Preparation of a Bombyx mori acetylcholinesterase enzyme reagent through chaperone protein disulfide isomerase co-expression strategy in Pichia pastoris for detection of pesticides

Li, Jiadong; Cai, Jun; Ma, Minting; Li, Liping; Lu, Linping; Wang, Yu; Wang, Chenglong; Yang, Jin-Yi; Xu, Zhen-Lin; Yao, Min; Shen, Xing; Wang, Hong

doi:10.1016/j.enzmictec.2020.109741

Cited by 9 publications

(2 citation statements)

References 39 publications

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“…Similar results were also reported in other studies. For instance, the enzyme activity of the rBmAChE2 with four potential intramolecular disulfide bonds was increased by almost 5 times after co-expression with PDI [ 30 ]. In addition, co-expression of HAC1, PDI, and ERO1 could increase the activity of mannanase ManA in the supernatant by 26%, 20%, and 15%, respectively [ 31 ].…”

Section: Discussionmentioning

confidence: 99%

Improved Production of Recombinant Carboxylesterase FumDM by Co-Expressing Molecular Chaperones in Pichia pastoris

Jiang

Guan

Liu

et al. 2023

Toxins

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Fumonisins (FBs) are mycotoxins that threaten public health and food safety worldwide. Enzymatic degradation of Fumonisin B1 (FB1) through decarboxylation has attracted much attention, whereas application of FB1 carboxylesterase in detoxification requires more effective expression of the recombinant carboxylesterase. In this study, the carboxylesterase FumDM from Sphingopyxis sp. ASAG22 was codon-optimized and co-expressed with five different molecular chaperones (PDI, CPR5, ERO1, HAC1, and Bip) in order to improve the expression level of FumDM in Pichia pastoris (also known as Komagataella phaffii) GS115. The co-expression of different chaperones caused varying degrees of improvement in FumDM activity for FB1. The enzyme activities of recombinant strains over-expressing PDI and CPR5 reached the highest levels of 259.47 U/mL and 161.34 U/mL, 635% and 357% higher than the original enzyme activity, respectively. Transcriptomic analysis of the two recombinant strains in comparison with the control strain showed that the correct folding of proteins assisted by molecular chaperones played a key role in the improvement of FumDM expression and its enzyme activity. This study demonstrated that co-expression of carboxylesterase FumDM and folding chaperones was an efficient strategy and therefore might inspire new perspectives on the improvement of carboxylesterase for detoxification of FB1.

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Section: Discussionmentioning

confidence: 99%

Improved Production of Recombinant Carboxylesterase FumDM by Co-Expressing Molecular Chaperones in Pichia pastoris

Jiang

Guan

Liu

et al. 2023

Toxins

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“…This strain is easy to use and requires neither extraordinary expertise nor expensive tissue culture facilities, and proteins expressed by this yeast will undergo series of eukaryotic post-translational modi cations, making it superior to bacterial expression systems (Karbalaei et al 2020). It is frequently reported that multiple proteins were co-expressed for both fundamental and applied research in this yeast (Cai et al 2017;Han et al 2021;Li et al 2021;Wu et al 2019;Zhao et al 2018), while rounds of gene insertion manipulations performed on a single strain were constrained due to the limited selectable markers therein.…”

Section: Introductionmentioning

confidence: 99%

Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins

Wang,

Han,

Zhu

et al. 2024

Biotechnol Lett

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ObjectiveThe objective was to develop a convenient strategy for co-expressing multiple proteins in Komagataella pha i via the Cre/loxP system without introducing any markers. ResultsA plasmid in this strategy was generated from pPICZαA with integration of lox71-Sh ble-lox66. Firstly, the plasmid was inserted with one target protein gene and then transformed into K. pha i KM71. Secondly, the auxiliary plasmid pPICZαA/cre/his4 containing CRE recombinase gene was further chromosomally inserted to Sh ble gene therein. Finally, methanol induction was conducted to produce CRE for Cre/loxP-mediated recombination, and consequently, the sequence between lox71 and lox66 was deleted, leading to recovery of Zeo R and His − markers. Then the resulted strain expressing the one target protein was used as the host to which another target protein gene could be inserted by the same procedures. ConclusionsWith easy manipulation, the method was effective in co-expressing multiple proteins with rescue of markers in this yeast.

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Recent Advances in Synthetic Biology Applications of Pichia Species

Sun

Zuo

Yao

et al. 2022

Synthetic Biology of Yeasts

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Preparation of a Bombyx mori acetylcholinesterase enzyme reagent through chaperone protein disulfide isomerase co-expression strategy in Pichia pastoris for detection of pesticides

Cited by 9 publications

References 39 publications

Improved Production of Recombinant Carboxylesterase FumDM by Co-Expressing Molecular Chaperones in Pichia pastoris

Improved Production of Recombinant Carboxylesterase FumDM by Co-Expressing Molecular Chaperones in Pichia pastoris

Recycling selectable markers via Cre/loxP system for constructing Komagataella phaffii strains co-expressing multiple proteins

Recent Advances in Synthetic Biology Applications of Pichia Species

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