Antonie van Leeuwenhoek

, Volume 99, Issue 1, pp 25–34

Pichia anomala: cell physiology and biotechnology relative to other yeasts

Authors

    • Yeast Research Group, School of Contemporary SciencesUniversity of Abertay Dundee
Original Paper

DOI: 10.1007/s10482-010-9491-8

Cite this article as:
Walker, G.M. Antonie van Leeuwenhoek (2011) 99: 25. doi:10.1007/s10482-010-9491-8

Abstract

Pichia anomala is a most interesting yeast species, from a number of environmental, industrial and medical aspects. This yeast has been isolated from very diverse natural habitats (e.g. in foods, insects, wastewaters etc.) and it also exhibits wide metabolic and physiological diversity. Some of the activities of P. anomala, particularly its antimicrobial action, make it a very attractive organism for biological control applications in the agri-food sectors of industry. Being a ‘robust’ organism, it additionally has potential to be exploited in bioremediation of environmental pollutants. This paper provides an overview of cell physiological characteristics (growth, metabolism, stress responses) and biotechnological potential (e.g. as a novel biocontrol agent) of P. anomala and compares such properties with other yeast species, notably Saccharomyces cerevisiae, which remains the most exploited industrial microorganism. We await further basic knowledge of P. anomala cell physiology and genetics prior to its fuller commercial exploitation, but the exciting biotechnological potential of this yeast is highlighted in this paper.

Keywords

Pichia anomalaPhysiologyBiotechnology

Introduction

Pichia spp. represent very interesting yeasts from both fundamental and applied perspectives. For example, from a cell biology viewpoint, they have proved most valuable in studies of organelle biogenesis, structure and function; and from an applied viewpoint, they have widespread biotechnological significance ranging from human therapeutic protein production, food fermentations, biocontrol agents and biofuel production. One particular species, Pichia anomala,1 exhibits great diversity with regard to its natural habitat, growth morphology, metabolism, stress-tolerance, and antimicrobial properties. It has been isolated from the following sources: flowering plants, fruit skins, insect intestinal tracts, human tissue and faeces, dairy and baked food products, contaminated oil, wastewaters, tree exudates, salted foods, and from the marine environment. This represents a wider range of habitats in Nature compared with the well-known Saccharomyces cerevisiae (brewer’s or baker’s yeast). P. anomala also differs from S. cerevisiae with regard to its mode of central carbon metabolism, in that it exhibits an insensitivity to glucose (i.e. is Crabtree negative). An interesting shared characteristic of both species relates to their killer yeast activity. However, do both yeasts kill other yeasts (or fungi) by the same mechanisms? The antifungal activity of P. anomala appears to be linked to cell wall hydrolysis (β glucanase-induced lysis) and/or to production of volatile metabolites (e.g. ethyl acetate), whereas S. cerevisae produces a killer toxin (e.g. K1 toxin peptide) that disrupts plasma membrane integrity. The antimycotic properties of P. anomala have led to this yeast being considered a valuable biocontrol agent against fungi of agronomical importance. Other potential biotechnological applications of P. anomala include environmental bioremediation, biopharmaceuticals and biofuels.

This paper reviews some of the unusual characteristics of P. anomala and will highlight these with reference to its potential biotechnological exploitation. Cell physiology of P. anomala will also be compared with better known yeast species, notably S. cerevisiae.

P. anomala in foods and beverages

P. anomala possesses several attributes with regard to food and beverage production and food/feed preservation, and some of these are summarised in Table 1. Benefits of P. anomala relate to its positive (flavour enhancing) roles in food and beverage fermentations and in food preservation. One particularly beneficial characteristic of P. anomala with regard to food and feedstuffs lies in its ability to liberate soluble phosphate from insoluble plant-derived phytic acid, which represents a form of non-utilisable phosphorus for monogastric animals. Phosphate solubilisation is facilitated by the activity of a thermostable phytase enzyme in P. anomala (Olstorpe et al. (2009a); Satyanarayana 2010).
Table 1

