Saccharomyces cerevisiae Promoter Engineering before and during the Synthetic Biology Era

Abstract

Synthetic gene circuits are made of DNA sequences, referred to as transcription units, that communicate by exchanging proteins or RNA molecules. Proteins are, mostly, transcription factors that bind promoter sequences to modulate the expression of other molecules. Promoters are, therefore, key components in genetic circuits. In this review, we focus our attention on the construction of artificial promoters for the yeast S. cerevisiae, a popular chassis for gene circuits. We describe the initial techniques and achievements in promoter engineering that predated the start of the Synthetic Biology epoch of about 20 years. We present the main applications of synthetic promoters built via different methods and discuss the latest innovations in the wet-lab engineering of novel promoter sequences.

Keywords: Saccharomyces cerevisiae; gene expression; promoter; synthetic biology; transcription factors.

Figure 1

Promoters in S. cerevisiae. (A) General structure of S. cerevisiae promoters. With respect to the TATA box, UASs are located between 100 and 1400 nt upstream, whereas TSSs lie from 40 up to 120 nt downstream. (B) The structure of the yeast CYC1 promoter. Two UASs are present upstream of three TATA boxes, whose sequences are reported in the figure, that activate at least six TSSs [27].

Figure 2

Hybrid promoters. (A) The CYC1 core promoter was merged to different UASs to construct either constitutive or inducible (here galactose-responsive) hybrid promoters [39]. (B) The PGK1 core promoter was turned, as well, into a galactose-inducible hybrid promoter when preceded by the UAS from pGAL1. The substitution of UAS_GAL1 with androgen-responsive elements (AREs) led to a new hybrid promoter induced by testosterone [41].

Figure 3

Synthetic promoters obtained by modifying the sequence of native promoters. Common methods to alter promoter sequence and activity are: point mutations via error-prone PCR [11], removal of functionless sequences [49,50], intron insertion along the 5′UTR [51], and nucleosome removal [52].

Figure 4

Minimal synthetic promoter design. The 8-nt-long TATA box is separated by three 10-nt-long UASs through a random spacer (30 nt). The TSS is placed 30 nt downstream of the TATA box (the sequence Optimal was designed to prevent nucleosome formation). This synthetic promoter, which is almost as strong as pGPD, has an overall length of only 116 nt [22].

Figure 5

Promoters regulated by bacterial proteins. (A) Repressed promoters demand to place the operator of a bacterial protein between the TATA box and the TSS. (B) To build activated promoters, operators shall be placed upstream of the TATA box. Moreover, a bacterial protein behaves as a yeast activator upon fusion to an activation domain. (C) The CRISPR-dCas9 system can be used to either activate (as in the figure) or repress transcription. The sgRNA binds a complementary sequence, along the promoter, that is followed by a protospacer adjacent motif (PAM).