Blocking methanol dissimilation pathway and incorporating RuMP to increase methanol utility in Komagataella phaffii

Abstract

Efficient methanol assimilation was crucial for methanol-based biomanufacturing of high-valued products. Given the low methanol utility in native Komagataella phaffii cells, we disrupted the genes encoding formaldehyde dehydrogenase and formate dehydrogenase and incorporated heterologous RuMP into K. phaffii to drive more flux into central metabolic pathways. We also performed transcriptome analysis to evaluate the metabolic impact of this genetic modification. The results showed that the biomass of Δfldh mutant strain was 8.3% higher than that of the wild type control strain. The ratio of biomass accumulation from methanol was respectively 5.754 and 6.209 in WT and Δfldh. Recombinant RuMP-Δfdh and hps-Δfdh rescued the unliving Δfdh and were able to growth in medium with methanol as sole carbon source. In addition, the transcription revealed the impact of disrupted fldh or fdh on TCA, PPP and the respiratory chain. Our results suggested that inactivation of fldh and expressing RuMP were beneficial for methanol utility. It was fdh rather than fldh that was indispensable for K. phaffii cell growth. This study provided new insights into how to reprogram K. phaffii to enhance its methanol assimilation rate, also the theoretical bases for the mechanism underlying the better methanol digestion.

Keywords

Komagataella phaffii / Methanol dissimilation / Gene knock-out / Transcriptome

References

[1] WangY, et al.. Synthetic Methylotrophy: A practical solution for Methanol-Based biomanufacturing. Trends Biotechnol, 2020, 38(6): 650-66

[2] SuiYF, et al.. Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus Niger. Microb Cell Fact, 2020, 191198

[3] VijayakumarVE, VenkataramanK. A systematic review of the potential of Pichia pastoris (Komagataella phaffii) as an alternative host for biologics production. Mol Biotechnol‌‌, 2024, 66(7): 1621-39

[4] RußmayerH, et al.. Customizing amino acid metabolism of Pichia pastoris for Recombinant protein production. Biotechnol J, 2023, 1812e2300033

[5] VozzaLA, et al.. Production of a Recombinant bovine enterokinase catalytic subunit in the Methylotrophic yeast Pichia pastoris. Biotechnol (Nature Publishing Company), 1996, 14(1): 77-81

[6] ChenCT, et al.. Synthetic methanol auxotrophy of Escherichia coli for methanol-dependent growth and production. Metab Eng, 2018, 49: 257-66

[7] TuyishimeP, et al.. Engineering Corynebacterium glutamicum for methanol-dependent growth and glutamate production. Metab Eng, 2018, 49: 220-31

[8] DaiZ, et al.. Metabolic construction strategies for direct methanol utilization in Saccharomyces cerevisiae. Bioresour Technol, 2017, 245: 1407-12

[9] WangG, et al.. Engineering yeast Yarrowia lipolytica for methanol assimilation. ACS Synth Biol, 2021, 10(12): 3537-50

[10] VanwonterghemI, et al.. Methylotrophic methanogenesis discovered in the archaeal phylum Verstraetearchaeota. Nat Microbiol, 2016, 1(170): 1-9

[11] KanehisaM, GotoS. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res, 2000, 28(1): 27-30

[12] Van der KleiIJ, et al.. The significance of peroxisomes in methanol metabolism in Methylotrophic yeast. Biochim Biophys Acta, 2006, 1763(12): 1453-62

[13] HeggesetTM, et al.. Genome sequence of thermotolerant Bacillus methanolicus: features and regulation related to Methylotrophy and production of L-lysine and L-glutamate from methanol. Appl Environ Microbiol, 2012, 78(15): 5170-81

[14] ChenAY, LanEI. Chemical production from methanol using natural and synthetic Methylotrophs. Biotechnol J, 2020, 156e1900356

[15] ZavecD, et al.. Beyond alcohol oxidase: the Methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2. FEMS Yeast Res, 2021, 212foab009

[16] LiP, et al.. Construction of efficient xylose utilizing Pichia pastoris for industrial enzyme production. Microb Cell Fact, 2015, 14: 221-10

[17] Bumjun LeeHY, SakaiY, KatoN. Physiological role of the glutathione dependent formaldehyde dehydrogenase in the Methylotrophic yeast Candida boidinii. Microbiology, 2002, 148: 2697-704