Important roles of Picha anomala in foods, feeds and beverages

Food/beverage applications

Examples of beneficial roles

References

Flavour enhancement

Volatile (e.g. esters) and savoury (e.g. nucleotides) flavours

Eun-Kyoung et al. (2003); Lee et al. (2003)

Food/feed bio-preservation

Biological control of fungi in fruits and cereals

Petersson and Schnürer (1995); Jijakli (2010); Olstorpe et al. (2009b)

Dairy fermentations

Probiotic effects

Mo et al. (2004)

Baking

Sourdough fermentations (not necessarily beneficial)

Daniel et al. (2010)

Winemaking

Volatile aromas, low alcohol wines, malic acid reduction

Ertin and Campbell (2001); Swangkeaw et al. (2009)

Enzymatic food/feed processing

Phytase, amylase, peptidase

Ray and Nanda (1996); Satyanarayana (2010)

Brewing

Anti-gushing potential in malting barley

Olstorpe et al. (2009a); Laitila et al. (2007; 2010)

There are some detrimental roles of P. anomala in relation to food production and storage (e.g. Deak 2008). As a food spoilage yeast, its contamination of yoghurts, bread, sugary cakes (Lanciotti et al. 1998), and wine (e.g. Rojas et al. 2001) can lead to taints commonly referred to as ‘chemical adulteration’. This may be due to the propensity of P. anomala to produce ethyl acetate (see below). In stored animal feeds such as silage, it can consume lactic acid (Jonsson and Pahlow 1984) and this may elevate pH thus reducing periods of safe feed storage.

Although there are some reports of nosocomial infections caused by P. anomala (e.g. Charkrabarti et al. 2001), with regard to food safety aspects P. anomala is classed at biosafety level 1 (De Hoog, 1996) and is considered safe for healthy individuals. In fact, P. anomala currently has QPS (qualified presumption of safety) status from EFSA (European Food Safety Authority) and this has benefits in terms of public perspectives of food biotechnology and acceptability of novel microorganisms in food (Sundh and Melin 2010).

P. anomala in the environment

With regard to the natural niche of P. anomala, this yeast has been isolated from very diverse sources. These include plants, soil, animals, insects, water, hospitals, and food (Table 2). In natural environments, P. anomala is believed to exist as a diploid yeast (Naumov et al. 2000). In relation to known growth extremes of P. anomala, this yeast has been found by Fredlund et al. (2002) to exhibit the following growth ranges: temperatures ranging from 3 to 37°C; pH values from 2 to 12; and osmotic conditions as low as aw 0.92 (NaCl) and 0.85 (glycerol). P. anomala is therefore quite ubiquitous in Nature and is able to tolerate a relatively wide range of environmental growth conditions. This contrasts with S. cerevisiae which is actually quite a rare yeast in natural environments (e.g. Martini 1993) and possesses a narrower range of growth extremes compared with P. anomala.
Table 2

Some diverse Picha anomala habitats

Isolation from:

References

Homo sapiens (human skin, faeces etc.)

Murphy et al. (1986); Mo et al. (2004)

Palm sugar

Nagatsuka et al. (2005)

Bread/fermenting dough

Lanciotti et al. (1998); Daniel et al. (2010)

Insects (e.g. Drosophila and malaria mosquito Anopheles)

Kurtzman (2001); Ricci et al. (2010)

Sea urchins

Kajikazawa et al. (2007)

Pharmaceutical wastewater

Recek et al. (2002)

Cereal silos and silage

Olstorpe (2008)

Oil-contaminated soil

El-Latif et al. (2006)

The sea

Wang et al. (2007)

Hospitals

Thuler et al. (1997); Chakrabarti et al. (2001); Reyes (2004)

P. anomala plays certain beneficial roles in the environment. For example, it exhibits (as do many other yeasts) saprophytic roles in the carbon cycle; it can help to alleviate pollution by bioremediation of recalcitrant chemicals/heavy metals in wastewaters; and it can act in the biological control of harmful microbes by combating biodeteriogenic fungi. Walker et al. (1995) showed that P. anomala was able to inhibit certain wood decay basidiomycetous fungi and it also displayed fungistatic acitivity against plant pathogenic fungi, including the causative agent of Dutch Elm disease, Ophiostoma novo ulmi. El-Latif Hesham et al. (2006) have shown that P. anomala can effectively degrade toxic chemicals such as the aromatic hydrocarbons naphthalene and benzopyrene, thus highlighting its potential role in environmental bioremediation processes (e.g. oil-contaminated industrial, terrestrial and marine environments).

