Overcoming Obstacles in Protein Expression in the Yeast Pichia pastoris: Interviews of Leaders in the Pichia Field

Ingram, Zoë; Patkar, Abha; Oh, Dahoon; Zhang, Kevin K.; Chung, Christina Z.; Lin‐Cereghino, Joan; Lin-Cereghino, Geoff P.

doi:10.56031/2576-215x.1010

Cited by 4 publications

(3 citation statements)

References 44 publications

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“…However, P. pastoris lacks the problem of harmful ethanol synthesis, but it cannot express any gene of interest. While specific proteins may have no issues with being expressed, others may have problems associated with glycosylation, secretion, and folding [ 69 , 70 ]. A recent study on recombinant overexpression by Xue, Yao [ 64 ] found excellent expression of cold-active esterase in the S. cerevisiae heterologous host, which was attributed to similarities between the yeast family to which the wild gene and S. cerevisiae belongs.…”

Section: Cold-active Lipase and Esterase Overexpression In Recombinan...mentioning

confidence: 99%

Cold-Active Lipases and Esterases: A Review on Recombinant Overexpression and Other Essential Issues

Matinja

Leow

Oslan

et al. 2022

IJMS

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Cold environments characterised by diverse temperatures close to or below the water freezing point dominate about 80% of the Earth’s biosphere. One of the survival strategies adopted by microorganisms living in cold environments is their expression of cold-active enzymes that enable them to perform an efficient metabolic flux at low temperatures necessary to thrive and reproduce under those constraints. Cold-active enzymes are ideal biocatalysts that can reduce the need for heating procedures and improve industrial processes’ quality, sustainability, and cost-effectiveness. Despite their wide applications, their industrial usage is still limited, and the major contributing factor is the lack of complete understanding of their structure and cold adaptation mechanisms. The current review looked at the recombinant overexpression, purification, and recent mechanism of cold adaptation, various approaches for purification, and three-dimensional (3D) crystal structure elucidation of cold-active lipases and esterase.

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Section: Cold-active Lipase and Esterase Overexpression In Recombinan...mentioning

confidence: 99%

Cold-Active Lipases and Esterases: A Review on Recombinant Overexpression and Other Essential Issues

Matinja

Leow

Oslan

et al. 2022

IJMS

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“…This led me to conclude that clonal variation is the most likely explanation for my findings. In addition to variation in integration site and copy number between transformants, P. pastoris is known to show variable protein expression between genetically identical cells [157][158][159]. This could explain why I observed both increases and decreases in protein concentrations between experiments, and why these changes occurred without any change in my method.…”

Section: Attempts To Detect Patk Expression and Activitymentioning

confidence: 98%

“…The work presented in Chapter 5 highlights some of the complications associated with protein expression in yeast. Some key challenges when working with P. pastoris are the relatively long culture times and variability between genetically identical cells [152,157,158]: together, these factors make optimisation of protein expression in P. pastoris very time-consuming. In particular, I found that even when protein is expressed by a particular P. pastoris clone, there can be significant variation between experiments.…”

Section: Pyrimidine Metabolism and Recombinant Protein Expressionmentioning

confidence: 99%

Establishing Molecular Biology Tools for Phytophthora agathidicida

Hayhurst¹

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Phytophthora – Greek for “the plant-destroyer” – is a genus of eukaryotic microorganisms that cause devastating plant diseases worldwide. Collectively, Phytophthora species cause billions of dollars in damages to crops and native ecosystems annually. Despite these extensive impacts, Phytophthora research is challenging due to a lack of molecular biology tools. Widely used techniques such as transformation and gene deletion remain challenging to establish in Phytophthora species. However, CRISPR-Cas genome editing was recently achieved in a small number of model Phytophthora species – but these breakthroughs have yet to reach most members of the genus. The pathogen Phytophthora agathidicida is one example. P. agathidicida causes kauri dieback, a disease that threatens kauri, one of our most important tree species in New Zealand. P. agathidicida was formally named in 2015 and is therefore an emergent threat. Consequently, key molecular methods such as transformation and CRISPR-Cas genome editing have yet to be established for this pathogen. The overarching goal of this thesis was to establish molecular biology methods for the study of P. agathidicida. The first aim was to identify a suitable target for genome editing with CRISPR-Cas and design guide RNAs for the target. A putative thymidine kinase gene from the P. agathidicida isolate 3770 genome was selected as a target, and eight guide RNA sequences were designed. All of the guide RNAs successfully directed the digestion of the target gene in vitro. The second aim was to establish a method to produce and transform P. agathidicida protoplasts. An optimised recovery medium was identified using a mock transformation method, which increased protoplast germination rates from 2.4% in a previously used medium to 15% in the new medium. Using the optimised conditions, P. agathidicida was transformed with plasmids encoding Cas nuclease-GFP fusion proteins. Transformants were both fluorescent and antibiotic resistant. The third aim was to express, purify, and characterise the putative thymidine kinase enzyme. Yeast species Pichia pastoris was used as a protein expression host, but neither protein expression nor kinase activity were consistently observed. In this thesis, P. agathidicida was successfully transformed for the first time. Transformation is a huge advancement and will greatly accelerate molecular biology research in P. agathidicida for years to come. In particular, transformation is a key step towards CRISPR-Cas genome editing in P. agathidicida. CRISPR-Cas will not only provide insights into its fundamental biology, but may guide the development of new control strategies for this devastating pathogen.

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Current achievements, strategies, obstacles, and overcoming the challenges of the protein engineering in Pichia pastoris expression system

Eskandari,

Nezhad,

Leow

et al. 2023

World J Microbiol Biotechnol

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Overcoming Obstacles in Protein Expression in the Yeast Pichia pastoris: Interviews of Leaders in the Pichia Field

Cited by 4 publications

References 44 publications

Cold-Active Lipases and Esterases: A Review on Recombinant Overexpression and Other Essential Issues

Cold-Active Lipases and Esterases: A Review on Recombinant Overexpression and Other Essential Issues

Establishing Molecular Biology Tools for Phytophthora agathidicida

Current achievements, strategies, obstacles, and overcoming the challenges of the protein engineering in Pichia pastoris expression system

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