[18] LeeB, et al.. Physiological role of the glutathione-dependent formaldehyde dehydrogenase in the Methylotrophic yeast Candida boidinii. Microbiology, 2002, 148(9): 2697-704

[19] SakaiY, et al.. Regulation of the formate dehydrogenase gene, FDH1, in the Methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, Methylamine, and choline. J Bacteriol, 1997, 179(14): 4480-5

[20] NakagawaT, et al.. Alcohol oxidase hybrid oligomers formed in vivo and in vitro. Yeast, 1999, 15(12): 1223-30

[21] SakaiY, et al.. Regulation and physiological role of the DAS1 gene, encoding dihydroxyacetone synthase, in the Methylotrophic yeast Candida boidinii. J Bacteriol, 1998, 180(22): 5885-90

[22] YurimotoH, et al.. Physiological role of S-formylglutathione hydrolase in C(1) metabolism of the Methylotrophic yeast Candida boidinii. Microbiology, 2003, 149(Pt 8): 1971-9

[23] YurimotoH, et al.. Alcohol dehydrogenases that catalyse Methyl formate synthesis participate in formaldehyde detoxification in the Methylotrophic Candida boidinii. Yeast, 2004, 21(4): 341-50

[24] YurimotoH, et al.. Regulation and evaluation of five methanol-inducible promoters in the Methylotrophic yeast Candida boidinii. Biochim Biophys Acta, 2000, 1493(1–2): 56-63

[25] HoriguchiH, et al.. Antioxidant system within yeast peroxisome. Biochemical and physiological characterization of CbPmp20 in the Methylotrophic yeast Candida boidinii. J Biol Chem, 2001, 276(17): 14279-88

[26] VartiainenE, et al.. Evaluation of synthetic formaldehyde and methanol assimilation pathways in Yarrowia lipolytica. Fungal Biology Biotechnol, 2019, 6: 271-13

[27] BerriosJ, et al.. Role of dissimilative pathway of Komagataella phaffii (Pichia pastoris): formaldehyde toxicity and energy metabolism. Microorganisms, 2022, 1071466

[28] YuYF, et al.. Comparative transcriptome and metabolome analyses reveal the methanol dissimilation pathway of Pichia pastoris. BMC Genomics, 2022, 23(1): 366-79

[29] BennettRK, et al.. Expression of heterologous non-oxidative Pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli Methylotroph. Metab Eng, 2018, 45: 75-85

[30] MurrayB, et al.. Methionine adenosyltransferases in liver cancer. World J Gastroentero, 2019, 25(31): 4300-19

[31] PascaleRM, et al.. Deregulation of methionine metabolism as determinant of progression and prognosis of hepatocellular carcinoma. Transl Gastroenterol Hepatol, 2018, 336.

[32] GouD, et al.. Gluconeogenic enzyme PCK1 supports S-adenosylmethionine biosynthesis and promotes H3K9me3 modification to suppress hepatocellular carcinoma progression. J Clin Invest, 2023, 13313e161713

[33] ShrivastavaPK, ShrivastavaSK. Dextran carrier macromolecule for colon specific delivery of celecoxib. Curr Drug Deliv, 2010, 7(2): 144-51

[34] ReynoldsTB. Going with the Flo: the role of Flo11-Dependent and independent interactions in yeast mat formation. J Fungi (Basel Switzerland), 2018, 4(132): 1-10

[35] RauxE, et al.. The role of Saccharomyces cerevisiae Met1p and Met8p in Sirohaem and cobalamin biosynthesis. Biochem J, 1999, 338(Pt 3): 701-8

[36] Ubiyvovk VM, et al. Cloning and functional analysis of the GSH1/MET1 gene complementing cysteine and glutathione auxotrophy of the Methylotrophic yeast Hansenula polymorpha. Ukrains’kyi Biokhimichnyi Zhurnal (1999). 2011;83(5):67–81.

[37] FiermonteG, et al.. The sequence, bacterial expression, and functional reconstitution of the rat mitochondrial dicarboxylate transporter cloned via distant homologs in yeast and Caenorhabditis elegans. J Biol Chem, 1998, 273(38): 24754-9

Funding

National Natural Science Foundation of China(32370054)

the national first-class discipline program of Light Industry Technology and Engineering(LITE2018-24)

RIGHTS & PERMISSIONS

Jiangnan University