P. anomala in industry

P. anomala has been shown to produce several metabolites that can be potentially exploited as biotechnological commodities (summarised in Table 3). These products range from bioremediation agents, biopharmaceuticals, biosurfactants, biofuels, biocides and biocontrol agents (BCAs). The latter are particularly attractive for large-scale applications in the agri-food sectors of industry, for example, to prevent fungal spoilage in fruit and cereals in post-harvest storage conditions (Jijakli 2010 and Olstorpe 2010, respectively).
Table 3

P. anomala products of biotechnological potential

Product

Potential application

Reference

Sophorolipids

Biosurfactants

Thaniyavarn et al. (2008)

γ-aminobutyric acid, GABA

Pharmaceuticals (GABA acts as a neurotransmitter, improves cerebral blood flow)

Kaku and Hagiwara (2008)

Volatile organic compounds

Fragrances

Buzzini et al. (2003)

Isobutanol

Biofuels

US Patent (2009)

Beverage starter culture

Low-alcohol wines; aromas

Ertin and Campbell (2001)

Panomycocin

Novel zymocidial agents

Izgü et al.(2006)

Antiviral agent

Influenza virus therapy

Conti et al. (2008)

Amoebicidal agent

Therapy of Acanthamoeba infections

Fiori et al. (2006)

Anti-Pneumocystis agent

Therapy of Pneumocystis carnii

Seguy et al. (1996)

Antibacterial agent

Therapy of Streptococcal infections

Conti et al. (2002)

Biocontrol/biopreservative

Stored grain, vines, fruit

Jijakli (2010); Olstorpe (2010)

Enzymes

Phytase, esterase, peptidase, β-glucosidase, amylase

Ray and Nanda (1996); Satyanarayana, T (2010)

Bioethanol (indirectly)

Maintenance of airtight stored grain (biofuels)

Passoth et al. (2009)

Antimicrobial activity of P. anomala

For a yeast, P. anomala exhibits very unusual broad spectrum antimicrobial properties. For example, it has been shown to suppress the growth of several fungi, yeast and bacterial species and viruses (Table 4).
Table 4

Summary of antimicrobial properties of P. anomala

Antimicrobial characteristic

Examples of microbes suppressed

References (examples)

Antifungal

Aspergillus, Botrytis, Penicillium, Fusarium

Jijakli and Lepoivre (1998); Masih et al. (2000); Jijakli (2010); Laitila et al. (2007)

Antizymal

Various yeasts, incl. C. albicans

Sawant et al. (1988)

Antibacterial

Erwinia spp.; Enterobacteriaceae; Streptococci

Polonelli and Morace (1986); Conti et al. (2002)

Antiviral

Influenza virus

Conti et al. (2008)

P. anomala is a killer yeast and a variety of killer toxins are known in P. anomala strains. With regard to the genetic basis of the killer phenomenon in P. anomala, the killer factor proteins are thought to be chromosomally inherited, unlike S. cerevisiae killer toxins (such as K1) which are encoded on double-stranded RNA virus-like extra-chromosomal elements, or Kluyveromyces lactis toxins which are encoded on linear DNA plasmids. P. anomala killer toxins also differ from those of other killer yeasts in that they exhibit diversity in terms of broad spectrum antimicrobial activity, variable molecular mass (e.g. from 3 to 300 kDa), and different pH and temperature optima (Passoth et al. 2006). Recently, De Ingeniis et al. (2009) have shown that a P. anomala killer toxin (peptide of ~8 kDa) possesses novel ubiquitin-like characteristics.

