Methylotrophic yeasts - Romanian Biotechnological Letters

Romanian Biotechnological Letters Copyright © 2010 University of Bucharest

Vol. 15, No.4, 2010 Printed in Romania. All rights reserved REVIEW

Methylotrophic yeasts: diversity and methanol metabolism Received for publication, September 15, 2009 Accepted, Juy 1, 2010 OANA NEGRUŢĂ1*, ORTANSA CSUTAK2, ILEANA STOICA2, ELENA RUSU3, TATIANA VASSU2 1 University of Bucharest, Faculty of Biology, Center for Research, Consulting and Training in Microbiology, Genetics and Biotechnology, Aleea Portocalilor 1-3, 060101 Bucharest, Romania 2 University of Bucharest, Faculty of Biology, Department of Genetics, Aleea Portocalilor 1-3, 060101 Bucharest, Romania 3 Titu Maiorescu University, Faculty of Dental Medicine, Street Gheorghe Petrascu, no. 67A, sector 3, 060101 Bucharest, Romania * [email protected]

Abstract Yeast identification has a real importance for selection of technological strains and also for studying biodiversity and population dynamics in the fermentation systems. Important studies on yeast population dynamics showed that Saccharomyces genus is dominant in alcoholic fermentation, while other genera like: Kloeckera, Candida, Pichia, Hansenula, Hanseniaspora and Metschnikowia are growing in the first step of the process. The purpose of this review was to emphasize the importance of the methylotrophic yeasts diversity, with the most important morpho-physiological characteristics, the complexity of the life cycle, and also the main pathway of the methanol metabolism, with the enzymes involved in this process.

Keywords: methylotrophic yeasts, methanol metabolism, Hansenula polymorpha

Introduction The yeasts pose great benefits for humankind by their use in the production of food, wines, beer, and a diversity of bio-chemicals. Preparation of beer and bread with the help of yeasts has been current practice for millennia. Speaking of food, they can also be used as an alimentary supplement because of their high content in vitamin B, amino acids and minerals. At present, they are used not only in the ‘classical’ industrial processes, but also as models for the study of gene regulation in eukaryotic cells, and also as bio-factories for homologous and heterologous proteins [7]. Methylotrophic yeasts (belonging to Hansenula, Candida, Pichia, Torulopsis genera) are capable to metabolise monocarbonic compounds like methanol and formaldehyde [17]. They are very prevalent in nature in particular in moulds, fruit and other vegetable products, exudates of trees and their barks [6]. A possible explanation for the existence of methylotrophic yeasts in this kind of habitats is that methanol can derive from the metoxi chain present in lignin from woods. Almost all tested methylotrophic yeasts are capable to grow on pectic medium, a polymer rich in the metoxi chain, found especially in fruit [1]. Factors like geographic localization and the climatic conditions may influence the diversity of yeast [31]. H. polymorpha and P. pastoris are intensively used in the study of biogenesis, assembly and degradation of the peroxisomes [26]. They were isolated for the first time decades ago by cultivation on media enriched with methanol [21]. The enzymology of the methanol dissimilation pathway was elucidated after 10 years and a lot of confusions were made on the way. The existence of the peroxisomes –induced by methanol in the medium – which contain the enzymes of methanol metabolism, like: alcohol oxidase (AOX), 5369

OANA NEGRUŢĂ, ORTANSA CSUTAK, ILEANA STOICA, ELENA RUSU, TATIANA VASSU

dihydroxyacetone sintase (DHAS) and catalase (CAT) is a very important characteristic of these yeasts. H. polymorpha and P. pastoris were successfully used for the production of heterologous proteins. Moreover, H. polymorpha is the species chosen for studying the genetic control of enzymes involved in methanol metabolism, nitrate assimilation, resistance to heavy metals, and to oxidative stress. Also, it has been considered the usage of methylotrophic yeasts in the treatment of the methanol and formaldehyde containing wastewater [16, 17]. Alcohol oxidase (AOX) The first enzyme involved in yeast methanol metabolism, is alcohol oxidase (AOX), localized in peroxisomes, a particular location [23]. It oxidizes methanol to formaldehyde and hydrogen peroxide.

