Synthetic Core Promoters as Universal Parts for Fine-Tuning Expression in Different Yeast Species

Abstract

Synthetic biology and metabolic engineering experiments frequently require the fine-tuning of gene expression to balance and optimize protein levels of regulators or metabolic enzymes. A key concept of synthetic biology is the development of modular parts that can be used in different contexts. Here, we have applied a computational multifactor design approach to generate de novo synthetic core promoters and 5′ untranslated regions (UTRs) for yeast cells. In contrast to upstream cis-regulatory modules (CRMs), core promoters are typically not subject to specific regulation, making them ideal engineering targets for gene expression fine-tuning. 112 synthetic core promoter sequences were designed on the basis of the sequence/function relationship of natural core promoters, nucleosome occupancy and the presence of short motifs. The synthetic core promoters were fused to the Pichia pastoris AOX1 CRM, and the resulting activity spanned more than a 200-fold range (0.3% to 70.6% of the wild type AOX1 level). The top-ten synthetic core promoters with highest activity were fused to six additional CRMs (three in P. pastoris and three in Saccharomyces cerevisiae). Inducible CRM constructs showed significantly higher activity than constitutive CRMs, reaching up to 176% of natural core promoters. Comparing the activity of the same synthetic core promoters fused to different CRMs revealed high correlations only for CRMs within the same organism. These data suggest that modularity is maintained to some extent but only within the same organism. Due to the conserved role of eukaryotic core promoters, this rational design concept may be transferred to other organisms as a generic engineering tool.

Engineered bidirectional promoters enable rapid multi-gene co-expression optimization

“…This vector contains integration sequences near the ARG4 locus and was linearized with Swa I to target insertion near the ARG4 locus, as had been well established for promoter characterizations in P. pastoris 30,39,41 . Also the following vectors described below were based on this vector backbone.…”

Effect of Plasmid Design and Type of Integration Event on Recombinant Protein Expression in Pichia pastoris

“…We aimed to investigate specific/nonspecific integration, hence we transformed small amounts of DNA to avoid the pronounced occurrence of multicopy integrations. Previous studies with reporter plasmids from the pPpT4 family (28)(29)(30) showed that 1 g of linearized vector DNA resulted in an even expression landscape, i.e., similarly expressing clones. Hence, we also used amounts equivalent to 1 g of the reference constructs from references 28-30.…”

“…While these integration events can have a profound influence on strain productivity and physiology, it remains partly unclear to what extent they occur, how they are influenced by expression cassette properties, and how they bias typical standard expression approaches in P. pastoris. For comparisons of different enzyme variants (e.g., from natural variants or protein engineering efforts) or promoter variants (e.g., to fine-tune expression [28][29][30]), it is necessary to perform multiple transformations for each clone expressing a different gene. Expression from the actual transformants used for comparisons may be biased by clonal variation to an unknown extent, possibly influencing the interpretation of the study if only a limited number of transformants are tested.…”

Engineering of alcohol dehydrogenase 2 hybrid‐promoter architectures in Pichia pastoris to enhance recombinant protein expression on ethanol

“…Promoter engineering includes approaches such as chimeric promoter design (Blazeck, Garg, Reed, & Alper, 2012;Blazeck, Liu, Redden, & Alper, 2011), random mutagenesis (Alper, Fischer, Nevoigt, & Stephanopoulos, 2005;Portela, Vogl, Ebner, Oliveira, & Glieder, 2018;Qin et al, 2011), modification of transcription factor binding sites (Ata et al, 2017;Hartner et al, 2008;Prielhofer et al, 2018), and synthetic promoter design (Curran et al, 2014;Portela et al, 2017;Redden & Alper, 2015;Vogl, Ruth, Pitzer, Kickenweiz, & Glieder, 2014). Synthetic biology extends on promoter engineering aiming to increase the transcription rate through designing regulatory circuit(s) by engineering binding of activating TF(s).…”