High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

Abad, Sandra; Nahálka, Jozef; Winkler, Margit; Bergler, Gabriele; Speight, Robert; Glieder, Anton; Nidetzky, Bernd

doi:10.1007/s10529-010-0456-9

Cited by 11 publications

(5 citation statements)

References 27 publications

(32 reference statements)

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“…[1-3]). It can be grown to high cell densities, is characterized by an efficient secretory system and many tools for molecular manipulation are available.…”

Section: Introductionmentioning

confidence: 99%

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

et al. 2012

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ΒackgroundThe methylotrophic yeast Pichia pastoris has become an important host organism for recombinant protein production and is able to use methanol as a sole carbon source. The methanol utilization pathway describes all the catalytic reactions, which happen during methanol metabolism. Despite the importance of certain key enzymes in this pathway, so far very little is known about possible effects of overexpressing either of these key enzymes on the overall energetic behavior, the productivity and the substrate uptake rate in P. pastoris strains.ResultsA fast and easy-to-do approach based on batch cultivations with methanol pulses was used to characterize different P. pastoris strains. A strain with MutS phenotype was found to be superior over a strain with Mut+ phenotype in both the volumetric productivity and the efficiency in expressing recombinant horseradish peroxidase C1A. Consequently, either of the enzymes dihydroxyacetone synthase, transketolase or formaldehyde dehydrogenase, which play key roles in the methanol utilization pathway, was co-overexpressed in MutS strains harboring either of the reporter enzymes horseradish peroxidase or Candida antarctica lipase B. Although the co-overexpression of these enzymes did not change the stoichiometric yields of the recombinant MutS strains, significant changes in the specific growth rate, the specific substrate uptake rate and the specific productivity were observed. Co-overexpression of dihydroxyacetone synthase yielded a 2- to 3-fold more efficient conversion of the substrate methanol into product, but also resulted in a reduced volumetric productivity. Co-overexpression of formaldehyde dehydrogenase resulted in a 2-fold more efficient conversion of the substrate into product and at least similar volumetric productivities compared to strains without an engineered methanol utilization pathway, and thus turned out to be a valuable strategy to improve recombinant protein production.ConclusionsCo-overexpressing enzymes of the methanol utilization pathway significantly affected the specific growth rate, the methanol uptake and the specific productivity of recombinant P. pastoris MutS strains. A recently developed methodology to determine strain specific parameters based on dynamic batch cultivations proved to be a valuable tool for fast strain characterization and thus early process development.

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“…[1-3]). It can be grown to high cell densities, is characterized by an efficient secretory system and many tools for molecular manipulation are available.…”

Section: Introductionmentioning

confidence: 99%

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

et al. 2012

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“…construct 2, 8 and Co-transformant) was monitored over time. Therefore, K. phaffii whole cells were cultivated, Ps LOX and Mt HPL production was induced with either MeOH or GY, and the whole-cell biocatalysts were frozen, stored at −20°C and thawed before use for improved permeability ( Chen, 2007 ; Abad et al, 2011 ; Rinnofner et al, 2019 ). All reactions were performed in baffled shake flasks with a reaction volume of approximately 25 ml for 80 h at 15°C ( Figure 6 ).…”

Section: Resultsmentioning

confidence: 99%

From linoleic acid to hexanal and hexanol by whole cell catalysis with a lipoxygenase, hydroperoxide lyase and reductase cascade in Komagataella phaffii

Hashem

Hochrinner

Bürgler

et al. 2022

Front. Mol. Biosci.

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Green leaf volatiles (GLVs) cover a group of mainly C6-and C9-aldehydes, -alcohols and -esters. Their name refers to their characteristic herbal and fruity scent, which is similar to that of freshly cut grass or vegetables. Lipoxygenases (LOXs) catalyze the peroxidation of unsaturated fatty acids. The resulting hydroperoxy fatty acids are then cleaved into aldehydes and oxo acids by fatty acid hydroperoxide lyases (HPLs). Herein, we equipped the yeast Komagataella phaffii with recombinant genes coding for LOX and HPL, to serve as a biocatalyst for GLV production. We expressed the well-known 13S-specific LOX gene from Pleurotus sapidus and a compatible HPL gene from Medicago truncatula. In bioconversions, glycerol induced strains formed 12.9 mM hexanal using whole cells, and 8 mM hexanol was produced with whole cells induced by methanol. We applied various inducible and constitutive promoters in bidirectional systems to influence the final ratio of LOX and HPL proteins. By implementing these recombinant enzymes in Komagataella phaffii, challenges such as biocatalyst supply and lack of product specificity can finally be overcome.

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“…Boer et al [ 57 ] assembled a synthetic gene encoding miraculin and ligated it into an expression vector of E. coli . The cloning results in the synthesis of recombinant miraculin, but a comprehensive characterization was not made [ 86–91 ].…”

Section: Miraculinmentioning

confidence: 99%

Bioprospecting and biotechnological insights into sweet-tasting proteins by microbial hosts―a review

Bilal

et al. 2022

Bioengineered

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Owing to various undesirable health effects of sugar overconsumption, joint efforts are being made by industrial sectors and regulatory authorities to reduce sugar consumption practices, worldwide. Artificial sweeteners are considered potential substitutes in several products, e.g., sugar alcohols (polyols), high-fructose corn syrup, powdered drink mixes, and other beverages. Nevertheless, their long-standing health effects continue to be debatable. Consequently, growing interest has been shifted in producing non-caloric sweetenersfrom renewable resources to meet consumers’ dietary requirements. Except for the lysozyme protein, various sweet proteins including thaumatin, mabinlin, brazzein, monellin, miraculin, pentadin, and curculin have been identified in tropical plants. Given the high cost and challenging extortion of natural resources, producing these sweet proteins using engineered microbial hosts, such as Yarrowia lipolytica, Pichia pastoris, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis represents an appealing choice. Engineering techniques can be applied for large-scale biosynthesis of proteins, which can be used in biopharmaceutical, food, diagnostic, and medicine industries. Nevertheless, extensive work needs to be undertaken to address technical challenges in microbial production of sweet-tasting proteins in bulk. This review spotlights historical aspects, physicochemical properties (taste, safety, stability, solubility, and cost), and recombinant biosynthesis of sweet proteins. Moreover, future opportunities for process improvement based on metabolic engineering strategies are also discussed.

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High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

Cited by 11 publications

References 27 publications

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

Recombinant protein expression in Pichia pastoris strains with an engineered methanol utilization pathway

From linoleic acid to hexanal and hexanol by whole cell catalysis with a lipoxygenase, hydroperoxide lyase and reductase cascade in Komagataella phaffii

Bioprospecting and biotechnological insights into sweet-tasting proteins by microbial hosts―a review

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