Figure. 1. Methanol metabolism pathway in methylotrophic yeasts. 1 – alcohol oxidase, 2 – catalase, 3 – dihydroxyacetone synthase, 4 – formaldehyde dehydrogenase, 5 – formate dehydrogenase, 6 – dihydroxyacetone kinase, GSH – glutathione, Xu5P – xylulose-5phosphate, FBP – fructose-1,6- bisphosphate. [10].

Being one of the key enzymes in methanol utilization, AOX is found in all methylotrophic yeasts [14]. In its mature active form AOX is a 600kD molecule, consisting of eight identical subunits of 74kD, with a cvasicubic orientation, each unit including one flavin adenin dinucleotid molecule (FAD) as prostetic group. The monomers are synthesized in cytosol and then are post-transcriptional imported into peroxisomes, where their assembly into the active octamer takes place [11], in contrast with other proteins that are imported as oligomeres. This might be related to certain favourable conditions met by the peroxisomes (such as pH value or certain ions concentrations) [30]. AOX is synthesized in high amounts to compensate the low affinity for oxygen, the AOX promotor being very attractive for the expression of the heterologous proteins, as it is tightly regulated and very easy induced by methanol [32]. The AOX promoter is one of the most powerful and the most precisely regulated promotors known [4]. The AOX genes from H. polymorpha, C. boidinii and P. pastoris were isolated and sequenced. H. polymorpha and C. boidinii possess only one AOX gene while P. pastoris has two AOX genes (AOX 1 and AOX 2) [23].The AOX gene from H. polymorpha contains a large promotor region of 1.5kb. AOX oxidizes not only methanol, but also other alcohols with short chain and formaldehyde. Therefore, it is used for determination of lower alcohols and formaldehyde, as 5370

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Methylotrophic yeasts: diversity and methanol metabolism

well as for the construction of sensors that detect alcohol, in conjugation with oxygen and hydrogen peroxide sensors. It is also very attractive for various biotechnological applications, including formaldehyde assay in food production, in waste waters and in pharmaceuticals. The rate of enzymatic oxidation of formaldehyde by AOX from H. polymorpha is twice lower compared to that corresponding to oxidation of the most appropriate substrate, methanol [27]. In cells, AOX is localized together with dihydroxiacetone synthase and catalase. AOX and dihydroxiacetone synthase are synthesized in very large amounts, depending on the growth conditions they can reach a volume of up to 70% of all cellular proteins. Therefore AOX forms crystalloids which give the characteristic shape of the peroxizomes. Catalase is located predominately at the periphery of the organelles, between the AOX crystalloids and the peroxisomal membrane. [34]. Catalase promotes the decomposition of hydrogen peroxide that is a powerful oxidant agent, toxic for the cells. Catalase is a homotetramer of approximately 241 kDa, which contains the heme group as cofactor [34]. The absence of catalase from peroxizomes determines the accumulation of toxic H2O2 and/or other reactive oxygen species (ROS), and the physiological importance of the enzyme is revealed by the fact that its deficiencies lead to acatalasemia (a disease which is genetically determined) [13]. The use of formaldehyde by the cells In methylotrophic yeast metabolism formaldehyde is situated at the branching point of the assimilation and dissimilation pathways. Because it is a very toxic compound its intracelular levels are strictly regulated [26]. AOX oxidates methanol to formaldehyde, that is subsequently subjected either to the action of a transketolase (peroxisomal dihydroxyacetone synthase) or to direct oxidation into the cytosol. Dihydroxiacetone synthase is a homodimer of 155kDa that catalyses the transfer of glycoaldehyde from xylulose - 5 phosphate as a donor to the formaldehyde molecule as acceptor, by a ‘ping-pong’ mechanism. If enough xylulose- 5- phosphate is present into peroxisomes then formaldehyde is immediately fixed by DHAS, otherwise formaldehyde diffuses into the cytosol where it is oxidized to CO2 by S- formyl gluthatione hydrolase and formate dehydrogenase (FDH) [34]. FDH is not essential, but it is still necessary for optimal growth on methanol since it plays a significant role in formaldehyde detoxification, energy production and the regulation of the glutathione level in cells [36]. FDH from bacteria and yeasts are some of the most stable enzymes. They are produced in small amounts. The FDH content from different methylotrophic bacteria and yeast strains can reach 10 to 18% of the total cellular proteins. The enzyme is a homodimer of 42kDa with both subunits containing an independent active centre [7, 19]. Formaldehyde might be oxidized to formate in a glutathione-dependent or glutathione-independent manner [25]. The enzyme has isoforms, some being NAD+ dependent [33]. FDH hydrolysis S- formylglutathione, resulting the complex enzyme- format, with the format molecule being subsequently oxidized to CO2. NADH is generated during formaldehyde oxidation [15]. Pichia angusta Current name: Pichia angusta (Teun., H.H. Hall & Wick.) Kurtzman, Antonie van Leeuwenhoek 50(3): 212 (1984) Pichia angusta has different synonyms: Hansenula angusta Teun., H.H. Hall & Wick., Mycologia 52(2): 185 (1961) [1960]; Hansenula polymorpha Morais & M.H. Maia, An. Esc. Sup. Quim. Univ. Recife 1: 16 (1959) Ogataea polymorpha (Morais & M.H. Maia) Y. Yamada, K. Maeda & Mikata, Biosc., Biotechn., Biochem. 58(7): 1254 (1994) [37]. Romanian Biotechnological Letters, Vol. 15, No. 3, 2010

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In spite of many descriptions of tested habitats (moulds, rotting fruit and vegetables, intestinal tract of pigs, soil, residues from the presses of olive oil, milk from cows ill of mastitis, soil irrigated with treated or untreated wastewater, corn flour, ropy orange juice a.o.) the complete ecology of this microorganism is still unknown. H. polymorpha presents a significant capacity to grow at high temperatures. H. polymorpha and C. boidinii as well as Torulopsis sonorensis (renamed C. sonorensis) were among the first identified, while P. pastoris was isolated from exudates of trees in North America and Europe. H. polymorpha is now designated as Pichia angusta with the „polymorpha” epithet being prioritary - even though it was first used for a species presently known as Ogataea polymorpha (Moraris and Maia)[20]. The Pichia genus (ex-Hansenula) belongs to Saccharomycetaceae Family, Hemiascomycetes Order, Ascomycota Fillum. The Pichia genus species are heterogeneous in some respects, with hat-shaped or spherical ascospores and different types of Q coenzyme. Pichia genus comprises 92 species being one of the richest taxons. They can be identified besides classical tests by using RFLP for ITS1, 5.8S, and ITS2 [3]. It has round small cells that may form lateral buds. Ascospores are small, hemispheric or hat-shaped and connected with each other, or of a planet Saturn form and usually unbound by fissure of the asca. H. polymorpha can ferment glucose, but not galactose, sucrose, maltose or raffinose, it does not assimilate carbon under aerobic conditions, and does not form petite colonies [12]. The life cycle of H. polymorpha is similar to that of S. cerevisiae. It encompasses a cell proliferation process whereby a cell generates two cells by mitotic division and also sporulation and mating of the haploids of opposite types (a and α, respectively) [35]. On a rich medium the haploid and diploid cells germinate. On a minimal (sporulation) medium the diploid cells form some ascus with four ascospores in meiosis and soprulation. Pichia pastoris Current name: Pichia pastoris (Guillierm.) Phaff [as 'pastori'], Antonie van Leeuwenhoek 22: 115 (1956) Pichia pastoris has different synonyms: Endomyces pastoris (Guillierm.) Zender [as 'pastori'], (1926), Komagataella pastoris (Guillierm.) Y. Yamada, M. Matsuda, K. Maeda & Mikata, Biosc., Biotechn., Biochem. 59(3): 444 (1995), Petasospora pastoris (Guillierm.) Boidin & Abadie [as 'pastori'], Bull. trimest. Soc. mycol. Fr. 70: 365 (1955) [1954], Saccharomyces pastoris (Guillierm.), Lodder & Kreger-van Rij [as 'pastori'], Yeasts, a taxonomic study, [Edn 1] (Amsterdam): 197 (1952), Zygosaccharomyces pastoris Guillierm. [as 'pastori'], C. r. hebd. Séanc. Mém. Soc. Biol. 71: 466-470 (1919), Zygowillia pastoris (Guillierm.) Kudryavtsev, (1954), Zymopichia pastoris (Guillierm.) E.K. Novák & Zsolt, Acta bot. hung. 7: 121 (1961) [38]. It has oval small cells with unipolar budding. On solid medium it forms white or cream-colored, non-filamentous colonies. It grows on various media, on a variety of simple carbon sources including glucose, glycerol, galactose, fructose, ethanol, methanol, alanine, lysine, succinate, etyliamine, mannitol, L-rhamnose and trehalose. P. pastoris does not grow on galactose, arabinose, ribose, maltose, sucrose, raffinose, melibiose, cellulose or starch. P. pastoris is homotalic, isolation of mutants being very difficult because of this characteristic. It often exhibits aberrant segregation, and low viability of the spores [22]. This suggests instability of the mating type which can be used in the process of selection of mutants, as it offers a chance of detecting some heterotalic strains. [29]. Pichia guiliermondii Current name: Pichia guilliermondii Wick., J. Bact. 92: 1269 (1966), and synonyms Yamadazyma guilliermondii (Wick.) Billon-Grand, Mycotaxon 35(2): 203 (1989) [39]. C. guilliermondii is a common species both in natural and clinical environment. Since the first description in 1917 made by Castellani (Endomyces guilliermondii), a big number of 5372