In addition to its activity as a classical killer yeast (i.e. displaying ability to kill other yeast species—Walker 1998), P. anomala also exhibits antifungal effects. For example, Masih et al.(2000) have shown that Botrytis cinerea (a major plant pathogen) displayed emptied hyphae in contact with P. anomala yeast cells. These workers showed that P. anomala protected vines (Vinus vinifera) against Botrytis infestation. Similar investigations by Mohamed and Saad (2009) have shown by scanning electron microscopy antagonistic effects of P. anomala cells interacting with the fungus Botryodiplodia theobromae, showing pitting and disruption in hyphal surfaces that were totally penetrated and killed by P. anomala. The antimycotic properties of P. anomala were originally described by Björnberg and Schnürer (1993) against grain-storage fungi, and whilst the mode of action is still unclear, it may be due to several factors acting singularly or in combination (Table 5). Jijakli and Lepoivre (1998) proposed that the suppression of B. cinerea by P. anomala is partly due to the activity of an exo-β-1,3-glucanase. Fredlund et al. (2004b) have shown that secretion of a volatile ester, namely ethyl acetate, by P. anomala may be responsible (possibly together with other metabolities such as ethanol) for its mode of antifungal activity, particularly against grain-storage moulds such as Penicillium spp. (Druvefors et al. 2005). Ester biosynthesis in P. anomala appears to differ from that in other yeast species such as S. cerevisiae with ethyl acetate being produced via an inverse esterase from acetate, rather than from acetyl CoA via ethanol acetyltransferase (Fredlund 2004). A recent study (Kurita 2008) has compared esterase activities in both S. cerevisiae and P. anomala. The secretion of ethyl acetate by P. anomala is an interesting (from an antifungal biocontrol perspective) and well-established characteristic, bearing in mind that the species was originally named Saccharomyces acetaethylicus by Beijerinick in 1892 (Lodder and Kreger-van Rij 1952).
Table 5

Antimycotic activity of P. anomala: candidate antifungal agents

Antifungal agents or modes of antifungal action

Likely relative contribution (ranging from ***** predominant to * lesser importance)

Killer toxins

*****

Hydrolytic enzymes (e.g. β-glucanase)

****

Volatile chemicals (e.g. ethyl acetate)

***

Nutrient competition

**

Media acidification

*

Carbon dioxide

*

Predation/mycoparasitism

*

Other antifungal agents

Unknown

In addition to its action against biodeteriogenic fungi in the agri-food areas, P. anomala also has potential applications in medical mycology. For example, P. anomala has long been recognised as possessing anti-Candida albicans activity (e.g. Hodgson et al. 1995; Polonelli et al. 1983; Sawant et al. (1988); Buzzini and Martini, 2001). Polonelli et al. (1990) were the first to show that P. anomala killer toxin was active in vivo in experimental mice. More recently, Izgü et al. (2006) have shown that the K5 killer toxin of P. anomala displays activity against selected dermatophytes (Microsporum spp. and Trichophyton spp). The K5 killer protein (named ‘panomycin’) was previously shown by Izgü and Altinbay (2004) to exhibit exo-β-1,3-glucanase activity. Magliani et al. (1997) and Polonelli et al. (2010) have discussed medical applications of P. anomala killer toxins, in particular the immunomodulatory activities of ‘antibiobodies’.

