Knockout of the DAS gene increases S-adenosylmethionine production in Komagataella phaffii

Liu, Taiyu; Liu, Baolin; Zhou, Huan; Zhang, Jianguo

doi:10.1080/13102818.2020.1837012

Cited by 6 publications

(4 citation statements)

References 27 publications

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“…Modifying the MUT pathway and the redistribution of metabolic flux for rProt expression in key metabolic steps directed to a target product has also been successful [37][38][39][40]. However, little attention has been paid in recent years to modifying this pathway to improve the production of different recombinant proteins than those abovementioned, and some high-value compounds, such as malic acid and S-adenosylmethionine, have been produced when a knockout of FDL and DAS was introduced [48,49]. Thus, the work presented by Zavec et al [39] and Tyurin and Kozlov [40] highlights the need to question the previously acknowledged and accepted premise of the MUT pathway behaviour and re-evaluate methanol metabolism from different perspectives.…”

Section: Engineering Of Co-substrate Catabolic Pathwaysmentioning

confidence: 99%

Advances in Cell Engineering of the Komagataella phaffii Platform for Recombinant Protein Production

et al. 2022

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Komagataella phaffii (formerly known as Pichia pastoris) has become an increasingly important microorganism for recombinant protein production. This yeast species has gained high interest in an industrial setting for the production of a wide range of proteins, including enzymes and biopharmaceuticals. During the last decades, relevant bioprocess progress has been achieved in order to increase recombinant protein productivity and to reduce production costs. More recently, the improvement of cell features and performance has also been considered for this aim, and promising strategies with a direct and substantial impact on protein productivity have been reported. In this review, cell engineering approaches including metabolic engineering and energy supply, transcription factor modulation, and manipulation of routes involved in folding and secretion of recombinant protein are discussed. A lack of studies performed at the higher-scale bioreactor involving optimisation of cultivation parameters is also evidenced, which highlights new research aims to be considered.

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Section: Engineering Of Co-substrate Catabolic Pathwaysmentioning

confidence: 99%

Advances in Cell Engineering of the Komagataella phaffii Platform for Recombinant Protein Production

et al. 2022

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“…Pichia pastoris (also known as Komagataella phaffii), a yeast strain with a Generally Regarded as Safe (GRAS) status [5,6], has become increasingly popular due to its advantages for use as a versatile yeast cell factory, including high protein secretion, low glycosylation levels, a Crabtree negative phenotype, robustness in high-density fermentation, and high methanol tolerance [5,7]. The availability of genetic manipulation tools [8], such as the CRISPR/Cas9 system, has allowed for effective editing and genetic manipulation of the P. pastoris genome [9].…”

Section: Introductionmentioning

confidence: 99%

Metabolic Engineering of Pichia pastoris for the Production of Triacetic Acid Lactone

Feng

et al. 2023

JoF

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Triacetic acid lactone (TAL) is a promising renewable platform polyketide with broad biotechnological applications. In this study, we constructed an engineered Pichia pastoris strain for the production of TAL. We first introduced a heterologous TAL biosynthetic pathway by integrating the 2-pyrone synthase encoding gene from Gerbera hybrida (Gh2PS). We then removed the rate-limiting step of TAL synthesis by introducing the posttranslational regulation-free acetyl-CoA carboxylase mutant encoding gene from S. cerevisiae (ScACC1*) and increasing the copy number of Gh2PS. Finally, to enhance intracellular acetyl-CoA supply, we focused on the introduction of the phosphoketolase/phosphotransacetylase pathway (PK pathway). To direct more carbon flux towards the PK pathway for acetyl-CoA generation, we combined it with a heterologous xylose utilization pathway or endogenous methanol utilization pathway. The combination of the PK pathway with the xylose utilization pathway resulted in the production of 825.6 mg/L TAL in minimal medium with xylose as the sole carbon source, with a TAL yield of 0.041 g/g xylose. This is the first report on TAL biosynthesis in P. pastoris and its direct synthesis from methanol. The present study suggests potential applications in improving the intracellular pool of acetyl-CoA and provides a basis for the construction of efficient cell factories for the production of acetyl-CoA derived compounds.

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“…A surplus of FLD activity was shown to result in higher theoretical NADH formation rates and finally also in significantly improved butanediol production rates when a P. pastoris strain overexpressing FLD and butanediol dehydrogenase was applied for whole-cell biotransformation [9]. Additionally, by deleting the genes coding for dihydroxyacetone synthase isoforms 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly, which led to an increase in ATP and higher S-adenosylmethionine production [8,10]. Therefore, an enhanced dissimilation pathway optimizes the energy distributions of methanol metabolism, guaranteeing more efficient NADH/product coupling and exploitation of both NADH steps for cofactor regeneration in whole-cell biotransformations [9].…”

Section: Introductionmentioning

confidence: 99%

Comparative transcriptome and metabolome analyses reveal the methanol dissimilation pathway of Pichia pastoris

Yang

Zhao

et al. 2022

BMC Genomics

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Background Pichia pastoris (Komagataella phaffii) is a model organism widely used for the recombinant expression of eukaryotic proteins, and it can metabolize methanol as its sole carbon and energy source. Methanol is oxidized to formaldehyde by alcohol oxidase (AOX). In the dissimilation pathway, formaldehyde is oxidized to CO2 by formaldehyde dehydrogenase (FLD), S-hydroxymethyl glutathione hydrolase (FGH) and formate dehydrogenase (FDH). Results The transcriptome and metabolome of P. pastoris were determined under methanol cultivation when its dissimilation pathway cut off. Firstly, Δfld and Δfgh were significantly different compared to the wild type (GS115), with a 60.98% and 23.66% reduction in biomass, respectively. The differential metabolites between GS115 and Δfld were mainly enriched in ABC transporters, amino acid biosynthesis, and protein digestion and absorption. Secondly, comparative transcriptome between knockout and wild type strains showed that oxidative phosphorylation, glycolysis and the TCA cycle were downregulated, while alcohol metabolism, proteasomes, autophagy and peroxisomes were upregulated. Interestingly, the down-regulation of the oxidative phosphorylation pathway was positively correlated with the gene order of dissimilation pathway knockdown. In addition, there were significant differences in amino acid metabolism and glutathione redox cycling that raised our concerns about formaldehyde sorption in cells. Conclusions This is the first time that integrity of dissimilation pathway analysis based on transcriptomics and metabolomics was carried out in Pichia pastoris. The blockage of dissimilation pathway significantly down-regulates the level of oxidative phosphorylation and weakens the methanol assimilation pathway to the point where deficiencies in energy supply and carbon fixation result in inefficient biomass accumulation and genetic replication. In addition, transcriptional upregulation of the proteasome and autophagy may be a stress response to resolve formaldehyde-induced DNA–protein crosslinking.

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Knockout of the DAS gene increases S-adenosylmethionine production in Komagataella phaffii

Cited by 6 publications

References 27 publications

Advances in Cell Engineering of the Komagataella phaffii Platform for Recombinant Protein Production

Advances in Cell Engineering of the Komagataella phaffii Platform for Recombinant Protein Production

Metabolic Engineering of Pichia pastoris for the Production of Triacetic Acid Lactone

Comparative transcriptome and metabolome analyses reveal the methanol dissimilation pathway of Pichia pastoris

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