new species and varieties phenotipically similar to C. guilliermondii was described. P. guilliermondii Wickerham represents a collection of sporogenous strains some of them belonging to the Candida guilliermondii sporogenous species. Therefore each C. guilliermonidii strain is theoretically capable to hybridise with any strain of P. guilliermondii. Sources of isolation are very diverse - plants, lake water, cow rumen, soil polluted with oil [28], it was isolated also from insects on Ulmus sp., unpolluted soil and water [41], and from shrimp [18] and other invertebrates but its most common habitat is the nectar of flowers were they are to be found together with Metschnikovia reukaufii. Strains from the sea were found in low salinity waters (e.g. the Adriatic) but also in sea with hipersalinity together with strains of Candida, Debaryomyces, and Metschnikowia. It is isolated from marine medium especially in the rainy seasons [9]. The species has a large geographic distribution [2]. The cells are heterogenous, many of them having an oblong shape (app. 2x10µ), sometimes forming a pseudomycelium, but not true hyphae. The ascae contain 1-2 hat-shaped ascospores which enhance their volume after being freed. All natural strains that were examined or collection strains of P. guilliermondii are heterothalic, mating types being designed mat+ and mat-. Homothalic strains are not described, but some of the meiotic segregates of the hybrids obtained by protoplast fusion develop homothalic phenotypes. Such phenotypes were designated as pseudohomothallic (mat+,-). It was hypothesized that pseudohomothallic (mat+,-) strains are aneuploids. Candida boidinii Current name: Candida boidinii C. Ramírez [as 'boidini'], Microbiol. esp. 6(3): 251 (1953), [40] also can be found under diverse synonyms: Candida koshuensis Yokotsuka & S. Goto, Candida methanolica Oki & Kounu, Candida olivarium Santa María, Candida ooitensis Kumamoto & Seriu, Candida queretana T. Herrera & Ulloa, Candida silvicola Shifrine & Phaff var. melibiosica Nowakowska-Waszczuk & Pietka, Hansenula alcoolica Urakami, Kloeckera boidinii nom. nud., Torulopsis enokii Urakami (http://www.cbs.knaw.nl). Yeast strains described as C. boidinii are usually anamorphic. Molecular techniques allow now the identification of teleomorphic Candida species [8]. It seems that the presence of Candida genus is a very important coastal pollution marker for marine environments. The isolation and identification of the yeasts from sand and sea water from Brazil showed the presence of 292 yeast strains belonging to 4 genera and 31 species, with Candida being dominant and many strains of C. boidinii being present [18]. Many strains of C. boidinii were isolated from fermented olives, together with other representatives of the genus Candida, as well as of Hansenula, Pichia, Torulopsis and Saccharomyces genera [5]. It has oval small cells with multilateral budding, simple or elaborated pseudohyphae, white or cream colonies. On solid medium it forms white or cream colonies. It grows on various media, on a variety of simple carbon sources including galactose, ribose, arabinose, sucrose, maltose, lactose, L-rhamnose, ethanol, methanol.

Conclusions In conclusion, the methylotrophic yeasts, and especially H. polymorpha and P. pastoris are used in the research area and also in biotechnological applications, one of the most important being the production of heterologous proteins of a large industrial and medicine importance. The increase of SCP (Single Cell Protein) requirements or the remediation of the polluted systems by making use of natural alternatives, represent important reasons for the necessity of characterizing the methylotrophic yeasts. Romanian Biotechnological Letters, Vol. 15, No. 3, 2010

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