In a Pichia

Culture of Pichia membranifaciens, from Tomas Linder.

In my previous post, I alluded to the revolutionary effect that DNA analysis had on the classification of bacteria. A similar thing happened for the study of yeasts. Previously, the taxonomy of yeasts (i.e. unicellular fungi) had suffered for the same reasons as bacterial taxonomy: a dearth of usable morphological features combined with uncertainty about the significance or otherwise of metabolic variations. With the availability of genetic information, the relations between yeast taxa became far easier to ascertain.

Needless to say, this lead to a significant shake-up in our understanding of individual yeast taxa. One of the harder-hit taxa was the genus Pichia, previously recognised as a large genus of close to 100 species. Molecular phylogenetic analyses showed that the various species of Pichia were widely scattered within the Saccharomycotina, a fungal clade that includes a large number of yeast species (including such familiar taxa as the brewer's or baker's yeast Saccharomyces cerevisiae). This probably did not come as a huge shock: part of the reason for Pichia's size was that it had not been very stringently defined. Members of this genus were characterised by multilateral budding (that is, buds could develop anywhere along the side of the yeast cell) on a narrow base. They could produce hyphae and/or pseudohyphae (except when they didn't), they might ferment sugars (except when they couldn't), and nitrate might be used as their sole source of nitrogen (except when it wasn't). Pichia spores might be hat-shaped, hemispheroidal or spherical, and they might or might not have a ledge or rim around the equator (Kurtzman 2011).

All of which adds up to a genus that probably tended to be defined as 'the genus that includes any yeast not belonging to these other genera'. In other words, the classic concept of a wastebasket taxon. As a result, the genus has been progressively pared down to a smaller array of species concentrated around the type, Pichia membranifaciens. This is a yeast commonly found as a spoilage organism on foods such as fruit or cheese. Among its other sins, it may grow as a film in the surface of wine, giving the wine an off taste. However, it's not all bad news: recently, P. membranifaciens has been studied as a potential biocontrol agent as it may produce a toxin that has an inhibitory effect on other contaminating fungi (Santos et al. 2009).

A growing culture of Komagataella pastoris, from here.

Somewhat unfortunately, one of the species to be expelled from Pichia is perhaps the best-studied: the yeast formerly known as Pichia pastoris (now supposed to be referred to as Komagataella pastoris though a quick Google Scholar search suggests that a great many authors are pretending that hasn't happened). This species can be grown using methanol as a sole carbon source, and protocols were developed in the 1970s for growing it in high densities at an industrial scale. The original plan was for it to be used for high-protein stock-feed using methanol produced as a by-product of oil refining (the modern agricultural industry has been described as the process of turning oil into food; this would have been a somewhat literal example). Rising oil prices rendered this proposal economically inviable but the P. pastoris industry was to have a reprieve, as the culture method was adopted as a means of producing active proteins (Cereghino & Cregg 2000). Procedures were developed for inserting foreign genes into the yeast, with the resulting pure methanol-based culture allowing the target protein to be generated at a greater rate and higher purity than might be possibly with a culture of the original source organism. Enzymes for laboratory studies, vaccines, medical products such as insulin: whatsitsname pastoris has been used in the production of them all.

REFERENCES

Cereghino, J. L., & J. M. Cregg. 2000. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiology Reviews 24: 45–66.

Kurtzman, C. P. 2011. Phylogeny of the ascomycetous yeasts and the renaming of Pichia anomala to Wickerhamomyces anomalus. Antonie van Leeuwenhoek 99: 13–23.

Santos, A., M. San Mauro, E. Bravo & D. Marquina. 2009. PMKT2, a new killer toxin from Pichia membranifaciens, and its promising biotechnological properties for control of the spoilage yeast Brettanomyces bruxellensis. Microbiology 155: 624–634.

Taxon of the Week: A Barely Pronounceable Yeast

"Yeast" is, admittedly, not one of the most tasteful-sounding words in the English language (it's right up there with "moist" and "sphagnum" in the category of words that sound crude without necessarily meaning anything offensive) but yeasts are an abundant component of our environment. They are most familiar as a vital component in making many foods (such as the baker's or brewer's yeast, Saccharomyces cerevisiae, in bread and beer) or less welcomely as opportunistic pathogens (such as Candida albicans, the cause of thrush). Technically speaking, yeasts are fungi that have abandoned the hyphal growth-form of most fungi and reverted to a more unicellular mode of life. Phylogenetically, yeasts are scattered throughout the fungal family tree, and it is this scattered distribution among more standard hyphal fungi that suggests that the yeast form has arisen multiple times independently.

