Pathway engineering facilitates efficient protein expression in Pichia pastoris

Liu, Chao; Gong, Jin‐Song; Su, Chang; Li, Hui; Li, Heng; Rao, Zhiming; Xu, Zhenghong; Shi, Jin‐Song

doi:10.1007/s00253-022-12139-y

Cited by 25 publications

(22 citation statements)

References 151 publications

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“…Fungi produce a broad diversity of primary and secondary metabolites with different bioactivities. Different biotechnological processes have been implemented with the yeasts Saccharomyces cerevisiae [ 41 , 42 , 43 ], Yarrowia lipolitica [ 44 , 45 ], and Komagataella phaffii (formerly Pichia pastoris [ 46 , 47 ]) [ 48 , 49 ]. The production of high-value metabolites through different biotechnological processes is also achieved by means of filamentous fungi, such as Aspergilli and Penicillium species, Acremonium chrysogenum , Blakeslea trispora, and Trichoderma reesei [ 50 , 51 , 52 ].…”

Section: Development Of Biotechnological Fungal Platforms For the Bio...mentioning

confidence: 99%

“…The application of new genetic engineering techniques at the molecular level has allowed a considerable increase in the production of recombinant proteins and secondary metabolites in yeast and filamentous fungi [ 49 , 63 , 64 , 65 , 66 , 67 ]. This has contributed to the design of fungal hosts for the heterologous expression of biosynthetic gene clusters that could work as efficient platforms to produce high-added-value secondary metabolites, such as cannabinoids.…”

Section: Development Of Biotechnological Fungal Platforms For the Bio...mentioning

confidence: 99%

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Biotechnological Fungal Platforms for the Production of Biosynthetic Cannabinoids

Kosalková

Barreiro

Sánchez-Orejas

et al. 2023

JoF

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Cannabinoids are bioactive meroterpenoids comprising prenylated polyketide molecules that can modulate a wide range of physiological processes. Cannabinoids have been shown to possess various medical/therapeutic effects, such as anti-convulsive, anti-anxiety, anti-psychotic, antinausea, and anti-microbial properties. The increasing interest in their beneficial effects and application as clinically useful drugs has promoted the development of heterologous biosynthetic platforms for the industrial production of these compounds. This approach can help circumvent the drawbacks associated with extraction from naturally occurring plants or chemical synthesis. In this review, we provide an overview of the fungal platforms developed by genetic engineering for the biosynthetic production of cannabinoids. Different yeast species, such as Komagataella phaffii (formerly P. pastoris) and Saccharomyces cerevisiae, have been genetically modified to include the cannabinoid biosynthetic pathway and to improve metabolic fluxes in order to increase cannabinoid titers. In addition, we engineered the filamentous fungus Penicillium chrysogenum for the first time as a host microorganism for the production of Δ9-tetrahydrocannabinolic acid from intermediates (cannabigerolic acid and olivetolic acid), thereby showing the potential of filamentous fungi as alternative platforms for cannabinoid biosynthesis upon optimization.

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Section: Development Of Biotechnological Fungal Platforms For the Bio...mentioning

confidence: 99%

Section: Development Of Biotechnological Fungal Platforms For the Bio...mentioning

confidence: 99%

Biotechnological Fungal Platforms for the Production of Biosynthetic Cannabinoids

Kosalková

Barreiro

Sánchez-Orejas

et al. 2023

JoF

View full text Add to dashboard Cite

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“…2−4 In some cases, the failure to produce ample amounts of recombinant proteins can be ascribed to one or multiple defects of the host cell (such as deficient translocating capability or extracellular protease-mediated degradation). 5,6 Nevertheless, to what extent the expression cassette itself could affect heterologous gene expression remains largely unknown.…”

Section: Introductionmentioning

confidence: 99%

“…However, the endeavors to heterologously express a gene are often strikingly simplified into placing the gene of interest between a selected pair of promoters and terminator. Although this strategy has proven to be very successful in expressing a lot of recombinant proteins in certain expression systems such as the widely used prokaryotic pET system, heterologous expression in many more systems has long been frustrating, as the expression level of recombinant proteins is often lower than that of the native ones. − In some cases, the failure to produce ample amounts of recombinant proteins can be ascribed to one or multiple defects of the host cell (such as deficient translocating capability or extracellular protease-mediated degradation). , Nevertheless, to what extent the expression cassette itself could affect heterologous gene expression remains largely unknown.…”

Section: Introductionmentioning

confidence: 99%

Exploiting Systematic Engineering of the Expression Cassette as a Powerful Tool to Enhance Heterologous Gene Expression in Trichoderma reesei

Ji,

Xu,

Sun

et al. 2024

J. Agric. Food Chem.

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Many endeavors in expressing a heterologous gene in microbial hosts rely on simply placing the gene of interest between a selected pair of promoters and terminator. However, although the expression efficiency could be improved by engineering the host cell, how modifying the expression cassette itself systematically would affect heterologous gene expression remains largely unknown. As the promoter and terminator bear plentiful cis-elements, herein using the Aspergillus niger mannanase with high application value in animal feeds and the eukaryotic filamentous fungus workhorse Trichoderma reesei as a model gene/host, systematic engineering of an expression cassette was investigated to decipher the effect of its mutagenesis on heterologous gene expression. Modifying the promoter, signal peptide, the eukaryotic-specific Kozak sequence, and the 3′-UTR could stepwise improve extracellular mannanase production from 17 U/mL to an ultimate 471 U/mL, representing a 27.7-fold increase in expression. The strategies can be generally applied in improving the production of heterologous proteins in eukaryotic microbial hosts.

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“…K. phaffi was also suggested to be used as a model organism for yeasts as it has undergone a slower evolution than Saccharomyces cerevisiae and retained more characteristics of ancient yeasts and other metazoan organisms [4]. Consequently, different aspects of general biology and especially the protein production and secretion pathway have been studied over the years (reviewed in [3][4][5][6][7][8]). In many of these studies, reverse transcription quantitative PCR (RT-qPCR) analyses are used to determine the transcript levels of native and heterologous genes [9][10][11][12][13][14][15][16][17][18][19][20].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of reference genes for transcript analyses in Komagataella phaffii (Pichia pastoris)

Besleaga

Vignolle

Kopp

et al. 2023

Fungal Biol Biotechnol

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Background The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools. Results We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays. Conclusion The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.

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Pathway engineering facilitates efficient protein expression in Pichia pastoris

Cited by 25 publications

References 151 publications

Biotechnological Fungal Platforms for the Production of Biosynthetic Cannabinoids

Biotechnological Fungal Platforms for the Production of Biosynthetic Cannabinoids

Exploiting Systematic Engineering of the Expression Cassette as a Powerful Tool to Enhance Heterologous Gene Expression in Trichoderma reesei

Evaluation of reference genes for transcript analyses in Komagataella phaffii (Pichia pastoris)

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