Tailored UPRE2 variants for dynamic gene regulation in yeast

Abstract

Genetic elements are foundational in synthetic biology serving as vital building blocks. They enable programming host cells for efficient production of valuable chemicals and recombinant proteins. The unfolded protein response (UPR) is a stress pathway in which the transcription factor Hac1 interacts with the upstream unfolded protein response element (UPRE) of the promoter to restore endoplasmic reticulum (ER) homeostasis. Here, we created a UPRE2 mutant (UPRE2m) library. Several rounds of screening identified many elements with enhanced responsiveness and a wider dynamic range. The most active element m84 displayed a response activity 3.72 times higher than the native UPRE2. These potent elements are versatile and compatible with various promoters. Overexpression of HAC1 enhanced stress signal transduction, expanding the signal output range of UPRE2m. Through molecular modeling and site-directed mutagenesis, we pinpointed the DNA-binding residue Lys60 in Hac1(Hac1-K60). We also confirmed that UPRE2m exhibited a higher binding affinity to Hac1. This shed light on the mechanism underlying the Hac1-UPRE2m interaction. Importantly, applying UPRE2m for target gene regulation effectively increased both recombinant protein production and natural product synthesis. These genetic elements provide valuable tools for dynamically regulating gene expression in yeast cell factories.

Keywords: Hac1; UPRE2 variants; responsive promoter; unfolded protein response.

Fig. 1.

UPRE2 flanking sequences influence stress responses. (A) Schematic diagram of UPRE2m library creation and screening (created with BioRender.com). (B) Response of hybrid promoter TDH3p CORE with the complementary nucleotide alterations in UPRE2 flanking sequences to 5 mM DTT treatment in WT and Δhac1 strains. Data shown are mean values ± SDs of biological duplicates of single clones.

Fig. 2.

Analysis of UPRE2m sequences. (A) Response of hybrid promoter UPRE2m-TDH3p CORE upon 5 mM DTT treatment. (B) GC content in different UPRE2m groups. The dashed line indicates GC content of the native UPRE2. (C) Free energy of UPRE2m in different groups. In the box plots, whisker lengths correspond to the dataset’s minimum and maximum values. The upper and lower whiskers extend to the maximum and minimum values, respectively. The lines within the box plot indicate the median and quartiles. (D) Phylogenetic analysis of UPRE2m.

Fig. 3.

Response of the high-activity hybrid promoter UPRE2m-TDH3p CORE to DTT. (A) Response of hybrid promoter UPRE2m-TDH3p CORE to 5 mM DTT treatment. (B) Response of hybrid promoter UPRE2m-TDH3p CORE to varying DTT concentrations (0.1, 0.5, 1, 3, 5, and 6 mM). (C) Cell imaging of hybrid promoter UPRE2m-TDH3p CORE with high activity upon 5 mM DTT treatment. Data shown are mean values ± SDs of biological duplicates of single clones.

Fig. 4.

The amino acid residue K60 in the basic region of Hac1 is required for UPRE2m recognition. (A) Response of hybrid promoter UPRE2m-TDH3p CORE in transcription factor knockout strains. (B) Response of UPRE2m-TDH3p CORE in the HAC1 overexpression strain. (C) Key amino acids in Hac1’s functional domains. (D) Relative responsiveness of hybrid promoter UPRE2m-TDH3p CORE in the Hac1-E39A, Hac1-K60A, and Hac1-E39A K60A strains, with the wild-type Hac1-complemented strain’s response activity set as a baseline at 1. (E) Binding of GST-Hac1 to UPRE2m as determined by EMSA. Data shown are mean values ± SDs of biological duplicates of single clones.

Fig. 5.

Modular assembly of UPRE2m. (A and B) Response of the hybrid promoter 2×UPRE2m-TDH3p CORE to 5 mM DTT treatment. (C and D) The element m94-m84 was assembled with core regions of different promoters and their responses to 5 mM DTT treatment. Data shown are mean values ± SDs of biological duplicates of single clones.

Fig. 6.

Position effects of UPRE2m and UPRE2m application in the synthetic responsive promoter in S. cerevisiae. (A and B) Influence of m94 positioning on TDH3p activity. (C and D) Effect of m94 insertion upstream of the target promoter on α-amylase production. (E and F) Impact of promoters (G1: TDH3p CORE, G2: m94-m84-TDH3p CORE) on regulating tHMG1 expression and subsequent squalene production in Δhac1, WT, and HAC1 overexpression strains; strains without tHMG1 expression cassettes served as controls; strains were cultured in YPD medium. Data shown are mean values ± SDs of biological duplicates or triplicates of single clones. The statistical significance was determined by a two-tailed homoscedastic (equal variance) t test. *P < 0.05; **P < 0.01.