Utilization of Methanol by Yeasts

Utilization of Methanol by Yeasts

YOSHIKI TANI,

NOBUOKATO, AND

HIDEAKIYAMADA Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto, Japan I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Dissimilation and Assimilation of Methanol in Methylotrophs Other than Yeast.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Dissimilation and Assimilation of Methanol in Yeasts . . . . . . . . A. Enzyme System for the Dissimilation of Methanol . . . . . . B. Assimilation of Methanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Cell Yield and the Metabolic Pathway, .................... V. Production of Cells and Metabolites . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

165 167 170 170 179 180 182 183

1. Introduction Methanol has attracted much attention as a convenient raw material for industrial fermentation. Consequently, investigations of microorganisms which grow on reduced C, compounds, e.g., methane and methanol, as the sole source of carbon and energy have increased. Investigations have been limited to studying the unique metabolic pathway of these compounds. Results of this research have made methanol of practical use in the fermentative production of cells and metabolites. This is a typical pattern in the development of applied microbiology, in which applied and fundamental studies are interrelated. The ability to assimilate reduced C , compounds was first reported by Sohngen (1906),who isolated an aerobic methane-utilizing bacterium Bacillus methanicus. No further information on the utilization of reduced C, compounds was forthcoming for the next 50 years, in spite of the wide occurrence of methane in nature. Microbiologists were interested only in methane-producing microorganisms. Sohngen’s strain was reisolated as a methane and methanol utilizer by Dworkin and Foster (1956) and named Pseudomonas methanica. Their studies showed the physiological peculiarity of methylotrophs. The microbial utilization of methane and methanol has since become the concern of several groups of scientists. Table I is a chronological list of researches on methylotrophs, showing that various kinds of methylotrophs 165 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 24 Copyright @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-0026244

166

YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA

TABLE I MICROBIAL UTILIZATION OF REDUCEDc ,COMPOUNDS First description of a reduced C, compound utilizer, Bacillus methanicus Reisolation of Sohngen's strain, Pseudomonas methanica Isolation of the facultative methylotroph, Pseudomonas PRL-W4, and identification of serine as the first stable intermediate in its metabolic pathway Outlining the serine pathway, Pseudomom AM1 Characterization of primary alcohol dehydrogenase, Pseudomonas M27 Outlining the ribulose monophosphate pathway, Pseudomonas methanica Isolation of a methanol-utilizing yeast, Kloeckera sp. 2201 Systematic characterization of methane-utilizing bacteria Completion of the id+-serine pathway, Pseudomonas MA Crystallization and characterization of alcohol oxidase, Kloeckera sp. 2201 Identification of hexose phosphate as the primary stable intermediate in yeast, Candidu N-16 Isolation of methanol-utilizing fungi, Trichodemna lignorum Identification of Durabino-3-hexulose phosphate, completion of the ribulose monophosphate pathway, Methylococcus capsulatus First International Symposium on Microbial Growth on C, Compounds Isolation of a methanol-utilizing actinomycete, Streptomyces sp. 239 Adoption of Methylomonadaceae in Bergey's Manual of Determinative Bacteriology Finding of the microbody in methanol-utilizing yeasts Second International Symposium on Microbial Growth on C, Compounds

Sohngen (1906) Dworkin and Foster (1956) Kaneda and Roxburgh (1959a,b,c) Large et al. (1962) Anthony and Zatman (1964) Johnson and Quayle (1965), Kemp and Quayle (1965) Ogata et al. (1969) Whittenbury et aZ. (1970a,b) Bellion and Hersh (1972) Tani et a/. (1972a,b) Fujii and Tonomura (1973) Tye and Willetts (1973) Kemp (1974) at Tokyo (1974) N. Kato et al. (1974a) Buchanan and Gibbons (1974) Fukui et al. (1975a), van Dijken et al. (1975a), Sahm et a / . (1975) at Moscow (1977)

have been isolated and that mechanisms which produce energy and which synthesize cell materials from reduced C compounds have been extensively investigated. These studies are related to the need for a global food supply, which has been a concern since the late 195Os, and to the consequent international trend to develop single-cell protein production. At present, the practical use of methylotrophs on an industrial scale is limited to cell production in a few countries. The utilization of reduced C1compounds by yeasts began with the investigations of Kloeckera sp. 2201 by Ogata et al. (1969). The history of research on methanol-utilizing yeasts is short in comparison with that on bacterial

