Systematic sequence engineering enhances the induction strength of the glucose-regulated GTH1 promoter of Komagataella phaffii.

The promoter of the high-affinity glucose transporter Gth1 (PGTH1) is tightly repressed on glucose and glycerol surplus, and strongly induced in glucose-limitation, thus enabling regulated methanol-free production processes in the yeast production host Komagataella phaffii. To further improve this promoter, an intertwined approach of nucleotide diversification through random and rational engineering was pursued. Random mutagenesis and fluorescence activated cell sorting of PGTH1 yielded five variants with enhanced induction strength.

Synthetic activation of yeast stress response improves secretion of recombinant proteins.

Yeasts, such as Pichia pastoris (syn Komagataella spp.), are particularly suitable expression systems for emerging classes of recombinant proteins. Among them, recombinant antibody fragments, such as single-chain variable fragments (scFv) and single-domain antibodies (VHH), are credible alternatives to monoclonal antibodies.

Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion.

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media.

Fine-Tuning of Transcription in Using dCas9 and RNA Scaffolds.

For metabolic engineering approaches, fast and reliable tools are required to precisely manipulate the expression of target genes. dCas9 can be fused 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 .

CRISPR/Cas9-Mediated Homology-Directed Genome Editing in Pichia pastoris.

State-of-the-art strain engineering techniques for the methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) include overexpression of endogenous and heterologous genes and deletion of host genes.

Superior protein titers in half the fermentation time: Promoter and process engineering for the glucose-regulated GTH1 promoter of Pichia pastoris.

Protein production in Pichia pastoris is often based on the methanol-inducible P promoter which drives the expression of the target gene. The use of methanol has major drawbacks, so there is a demand for alternative promoters with good induction properties such as the glucose-regulated P promoter which we reported recently. To further increase its potential, we investigated its regulation in more details by the screening of promoter variants harboring deletions and mutations.

GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris.

Background: State-of-the-art strain engineering techniques for the host Pichia pastoris (syn. Komagataella spp.) include overexpression of homologous and heterologous genes, and deletion of host genes.

Transcriptional engineering of the glyceraldehyde-3-phosphate dehydrogenase promoter for improved heterologous protein production in Pichia pastoris.

The constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (P ), which is one of the benchmark promoters of Pichia pastoris, was analyzed in terms of putative transcription factor binding sites. We constructed a synthetic library with distinct regulatory properties through deletion and duplication of these putative transcription factor binding sites and selected transcription factor (TF) genes were overexpressed or deleted to understand their roles on heterologous protein production. Using enhanced green fluorescent protein, an expression strength in a range between 0.

Implications of evolutionary engineering for growth and recombinant protein production in methanol-based growth media in the yeast Pichia pastoris.

Background: Pichia pastoris is a widely used eukaryotic expression host for recombinant protein production. Adaptive laboratory evolution (ALE) has been applied in a wide range of studies in order to improve strains for biotechnological purposes. In this context, the impact of long-term carbon source adaptation in P.

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level.

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol.

Pichia pastoris: protein production host and model organism for biomedical research.

Pichia pastoris is the most frequently used yeast system for heterologous protein production today. The last few years have seen several products based on this platform reach approval as biopharmaceutical drugs. Successful glycoengineering to humanize N-glycans is further fuelling this development.

Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris.

Background: Inducible high-level expression is favoured for recombinant protein production in Pichia pastoris. Therefore, novel regulated promoters are desired, ideally repressing heterologous gene expression during initial growth and enabling it in the production phase. In a typical large scale fed-batch culture repression is desired during the batch phase where cells grow on a surplus of e.