The largest grouping of yeasts is the class Saccharomycetes. While morphologically fairly uniform (lets face it, there's only so much you can do with a relatively unornamented unicell), Saccharomycetes show a reasonable degree of diversity in biochemistry. The most recent (2006) edition of the Outline of Ascomycota (Eriksson, 2006 - the kind-of-sort-of official classification of ascomycete fungi) places all Saccharomycetes in a single order, the Saccharomycetales, but I personally prefer the admittedly less popular idea of dividing Saccharomycetes into a number of orders (as was done, for instance, by Schweigkofler et al., 2002) as I feel that this (a) better reflects said chemical diversity, and (b) reduces the redundant uninformativeness of having only a single order in a single class.

Within the Saccharomycetales (however you want to define it), the genus Metschnikowia contains spherical to cylindrical yeasts that reproduce by multilateral budding and produce elongate to club-shaped asci containing one or two needle-shaped spores each (Miller et al., 1967 - if you're wondering what an ascus is, feel free to look it up on Palaeos). Successive rounds of budding may lead to the formation of a pseudomycelium (the image at the top of the page shows Metschnikowia pulcherrima and comes from the INRA). The presence of only two instead of the expected four spores per ascus is due to the spores originating after meiosis stage I (segregation of homologous chromosomes) but before meiosis II (separation of sister chromatids). Meiosis II occurs as per normal after spore division, but one of the resultant nuclei is degraded in each cell (Marinoni et al., 2003). However, I must confess ignorance of what happens in those species with only a single spore per ascus.



Opened ascus of Metschnikowia zobellii showing needle-like spores. From Miller et al., 1967.

Metschnikowia was first discovered as a parasite of water fleas (Daphnia magna, small freshwater crustaceans) but various species and their Candida anamorphs* have since been found in association with a wide range of arthropods and plants (sometimes both - Candida kunwiensis, the anamorph of Metschnikowia kunwiensis - Brysch-Herzberg, 2004 - was isolated in a Korean laboratory from flowers of Ipomoea batatas (kumara) at the same time as German researchers independently isolated it from the bodies of pollinating bumblebees - Hong et al., 2003). Most are probably commensals on their hosts as opposed to parasites.

*An "anamorph" is the asexually reproducing form of a fungus, as opposed to the sexually reproducing teleomorph. Someday I shall write about the complicated and confusing system of double nomenclature attendant on fungi whereby asexual forms are classified separately from sexual forms, and the combination of history, theory and a certain degree of pragmatism that has lead to this system. Today, however, is not that day.

Recently certain Metschnikowia species have gained a bit of attention as potential biocontrol agents, as plant-associated forms have been demonstrated to inhibit the growth of other micro-organisms on their hosts. Metschnikowia yeasts may be very useful in inhibiting the growth of mould on harvested fruit, and at least one patent exists suggesting coating harvested fruit in a mixture containing isolated Metschnikowia culture.

REFERENCES

Brysch-Herzberg, M. 2004. Metschnikowia kunwiensis comb. nov., the teleomorph of Candida kunwiensis. FEMS Yeast Research 4: 605-607.

Eriksson, O. E. (ed.) 2006. Outline of Ascomycota - 2006. Myconet 12: 1-82.

Hong, S. G., K. S. Bae, M. Herzberg, A. Titze & M.-A. Lachance. 2003. Candida kunwiensis sp. nov., a yeast associated with flowers and bumblebees. International Journal of Systematic and Evolutionary Microbiology 53: 367-372.

Marinoni, G., J. Piskur & M. A. Lachance. 2003. Ascospores of large-spored Metschnikowia species are genuine meiotic products of these yeasts. FEMS Yeast Research 3 (1): 85-90.

Miller, M. W., E. R. Barker & J. I. Pitt. 1967. Ascospore numbers in Metschnikowia. Journal of Bacteriology 94 (1): 258-259.

Schweigkofler, W., K. Lopandic, O. Molnár & H. Prillinger. 2002. Analysis of phylogenetic relationships among Ascomycota with yeast phases using ribosomal DNA sequences and cell wall sugars. Organisms, Diversity and Evolution 2: 1-17.