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Synergistic modular promoter and gene optimization to push cellulase secretion by Pichia pastoris beyond existing benchmarks

Paper ID Volume ID Publish Year Pages File Format Full-Text
23014 43409 2014 9 PDF Available
Title
Synergistic modular promoter and gene optimization to push cellulase secretion by Pichia pastoris beyond existing benchmarks
Abstract

•Systematic codon optimization of cellulase gene for secretory expression by Pichia pastoris.•Expression of 48 different genes with three different promoters.•Cellulase titers for the first time crossing the 15 g/l benchmark.•Synergies of codon optimization, multi-copy integration and AOX1 promoter variants.•Codon optimization can compensate for multi-copy integration.

Although successfully used for heterologous gene expression for more than twenty years, general knowledge about all factors influencing protein expression by Pichia pastoris is still lacking. For high titers of protein clones are optimized individually for each target protein. Optimization efforts in this study were focused on the DNA level, evaluating a set of 48 different individual synthetic genes (TrCBH2) coding for the same protein sequence of a Trichoderma reesei cellulase in combination with three different promoter sequences: PGAP (constitutive) and the synthetic AOX1 promoter variants PDeS (derepressed) and PEn (enhanced, inducible). Expression of active secreted enzyme varied from undetectable to ∼300% of the best known gene, as determined by secreted enzyme activity analyses of supernatants from 96 well plate and bioreactor cultivations. Finally, the best optimized gene and new promoters were combined to engineer highly productive P. pastoris CBH2 expression strains. Although no methanol was used for induction a final titer of more than 18 g/l of secreted protein was produced under controlled conditions in small scale bioreactor cultivations after 60–70 h of growth limiting glycerol feed. This is the highest concentration of secreted enzyme in P. pastoris published so far and single parts of the expression cassette could be independently optimized showing additive effects for improvements in protein production by P. pastoris.

Keywords
Pichia pastoris; Synonymous codon optimization; Synthetic promoters; Gene dosage; Cellulase secretion
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Synergistic modular promoter and gene optimization to push cellulase secretion by Pichia pastoris beyond existing benchmarks
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 191, 10 December 2014, Pages 187–195
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us