An outlook to sophisticated technologies and novel developments for metabolic regulation in the Saccharomyces cerevisiae expression system

Abstract

Saccharomyces cerevisiae is one of the most extensively used biosynthetic systems for the production of diverse bioproducts, especially biotherapeutics and recombinant proteins. Because the expression and insertion of foreign genes are always impaired by the endogenous factors of Saccharomyces cerevisiae and nonproductive procedures, various technologies have been developed to enhance the strength and efficiency of transcription and facilitate gene editing procedures. Thus, the limitations that block heterologous protein secretion have been overcome. Highly efficient promoters responsible for the initiation of transcription and the accurate regulation of expression have been developed that can be precisely regulated with synthetic promoters and double promoter expression systems. Appropriate codon optimization and harmonization for adaption to the genomic codon abundance of S. cerevisiae are expected to further improve the transcription and translation efficiency. Efficient and accurate translocation can be achieved by fusing a specifically designed signal peptide to an upstream foreign gene to facilitate the secretion of newly synthesized proteins. In addition to the widely applied promoter engineering technology and the clear mechanism of the endoplasmic reticulum secretory pathway, the innovative genome editing technique CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated system) and its derivative tools allow for more precise and efficient gene disruption, site-directed mutation, and foreign gene insertion. This review focuses on sophisticated engineering techniques and emerging genetic technologies developed for the accurate metabolic regulation of the S. cerevisiae expression system.

Keywords: CRISPR/Cas9; codon optimization; fusion partner; genomic engineering; metabolic regulation; promoter engineering.

FIGURE 1

Promising promoter expression systems. (A). The bidirectional promoter that is generated from yeast initiates transcription in both orientations as transcription start sites (TSSs) are located in the same nucleosome-depleted region (NDR) (Xu et al., 2009; Wei et al., 2011), which is responsible for the assembly and origination of gene transcription. Construction of a double promoter expression system by the fusion of two powerful promoters based on the bidirectional promoters from S. cerevisiae (Miller et al., 1998; Yuan et al., 2021). (B). An ideal consecutively double promoter expression system that can initiate the transcription of each gene under different treatments, (C). An ideal simultaneous double promoter expression system that simultaneously initiates the transcription of two genes under the same condition.

FIGURE 2

The mechanism by which IRE1, PERK, and BIP combat secretory demand. The stress sensor IRE1 is activated when the aggregation of unfolded peptides occurs in the ER lumen, leading to the initiation of the unfolded protein response (UPR) to aid the folding of the nascent peptides and ERAD to degrade the malfolded proteins. Once the UPR is activated, the expression level of the UPR target genes (e.g., resident chaperone Bip and lectin proteins, folding factors, etc.) is upregulated to buffer the ER stress.

FIGURE 3

Mechanism of CRISPR/Cas9 in Saccharomyces cerevisiae.

FIGURE 4

The mechanisms and applications of CRISPR activation(a), interference (i), and deletion (d). CRISPRa is expected to enhance both native and foreign gene expression by fusing an activation domain to the nuclease-deficient Cas protein (Shaw et al., 2022; Sugianto et al., 2023). By fusing a repression domain to the nuclease-deficient Cas protein, CRISPRi is applied to inhibit the metabolic competing pathways, leading to an increase in the biosynthesis of natural metabolites and chemicals (Ni et al., 2019; Lim et al., 2021; Morita et al., 2022). Overexpression of target genes is always not allowed in most microorganisms due to the endogenous defensive mechanism and complex metabolic networks, thus using CRISPRd to remove the competing pathways and factors limiting protein secretion always can increase target gene production (Wang et al., 2022; Yang et al., 2022).