Molecular strategies to increase the levels of heterologous transcripts inKomagataella phaffiifor protein production

Piva, Luiza Cesca; Bentacur, Maritza Ocampo; Reis, Viviane Castelo Branco; Marco, Janice Lisboa De; Moraes, Lídia Maria Pepe de; Torres, Fernando Araripe Gonçalves

doi:10.1080/21655979.2017.1296613

Cited by 9 publications

(6 citation statements)

References 40 publications

(42 reference statements)

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“…This study also exceeds the recently reported values in a fermenter of 2 L (97.34 U•mL −1 or 97.34 mg•L −1 ) and 30 L (62.64 U•mL −1 or 62.64 mg•L −1 ) of r-TmDEX49A produced in K. phaffii in the optimized fermentation (100 h) methanol-induced manner with a productivity of 0.62 mg•L −1 •h −1 [21]. Despite the wide use of the AOX1 promoter, it has some limitations, as methanol is a highly dangerous compound and even more so when working in large volumes, and storage can also be risky in industry [46,47]. Additionally, being a derivative from petrochemical sources, it may require purification steps to produce food and food additive products [48].…”

Section: High Cell Density Fermentation Of K Phaffii Dex49a-δsp-δn30 ...mentioning

confidence: 99%

Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

et al. 2022

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In the sugar industry, dextran generates difficulties in the manufacturing process. Using crude dextranase (EC 3.2.1.11) to eliminate dextran in sugar is an effective practice. In this study, a synthetic dextranase-encoding gene of the filamentous fungus Talaromyces minioluteus, lacking its putative native signal peptide (1–20 amino acids) and the next 30 amino acids (r–TmDEX49A–ΔSP–ΔN30), was fused to the Saccharomyces cerevisiae prepro α–factor (MFα–2) signal sequence and expressed in Komagataella phaffii under the constitutive GAP promoter. K. phaffii DEX49A–ΔSP–ΔN30, constitutively producing and secreting the truncated dextranase, was obtained. The specific activity of the truncated variant resulted in being nearly the same in relation to the full-length mature enzyme (900–1000 U·mg−1 of protein). At shaker scale (100 mL) in a YPG medium, the enzymatic activity was 273 U·mL−1. The highest production level was achieved in a fed-batch culture (30 h) at 5 L fermenter scale using the FM21–PTM1 culture medium. The enzymatic activity in the culture supernatant reached 1614 U·mL−1, and the productivity was 53,800 U·L−1·h−1 (53.8 mg·L−1·h−1), the highest reported thus far for a DEX49A variant. Dextran decreased r–TmDEX49A–ΔSP–ΔN30 mobility in affinity gel electrophoresis, providing evidence of carbohydrate–protein interactions. K. phaffii DEX49A–ΔSP–ΔN30 shows great potential as a methanol-free, commercial dextranase production system.

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Section: High Cell Density Fermentation Of K Phaffii Dex49a-δsp-δn30 ...mentioning

confidence: 99%

Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

et al. 2022

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“…This study also exceeds the recently reported values in a fermenter of 2 L (97.34 U.mL -1 or 97.34 mg.L -1 ) and 30 L (62.64 U.mL -1 or 62.64 mg.L -1 ) of r-TmDEX49A produced in K. phaffii in the optimized fermentation (100 h) methanol-induced manner with a productivity of 0.62 mg.L -1 .h -1 [21]. Despite the wide use of the AOX1 promoter, it has some limitations as methanol is a highly dangerous compound and even more so when working in large volumes, and storage can also be risky in industry [45,46]. Also, being a derivative from petrochemical sources, it may require purification steps to produce food and food additive products [47].…”

Section: High Cell Density Fermentation Of K Phaffii Dex49a-δsp-δn30 ...mentioning

confidence: 99%

Constitutive High Expression Level of A Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r-TmDEX49A-ΔSP-ΔN30) in Komagataella phaffii (Pichia pastoris)

