Positive selection of efficient ethanol producers from xylose at 45 °C in the yeast Ogataea polymorpha

Abstract

This research presents a method for the positive selection of mutants with improved xylose and L-arabinose fermentation in the thermotolerant, naturally xylose-utilizing yeast Ogataea polymorpha which is based on isolation of the mutants growing on L-arabinose as sole carbon and energy source. Whole-genome sequencing of the most efficient xylose-fermenting strain, A107, revealed mutations in the API1 and IRA1 genes, which are homologous to bacterial arabinose-5-phosphate isomerase and the Ras-GTPase activating domain in Saccharomyces cerevisiae, respectively. Disruption of the IRA1 gene increased ethanol production during the fermentation of xylose and L-arabinose in O. polymorpha at 45 °C. Overexpression of the API1 gene specifically enhanced L-arabinose fermentation without affecting xylose fermentation. The most productive mutant strain accumulated 20.91 g/L of ethanol in a xylose-containing medium at 45 °C, exceeding the ethanol accumulation level of the wild-type strain (0.40 g/L) by over 50 times. This strain holds potential for application in simultaneous saccharification and fermentation (SSF) processes.

Keywords: API1 gene; IRA1 gene; Alcoholic fermentation; L-arabinose; Reverse genetics.

Fig. 1

Growth, ethanol production, and selection of O. polymorpha mutants on different carbon sources. (a) Plating O. polymorpha UV-irradiated cells on a medium containing 15% L-arabinose. (b) Biomass accumulation of BEP/cat8Δ/DAS1/TAL2 and BEP/cat8Δ/DAS1/TAL2/107 mutant in YNB medium supplemented with 2% xylose, 2% L-arabinose, 2% glucose, or 50% bagasse hydrolysate during a growth test at 37 °C. (c) Ethanol production (g/L) by the parental O. polymorpha BEP/cat8Δ/DAS1/TAL2 strain and one of the best UV-mutants BEP/cat8Δ/DAS1/TAL2/107 during high-temperature alcoholic fermentation (45 °C) with 10% xylose, 10% L-arabinose, 10% glucose, or 50% bagasse hydrolysate. Error bars represent the standard error of the mean (SE), n = 3. In some cases, error bars are not visible due to the small magnitude of the error.

Fig. 2

Growth, ethanol production, and selection of O. polymorpha mutants on different carbon sources. (a) Stepwise selection of BEP/cat8Δ/DAS1/TAL2/107 mutants resistant to 2-DG and BrPA on a medium containing 15% L-arabinose. (b) Biomass accumulation of BEP/cat8Δ/DAS1/TAL2/107, BEP/cat8Δ/DAS1/TAL2/107/2-DG and BEP/cat8Δ/DAS1/TAL2/107/2-DG/BrPA (A107) mutants in YNB medium supplemented with 2% xylose, 2% L-arabinose, 2% glucose, or 50% bagasse hydrolysate during a growth test at 37 °C. (c) Ethanol production (g/L) by the O. polymorpha selected mutants BEP/cat8Δ/DAS1/TAL2/107, BEP/cat8Δ/DAS1/TAL2/107/2-DG and BEP/cat8Δ/DAS1/TAL2/107/2-DG/BrPA (A107) during high-temperature alcoholic fermentation (45 °C) of 10% xylose, 10% L-arabinose, 10% glucose, or 50% bagasse hydrolysate. Error bars represent the standard error of the mean (SE), n = 3. In some cases, error bars are not visible due to the small magnitude of the error.

