A synthetic promoter system for well-controlled protein expression with different carbon sources in Saccharomyces cerevisiae

Abstract

Background: Saccharomyces cerevisiae is an important synthetic biology chassis for microbial production of valuable molecules. Promoter engineering has been frequently applied to generate more synthetic promoters with a variety of defined characteristics in order to achieve a well-regulated genetic network for high production efficiency. Galactose-inducible (GAL) expression systems, composed of GAL promoters and multiple GAL regulators, have been widely used for protein overexpression and pathway construction in S. cerevisiae. However, the function of each element in synthetic promoters and how they interact with GAL regulators are not well known.

Results: Here, a library of synthetic GAL promoters demonstrate that upstream activating sequences (UASs) and core promoters have a synergistic relationship that determines the performance of each promoter under different carbon sources. We found that the strengths of synthetic GAL promoters could be fine-tuned by manipulating the sequence, number, and substitution of UASs. Core promoter replacement generated synthetic promoters with a twofold strength improvement compared with the GAL1 promoter under multiple different carbon sources in a strain with GAL1 and GAL80 engineering. These results represent an expansion of the classic GAL expression system with an increased dynamic range and a good tolerance of different carbon sources.

Conclusions: In this study, the effect of each element on synthetic GAL promoters has been evaluated and a series of well-controlled synthetic promoters are constructed. By studying the interaction of synthetic promoters and GAL regulators, synthetic promoters with an increased dynamic range under different carbon sources are created.

Keywords: Carbon sources; Protein expression; Saccharomyces cerevisiae; Synthetic promoter.

Fig. 1

UASs and their impact on GAL promoter strength in S. cerevisiae. a The architecture of the native GAL1 promoter. Upstream activating sequences (UAS_GAL1) and core promoter (cP_GAL1) are shown. U1, U2, U3, and U4 represented the four UASs of P_GAL1; M1 and M2 represent repressor Mig1p binding sites, which was responsible for glucose repression. b The sequence of UASs used in this study. U1-U4 and U4g are from ScP_GAL1 (S. cerevisiae, green), U5-U7 from SkP_GAL2 (S. kudriavzevii, purple) and U8 from ScP_GAL7 (S. cerevisiae, red). (c-d) The effect of UASs on the activities of synthetic promoters when cP_GAL1 (c) and cP_CYC1 (d) were used as core promoter. The activities of all synthetic promoters were tested after induction with 2% galactose for 24 h. Normalized fluorescence = Fluorescence intensity/OD₆₀₀. Data are mean ± SD (standard deviation) from three biological replicates

Fig. 2

Characterization of the synthetic promoters by core promoter replacement. The normalized fluorescence for constructs with only the core promoter (a) and with UAS_GAL1 fusion (b). The activities of all synthetic promoters were tested after cultivation with 2% glucose (grey) or galactose (orange) for 24 h. Data are mean ± SD from three biological replicates

Fig. 3

GAL80 deletion affected the activities of synthetic promoters under different carbon sources. a The regulation mechanism of GAL1 promoter under galactose and glucose condition. Gal4p: the transcriptional activator responsible for galactose induction; Gal80p: the repressor of Gal4p; Mig1p: the repressor that responds to glucose. WT: Wildtype strain; Δgal80: GAL80 deletion strain. b, c GAL80 deletion improved the activities of GAL promoters in the presence of 2% galactose (b) or 2% glucose (c). The promoter activities were continuously monitored for 36 h. d Glucose inhibition profile of promoters. Synthetic promoters were incubated with 2% raffinose with a glucose gradient from 0 to 2% for 5 h with initial OD₆₀₀ at 0.1. After inhibition, mean fluorescence intensity of cells was analyzed by flow cytometry. e Galactose induction profile of native and synthetic promoters. 2% raffinose was used as background carbon source with galactose gradient concentration from 0 to 2% for 5 h with initial OD600 at 0.1. After induction, mean fluorescence of intensity of cells was analyzed by flow cytometry. Data are mean ± SD from three biological replicates and the shadow patterns of the curve represents errors as standard deviation

Fig. 4

The effect of GAL1 and GAL80 double deletion on synthetic promoters. a Schematic diagram of galactose metabolism. GAL1: Galactokinase; GAL7: Galactose-1-phosphate uridyl transferase; GAL10: UDP-glucose-4-epimerase; GAL5: Phosphoglucomutase. UDP: Uridine diphosphate; EMP: Embden-Meyerhof-Parnas. In this study, GAL1 was deleted to block galactose metabolism. b Double deletion of GAL1 and GAL80 enhanced GAL promoter activity under glucose growth condition. c Double deletion of GAL1 and GAL80 improved synthetic promoter activity under raffinose or fructose conditions. d Galactose induction profile of promoters in GAL1/GAL80 double deletion strain. e Characterization of the synthetic promoter system under different carbon sources. Data are mean ± SD from three biological replicates and the shadow patterns of the curve represents errors as standard deviation

Fig. 5

The synthetic promoter system significantly improved β-glucosidase secretion (a) and surface display (b). SED1, a gene encoding yeast cell wall protein which is a commonly used surface-displayed system. Enzyme activities were measured under glucose or galactose growth conditions for 12 h and 24 h, respectively. + represents in the presence of GAL1 or GAL80;—represents the deletion of GAL1 or GAL80.The enzyme activity was quantified by pNPG assay and the data are mean ± SD from three biological replicates