A modular Golden Gate toolkit for Yarrowia lipolytica synthetic biology

Abstract

The 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.

Figure 1

Schematic drawing of the 3‐TU GG assembly with letters indicating the 4‐nt overhang flanking each GG brick. The sequences of each 4‐nt overhang are available in Fig. S1. InsUp and InsDown: sequences for insertion in the genome; Prom: promoter; Ter: terminator.

Figure 2

Expression associated with the nine promoters using RedStarII as a reporter gene in position 1 (first TU). A. Constitutive promoters, using glucose as the carbon source. B. Inducible promoters, using glucose (blue bars) and erythritol (orange bars) as the carbon sources. Promoters were tested on the strain JMY1212, and a construct‐free strain (JMY1350; Table S1) was the control. The values correspond to the mean of two independent clones that randomly integrated each construct. Error bars represent standard deviations.

Figure 3

Impact of different terminators on gene expression levels using RedStarII as a reporter gene under the pTEF promoter. The bars correspond to mean specific red fluorescence (position 1). The values correspond to the mean of five to seven independent clones that randomly integrated each construction. JMY1212 strain was used. Error bars represent standard deviations.

Figure 4

Examples of successful integration at the locus sites. A. Detection of lipase activity on a tributyrin plate. When the colonies were surrounded by a clear zone, they were capable of lipase production. The GGA was used to transform a JMY195 strain, and the results were compared with those for the wild type, W29, and the lipase defective strain, JMY1212. B. Detection of glycogen synthase activity using Lugol's solution. When yellow wells were present, it indicated a defect in glycogen synthase, which is a characteristic of the Δgsy1 mutant strain (Bhutada et al., 2017). In this case, the JMY1212 strain was used to test the integration at gsy locus. These results were compared with those for the wild type, W29. C. Impact of integration site on gene expression levels using RedStarII as a reporter gene. The bars correspond to mean specific red fluorescence. The values correspond to the mean of four independent clones that have a correct locus integration. Error bars represent standard deviations.

Figure 5

A. Mean specific fluorescence of strains expressing a single fluorescent protein – RedStarII, YFP or mTurquoise – in position 1 of a 1‐TU GG vector or of strains expressing all three fluorescent proteins in a 3‐TU GG vector. In G1G2G3, RedStarII is in position 1, YFP is in position 2, and mTurquoise is in position 3. JMY1212 was the control strain. The values correspond to the average for 8–10 independent clones that randomly integrated each construct. Error bars represent standard deviations. B. Schematic of the construct used, all of them were transformed into JMY1212 strain.

Figure 6

Synthetic pathway assembly using the Golden Gate toolkit and the resulting expression in Y. lipolytica. A. Schematic representation of a three‐gene assembly, composed of Y. lipolytica xylulokinase (ylXK), Y. lipolytica xylitol dehydrogenase (ylXDH) and Y. lipolytica xylose reductase (ylXR) genes, that allows Y. lipolytica to grow on xylose. B. Y. lipolytica clones after transformation with the xylose cassette that were grown in a plate containing only xylose as the carbon source; clones were grown in a plate containing glucose as the carbon source for the control. The xylose cassette was correctly expressed in 79% of JMY1212 transformants. WT: wild type. Xyl: xylose. Glc: glucose. Ctrol+: positive control, which was the Y. lipolytica strain expressing the three genes developed via the standard cloning method and whose xylose utilization was verified (Ledesma‐Amaro et al., 2016).