Improvement of D-lactic acid production from methanol by metabolically engineered Komagataella phaffii via ultra-violet mutagenesis

Abstract

Methanol has attracted attention as an alternative carbon source to petroleum. Komagataella phaffii, a methanol-assimilating yeast, is a useful host for the chemical production from methanol. A previous study successfully constructed a metabolically engineered K. phaffii GS115/S8/Z3 strain capable of producing D-lactic acid from methanol. In this study, we aimed to develop a strain with improved D-lactic acid production by applying ultra-violet mutagenesis to the D-lactic acid-producing strain, GS115/S8/Z3. The resulting mutant strain DLac_Mut2_221 produced 5.38 g/L of D-lactic acid from methanol, a 1.52-fold increase compared to the parent strain GS115/S8/Z3. The transcriptome analysis of the mutant DLac_Mut2_221 identified 158 differentially expressed genes, providing insights into key mechanisms contributing to enhanced D-lactic acid production. Metabolic engineering strategies for K. phaffii based on the knowledge gained from this study will contribute to improving the productivity of various useful chemicals from methanol.

Keywords: D-lactic acid; Komagataella phaffii; Methanol; Transcriptome analysis; Ultra-violet mutagenesis; Yeast.

Fig. 1

D-Lactic acid concentration after 48-h microplate cultivation of mutants obtained by UV mutagenesis of GS115/S8/Z3. Values are displayed in ascending order from left to right.

Fig. 2

Time-course changes in (A) methanol concentration, (B) OD₆₀₀, (C) D-lactic acid concentration, and (D) maximum D-lactic acid production in GS115/S8/Z3 and seven mutants. Data are presented as the average of three independent experiments. Error bars represent mean ± standard deviation. The statistical significance of the maximum D-lactic acid production compared to the parent strain GS115/S8/Z3 was evaluated using a two-tailed, unpaired, homoscedastic Student's t-test (∗, p < 0.05).

Fig. 3

D-Lactic acid concentration after 48-h microplate cultivation of mutants obtained by UV mutagenesis of DLac_Mut1_25. Values are displayed in ascending order from left to right.

Fig. 4

Time-course changes in (A) methanol concentration, (B) OD₆₀₀, (C) D-lactic acid concentration, and (D) maximum D-lactic acid production of eight mutants. Gray line in (D) indicates D-lactic acid production in DLac_Mut1_25 (4.45 g/L). Data are presented as the average of three independent experiments. Error bars represent mean ± standard deviation. The statistical significance of the maximum D-lactic acid production compared to the parent strain DLac_Mut1_25 was evaluated using a two-tailed, unpaired, homoscedastic Student's t-test (∗, p < 0.05).

Fig. 5

Volcano plot of genes for mutant Dlac_Mut2_221. Vertical lines represent ±1.5-fold change (log₂ fold change = ± 0.59). Horizontal line indicates a p-value of 0.01 (-log₁₀ p-value = 2.0). Red and blue plots represent DEG-UP and DEG-DOWN, respectively, in yeast. Black plots represent non-DEGs (i.e., genes with no change in transcription level).

Fig. 6

Gene Ontology enrichment analysis. (A) Up-regulated and (B) down-regulated differentially expressed genes. BP, biological process; MF, molecular function; CC, cellular component.

Fig. 7

KEGG pathway enrichment analysis. (A) Up-regulated and (B) down-regulated differentially expressed genes.

Fig. 8

Schematic of methanol utilization pathway in K. phaffii. ACO: Aconitase, AOX: Alcohol oxidase; CS: Citrate synthase; DHA: Dihydroxyacetone, DHAP: Dihydroxyacetone Phosphate, F6P: Fructose-6-phosphate, FBP: Fructose-1,6-Bisphosphate, FDH: Formate dehydrogenase; FGH: S-formylglutathione hydrolase; FLD: Formaldehyde dehydrogenase; GAP: Glyceraldehyde 3-phosphate; ICL: Isocitrate lyase; MDH: Malate dehydrogenase; MLS: Malate synthase; Xu5P: Xylulose 5-phosphate. Solid lines represent a single reaction, whereas dashed lines represent multiple reactions. Enzyme genes marked in red represent upregulated genes.