Recombinant protein expression and metabolic engineering with the yeast pichia pastoris (K. phaffii)

BioGrammatics supports the production of proteins and metabolites using a eukaryotic microbial production system, primarily yeast, Pichia. We send reagents (strains, plasmids, medium) and provide guidance for wet lab work in your lab, as well as conduct contract services at our lab in Carlsbad, CA.

Every client is important, we have provided personalized solutions for scientists in academia and industry, from small, virtual start-ups, to large pharmaceutical companies, from a simple phone call to multi-year projects. Most of our strain engineering "tools" are entirely owned by BioGrammatics; however, we partner with Pichia labs all over the world to find solutions for making more protein, and better protein products. To support the Pichia community and expand the number of "Pichia People", BioGrammatics has co-hosted international Pichia meetings with Research Corporation Technologies (RCT) for decades (our first Pichia conference was in La Jolla CA, in 1985). Pichia 2026 in Mallorca, Spain, was wonderful; and we plan to host Pichia 2028 back in California again.

BioGrammatics primary focus is on strain development; however, we also support process development and scale up with 1.5 and 3 liter fermentors, MACS flow cytometry, tangential flow filtration (TFF) and chromatography systems, with a capacity to process up to 40 liters of supernatant. Broader process development, and scale up is performed with experienced partners (e.g. with higher thru-put process development, to production at the 100k+ liter level).

Our "Strain Construction and Expression Testing" tools and services include: Wild type and modified Pichia strains such as methanol utilization sensitive strains (aox1-, or aox1- & aox2-), protease deficient strains (pep4-, sub2-, prb1-, yps1-, kex2- and others), and auxotrophic mutant strains (ade-, ura- , his-, leu-, arg-). BioGrammatics Pichia strains can be provided as viable cells, and as cells competent to take up DNA (E-comp); our transformation efficiencies can be > 10E6/ug of transformed plasmid DNA to support production of libraries, or co-transformation processes. Our expression system design considers, the optimal codons, promoters, secretion signals, and other elements. We have:

• codon optimization algorithms
• multiple dominant selection systems, e.g. genes resistant to G418, Nourseothricin/Nat, hygromycin, zeocin and blasticidin.
• systems to manipulate the Pichia genome (cre/lox and flp/frt12), e.g. to Knock-out, or remove markers/"backbone" DNA (E. coli ori, etc.).
• different sites for targeted integration (T-sites, e.g. 12 sites known not to interfere with growth or transformation competency).
• Pichia Gold™ system, using our ade- and ura- strain for rapid library production, and screening "generally regarded as save" strains (GRAS).
• Genome and transcriptome sequencing services to support strain/process development and regulatory applications (genomic workbench program solutions).
• Robotic liquid handling and colony picking to support high through put (HTP) screening projects (Singer Instruments PIXL).
• A growing library of Helper Plasmids, to further support protein expression and usher proteins through the Pichia secretory pathway, including chaperones, transcription factors, protein disulfide isomerase (PDI) and other genes (many, under the control of multiple different promoters for optimal levels).
• a collection of Knock Out (KO) strains, 400 and rising.

www.biogrammatics.com