight mass spectrometry was used to determine the intracellular pools of metabolites from the central carbon pathways, energy metabolism pathways, and the levels of twenty amino acids. When xylose instead of glucose was metabolized by MA-R4, glycolytic metabolites including 3- phosphoglycerate, 2- phosphoglycerate, phosphoenolpyruvate, and pyruvate were dramatically reduced, while conversely, most pentose phosphate pathway metabolites such as sedoheptulose 7- phosphate and ribulose 5-phosphate were greatly increased. These results suggest that the low metabolic activity of glycolysis and the pool of pentose phosphate pathway intermediates are potential limiting factors in xylose utilization. It was further demonstrated that during xylose fermentation, about half of the twenty amino acids declined, and the adenylate/guanylate energy charge was impacted due to markedly decreased adenosine triphosphate/adenosine monophosphate and guanosine triphosphate/guanosine monophosphate ratios, implying that the fermentation of xylose leads to an inefficient metabolic state where the biosynthetic capabilities and energy balance are severely impaired. In addition, fermentation with xylose alone drastically increased the level of citrate in the tricarboxylic acid cycle and increased the aromatic amino acids tryptophan and tyrosine, strongly supporting the view that carbon starvation was induced. Interestingly, fermentation with xylose alone also increased the synthesis of the polyamine spermidine and its precursor S-adenosylmethionine. Thus, differences in carbon substrates, including glucose and xylose in the fermentation medium, strongly influenced the dynamic metabolism of MA-R4. These results provide a metabolic explanation for the low ethanol productivity on xylose compared to glucose

Identification and Characterization of a Novel Issatchenkia orientalis GPI-Anchored Protein, IoGas1, Required for Resistance to Low pH and Salt Stress.

The use of yeasts tolerant to acid (low pH) and salt stress is of industrial importance for several bioproduction processes. To identify new candidate genes having potential roles in low-pH tolerance, we screened an expression genomic DNA library of a multiple-stress-tolerant yeast, Issatchenkia orientalis (Pichia kudriavzevii), for clones that allowed Saccharomyces cerevisiae cells to grow under highly acidic conditions (pH 2.0). A genomic DNA clone containing two putative open reading frames was obtained, of which the putative protein-coding gene comprising 1629 bp was retransformed into the host. This transformant grew significantly at pH 2.0, and at pH 2.5 in the presence of 7.5% Na2SO4. The predicted amino acid sequence of this new gene, named I. orientalis GAS1 (IoGAS1), was 60% identical to the S. cerevisiae Gas1 protein, a glycosylphosphatidylinositol-anchored protein essential for maintaining cell wall integrity, and 58-59% identical to Candida albicans Phr1 and Phr2, pH-responsive proteins implicated in cell wall assembly and virulence. Northern hybridization analyses indicated that, as for the C. albicans homologs, IoGAS1 expression was pH-dependent, with expression increasing with decreasing pH (from 4.0 to 2.0) of the medium. These results suggest that IoGAS1 represents a novel pH-regulated system required for the adaptation of I. orientalis to environments of diverse pH. Heterologous expression of IoGAS1 complemented the growth and morphological defects of a S. cerevisiae gas1Δ mutant, demonstrating that IoGAS1 and the corresponding S. cerevisiae gene play similar roles in cell wall biosynthesis. Site-directed mutagenesis experiments revealed that two conserved glutamate residues (E161 and E262) in the IoGas1 protein play a crucial role in yeast morphogenesis and tolerance to low pH and salt stress. Furthermore, overexpression of IoGAS1 in S. cerevisiae remarkably improved the ethanol fermentation ability at pH 2.5, and at pH 2.0 in the presence of salt (5% Na2SO4), compared to that of a reference strain. Our results strongly suggest that constitutive expression of the IoGAS1 gene in S. cerevisiae could be advantageous for several fermentation processes under these stress conditions

Comparison of the metabolites in central carbon metabolism (including glycolysis, the pentose phosphate pathway, and tricarboxylic acid cycle pathway) of MA-R4 during the fermentation of glucose alone (G1 and G2 stages), mixed sugars of glucose and xylose (M1, M2, M3, and M4 stages), and xylose alone (X1 and X2 stages).

<p>Each bar represents the mean amounts of the metabolites (pmol/OD600⋅mL) in each sampling stage (G1, G2, M1, M2, M3, M4, X1, and X2). These eight sampling points during the fermentation experiments are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069005#pone-0069005-g001" target="_blank">Figure 1</a>. Error bars represents the standard deviations from three independent experiments. The arrowheads in the figure represent the direction of enzymatic reactions. Abbreviations: GLC, glucose; XYL, xylose; XYLI, xylitol; XYLU, xylulose; X5P, xylulose 5-phosphate; G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; FBP, fructose 1,6−bisphosphate; GA3P, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; G3P, glycerol 3-phosphate; GLY, glycerol; 6PG, 6-phosphogluconate; RL5P, ribulose 5-phosphate; R5P, ribose 5-phosphate; S7P, sedoheptulose 7- phosphate; E4P, erythrose 4-phosphate; 3-PGA, 3- phosphoglycerate; 2-PGA, 2- phosphoglycerate; PEP, phosphoenolpyruvate; PYR, pyruvate; LAC, lactate; ACD, acetaldehyde; ACE, acetate; ETOH, ethanol; Ac-CoA, acetyl coenzyme A; CoASH, coenzyme A; CIT, citrate; ACO, aconitate; ICT, isocitrate; 2-OG, 2-oxoglutarate; Suc-CoA, succinyl coenzyme A; SUC, succinate; FUM, fumarate; MAL, malate; OAA, oxaloacetate.</p

Summary of 48-h fermentations in different media by <i>S. cerevisiae</i> strain MA-R4.

<p>Values are the averages of three independent experiments ± standard deviation.</p><p>ND, not detectable.</p