UTILIZATION OF METHANOL BY YEASTS

167

methylotrophs. However, after the brief review by Cooney and Levine (1972), research on methanol utilization by yeasts has made rapid progress (Sahm, 1977). In the present review, metabolic features of the methanolutilizing yeasts will be compared with those of other microorganisms. II. Dissimilation and Assimilation of Methanol in Methylotrophs Other than Yeast There are many unique features of the metabolic pathway for methanol, especially in the early steps of the dissimilation and assimilation pathways. A large part of this unique metabolic pathway has been made clear by studies with bacterial methylotrophs (Quayle, 1972). The dissimilation pathway provides electrons for the respiratory chain to produce ATP. Each step in the reaction sequence leading to the complete oxidation of methanol to C 0 2 through formaldehyde and formate has been demonstrated enzymatically. Enzymes which oxidize methanol in bacteria are listed in Table 11. Methane is introduced into the sequence after oxidation to methanol. Phenazine methosulfate-dependent primary alcohol dehydrogenase (EC 1.1.99.8) is characteristic in bacterial methanol oxidation and has been thoroughly investigated. This enzyme also oxidizes formaldehyde, although the K , value for formaldehyde is larger than that of methanol (Sperl et al., 1974). Almost all reported bacterial methylotrophs have this enzyme. Methane-utilizing bacteria are divided into two groups according to the localization of this enzyme in the cell fraction (Pate1and Felix, 1976), which coincides with the arrangement of their intracytoplasmic membranes. The electron acceptor of this enzyme reaction in uivo has been suggested to be cytochrome c (Anthony, 1975; Netrusov et a l . , 1977). The prosthetic group of the enzyme is thought to be a pteridine derivative (Anthony and Zatman, 1967), but the structure of the compound is still unknown. There are only a few examples of methanol oxidation by fungi. Activities of NAD-linked alcohol dehydrogenase and formaldehyde dehydrogenase and of methylene blue-linked methanol dehydrogenase have been detected in cell extracts of Paecilomyces varioti and Gliocladium deliquescens (Sakaguchi et al., 1975). Streptomyces sp. 239, which is the only known actinomycete which utilizes reduced C, compounds, has a different system for methanol oxidation (Kato et a l . , 1975). The oxidation of methanol, formaldehyde, and formate by the cell-free extract requires the presence of phenazine methosulf;?te-2,6-dichlorophenolindophenol or cytochrome c . The incorporation of methanol into cell constituents has been investigated chiefly with bacterial systems. A first oxidation product of methanol, formaldehyde, enters into two different assimilation pathways. One is the ribulose

TABLE I1 OXIDATION OF METHANOL IN BACTERIA Step CH,OH

4

HCHO

Enzymes

Strains

References

(1) Primary alcohol dehydrogenase electron acceptor; phenazine methosulhte (cytochrome c ) :cofactor; pteridine compound, ammonium ion

Pseudomonas M27 Pseudomonas AM1 Methylococcus capsulatus Hyphomicrobium WC Methylosinus sporiurn Pseudomonas C Pseudomonas sp. 2941 Pseudomonas PRL-W4

Anthony and Zatman (1964) Johnson and Quayle (1964) Patel et al. (1972) Sperl et al. (1974) Patel and Felix (1976) Goldberg (1976) Yamanaka and Matsumoto (1977) Kaneda and Roxburgh (1959b)

Pseudomonas methanica

Harrington and Kallio (1960)

Methylococcus capsulatus Methylobacter capsulatus Methylomonas methanica Pseudomonas AM1 Methylococcus capsulatus Pseudomonas methanica Pseudomanas M27 Hyphomicrobium WC Methylosinus sporium Pseudomonas C Pseudomonas sp. 2941 Pseudomonas methanica

Wadzinski and Ribbons (1975) Patel and Felix (1976)

Pseudomonas AM 1

Johnson and Quayle (1964)

Pseudomonas AM1 Methylococcus capsulatus

Johnson and Quayle (1964) Patel and Hoare (1971)

Methanol dehydrogenase electron acceptor; NAD Peroxidase (in the presence of glucose and glucose oxidase) (4) Methanol oxidase present in particulate fraction HCHO 4 HCOOH

HCOOH

-+

COP

(5) Aldehyde dehydrogenase the same enzyme with primary alcohol dehydrogenase

(6) Formaldehyde dehydrogenase electron acceptor; NAD: glutathione dependent (7) Aldehyde dehydrogenase electron acceptor; 2,6-dichlorophenol indophenol (8) Formate dehydrogenase electron acceptor; NAD

Heptinstd and Quayle (1970) Patel and Hoare (1971) Patel et al. (1972) Sperl et al. (1974) Patel and Felix (1976) Goldberg (1976) Yamanaka and Matsumoto (1977) Harrington and Kallio (1960)

