EasyClone: method for iterative chromosomal integration of multiple genes 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.

Engineering prokaryotic transcriptional activators as metabolite biosensors in yeast

“…EasyClone plasmids used in this work are described ref. 49. E. strain DH5α was used as a host for cloning and plasmid propagation.…”

“…S. cerevisiae cells were grown at 30 °C in synthetic complete medium as well as drop-out medium, and agar plates were prepared using pre-mixed drop-out powders (Sigma-Aldrich). Mineral medium was freshly prepared as described previously 49 . For all media containing diacids, 1.4 mM of the individual diacids were dissolved in mineral medium and pH adjusted to 4.5 before sterile filtration.…”

“…All gene fragments and correct overhangs for USER cloning were amplified by PCR using oligonucleotides listed and described in Supplementary Table 5. Unless otherwise stated, the amplified products were USER-cloned into EasyClone integrative plasmids 49 , and confirmed by sequencing.…”

Establishing a synthetic pathway for high-level production of 3-hydroxypropionic acid in Saccharomyces cerevisiae via β-alanine

“…cerevisiae CEN.PK strains were obtained from Peter Kötter (Johann Wolfgang Goethe-University Frankfurt, Germany). EasyClone plasmids used in this work are described in Jensen et al (2014) (Liao et al, 2010). The chemicals were from Sigma-Aldrich.…”

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

“…Correct insertions of TAL genes were verified by yeast colony PCR by primers pIntFwdU and XI-5 down_out. Alternatively, for plasmid-borne expression, 12 genes, including those encoding HaTAL1 and FjTAL optimized for S. cerevisiae (see Table S1 in the supplemental material), were amplified using the oligonucleotide also listed in Table 1 and inserted by uracil excision into the vector pCfB132 together with the PPGK1 promoter amplified by primers PPGK1_fw and PPGK1_rv (49). The finished plasmids were transformed into S. cerevisiae CEN.PK102-5B selecting for growth on synthetic dropout medium plates lacking uracil.…”