PatentDOI
Total synthesis and functional overexpression of a candida rugosa lip1 gene coding for a major industrial lipase
TLDR
In this paper, the LIP1 gene was completely synthesised with an optimised nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation, and the recombinant CRL was produced at high level and purity, accounting for 90-95 % of the secreted proteins.Abstract:
The dimorphic yeast Candida rugosa has an unusual codon-usage which hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Lipases produced by this yeast are extensively used in industrial bioconversions, but commercial lipase samples contain several different isoforms encoded by the LIP genes family. In a first laborious attempt the LIP1 gene, encoding the major isoform of the C. rugosa lipases (CRLs), was systematically modified by site-directed mutagenesis to gain functional expression in S. cerevisiae. As alternative approach, the gene (1688 bp) was completely synthesised with an optimised nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally overexpressed in Pichia pastoris. The recombinant CRL was produced at high level and purity, accounting for 90-95 % of the secreted proteins. The physical-chemical and catalytic properties of the recombinant lipase were compared with those of a commercial, non-recombinant C. rugosa lipase preparation.read more
Citations
More filters
Journal ArticleDOI
Heterologous protein expression in the methylotrophic yeast Pichia pastoris
TL;DR: This paper reviews the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced and describes new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.
Journal ArticleDOI
Production, purification, characterization, and applications of lipases.
TL;DR: The production, recovery, and use of microbial lipases are discussed; issues of enzyme kinetics, thermostability, and bioactivity are addressed; and production of recombinant lipases is detailed.
Journal ArticleDOI
Heterologous protein production using the Pichia pastoris expression system.
TL;DR: The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins and the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed.
Journal ArticleDOI
Recombinant protein expression in Pichia pastoris.
TL;DR: Major advances in the development of new strains and vectors, improved techniques, and the commercial availability of these tools coupled with a better understanding of the biology of Pichia species have led to this microbe’s value and power in commercial and research labs alike.
Journal ArticleDOI
Overview of fungal lipase: a review.
TL;DR: An overview regarding the fungal lipase production, purification, and application is discussed, and various industrial applications of lipase in pulp and paper, food, detergent, and textile industries are described.
References
More filters
Journal ArticleDOI
Codon usage patterns in Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Drosophila melanogaster and Homo sapiens; a review of the considerable within-species diversity
Paul M. Sharp,Elizabeth Cowe,Desmond G. Higgins,Denis C. Shields,Kenneth H. Wolfe,Frank Wright +5 more
TL;DR: These trends for codon usage are illustrated for six species whereCodon usage has been examined in detail, by presenting the pooled codon used for the 10% of genes at either end of the major trend.
Journal ArticleDOI
Relationship between sequence conservation and three‐dimensional structure in a large family of esterases, lipases, and related proteins
Miroslaw Cygler,Joseph D. Schrag,Joel L. Sussman,Michal Harel,Israel Silman,Mary K. Gentry,Bhupendra P. Doctor +6 more
TL;DR: An improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained, and 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved.
Journal ArticleDOI
Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae
Anthony J. Brake,James P Merryweather,Doris Coit,Ulrike Heberlein,Frank R. Masiarz,Guy T. Mullenbach,Mickey S. Urdea,Pablo Valenzuela,Philip J. Barr +8 more
TL;DR: Saccharomyces cerevisiae cells were transformed with plasmids containing hybrid genes in which the sequence encoding mature human epidermal growth factor was joined to sequences encoding the leader region (preprosegment) of the precursor of the yeast mating pheromone alpha-factor.
Journal ArticleDOI
Insights into interfacial activation from an open structure of Candida rugosa lipase.
Pawel Grochulski,Yunge Li,Joseph D. Schrag,Francois Bouthillier,P. Smith,D. Harrison,B. Rubin,Miroslaw Cygler +7 more
TL;DR: The structure of the Candida rugosa lipase determined at 2.06-A resolution reveals a conformation with a solvent-accessible active site and provides a new image of the substrate binding region and active site access, which is different from that inferred from theructure of the "closed" form of the G. candidum lipase.
Journal ArticleDOI
High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene.
TL;DR: In this paper, the tetanus toxin fragment C was induced to 27% of total cell protein or about 12 g/l of culture in high biomass fermentations and the level of expression was largely independent of the site of chromosomal integration of the gene (AOX1 or HIS4), the type of integrant (insertion or transplacement), and the methanol utilisation phenotype of the host strain (Mut+ or Muts).