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United States Department of Agriculture

Agricultural Research Service

Title: Expression of a lipid-inducible, self-regulating form of Yarrowia lipolytica lipase LIP2 in Saccharomyces cerevisiae

Authors
item Shockey, Jay
item Chapital, Dorselyn
item Gidda, Satinder -
item Mason, Catherine
item Davis, Gaynelle -
item Klasson, K Thomas
item Cao, Heping
item Mullen, Robert -
item Dyer, John

Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 19, 2011
Publication Date: December 15, 2011
Citation: Shockey, J., Chapital, D., Gidda, S., Mason, C., Davis, G., Klasson, K.T., Cao, H., Mullen, R., Dyer, J. 2011. Expression of a lipid-inducible, self-regulating form of Yarrowia lipolytica lipase LIP2 in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 92(6):1207-1217.

Interpretive Summary: Many American industries are often left with large excesses of commodities or byproducts from manufacturing processes. Soybean oil and soap stocks are two examples of products that sometimes go to waste or must be disposed of by expensive methods. This lost productivity drags down the profitability of the original product. The current manuscript describes the initial steps to create a baker’s yeast strain that take up leftover oils and other similar products and convert them to new high-value designer oils that can be used for other industrial manufacturing. The main focus is the use of baker’s yeast to produce a protein from another yeast species, Yarrowia lipolytica. This gene is produced internally, and pumped outside the cells to act on the oils that the yeast is grown in. The protein is turned on by exposure of the yeast to oils, and turned off when the oils are used up. This protein will be a valuable tool in the conversion of baker’s yeast into a safe, easily contained “minifactory” for production of useful high-value oil products.

Technical Abstract: The Yarrowia lipolytica lipase 2 gene (YlLIP2) was cloned into galactose- and fatty acid-inducible Saccharomyces cerevisiae expression vectors and used to generate yeast strains that secrete active LIP2 enzyme activity, as evidenced by results from gene expression analysis and tributyrin turbidity clearance assays. Alteration of the amino-terminal targeting sequences of the LIP2 protein produced differing levels of enzyme activity. Expression of the LIP2 gene in constructs driven by the Saccharomyces cerevisiae PEX11 promoter was induced by free fatty acids or triacylglycerol in the culture medium. These data provided evidence of the creation of a self-regulating positive control feedback loop that should allow the cells to upregulate LIP2 production when complex lipids are present in the media, but allow for downregulation when lipids are absent. Autonomous production of extracellular lipase is a necessary step towards the generation of yeast strains that can serve as biocatalysts for conversion of low-value triacylglycerols to novel lipid products.

Last Modified: 10/1/2014
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