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Beyond alcohol oxidase: the methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2.

ABSTRACT: Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut- phenotype. The Mut- phenotype is commonly regarded as unable to utilize methanol. In contrast to the literature, we found that the Mut- strain can consume methanol. This ability was based on the promiscuous activity of alcohol dehydrogenase Adh2, an enzyme ubiquitously found in yeast and normally responsible for ethanol consumption and production. Using 13C labeled methanol as substrate we could show that to the largest part methanol is dissimilated to CO2 and a small part is incorporated into metabolites, the biomass, and the secreted recombinant protein. Overexpression of the ADH2 gene in K. phaffii Mut- increased both the specific methanol uptake rate and recombinant protein production, even though the strain was still unable to grow. These findings imply that thermodynamic and kinetic constraints of the dehydrogenase reaction facilitated the evolution towards alcohol oxidase-based methanol metabolism in yeast.

SUBMITTER: Zavec D

PROVIDER: S-EPMC7972947 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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Beyond alcohol oxidase: the methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2.

Zavec Domen D Troyer Christina C Maresch Daniel D Altmann Friedrich F Hann Stephan S Gasser Brigitte B Mattanovich Diethard D

FEMS yeast research 20210301 2

Methylotrophic yeasts are considered to use alcohol oxidases to assimilate methanol, different to bacteria which employ alcohol dehydrogenases with better energy conservation. The yeast Komagataella phaffii carries two genes coding for alcohol oxidase, AOX1 and AOX2. The deletion of the AOX1 leads to the MutS phenotype and the deletion of AOX1 and AOX2 to the Mut- phenotype. The Mut- phenotype is commonly regarded as unable to utilize methanol. In contrast to the literature, we found that the Mu ...[more]

PMID: 33599728

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Project description:BackgroundBioconversion of methanol derived from CO2 reduction into value-added chemicals provides a unique approach for mitigating global warming and reducing fossil fuels dependence. Production of 3-hydroxypropionic acid (3-HP), a key building block for the development of biobased products such as acrylates and 1,3-propanediol, has been successfully achieved using methanol as the sole carbon and energy source in the methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris). However, challenges remain in meeting commercially relevant concentrations, yields and productivities of 3-HP, prompting further strain optimization. In the present study, we have combined metabolic engineering strategies aiming at increasing metabolic precursors supply and redirecting carbon flux towards 3-HP production.ResultsA combinatorial metabolic engineering strategy targeting precursors supply and 3-HP export was applied to the original 3-HP producing K. phaffii strain harboring the synthetic β-alanine pathway and a mutated NADP-dependent formate dehydrogenase from Pseudomonas sp. 101 (PseFDH(V9)). To do so, several genes encoding enzymes catalyzing reactions immediately upstream of the β-alanine pathway were overexpressed to enhance precursors availability. However, only the overexpression of the pyruvate carboxylase PYC2 gene significantly increased the 3-HP yield on biomass (YP/X) in small-scale cultivations. Co-overexpression of PYC2 and the lactate permeases ESBP6 and JEN1 genes led to a 55% improvement in 3-HP titer and product yield in methanol deep-well plate cultures compared to the reference strain, mostly due to Esbp6 activity, proving its effectiveness as a 3-HP transporter. Deletion of the native formate dehydrogenase gene FDH1 did not increase methanol flux entering the assimilatory pathway. Instead, knockout strains showed severe growth defects due to toxic intermediates accumulation. Co-expression of the PseFDH(V9) encoding gene in these strains failed to compensate for the loss of the native FDH. The strain combining PYC2, ESBP6, and JEN1 overexpression was further tested in fed-batch cultures at pH 5, achieving a 3-HP concentration of 27.0 g l- 1, with a product yield of 0.19 g g- 1, and a volumetric productivity of 0.56 g l- 1 h- 1 for the methanol feeding phase of the cultivations. These results represent a 42% increase in final concentration and over 20% improvement in volumetric productivity compared to the original 3-HP-producing strain. Furthermore, bioreactor-scale cultivations at pH 3.5 revealed increased robustness of the strains overexpressing monocarboxylate transporters.ConclusionsOur results point out the potential of lactate transporters to efficiently drive 3-HP export in K. phaffii, leading to higher titers, yields, and productivities, even at lower pH conditions.

Project description:BackgroundMethanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways.ResultsIn this study, production of 3-HP via the synthetic β-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus, and ydfG from Escherichia coli K-12. The expression of these key enzymes allowed a production of 1.0 g l-1 of 3-HP in small-scale cultivations using methanol as substrate. The addition of a second copy of the panD gene and selection of a weak promoter to drive expression of the ydfG gene in the PpCβ21 strain resulted in an additional increase in the final 3-HP titer (1.2 g l-1). The 3-HP-producing strains were further tested in fed-batch cultures. The best strain (PpCβ21) achieved a final 3-HP concentration of 21.4 g l-1 after 39 h of methanol feeding, a product yield of 0.15 g g-1, and a volumetric productivity of 0.48 g l-1 h-1. Further engineering of this strain aiming at increasing NADPH availability led to a 16% increase in the methanol consumption rate and 10% higher specific productivity compared to the reference strain PpCβ21.ConclusionsOur results show the potential of K. phaffii as platform cell factory to produce organic acids such as 3-HP from renewable one-carbon feedstocks, achieving the highest volumetric productivities reported so far for a 3-HP production process through the β-alanine pathway.

Dataset Information

Beyond alcohol oxidase: the methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2.

Publications

Beyond alcohol oxidase: the methylotrophic yeast Komagataella phaffii utilizes methanol also with its native alcohol dehydrogenase Adh2.

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