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  • A short history of Pichia pastoris

    The two distinct, but related yeast species known today colloquially as Pichia pastoris were both isolated from the wild during the first half of the twentieth century. Unlike Saccharomyces cerevisiae and Schizosaccharomyces pombe, there is no recorded historical use of Pichia pastoris by humans. In fact, based on the two independent isolates from southern France and northern California, it is likely that the species were found in an attempt to identify contaminating wild yeasts that were competing against productive viniculture strains. The genus Pichia was originally established by Emil Hansen in 1904 in honor of the nineteenth century scientist P. Pichi. In 1919, Guilliermond isolated a wild yeast from a French chestnut tree. This isolate, however, was not initially categorized under the genus Pichia, but rather classified as Zygosaccharomyces pastori. As scientists began to better understand yeast mating, conjugation and spore formation, Zygosaccharomyces pastori was eventually reclassified as Saccharomyces pastori. By mid-twentieth century taxonomic criteria, several yeast isolates from the Yosemite region of California were used by Herman Phaff to establish a new Pichia species, Pichia pastoris. Since Phaff, using 1940's technology, was unable to distinguish between his new species and the Saccharomyces pastori isolate of Guilliermond, the older species was merged into the newer one, resulting in two yeasts that had evolved in distinct environments being classified as a single species.

    During the 1960s it was discovered that some yeast species could grow on methanol as their sole carbon and energy source. Phillips Petroleum started a commercial development program to chemically oxidize methane gas and then utilize the resulting methanol to generate animal feed grade single–cell protein by yeast fermentation. A variety of yeast species known at the time to grow on methanol were initially tried, but the program eventually settled on Pichia pastoris for commercial scale–up. Phillips was successful with their fermentation process development, generating the capacity to produce Pichia pastoris at >100,000 liter fermentation scale at cell densities approaching 500 grams of wet cell pellet / liter. As part of the patent application process, strains that had originally been isolated by Guilliermond and Phaff were deposited in the NRRL yeast culture collection with new strain numbers. Unfortunately for Phillips, the increases in natural gas costs during the 1970s resulted in the program becoming economically unfeasible as a simple single–cell protein process. The development of molecular techniques to manipulate yeast genomes during the 1980s allowed Phillips to rethink their approach, and they initiated a research program to utilize Pichia pastoris as a heterologous protein expression host, with the goal of producing animal feed grade single–cell protein that contained recombinant growth factors–significantly increasing the value of the Pichia biomass.

    Phillips funded research at the Salk Institute BioTechnology/Industrial Associates (SIBIA, a commercial spin–out of The Salk Institute in La Jolla, CA) to develop the molecular biology tools necessary for heterologous protein expression in Pichia. The methanol assimilation / dissimilation pathway provided excellent candidates for regulated promoters that could be used for protein expression. In particular, alcohol oxidase, a peroxisomal enzyme responsible for converting methanol into formaldehyde, was essentially absent from cells when grown on glucose or glycerol but induced to 30% of total cell protein when Pichia was grown on methanol. By the late 1980s, vector constructs had been developed that allowed tightly regulated expression of heterologous proteins from the alcohol oxidase (AOX1) promoter (see Tschopp et al., 1987). Pichia has proved especially useful for the expression of human secretory proteins, since the fundamental mechanisms of protein secretion are highly conserved from lower eukaryotes such as Pichia through to plants and animals.

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