Fig. 3

Biomass accumulation by BEP/cat8Δ, BEP/cat8∆/api1∆ and BEP/cat8∆/API1*, BEP/cat8Δ/ira1Δ and BEP/cat8Δ/IRA1* and ethanol production by these strains. (a) Biomass accumulation by BEP/cat8Δ, BEP/cat8∆/api1∆ and BEP/cat8Δ/API1* in minimal medium supplemented with 2% xylose, 2% L-arabinose, 2% glucose, and 50% bagasse hydrolysate at 37 °C and (b) ethanol production by these strains during the alcoholic fermentation of 10% xylose, 10% L-arabinose, 10% glucose, and 50% bagasse hydrolysate at 45 °C. (c) Biomass accumulation by BEP/cat8Δ, BEP/cat8∆/ira1∆, BEP/cat8∆/IRA1* and A107 in minimal medium supplemented with 2% xylose, 2% L-arabinose, 2% glucose, and 50% bagasse hydrolysate at 37 °C and (d) ethanol production by these strains during the alcoholic fermentation of 10% xylose, 10% L-arabinose, 10% glucose, and 50% bagasse hydrolysate at 45 °C. Error bars represent the standard error of the mean (SE), n = 3. In some cases, error bars are not visible due to the small magnitude of the error.

Fig. 4

Growth kinetics in conditions of elevated xylose concentrations. Growth kinetics of parental BEP/cat8Δ strain, A107 mutant strain and yeast transformants O. polymorpha BEP/cat8∆/API1* and BEP/cat8∆/ira1∆ in conditions of elevated xylose concentrations, performed at 37 °C for (a) 14 h; (b) 24 h; (c) 48 h; (d) 72 h. Error bars represent the standard error of the mean (SE). In some cases, error bars are not visible due to the small magnitude of the error. Statistical significance is indicated by asterisks: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, calculated using unpaired two-tailed t-tests (n = 3). Dashed brackets denote the specific pairs of strains that were statistically compared.

Fig. 5

Biomass accumulation of four O. polymorpha strains at different temperatures and pH conditions. (a) Biomass accumulation of three O. polymorpha strains (BEP/cat8Δ, A107, BEP/cat8Δ/IRA1*, and BEP/cat8Δ/ira1Δ) in media containing 2% xylose, (b) 2% L-arabinose, (c) and 2% glucose at different temperatures (28 °C, 37 °C, 45 °C, 47 °C). Biomass accumulation of four O. polymorpha strains (BEP/cat8Δ, A107, BEP/cat8Δ/IRA1*, and BEP/cat8Δ/ira1Δ) in media containing (d) 2% xylose, (e) 2% L-arabinose and (f) 2% glucose at different pH conditions (4, 6, or 7.2). For medium with initial pH 4, the pH dropped to 2, for pH 6 it dropped to 3.2, and for pH 7.2, the pH reached 4.4 by the 96th hour of the experiment. Error bars represent the standard error of the mean (SE). In some cases, error bars are not visible due to the small magnitude of the error.

Fig. 6

Genes involved in the regulation of pentose sugars alcoholic fermentation in O. polymorpha yeast. The schematic illustrates the metabolic pathways and key regulatory factors involved in alcoholic fermentation of pentose sugars in O. polymorpha. Enzymes and transporters include XYL1 (xylose reductase), XYL2 (xylitol dehydrogenase), XYL3 (xylulokinase), ADH1 (alcohol dehydrogenase), PDC1 (pyruvate decarboxylase), HXT1 (low-affinity hexose transporter), and HXS1 (hexose-sensing receptor). Transcriptional regulators include CAT8 and AZF1 (transcription activators), MIG1, MIG2, TUP1, and HAP4 (transcription factors). DAS1 (dihydroxyacetone synthase) and TAL2 (peroxisomal transketolase and transaldolase, respectively) play roles in pentose metabolism. The diagram also represents signal transduction pathways related to IRA1 deletion and overexpression. Ras1/2 proteins mediate intracellular glucose signaling, while Cyr1 (adenylate cyclase) converts ATP to cAMP, activating PKA (protein kinase A), which regulates growth, metabolism, and stress responses. IRA1 acts as a negative regulator of Ras1/2, facilitating the transition from the active (GTP-bound) to inactive (GDP-bound) state. The API1 gene was identified in O. polymorpha as a gene involved in growth on L-arabinose, though its precise functional role remains to be fully elucidated. Arrows indicate regulatory interactions: red signifies inhibition/downregulation, green represents activation/upregulation, and blue highlights metabolic processes.