Selection of Heterologous Protein-Producing Strains in Yarrowia lipolytica
Yarrowia lipolytica has emerged as an alternative expression system for heterologous protein production and enzyme evolution. Several different expression systems dedicated for this species have been developed, ranging from the simple cloning of expression vectors to recently developed high-throughput methodologies using efficient cloning and assembly such as Gateway and Golden Gate strategies. The latter strategies, due to their modular character, enable multiple vector construction and the construction of expression cassettes containing different genes or a gene under different promoters of various strengths.
Here, we present the Golden Gate cloning strategy for the construction of multiple expression cassettes, the transformation into Y. lipolytica, and the selection of efficient enzyme-producing strains using an insect alpha-amylase as a reporter detected via a thermal cycler-based microassay.
Key wordsYarrowia lipolytica Heterologous protein Secretion Golden Gate Gateway Gene assembly Inducible promoter Targeting sequence High-throughput techniques
The experiments conducted by EC were financially supported by the Polish Ministry of Sciences and Higher Education (Project No IP2015 011074).
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