Fine-Tuning of Transcription in Pichia pastoris Using dCas9 and RNA Scaffolds

Abstract

For metabolic engineering approaches, fast and reliable tools are required to precisely manipulate the expression of target genes. dCas9 can be fused via RNA scaffolds to trans-activator domains and thus regulate the gene expression when targeted to the promoter region of a gene. In this work we show that this strategy can be successfully implemented for the methylotrophic yeast Pichia pastoris . It is shown that the thiamine repressible promoter of THI11 can be activated under repression conditions using a scgRNA/dCas9 construct. Furthermore, the RIB1 gene required for riboflavin production was activated, leading to increased riboflavin production exceeding the riboflavin titers of a conventional RIB1 overexpression with a pGAP promoter.

Treatment with surfactants enables quantification of translational activity by O-propargyl-puromycin labelling in yeast

“…Cultivations were done either in YP (20 g L − 1 peptone and 10 g L − 1 yeast extract) or in synthetic minimal media (ASMv6 [ 51 ]). For the cultivation of gcn2 Δ, ASMv6 with less nitrogen was used, which contained only 2.50 g (NH 4 ) 2 HPO 4 , 0.32 g (NH 4 ) 2 SO 4 and 8 mL NH 4 OH (25%).…”

A programmable high-expression yeast platform responsive to user-defined signals

“…2A ). Since the initial report in 2013 ( 38 , 39 ), CRISPRi-mediated gene regulation has been applied successfully in various species, including P. pastoris ( 36 , 37 ). We designed a CRISPRiD with expression of a human codon-optimized dCas9 protein ( Streptococcus pyogenes Cas9 nuclease deficient of D10A and H840A) ( 38 ) from a strong constitutive promoter P GAP and giRNA (single-guide RNA for interference) from defined input promoters (P R ) in P. pastoris .…”

“…Because of the fact that P. pastoris was used as an expression host but not a model organism, limited research was conducted to understand its fundamental functions. Recently, the progress made in transcriptional regulation mechanisms (28)(29)(30)(31) and gene editing methods (32)(33)(34)(35)(36)(37) by our and other groups has made it possible to construct a fully engineered expression platform for this host. Moreover, as a breakthrough in biotechnology, clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene transcriptional regulation has attracted much attention because of its flexibility, high efficiency, and programmability (38).…”

Established tools and emerging trends for the production of recombinant proteins and metabolites in Pichia pastoris

“…Recent development and fine-tuning of the CRISPR/Cas9 technology in P. pastoris allows for marker free integration of expression cassettes (46)(47)(48). While the technology still has its limitations, a large amount of research has been directed in improving the technology leading to higher efficiencies, possibility of multiple genomic integrations as well as application of deactivated Cas9 (dCas9) for targeted gene interference (49).…”