OPENPichia: licence-free Komagataella phaffii chassis strains and toolkit for protein expression.

The industrial yeast Komagataella phaffii (formerly named Pichia pastoris) is commonly used to synthesize recombinant proteins, many of which are used as human therapeutics or in food. However, the basic strain, named NRRL Y-11430, from which all commercial hosts are derived, is not available without restrictions on its use. Comparative genome sequencing leaves little doubt that NRRL Y-11430 is derived from a K. phaffii type strain deposited in the UC Davis Phaff Yeast Strain Collection in 1954. We analysed four equivalent type strains in several culture collections and identified the NCYC 2543 strain, from which we started to develop an open-access Pichia chassis strain that anyone can use to produce recombinant proteins to industry standards. NRRL Y-11430 is readily transformable, which we found to be due to a HOC1 open-reading-frame truncation that alters cell-wall mannan. We introduced the HOC1 open-reading-frame truncation into NCYC 2543, which increased the transformability and improved secretion of some but not all of our tested proteins. We provide our genome-sequenced type strain, the hoc1tr derivative that we named OPENPichia as well as a synthetic, modular expression vector toolkit under liberal end-user distribution licences as an unencumbered OPENPichia resource for the microbial biotechnology community.

Process development for an effective COVID-19 vaccine candidate harboring recombinant SARS-CoV-2 delta plus receptor binding domain produced by Pichia pastoris.

Recombinant protein-based SARS-CoV-2 vaccines are needed to fill the vaccine equity gap. Because protein-subunit based vaccines are easier and cheaper to produce and do not require special storage/transportation conditions, they are suitable for low-/middle-income countries. Here, we report our vaccine development studies with the receptor binding domain of the SARS-CoV-2 Delta Plus strain (RBD-DP) which caused increased hospitalizations compared to other variants. First, we expressed RBD-DP in the Pichia pastoris yeast system and upscaled it to a 5-L fermenter for production. After three-step purification, we obtained RBD-DP with > 95% purity from a protein yield of > 1 g/L of supernatant. Several biophysical and biochemical characterizations were performed to confirm its identity, stability, and functionality. Then, it was formulated in different contents with Alum and CpG for mice immunization. After three doses of immunization, IgG titers from sera reached to > 106 and most importantly it showed high T-cell responses which are required for an effective vaccine to prevent severe COVID-19 disease. A live neutralization test was performed with both the Wuhan strain (B.1.1.7) and Delta strain (B.1.617.2) and it showed high neutralization antibody content for both strains. A challenge study with SARS-CoV-2 infected K18-hACE2 transgenic mice showed good immunoprotective activity with no viruses in the lungs and no lung inflammation for all immunized mice.

Deletion analysis of Pichia pastoris alcohol dehydrogenase 2 (ADH2) promoter and development of synthetic promoters.

Pichia pastoris (Komagataella phaffii) is a non-conventional Crabtree-negative yeast with the capability of reaching very high cell densities in a fed-batch fermentation process. The alcohol dehydrogenase (ADH) genes of P. pastoris involved in ethanol metabolism were identified and were previously characterized. This work aimed to extend current knowledge of the regulation of the ADH2 promoter. To this end, we first determined the upstream activator (UAS) and repressor (URS) sequences of the promoter by deletion assays. Two upstream activator sites have been identified, positioned between -900 and -801 bp, and -284 and -108 bp upstream of the ADH2 transcription start site. The sequences positioned between -361 and -262 bp had a negative effect on the promoter activity and designated a repressor sequence (URS). We then demonstrated that Mxr1 (methanol expression regulator 1) transcription factor activates the ADH2 promoter through the direct interaction with UAS regions in response to ethanol. Furthermore, five different synthetic promoters were constructed by adding or deleting the regulatory sites. These synthetic promoters were tested for extracellular xylanase production at shake flask level by inducing with ethanol. These promoter variants improved the xylanase production ranging between 165% and 200% of the native promoter. The synthetic promoter 5 (SNT5) that displayed the highest activity was further evaluated at the fermenter scale. The modification in the promoter features might have several implications for industrial processes where decoupling the cell growth and product formation is advantageous.

Identification of major ADH genes in ethanol metabolism of Pichia pastoris.

The methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii) is a successful host widely used in recombinant protein production. The widespread use of a methanol-regulated alcohol oxidase 1 (AOX1) promoter for recombinant protein production has directed studies particularly about methanol metabolism in this yeast. Although there is comprehensive knowledge about methanol metabolism, there are other mechanisms in P. pastoris that have not been investigated yet, such as ethanol metabolism. The gene responsible for the consumption of ethanol ADH2 (XM_002491337, known as ADH3) was identified and characterized in our previous study. In this study, the ADH genes (XM_002489969, XM_002491163, XM_002493969) in P. pastoris genome were investigated to determine their roles in ethanol production by gene disruption analysis. We report that the ADH900 (XM_002491163) is the main gene responsible for ethanol production in P. pastoris. The ADH2 gene, previously identified as the only gene responsible for ethanol consumption, also plays a minor role in ethanol production in the absence of the ADH900 gene. The investigation of the carbon source regulation mechanism has also revealed that the ADH2 gene exhibit similar expression behaviours with ADH900 on glucose, glycerol, and methanol, however, it is strongly induced by ethanol.

Pichia pastoris Promoters.

The methylotrophic yeast Pichia pastoris (Komagataella phaffii) is used as an expression system for recombinant protein production for a variety of applications. It grows rapidly on inexpensive media containing methanol, glucose, glycerol, or ethanol as a sole carbon source. P. pastoris makes many posttranslational modifications and produces recombinant proteins either intracellularly or extracellularly. Because of these properties, P. pastoris has become a highly preferred host organism for biotechnology, pharmaceutical industry, and researchers.Recombinant protein production is usually performed under the control of the promoter of the alcohol oxidase gene I (AOX1). The AOX1 promoter is induced by methanol and repressed by glucose and ethanol. The regulation mechanisms of the AOX1 promoter have been studied in recent years. Another promoter used in recombinant protein production is derived from glyceraldehyde 3-phosphate dehydrogenase (GAP). It is a constitutive promoter. Recent literature showed that newly identified promoters of P. pastoris are promising as well, in addition to pAOX1 and pGAP.In this chapter, the regulation mechanisms of inducible pAOX1 and constitutive pGAP promoters are discussed. In addition, here we present an overview about the novel ADH3 promoter and alternative promoters of P. pastoris.

A quantitative polymerase chain reaction assay for the detection of equine (horse and donkey)-originated meat in processed bovine meat products.

Meat is one of the most important basic foodstuffs in human nutrition. Nowadays, adulteration and authenticity are common problems for meat products. Identification of meat species is important in terms of consumer protection and prevention of adulteration. There are different methods to determine adulteration of meat and meat products. These methods are histological controls, serological tests, and quantitative polymerase chain reaction. In this study, species identification and quantification analysis of meat and meat products were done by using horse-, donkey-, and bovine-specific primers with quantitative polymerase chain reaction method. Triple meat mixtures containing horse and donkey meat ranging from 0.1 to 50% levels were prepared within a bovine mixture for using species identification and quantification analysis. The method specificity was confirmed by melting curve analysis. In conclusion, quantitative polymerase chain reaction is an easy, rapid, and reliable method for meat species identification, and with this study an applicable method was developed for the detection and quantification of equine-originated meat in bovine meat products.