Enabling growth-decoupled recombinant protein production based on the methanol-free P promoter.

The current transition towards the circular bioeconomy requires a rational development of biorefineries to sustainably fulfill the present demands. The use of () can meet this challenge, since it has the capability to use crude glycerol as a carbon-source, a by-product from the biodiesel industry, while producing high- and low-added value products. Recombinant protein production (RPP) using has often been driven either by the methanol induced promoter (P ) and/or the constitutive promoter (P ).

CRISPR/Cas9 Tool Kit for Efficient and Targeted Insertion/Deletion Mutagenesis of the Komagataella phaffii (Pichia pastoris) Genome.

Efficient targeted genome engineering of Komagataella phaffii requires balanced expression of Cas9 nuclease and a target-specific guide RNA (gRNA). In addition, correct processing of the transcribed RNA to provide the designed gRNA as a target selective partner of targeted Cas9 protein for binding to genomic DNA is essential for efficient genome engineering. This method describes a step-by-step procedure and recommended tools for simple and efficient design of gRNAs to introduce insertions or deletions at targeted sites by CRISPR/Cas9-directed double-strand breaks, followed by error-prone nonhomologous end-joining repair.

Orthologous promoters from related methylotrophic yeasts surpass expression of endogenous promoters of Pichia pastoris.

Methylotrophic yeasts such as Komagataella phaffii (syn. Pichia pastoris, Pp), Hansenula polymorpha (Hp), Candida boidinii (Cb) and Pichia methanolica (Pm) are widely used protein production platforms. Typically, strong, tightly regulated promoters of genes coding for their methanol utilization (MUT) pathways are used to drive heterologous gene expression.

Methanol Independent Expression by Pichia Pastoris Employing De-repression Technologies.

Methanol is a well-established carbon source and inducer for efficient protein production employing Pichia pastoris (P. pastoris) as a host on micro-, lab and industrial scale. However, due to its toxicity and flammability, there is a desire to avoid methanol while maintaining the high productivity of P.

Publisher Correction: Engineered bidirectional promoters enable rapid multi-gene co-expression optimization.

The original version of this Article was updated after publication to add the ORCID ID of the author Thomas Vogl, which was inadvertently omitted, and to include a corrected version of the 'Description of Additional Supplementary Files' which originally lacked legends for each file.

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

Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization.