Pichia membranifaciens

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1. Classification

a. Higher order taxa

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2. Description and significance

Pichia membranifaciens is a yeast that is commonly found on various crops and plants and is known to be involved in alcohol fermentation[2]. It also has an anamorph species called Candida valida and a sister species called Pichia manchuria [3]. P. membranifaciens prevents food spoilage and contamination with its capability to kill competitor microbes that spoil commercial crops and fermented liquids like wine[4]. Due to its osmotolerance, its killer activity towards other yeasts and microbes it competes with, along with its fermentation capabilities, it has recently garnered interest in the scientific community as a potential non-chemical fungicide[2],[5],[6]. Additionally, the yeast’s ability to form bioflocculants in wastewater and capability to ferment sugars into ethanol at a high capacity are currently being investigated as potential affordable and sustainable biological solutions to the global water and energy crisis[7],[8].

3. Genome structure

Pichia membranifaciens is reported to have somewhere between 2-8 chromosomes[3]. The genome size is 11.58 mega base pairs and 279 contigs[9]. Though very limited research about the genomics of P. membranifaciens has been done, some genes have been isolated such as its acetate resistance genes[10]. P. membranifaciens lack genes encoding alcohol oxidases and dihydroxyacetone synthases, inhibiting their ability to metabolize methanol[11]. P. membranifaciens killer toxins, PMKT and PMKT2 are encoded by the genome in strains CYC 1106 and CYC 1086 respectively[12].

4. Cell structure

The cell shape of Pichia membranifaciens is ovoidal and cylindrical, and their filaments are called pseudohyphae[4]. Their colony has been observed to be creamy or yellow in color with smooth and tube-like appearances[4]. The cell wall of P. membranifaciens are primarily comprised of free and protein linked carbohydrates[13]. These cell wall components include (1→3)-β-d-glucans with (1→6)-β-linked branches and a mannoprotein, (1→6)-β-d-glucans with (1→3)-β-linked branches and chitin[13]. These cell surface polysaccharides serve as receptors for proteins as well as for other bacteria, viruses, and toxins that determine cell distribution and turnover[13].

5. Metabolic processes

Pichia membranifaciens carries out oxidative metabolism on the surface of wine and produces organic acids, acetaldehyde, ethyl acetate, and isoamyl acetate[10]. To aid in oxidative metabolism, P. membranifaciens possesses a coenzyme called Q7 which is necessary for ubiquinone synthesis and therefore for respiration[14]. The respiration pathway that this fungal species partakes in is the Cyanide Resistant Respiration (CRR), which is a common metabolic pathway in yeasts[15]. Additionally, P. membranifaciens does not utilize the lactic acid that is produced by the bacteria around it (which helps keep the environment acidic), but does utilize a substance called oleuropein, a phenolic substance dominant in olives[16]. Like many other yeasts, P. membranifaciens also secretes killer toxins to eliminate competing microbes in the environment[5]. These killer toxins, named Pichia membranifaciens killer toxin (PMKT) and PMKT2, are known to eliminate fungi such as Candida boidinii, Botrytis cinerea, Brettanomyces bruxellensis, etc. that are sensitive to these toxins[5],[6],[12],[13].

6. Ecology

P. membranifaciens can be found in a various types of environments. While Pichia membranifaciens are most notably known for creating biofilms on various alcohol products, they can also be found in fruit skins, cheese, olive brines, and baking products[4],[17],[18]. With an ethanol tolerance of 11%, they often reside in alcohol distilleries and are involved in all stages of the fermentation process[2]. Considering the fact that this yeast is commonly found in outdoor environments, it is a mesothermophile which has an optimal growth temperature of 20℃[5]. P. membranifaciens is a well-known halotolerant for it optimally grows at a sodium chloride concentration of 3M[5]. For this reason, Pichia membranifaciens is commonly found in olive brines[16]. The following species is also osmotolerant and acidophilic, with its optimal pH conditions being around a 4.0[5]. It has also been demonstrated that P. membranifaciens is capable of growing in the presence of common growth inhibitors such as acetate[10].

7. Pathology

Pichia membranifaciens, like many species in the Pichia genus, is capable of secreting toxins which kill other yeasts species that are sensitive to these toxins. Pichia membranifaciens themselves are immune to the lethality of these toxins, allowing them to thrive[5]. These killer toxins, PKMT and PKMT2, help eliminate similar yeast species that compete with P. membranifaciens[17]. Because of this adaptation, P. membranifaciens remains a dominant yeast in many different fermentation processes, especially in grapes and olives[6],[16]. PMKT and PMKT2 kill other yeast and filamentous fungi by binding to β-D-glucans and mannoproteins respectively on the host cell’s surface[12,[13]]. PMKT also triggers a secondary receptor called Cwp2p (a plasma membrane receptor) in the cytoplasm of sensitive cells[12]. These toxins act by lowering the intracellular pH, hence triggering the High Osmolarity Glycerol (HOG) pathway, which in turn creates pores that allow for an influx of ions into the cytoplasm[12]. Low concentrations of either toxin (PMKT/PMKT2) results in cell death; however, high concentrations of PMKT2 does not trigger apoptosis but arrests yeast cells in early S-phase instead[12]. Contrary to the species’ high halotolerance, P. membranifaciens’ killer activity correlates with significantly lower salt concentrations, with its optimal killer activity being at a salt concentration of 0-0.5M[5]. Pichia membranifaciens is also sensitive to the toxins of various other killer yeasts. Some of these killer yeasts include Pichia jadinii, Kluyveromyces lactis, and Pichia anomala, all of which are considered highly active killer yeasts[5].

8. Current Research

One of the most prevalent areas of research on P. membranifaciens is on its killer activity and the potential applications of this activity in agricultural settings. In addition to PMKTs, scientists are characterizing numerous other enzymes that P. membranifaciens secretes. There have been multiple studies that have demonstrated that the diversity of metabolites that P. membranifaciens is able to secrete is what enables it to be a versatile and effective antimicrobial against a vast range of plant and fruit pathogens including tomatoes, wine, olives,etc[5],[12]. For instance, P. membranifaciens has been shown to control the growth of Botrytis cinerea on post-harvest pears by secreting exo-glucanases. Meanwhile it can also inhibit Penicillium expansum growth on post-harvest peaches by competing with the microbe for nutrients and space[12]. Therefore, the agro-food industry has been keen to harness P. membranifaciens as a natural alternative to chemical antimicrobials and fungicides[12]. Nonetheless, when it comes to fermentation, P. membranifacien’s killer activity does not allow for other microbes that are not pathogenic to grow, thus preventing diversity of flavors in the food, and eliminating the potential benefits other microbes can offer[6].

In addition to characterizing the toxins that P. membranifaciens secretes, there is research being conducted on the mechanisms of these toxins on the microbes affected[12]. Understanding these mechanisms can eventually enable the agricultural industry to manipulate the killer activity’s efficacy under various desired environmental conditions[12]. These mechanism studies also have valuable implications in clinical settings; if P. membranifaciens is able to produce a stable toxin at physiological conditions, then this yeast can be leveraged as an antimicrobial for humans as well[12].

Due to the global warming and energy crisis currently being experienced worldwide, alternative forms of energy (especially bioethanol) are being considered, yet ethanol production in factory settings generate low yields and are complicated to implement[8]. Therefore, recent studies have also investigated the prospect of exploiting thermotolerant yeasts like Pichia membranifaciens for ethanol production[8]. Despite this prospect of using P. membranifaciens in energy production being suggested ,the practicality of the idea and the optimal conditions at which these fermentation processes can occur still needs to be investigated[8].

9. References

[1] [National Center for Biotechnology Information. (n.d.). Pichia membranifaciens. Retrieved October 22,2018, from NCBI:Taxonomy Browser website: https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&id=4926&lvl=3&lin=f&keep=1&srchmode=1&unlock]

[2] [Lachance, M.A., 1995. Yeast communities in a natural tequila fermentation. Antonie van Leeuwenhoek 68:151-160.]

[3] [Naumov, G.I., and Naumova E.S. 2009. Microbiology Chromosomal differentiation of the sibling species Pichia membranifaciens and Pichia manshurica. Microbiology 78:214-217.]

[4] [Masih, E I, et al. “Characterisation of the Yeast Pichia Membranifaciens and Its Possible Use in the Biological Control of Botrytis Cinerea, Causing the Grey Mould Disease of Grapevine.” FEMS Microbiology Letters, Federation of European Microbiological Societies, 25 July 2001.]

[5] [Aguiar, C. and Lucas, C. 2000. Yeasts killer/sensitivity phenotypes and halotolerance. Food Technol. Biotechnol. 38(1):39–46]

[6] [Santos, A, and D Marquina. “Killer Toxin of Pichia membranifaciens and Its Possible Use as a Biocontrol Agent against Grey Mould Disease of Grapevine.” Microbiology, Department of Microbiology, Biology Faculty, Complutense University of Madrid, Madrid, Spain, 2004.]

[7] [Zhang, Fan, et al. "Culture Condition Effect on Bioflocculant Production and Actual Wastewater Treatment Application by Different Types of Bioflocculants." Biodegradation and Bioremediation of Polluted Systems, IntechOpen, 2015.]

[8][Jutakanoke, Rumpa, et al. "Characterization and ethanol fermentation of Pichia and Torulaspora strains." Journal of Applied Pharmaceutical Science, vol. 4, no. 4, Apr. 2014, pp. 52-56.]

[9][Konishi, Masaaki, et al. "Draft genome sequencing of ascomycetes yeast Pichia membranifaciens KS47-1, which shows high acetate resistance in lignocellulosic feedstock hydrolysate."Genome Announcement, vol. 5, no. 8, 2017.]

[10][Oliveira, M., Brito, D., Catulo, L. et alc. 2004. Biotechnology of olive fermentation of ’Galega’ Portuguese variety. Grasas y Aceites 55.( 3):219-226.’’]

[11][Veiga, Alexandra, et al. “Energy Conversion Coupled to Cyanide-Resistant Respiration in the Yeasts Pichia Membranifaciens and Debaryomyces Hansenii.” OUP Academic, Oxford University Press, 1 Apr. 2003.]

[12][Santos, A., et al. “(1→ 6)-ß-D-Glucan as Cell Wall Receptor for Pichia Membranifaciens Killer Toxin.” Applied and Environmental Microbiology, American Society for Microbiology, May 2000.]

[13][Belda, Ignacio, et al. “The Biology of Pichia Membranifaciens Killer Toxins.” Current Neurology and Neuroscience Reports., U.S. National Library of Medicine, Apr. 2017.]

[14][Kurtzman, Cletus P. “Phylogeny of the Ascomycetous Yeasts and the Renaming of Pichia Anomala to Wickerhamomyces Anomalus.” United States Department of Agriculture, 2010, pubag.nal.usda.gov/download/48949/PDF/]

[15][Oliveira, M., Brito, D., Catulo, L. et alc. 2004. Biotechnology of olive fermentation of ’Galega’ Portuguese variety. Grasas y Aceites 55.( 3):219-226.]

[16][Riley R, Haridas S, Wolfe KH, et al. Comparative genomics of biotechnologically important yeasts. Proc Natl Acad Sci U S A. 2016;113(35):9882. doi: 10.1073/pnas.1603941113.]

[17][Yamada, Y, Kondo, K, Coenzyme Q system in the classification of the yeast genera Rhodotorula and Cryptococcus and the yeast-like genera Sporobolomyces and Rhodosporidium. J Gen Appl Microbiol 19, 59–77, 1973.]

Edited by [Alina Chenausky, Pulkit Mittal, Amilcar Pojoy, Darshi Shah, Maisha Savani], student of Jennifer Talbot for BI 311 General Microbiology, 2016, Boston University.