Stress tolerance of P. anomala

Figure 1 summarises major physicochemical and biotic stresses to which yeasts, including P. anomala, are exposed to when exploited in industry, or when used in environmental biocontrol applications. P. anomala responds to such stressful conditions by: accumulating trehalose and secreting ethyl acetate under oxygen limitation; synthesising glycerol (at start) and arabitol (at end) during salt stress; inducing biosynthesis of heat/cold shock proteins and stress enzymes; and altering structure of cell membranes. These stress responses are also observed in other yeasts (Walker and Van Dijck 2006), but arabitol accumulation in salt stressed cells of P. anomala (Bellinger and Larher 1988) is not an observable phenomenon in S. cerevisiae. Regarding hypoxic stress, Fredlund (2004) has proposed that trehalose accumulation in P. anomala is involved as a specific response to oxygen limitation. The ethyl acetate-secreting capabilities of P. anomala have been proposed by Fredlund (2004) to act as a stress protection measure, by preventing intracellular accumulation of toxic acetic acid (and at the same time suppressing the growth of competitor microbes). Although there are some conflicting reports of ethanol tolerance of P. anomala (e.g. Kalathenos et al. 1995; Stratford, 2006), this yeast is generally regarded as a resilient or ‘robust’ yeast (Fredlund et al. 2002; Passoth et al. 2006; Lahlali et al. 2008), and the stress adaptation mechanisms (both general and specific) it adopts must be very efficient. As testament to the inherent stress tolerance of P. anomala, Melin et al. (2005, 2007) and Mokiou and Magan (2008) have successfully preserved and stabilised this yeast at high viabilities in both liquid and desiccated formulations for environmental biocontrol applications.
https://static-content.springer.com/image/art%3A10.1007%2Fs10482-010-9491-8/MediaObjects/10482_2010_9491_Fig1_HTML.gif
Fig. 1

Major environmental stress factors impacting on P. anomala. Such stresses may be experienced by the yeast during growth in the natural environment or in industrial situations

Cell physiological aspects of P. anomala

The morphology of P. anomala exhibits diversity in terms of various cellular shapes with budding cells and branched pseudohyphae being evident in both liquid and solid culture media (Kurtzman 1998). As with other yeasts, it is possible that Quorum-sensing mechanisms may be involved in governing morphological changes and cell density related growth inhibition in this yeast (Walker 1998). Sexual reproduction in P. anomala is characterised by formation of hat-shaped spores (Kurtzman 1998).

P.anomala exhibits wide diversity regarding the metabolism of carbon and nitrogen sources (Table 6) and Fredlund et al. (2002) showed that this yeast can grow on selective media with solely starch and nitrate as C and N sources, respectively. It has also been reported to grow in vitamin-free media. Concerning oxygen requirements of P. anomala, this organism may be regarded as a facultative yeast, being able to grow in both oxygen-replete and oxygen-limited conditions. Respiratory growth of P. anomala is favoured under aerobic conditions and alcoholic fermentation is only induced by oxygen limitation. Fredlund et al. (2004a) reported that P. anomala exhibited growth rates of 0.22 and 0.056 h−1 and biomass yields of 0.59 and 0.11 g/g glucose under aerobic and anaerobic conditions, respectively. When shifted to oxygen limitation, P. anomala rapidly induced key fermentative enzymes (pyruvate decarboxylase and alcohol dehydrogenase—Fredlund et al. 2006) and also lowered flux through the pentose phosphate pathway. S. cerevisiae is also regarded as a facultative yeast, but in this organism, glucose (rather than oxygen) governs central carbon metabolism. Fredlund et al. (2004a) have shown that in aerobic conditions, pyruvate flux into P. anomala mitochondria is ~60% (c.f. only 7% under oxygen limitation) and that glucose consumption rate is faster under anaerobic conditions (4.6 c.f. 2.1 mmol/g biomass/h, respectively). All of this is demonstrative of the Pasteur Effect in P. anomala and an absence of the Crabtree Effect (Walker 1998). This represents a major difference with S. cerevisiae regarding glucose catabolism under conditions of altered oxygen and glucose availability. For example, if glucose concentrations are high, P. anomala will respire under aerobic conditions, unlike S. cerevisiae which is a Crabtree-positive yeast that will predominantly ferment high glucose levels, even in the presence of oxygen (due to catabolite repression/inactivation of mitochondrial oxidative functions). Only when P. anomala is transferred to O2-limited conditions will it concomitantly transfer to a fermentative mode of metabolism (Fredlund et al. 2004a).
Table 6

Carbon and nitrogen growth source diversity in P. anomala

Carbon sources

Nitrogen sources

Saccharides: hexoses (glucose, galactose, fructose); pentoses (arabinose, xylose); disaccharides (sucrose, lactose), polysaccharides (starch; β-glucans)

Nitrate

Alcohols: ethanol, glycerol

Nitrite

Organic acids: acetate, citrate, lactate, malate, succinate

Urea

Fatty acids: oleate, palmitate

L-glutamine

Aromatics: naphthalene, benzopyrene

L-histidine

The practical manifestation of these metabolic differences means that P. anomala can grow aerobically with high sugar concentrations at a relatively high growth rate and to higher cell densities than S. cerevisiae. The lack of a Crabtree Effect in P. anomala means that (unlike S. cerevisiae), there is no real necessity to keep sugar levels low and consequently no need to conduct fed-batch yeast propagation systems to control sugar feeding rates when attempting to maximise yeast biomass production.

