Construction of new Pichia pastoris X-33 strains for production of lycopene and β-carotene

Araya-Garay, José Miguel; Feijoo-Siota, Lucía; Rosa-Dos-Santos, F.; Veiga‐Crespo, Patricia; Villa, Tomás G.

doi:10.1007/s00253-011-3764-7

Cited by 78 publications

(54 citation statements)

References 41 publications

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“…However, developments in these fields may also be relevant for constructing improved host strains dedicated for protein production. There are several recent reviews and research articles describing advances in these fields in detail (Abad et al 2010; Araya-Garay et al 2012; Wriessnegger and Pichler 2013). …”

Section: Future Perspectives — Outlookmentioning

confidence: 99%

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Ahmad

Hirz

Pichler

et al. 2014

Appl Microbiol Biotechnol

801

564

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Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. pastoris to such high titres. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation. The last few years have been particularly fruitful because of numerous activities in improving the expression of such complex proteins with a focus on either protein engineering or on engineering the protein expression host P. pastoris. This review refers to established tools in protein expression in P. pastoris and highlights novel developments in the areas of expression vector design, host strain engineering and screening for high-level expression strains. Breakthroughs in membrane protein expression are discussed alongside numerous commercial applications of P. pastoris derived proteins.

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Section: Future Perspectives — Outlookmentioning

confidence: 99%

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Ahmad

Hirz

Pichler

et al. 2014

Appl Microbiol Biotechnol

801

564

View full text Add to dashboard Cite

show abstract

“…For example, the efficient biosynthesis of high-value carotenoids, such as lycopene [1.141 μg g −1 (dry weight)] and β-carotene [339 μg g −1 (dry weight)], was recently described in Ko. pastoris (Araya-Garay et al ., 2012). Three carotenogenic enzymes were expressed for the production of lycopene, geranylgeranyl diphosphate synthase (crtE), phytoene synthase (crtB), and phytoene desaturase (crtI) from Erwinia uredovora .…”

Section: Genetic Modificationmentioning

confidence: 99%

Improving industrial yeast strains: exploiting natural and artificial diversity

Steensels¹,

Snoek²,

Meersman³

et al. 2014

FEMS Microbiol Rev

395

288

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Yeasts have been used for thousands of years to make fermented foods and beverages, such as beer, wine, sake, and bread. However, the choice for a particular yeast strain or species for a specific industrial application is often based on historical, rather than scientific grounds. Moreover, new biotechnological yeast applications, such as the production of second-generation biofuels, confront yeast with environments and challenges that differ from those encountered in traditional food fermentations. Together, this implies that there are interesting opportunities to isolate or generate yeast variants that perform better than the currently used strains. Here, we discuss the different strategies of strain selection and improvement available for both conventional and nonconventional yeasts. Exploiting the existing natural diversity and using techniques such as mutagenesis, protoplast fusion, breeding, genome shuffling and directed evolution to generate artificial diversity, or the use of genetic modification strategies to alter traits in a more targeted way, have led to the selection of superior industrial yeasts. Furthermore, recent technological advances allowed the development of high-throughput techniques, such as ‘global transcription machinery engineering’ (gTME), to induce genetic variation, providing a new source of yeast genetic diversity.

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“…Therefore, successive genetic manipulation steps, e.g. for the construction of biosynthetic pathways, have been comparatively challenging in P. pastoris and only a few applications have been reported so far3031323334. To date, the humanisation of the N-glycosylation pathway in P. pastoris via multiple consecutive cloning steps has been the most sophisticated and successful genetic engineering endeavour1335.…”

mentioning

confidence: 99%

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

Schwarzhans

Wibberg

Winkler

et al. 2016

Sci Rep

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The non-conventional yeast Pichia pastoris is a popular host for recombinant protein production in scientific research and industry. Typically, the expression cassette is integrated into the genome via homologous recombination. Due to unknown integration events, a large clonal variability is often encountered consisting of clones with different productivities as well as aberrant morphological or growth characteristics. In this study, we analysed several clones with abnormal colony morphology and discovered unpredicted integration events via whole genome sequencing. These include (i) the relocation of the locus targeted for replacement to another chromosome (ii) co-integration of DNA from the E. coli plasmid host and (iii) the disruption of untargeted genes affecting colony morphology. Most of these events have not been reported so far in literature and present challenges for genetic engineering approaches in this yeast. Especially, the presence and independent activity of E. coli DNA elements in P. pastoris is of concern. In our study, we provide a deeper insight into these events and their potential origins. Steps preventing or reducing the risk for these phenomena are proposed and will help scientists working on genetic engineering of P. pastoris or similar non-conventional yeast to better understand and control clonal variability.

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Construction of new Pichia pastoris X-33 strains for production of lycopene and β-carotene

Cited by 78 publications

References 41 publications

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production

Improving industrial yeast strains: exploiting natural and artificial diversity

Non-canonical integration events in Pichia pastoris encountered during standard transformation analysed with genome sequencing

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