Protein expression and secretion in the yeast

Abstract

Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform. To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence. We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers. The wild-type URA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome. These vectors were used to express the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml(-1) in shake flasks, while a strain containing multiple integrations reached 2000 U ml(-1) in shake flasks, 11500 U ml(-1) in batch and 90500 U ml(-1) in fed batch. Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml(-1) in batch with a mono-copy lapA integrant and 28000 mU ml(-1) in fed batch with a multi-copy transformant.

Cloning, expression, and purification of insect (Sitophilus oryzae) alpha-amylase, able to digest granular starch, in Yarrowia lipolytica host

“…According to the characteristics provided by Madzak et al (2004), hp4d recombinant promoter is almost independent from environmental conditions and is able to drive a strong expression in virtually any medium, promoting growth-phase-dependent gene expression. It was found that hp4d-driven heterologous gene expression occurs mostly at the beginning of the stationary phase (Nicaud et al 2002), which we have also observed in our study. However, the increase in the pH value obviously hampered further accumulation of the recombinant α-amylase.…”

“…As demonstrated by Nicaud et al (2002), the heterologous gene copy number was a strong determinant of the amount of an enzyme produced, contributing to up to 8-fold increase in the enzyme activity, solely due to gene amplification. The technique applied in this study indicated the presence of 4 copies of the Amy1 gene per genome of Y. lipolytica 1.18 strain.…”

“…Although the ultimate values of the results cited above cannot be directly compared, due to different enzymatic activities being determined, it appears that the potential of Y. lipolytica was not fully explored in this study, and further research on improvement of the recombinant α-amylase production will be carried out. As demonstrated by Nicaud et al (2002), fed-batch cultures were highly beneficial for overexpression of heterologous proteins in Y. lipolytica strains, as illustrated by 7.9-fold or 11-fold increase in lipase and leucine amino peptidase production, respectively, when compared to batch cultures. In another study, rice α-amylase was efficiently produced in Y. lipolytica transformant using cyclic fed-batch culture mode, reaching high cell density culture (Chang et al 1998).…”

Robust succinic acid production from crude glycerol using engineered Yarrowia lipolytica

“…The auxotrophic strain Po1f (Leu − , Ura − ) and Po1g (Leu − ) derived from the wild type strain W29 (ATCC 20460) were kindly provided by Professor Catherine Madzak (Institut National de la Recherche Agronomique, AgroParisTech, France) [42, 43]. Po1f was used as a recipient of strain engineering and Po1g was used as the control strain.…”

Avaliação Da Produção De Inulinase Por Yarrowia Lipolytica

“…Além disso, excreta várias enzimas, como proteases, lipases, esterases e fosfatases, todas de grande interesse biotecnológico (Nicaud et al, 2002).…”