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Najla Gasmi Franck Fudalej Héla Kallel Jean-Marc Nicaud 《Applied microbiology and biotechnology》2011,89(1):109-119
In this work, we investigated the effect of codon bias and consensus sequence (CACA) at the translation initiation site on
the expression level of heterologous proteins in Yarrowia lipolytica; human interferon alpha 2b (hIFN-α2b) was studied as an example. A codon optimized hIFN-α2b gene was synthesized according
to the frequency of codon usage in Y. lipolytica. Both wild-type (IFN-wt) and optimized hIFN-α2b (IFN-op) genes were expressed under the control of a strong inducible promoter
acyl-co-enzyme A oxidase (POX2). Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2):
pre–proLIP2. Codon optimization increased protein production by 11-fold, whereas the insertion of CACA sequence upstream of
the initiation codon of IFN-op construct resulted in 16.5-fold increase of the expression level; this indicates that translational
efficiency plays an important part in the increase of hIFN-α2b production level. The replacement of the pre–proLIP2 signal
secretion with the LIP2 pre-region sequence followed by the X-Ala/X-Pro stretch but without the pro-region also increased
the secretion of the target protein by twofold, suggesting therefore that the LIP2 pro-region is not necessary for extracellular
secretion of small heterologous proteins in Yarrowia lipolytica. 相似文献
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Yarrowia lipolytica is widely used as a microbial producer of lipids and lipid derivatives. Here, we exploited this yeast’s potential to generate aromatic amino acids by developing chassis strains optimized for the production of phenylalanine, tyrosine and tryptophan. We engineered the shikimate pathway to overexpress a combination of Y. lipolytica and heterologous feedback-insensitive enzyme variants. Our best chassis strain displayed high levels of de novo Ehrlich metabolite production (up to 0.14 g l−1 in minimal growth medium), which represented a 93-fold increase compared to the wild-type strain (0.0015 g l−1). Production was further boosted to 0.48 g l−1 when glycerol, a low-cost carbon source, was used, concomitantly to high secretion of phenylalanine precursor (1 g l−1). Among these metabolites, 2-phenylethanol is of particular interest due to its rose-like flavour. We also established a production pathway for generating protodeoxyviolaceinic acid, a dye derived from tryptophan, in a chassis strain optimized for chorismate, the precursor of tryptophan. We have thus demonstrated that Y. lipolytica can serve as a platform for the sustainable de novo bio-production of high-value aromatic compounds, and we have greatly improved our understanding of the potential feedback-based regulation of the shikimate pathway in this yeast. 相似文献
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L. Gray N. Mackman J. -M. Nicaud I. B. Holland 《Molecular & general genetics : MGG》1986,205(1):127-133
Summary As a first step in the detailed analysis of the mechanism of secretion of haemolysin, we sought to identify sequences or domains
within haemolysin A (HlyA) that are essential for its secretion. For this purpose we examined the properties of a deletion
and Tn5 insertions into the region of theHlyA gene encoding the C-terminal part of the protein, since both of these are relatively simple to generate. We showed that removal
of 27 amino acids from the C-terminus of HlyA is sufficient to inhibit secretion drastically, although the residual polypeptide
is still haemolytically active. Cellular fractionation studies showed that haemolytic activity does not accumulate in large
amounts within the periplasmic space during normal secretion. More significantly, activity does not appear to accumulate within
this compartment when the export functionshlyB andhlyD are removed. These results are consistent with a mechanism in which interaction of the C-terminus of HlyA with the secretion
machinery, located in the inner membrane, is followed by direct transfer of haemolysin to the medium. 相似文献