Abstract
SummaryThe oleaginous yeast Yarrowia lipolytica is an established host for the bio‐based production of valuable compounds and an organism for which many genetic tools have been developed. However, to properly engineer Y. lipolytica and take full advantage of its potential, we need efficient, versatile, standardized and modular cloning tools. Here, we present a new modular Golden Gate toolkit for the one‐step assembly of three transcription units that includes a selective marker and sequences for genome integration. Perfectly suited to a combinatorial approach, it contains nine different validated promoters, including inducible promoters, which allows expression to be fine‐tuned. Moreover, this toolbox incorporates six different markers (three auxotrophic markers, two antibiotic‐resistance markers and one metabolic marker), which allows the fast sequential construction and transformation of multiple elements. In total, the toolbox contains 64 bricks, and it has been validated and characterized using three different fluorescent reporter proteins. Additionally, it was successfully used to assemble and integrate a three‐gene pathway allowing xylose utilization by Y. lipolytica. This toolbox provides a powerful new tool for rapidly engineering Y. lipolytica strains and is available to the community through Addgene.
Highlights
Yarrowia lipolytica is the most well-developed and wellresearched yeast in the domain of oleochemical production (Beopoulos et al, 2009; Ledesma-Amaro and Nicaud, 2016; Lazar et al, 2018)
Laboratory-scale production of ricinoleic acid has required the overexpression of three genes and the deletion of up to 10 genes, mainly due to the redundancy of genes involved in lipid metabolism in oleaginous microorganisms (Beopoulos et al, 2014)
We present the development and release of a new modular toolkit dedicated to Y. lipolytica that is based on the popular Golden Gate (GG) strategy (Engler et al, 2009)
Summary
Yarrowia lipolytica is the most well-developed and wellresearched yeast in the domain of oleochemical production (Beopoulos et al, 2009; Ledesma-Amaro and Nicaud, 2016; Lazar et al, 2018). It is considered to be a GRAS organism (Groenewald et al, 2013) and has an established history within the biotechnology industry It has been intensively used for various applications, ranging from biofuel to vaccine production (Madzak, 2018). For Y. lipolytica to produce omega-3 eicosapentaenoic acid at industrial levels, it has been necessary to integrate up to 30 copies of nine different genes and carry out one deletion (Xue et al, 2013). Laboratory-scale production of ricinoleic acid has required the overexpression of three genes and the deletion of up to 10 genes, mainly due to the redundancy of genes involved in lipid metabolism in oleaginous microorganisms (Beopoulos et al, 2014). Strain reconstruction requires multiple steps that sometimes lead to a final construct that does not display the expected phenotype or production yield because the transformation process results in the accumulation of potential trade-offs resulting from random insertions for example
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