Aristicas¹,

Valdés²,

Gámez³

et al. 2022

Preprint

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In the sugar industry, dextran generates difficulties in the manufacturing process. Crude dextranase (EC 3.2.1.11) to eliminate dextran in sugar is an effective practice. In this study, a synthetic dextranase encoding gene of the filamentous fungus Talaromyces minioluteus, lacking its putative native signal peptide (1-20 amino acids) and the next 30 amino acids (r-TmDEX49A-ΔSP-ΔN30), was fused to the Saccharomyces cerevisiae prepro -factor (MF-2) signal sequence and expressed in Komagataella phaffii under the constitutive GAP promoter. K. phaffii DEX49A-ΔSP-ΔN30, constitutively producing and secreting the truncated dextranase was obtained. The specific activity of the truncated variant resulted nearly the same in relation to the full-length mature enzyme (900-1000 U.mg-1 of protein). At shaker scale (100 mL) in YPG medium, the enzymatic activity was 273 U.mL-1. The highest production level was achieved in a fed-batch culture (30 h) at 5 L fermenter scale using the FM21-PTM1 culture medium. The enzymatic activity in the culture supernatant reached 1614 U.mL-1 and the productivity was 53800 U.L-1.h-1 (53.8 mg.L-1.h-1), the highest reported so far for a DEX49A variant. Dextran decreased r-TmDEX49A-ΔSP-ΔN30 mobility in affinity gel electrophoresis, providing evidence of carbohydrate-protein interactions. K. phaffii DEX49A-ΔSP-ΔN30 shows great potential as a methanol-free, commercial dextranase production system.

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“…After small-scale pilot experiments, there are multiple ways to improve the efficiency of recombinant protein production in K. phaffii for higher levels of protein yield. For example, it is possible to generate a strain with multiple expression cassettes [ 31 , 32 , 33 ]. This often includes the use of multiple selectable markers or dosage sensitive markers [ 32 , 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

“…For example, it is possible to generate a strain with multiple expression cassettes [ 31 , 32 , 33 ]. This often includes the use of multiple selectable markers or dosage sensitive markers [ 32 , 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains

Makeeva,

Muzaev,

Shubert

et al. 2023

Microorganisms

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Komagataella phaffii (Pichia pastoris) is a widely known microbial host for recombinant protein production and an emerging model organism in fundamental research. The development of new materials and techniques on this yeast improves heterologous protein synthesis. One of the most prominent ways to enhance protein production efficiency is to select K. phaffii strains with multiple expression cassettes and generate MutS strains using various vectors. In this study, we demonstrate approaches to expand the applications of pPICZ series vectors. Procedures based on PCR amplification and in vivo cloning allow rapid exchange of selectable markers. The combination of PCR amplification with split-marker-mediated transformation allows the development of K. phaffii MutS strains with two expression cassettes using pPICZ vectors. Both PCR-based approaches were applied to efficiently produce interleukin-2 mimetic Neo-2/15 in K. phaffii. The described techniques provide alternative ways to generate and improve K. phaffii strains without the need for obtaining new specific vectors or additional cloning of expression cassettes.

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Molecular strategies to increase the levels of heterologous transcripts inKomagataella phaffiifor protein production

Cited by 9 publications

References 40 publications

Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

Constitutive High Expression Level of a Synthetic Deleted Encoding Gene of Talaromyces minioluteus Endodextranase Variant (r–TmDEX49A–ΔSP–ΔN30) in Komagataella phaffii (Pichia pastoris)

Constitutive High Expression Level of A Synthetic Deleted Encoding Gene of <i>Talaromyces minioluteus</i> Endodextranase Variant (r-TmDEX49A-ΔSP-ΔN30) in <i>Komagataella phaffii</i> (<i>Pichia pastoris</i>)

Alternative PCR-Based Approaches for Generation of Komagataella phaffii Strains

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