Cyanamide-inducible expression of homing nuclease I- Sce I for selectable marker removal and promoter characterisation in Saccharomyces cerevisiae

Abstract

In synthetic biology, microbial chassis including yeast Saccharomyces cerevisiae are iteratively engineered with increasing complexity and scale. Wet-lab genetic engineering tools are developed and optimised to facilitate strain construction but are often incompatible with each other due to shared regulatory elements, such as the galactose-inducible (GAL) promoter in S. cerevisiae. Here, we prototyped the cyanamide-induced ^I- SceI expression, which triggered double-strand DNA breaks (DSBs) for selectable marker removal. We further combined cyanamide-induced ^I- SceI-mediated DSB and maltose-induced MazF-mediated negative selection for plasmid-free in situ promoter substitution, which simplified the molecular cloning procedure for promoter characterisation. We then characterised three tetracycline-inducible promoters showing differential strength, a non-leaky β-estradiol-inducible promoter, cyanamide-inducible DDI2 promoter, bidirectional MAL32/MAL31 promoters, and five pairs of bidirectional GAL1/GAL10 promoters. Overall, alternative regulatory controls for genome engineering tools can be developed to facilitate genomic engineering for synthetic biology and metabolic engineering applications.

Keywords: Genetic circuit; Genome engineering; Metabolic engineering; Promoter engineering; Synthetic biology; Yeast engineering.

Fig. 1

Cyanamide-inducible expression of ^I−SceI facilitating selectable marker removal for iterative strain engineering. (A) Integration of cyanamide-inducible ^I−SceI cassette at ho locus and selectable marker removal. (B) Integration of tetracycline-inducible GAL80 module for tetracycline-mediated repression on galactose-inducible promoters and selectable marker removal. (C) Integration of the mevalonate pathway modules and selectable marker removal. Negative, the KanMX4 marker not being removed; Positive, the KanMX4 marker being removed. P_XXX, the promoter of gene XXX; T_XXX, the terminator of gene XXX. T_synth3, a synthetic minimal terminator. NLS, nucleus-localising sequence. TetR-HPV16L^NLS-TUP1, encoding a tetracycline-depressible repressor. TetO, TetR-binding sequence. Metabolic pathway genes for terpene precursor synthesis: ERG12, ERG8, MVD1, IDI1, EfmvaS, ACS2, and EfmvaE. GAL, the genes involving in galactose metabolism and regulation. M, Thermo Scientific GeneRuler DNA Ladder Mix (Thermo Scientific#SM0331). #1, yeast clones replicated on the YPD agar supplemented with 300 μg ml⁻¹ G418. The yeast colony PCR results are shown in DNA agarose gel images.

Fig. 2

Diagrams of in situ gene replacement design for plasmid-free cloning of heterologous promoters into yeast genome. MazF, encoding an E. coli interferase toxin.

Fig. 3

Characterisation of regulatory promoters in S. cerevisiae. (A) Tetracycline-inducible TEF1[4×TetO] promoters: truncating the upstream activation sequence (UAS) resulted in decreased expression outputs. TetR-HPV16L^NLS-Tup1, an artificial trans-repressor binds to TetO sequences. Tetracycline binding to TetR disassociates the TetR-TetO interaction. (B) Cyanamide-inducible DDI2 promoter. (3) Estrogen (β-estrodiol)-inducible promoters. Zif268-hER-VP16, an artificial trans-activator targeting to Z268 sequences. β-Estrodiol binding results in nuclear translocation of hER (human estrogen receptor). LmrA-SV40^NSL-Mig1^C, an artificial trans-repressor binding to the LmrO sequence. (D) Maltose-inducible promoters. (E) Galactose-inducible promoters. (D & E) The bold lines show the fluorescence level of yEGFP under the control of the TEF1 promoter, and dashed lines were of E2Crimson. Cells were grown in YNB-glucose media (A and C), YP-glucose media (B & D), YP-maltose media (D), or YNB-galactose media (E). Mean values ± standard deviation were shown (n = 3 biological replicates).