ArticleScientific reports2017
Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris.
Article in Scientific reports, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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15 citing papers in PubMed, 35 citations in OpenAlex.
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- A novel CRISPR/Cas9 system with high genomic editing efficiency and recyclable auxotrophic selective marker for multiple-step metabolic rewriting inSynthetic and systems biotechnology · 2023Article
- Current advances ofFrontiers in microbiology · 2022Review
- Strains and Molecular Tools for Recombinant Protein Production in Pichia pastoris.Methods in molecular biology (Clifton, N.J.) · 2022Article
- Fhl1p protein, a positive transcription factor in Pichia pastoris, enhances the expression of recombinant proteins.Microbial cell factories · 2019Article
- High production of valencene in Saccharomyces cerevisiae through metabolic engineering.Microbial cell factories · 2019Article
- Advances in targeting and heterologous expression of genes involved in the synthesis of fungal secondary metabolites.RSC advances · 2019Review
- CRISPR-Cas9-mediated genomic multiloci integration in Pichia pastoris.Microbial cell factories · 2019Article
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Authors and funding
9 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Pichia pastoris is a widely used heterologous protein production workhorse. However, with its multiple genetic modifications to solve bottlenecks for heterologous protein productivity, P. pastoris lacks selectable markers. Existing selectable marker recycling plasmids have drawbacks (e.g., slow growth and conditional lethality). Here, zeocin-resistance marker recycling vectors were constructed using the Cre/loxP recombination system. The vectors were used to (i) knock in heterologous phytase, xylanase and lipase expression cassettes, (ii) increase the phytase, xylanase and lipase gene copy number to 13, 5, and 5, respectively, with vector introduction and (iii) engineer the secretion pathway by co-overexpressing secretion helper factors (Sly1p and Sec1p) without introducing selectable markers, giving a phytase field of 0.833 g/L. The vectors allow selectable marker recycling and would be a useful tool to engineer P. pastoris for high heterologous protein productivity.
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