EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae

Abstract

Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out.

Keywords: Saccharomyces cerevisiae; USER cloning; genome editing; integrative vectors; metabolic engineering.

Figure 1

Overview of the procedure for cloning genes into EasyClone vectors. Detailed protocol can be found as Supplementary Material (Fig. S1).

Figure 2

Plasmid construction. (a) Episomal vectors are based on the pESC vector (Agilent), where the multiple cloning sites and galactose-induced promoters were replaced by uracil excision-based cloning cassette. (b) Integrative vectors are based on the vectors described in Mikkelsen et al. (2012). The URA3 selection cassette flanked by direct repeats was exchanged with the different selective markers indicated, all of which are flanked by LoxP sites allowing Cre-mediated marker loop out. (c) Integration sites were organized on chromosomes X, XI, and XII. All integration sites (yellow boxes) are separated by either genetic elements that are essential for growth or by genes essential for maintaining wild-type growth rates (red boxes). Integration sites encircled in red provide good level of gene expression, have minimum risk of spontaneous loop out or rearrangements, and do not impair growth.

Figure 3

Experimental setup. CFP, YFP, and RFP were cloned into either episomal or integration vectors under the control of the strong TEF1 promoter. Saccharomyces cerevisiae was transformed with either three episomal or three integration vectors followed by flow cytometric analysis for presence of the three fluorescent proteins.

Figure 4

Flow cytometry on Saccharomyces cerevisiae strains co-expressing YFP, RFP, and CFP from either three episomal plasmids (top panel) or from triple integrations on the genome (bottom panel). The Log₁₀ mean value ± 15% for each color is indicated with a deep red vertical line and a light red shading, respectively.

Figure 5

3D plot of the fluorescence levels of cells expressing YFP, RFP, and CFP from either triple genomic integrations (left box) or from episomal plasmids (right box). Each dot in the plot represents a cell with a certain fluorescence signal from YFP, RFP, and CFP on the x-, y-, and z- axes, respectively. Red dots represent cells having fluorescence intensities for all three fluorophores being within Log₁₀ mean ± 15% for each color, and black dots represent cells with one or several fluorescence levels being outside of mentioned interval.