Engineering an Efficient Expression Using Heterologous GAL Promoters and Transcriptional Activators in Saccharomyces cerevisiae

Abstract

Galactose-inducible (GAL) promoters have been widely used in metabolic engineering in Saccharomyces cerevisiae for production of valuable products. Endogenous GAL promoters and GAL transcription factors have often been engineered to improve GAL promoter activities. Heterologous GAL promoters and GAL activator (Gal4p-like transcriptional activators), although existing in other yeasts or fungi, have not been well explored. In this study, we comprehensively characterized the activation effects of Gal4p activators from different yeasts or fungi on a variant of GAL promoters. Overexpressing endogenous Gal4p driven by PHHF1 increased the activities of native PGAL1 and heterologous P SkGAL2 by 131.20% and 72.45%, respectively. Furthermore, eight transcriptional activators from different organisms were characterized and most of them exhibited functions that were consistent with ScGal4p. Expression of KlLac9p from Kluyveromyces lactis further increased the activity of P ScGAL1 and P SkGAL2 by 41.56% and 100.63%, respectively, compared to ScGal4p expression, and was able to evade Gal80p inhibition. This optimized GAL expression system can be used to increase the production of β-carotene by 9.02-fold in S. cerevisiae. Our study demonstrated that a combination of heterologous transcriptional activators and GAL promoters provided novel insights into the optimization of the GAL expression system.

Hybrid Boolean gates show that Cas12c controls transcription activation effectively in the yeast S. cerevisiae

“…This is the simplest design of a hybrid gate since it demands a single TU to process two different input signals. The promoter choice fell on GAL1 (pGAL1, induced by galactose), CUP1 (pCUP1, induced by copper), and MET25 (pMET25, repressed by methionine) promoter ( Kusen et al, 2017 ; Møller et al, 2017 ; Wu et al, 2023 ). pGAL1 and pCUP1 alone mimic a YES (or buffer) gate (i.e., the output is ‘1’ when the input is ‘1’ too), whereas pMET25 and tc1/tc2 are NOT gates (i.e., the output is ‘1’ when the input is ‘0’).…”

Two-Phase Fermentation Systems for Microbial Production of Plant-Derived Terpenes

“…This is because the introduction of the inducer activates specific proteins within the cells, initiating the biosynthesis of the product [ 296 ]. Similarly, in the S. cerevisiae system, organic solvents are introduced along with inducers, such as galactose, required for the GAL promoters [ 297 ], past a certain level of cell proliferation. In some cases, if the constitutive promoters are employed in S. cerevisiae , the addition of organic solvents is timed to coincide with a particular OD value when product biosynthesis commences.…”