A molecular approach to optimize hIFN α2b expression and secretion in <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

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.     

Evaluation of Bacillus subtilis SPB1 Lipopeptide Biosurfactant Toxicity Towards Mice

The in vivo potential toxicity of SPB1 lipopeptide biosurfactant towards male mice was evaluated. An LD50 value (defined as the dose required to kill half the members of a tested population) was determined to be about 475 mg/kg. Results show that daily administration of SPB1 biosurfactant did not show any death cases at any dose. Also, no unusual changes in behavior and no intoxication were observed during the 28 days period of treatment. Analysis proved that there were no significant differences in the serum glucose concentration levels, plasma total cholesterol, aspartate aminotransferase activity and bilirubin concentration among the control and experimental groups. In contrast, a little enhancement of alanine aminotransferase activities was observed for mice treated by the highest dose of the biosurfactant corresponding to 47.5 mg/kg of body weight which indicated the necrosis of hepatocyte. A daily intake of doses lower than 47.5 mg/kg of body weight had no significant adverse effect on hematological parameters and serum biochemical data. These results proved that SPB1 biosurfactant could be of a great interest as an additive in food, cosmetic and pharmaceuticals fields.