Although these responses to oxygen and glucose represent major metabolic differences between S. cerevisiae and P. anomala, certainly similarities between the two yeasts do exist with regard to oxygen availability in that both species are unable to grow under strictly anaerobic conditions. This is because oxygen is required as an absolutely essential growth factor for sterol (ergosterol) and unsaturated fatty acid (oleic) synthesis (Walker 1998) during plasma membrane biogenesis in S. cerevisiae and P. anomala. Nevertheless, S. cerevisiae grows at similar rates under aerobic and sterol/fatty acid-supplemented anaerobic conditions, whilst P. anomala grows slower under the latter conditions. It is apparent that both yeasts respond to available oxygen in different ways. Table 7 summaries the major differences and similarities between P. anomala and S. cerevisiae in terms of metabolism and cell physiology.
Table 7

Cell physiological and other characteristic differences between P. anomala and S. cerevisiae

P. anomala

S. cerevisiae

Budding/pseudomycelia

Mainly budding

Crabtree negative

Crabtree positive

Predominantly respiratory

Predominantly fermentative

Oxygen sensitive

Glucose sensitive

Glucose uptake by H+ symport

Facilitated glucose diffusion

Malic acid utilisation

Malate only utilised with glucose

Several enzymes secreted

Few enzymes secreted

Antifungal action

Rarely antifungal (some strains)

High ethyl acetate

Low ethyl acetate

Widespread in Nature

Not widespread in Nature

Halotolerant

Not very halotolerant

Moderate ethanol tolerance

Ethanol tolerant

QPS (EFSA)

GRAS (FDA)

Opportunistic pathogen (some strains)

Doubtful opportunistic pathogenicity

Conclusions and future perspectives

Pichia anomala exhibits interesting and potentially exploitable physiological and metabolic characteristics. These include: morphological diversity (budding, pseudomycelial); stress tolerance (to low pH, high osmotic pressure, low O2, low aw); enzyme secretion (invertase, lipase, peptidase, amylase, phytase); nutritional diversity (range of C, N, and P sources); biodegradation (of polyaromatic hydrocarbons, naphthalene, benzopyrene,); Crabtree negativity (glucose insensitivity); antimicrobial activity (yeasts, fungi, bacteria, viruses); and production of potential commercial metabolites.

Although S. cerevisiae remains the world’s most exploited organism in industrial bioprocesses, other non-Saccharomyces yeasts like Pichia spp. have fantastic potential in biotechnology. Nevertheless, we still have much to learn about physiology and metabolism in non-Saccharomyces yeasts, including P. anomala, and enhancement of cell physiological knowledge in this yeast is a prerequisite for its fuller industrial exploitation. There are still several unresolved questions regarding carbon metabolism and its regulation in P. anomala. For example, it is conceivable that there is variability of the expression of the Crabtree effect in P. anomala strains, as previously demonstrated in Kluyveromyces lactis by Liti et al. (2001), and the underlying mechanisms of such metabolic phenomena and their practical significance require further research. Other areas of P. anomala research worthy of future investigation include: determination of modes of antimicrobial/antiviral action; and molecular understanding of the underlying reasons for stress tolerance. Stress tolerance and antimicrobial action are especially important P. anomala characteristics that can be exploited for future biotechnological applications.

Footnotes
1

Throughout this paper the species will be referred to as Pichia anomala, rather than Wickerhamomyces anomalus (see Kurtzman et al. 2008 and Kurtzman, 2010), mainly because it was the nomenclature used in the symposium from which this manuscript emanated (1st International Pichia anomala mini-Symposium).

 

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