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.

Engineering Cell Polarization Improves Protein Production in Saccharomyces cerevisiae

“…α-Amylase with or without the SED1 sequence under the control of promoter TPI1 or TDH3 was inserted into pCPOT using the Gibson assembly method to form plasmids pCPOT1, pCPOT2, pCPOT3, and pCPOT4. To overexpress genes involved in cell polarization, a centromeric copy plasmid pPOT2 [ 28 ] with a UAR3 marker was used as the backbone, and the TDH3 promoter, CYC1 terminator, and genes of cell polarization were inserted into pPOT2 using the Gibson assembly method.…”

“…The following supporting information can be downloaded at: , Figure S1: The expression of eGFP driven by TPI1p and TDH3p ; Figure S2: Cell growth of surface-displaying (A) and secreting (B) strains with the genomic expression of BUD1 or BUD10 ; Figure S3: The effect of combinatorial modifications on the production of α-amylase and cell growth; Figure S4: The growth curve (A) and the maximum specific growth rate of engineered strains. Table S1: Strains used in this study [ 23 ]; Table S2: Plasmids used in this study [ 26 , 27 , 28 , 49 ]; Table S3: Primers used in this study.…”

Metabolic Engineering: Methodologies and Applications

“…156 In another study, several UASs were inserted in front of the core GAL1 promoter from S. cerevisiae, creating a library of synthetic galactose-inducible promoters with an expanded dynamic range, in which the best promoter had 2fold higher activity than the wild-type GAL1 promoter under a variety of different carbon sources. 157 Furthermore, the activity of promoter Pylb from B. subtilis was optimized by substituting −35, −10 core region and upstream sequence with consensus sequences, resulting in an engineered promoter that exhibited 195-fold increase in superfolded green fluorescent protein (GFP) expression compared to the wild-type promoter. 158 In addition to hybrid promoters, promoter engineering can be used to develop minimal promoters, which can reduce the DNA burden of the plasmid construction process.…”

Saccharomyces cerevisiae cell surface display technology: Strategies for improvement and applications

“…The upstream activating sequences (UASs) of different promoters were combined with core promoters to construct a synthetic promoter library. Using this approach, galactose-inducible promoters stronger than P GAL1 were successfully constructed by fusing UAS GAL1 to the core promoters of TDH3 and TEF1 ( Deng et al, 2021 ). The selection of efficient signal peptides helps to enhance protein secretion and increase the number of proteins displayed on the cell surface.…”