169

UTILIZATION OF METHANOL BY YEASTS

monophosphate pathway (or the pentose monophosphate pathway), in which formaldehyde is condensed with ribulose 5-phosphate to form a unique compound, ~urabino-3-hexulose6-phosphate, which undergoes isomerization to fructose 6-phosphate (Strem et al., 1974) (Fig. 1). The second is the serine pathway, in which formaldehyde reacts with glycine to form L-serine by the catalysis of serine transhydroxymethylase (Quayle, 1972) (Fig. 2). Another pathway, which fixes C 0 2instead of formaldehyde, is the ribulose diphosphate pathway. This is usually found in autotrophs (Quayle, 1972). The product of the hexulose phosphate synthase reaction, the first step in the ribulose monophosphate pathway, has recently been identified (Kemp, 1974) and the enzyme has been extensively purified (Ferenci et al., 1974; Sahm et a l . , 1976; Kato et a l . , 1978). Now studies are focusing on metabolic control in the pathway. This may involve cyclic oxidation of formaldehyde through 6-phosphogluconate as a dissimilation pathway (Strpm et al., 1974; Colby and Zatman, 1975a,b; Ben-Bassat and Goldberg, 1977). One undetermined part of the serine pathway was the regeneration system ofglyoxylate and glycine. Bellion and Hersh (1972)showed the presence of isocitrate lyase, malate thiokinase (malate + ATP + CoA 4 malyl-CoA ADP + Pi), and malyl-CoA lyase (malyl-CoA + glyoxylate acetyl-CoA)in Pseudomonas MA. This led to the completion of “the icl +-serine pathway.” However, neither isocitrate lyase nor malate thiokinase activity has been detected in some methylotrophs, including Pseudomonas AM1, a typical strain possessing the id-serine pathway (Salem et al., 1973). Fungi assimilate methanol through the ribulose monophosphate pathway (Tye and Willetts, 1973) or through the serine pathway (Sakaguchi et al., 1975). An actinomycete, Streptomyces sp. 239, is thought to use both pathways. Activities of hydroxpyruvate reductase and hexulose phosphate synthase, respectively the key enzymes of the ribulose monophosphate and serine pathways, have been detected in this strain (Kato et al., 1977a).

+

+

Fructose 1.6-PZ 4 6-P-Gluconate

acetone- P

FIG. 1. The rihulose monophosphate pathway in methylotrophs. @ hexulose phosphate synthase, phosphohexulose isomerase.

@

170

YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA

@ @

@

FIG. 2. The icl +-serine pathway in rnethylotrophs: serine transhydroxyrnethykse, hydroxypyruvate reductase, malate thiokinase, rnalyl-CoA lyase, iswitrate lyase.

@

0

Ill. Dissimilation and Assimilation of Methanol in Yeasts A number of methanol-utilizing yeasts has been found since the first isolation by Ogata et al. (1969), and a number of type strains of yeasts has also been shown to utilize methanol (Hazeu et al., 1972). These species are limited to several genera which include both ascomycetous and asporogenous yeasts (Table 111).All are facultative methylotrophs. Multipolar budding and a requirement for biotin and/or thiamine are common features of many of these yeasts. These peculiar characteristics of methanol-utilizing yeasts may lead to a new classification system, such as that of Methylomonadaceae for bacterial methylotrophs (Buchanan and Gibbons, 1974). More detailed studies of classification using chemotaxonomy are needed. Candida sp. WY-3, which grows on secondary and tertiary amines, is another type of reduced C, compound-utilizing yeast (Yamada et al., 1976). This yeast may dissimilate the methyl group of the amines in a pathway which affords formaldehyde (Colby and Zatman, 1973). The principal parts of the dissimilation and assimilation pathways of methylotrophs have been shown in studies with bacteria. Knowledge of the enzyme systems in yeasts has accumulated rapidly because of extensive studies with bacteria and because of the usefulness of yeasts in single-cell protein production. THE DISSIMILATION OF METHANOL

A. ENZYME SYSTEMFOR

Each step in the reactions involved in methanol oxidation by yeasts has been investigated at the enzymatic level. Table IV gives the enzymes responsible for the oxidation of methanol to COz through formaldehyde and formate.

TABLE I11 CHARACTERISTICS OF METHANOL-UTILIZING YEASTS Strains Ascomycetous yeasts Hansenula (9)a H . pdymorpha H . ofunaensis Pichia (6) P . methanolica P . lindnerii P. inethanothenno Saccharornyces (2) Asporogenous yeasts Candida (7) C. N-16 C. methanolica C.boidinii Kloeckera (1) K. sp. 2201 Rhodotomla (1) Twulopsis (12) T . methanolooescens T . methanoswbosa T . methanodonnercqii T . nagoyaensis

Budding

Methylotrophism

Growth factor

Multipolar Multipolar

Facultative Facultative

Biotin, thiamine

Multipolar Multipolar Multipolar

Facultative Facultative Facultative

Biotin Biotin, thiamine

Multipolar Multipolar Mu1tipolar

Facultative Facultative Facultative

Biotin Biotinb Biotin

Tonomura et al. (1972) Oki et al. (1972) Sahm and Wagner (1972)

Bipolar

Facultative

Thiamineb

Ogata et al. (1969, 1970)

Multipokr Multipolar Multipolar Multipolar

Facultative Facultative Facultative Facultative

Biotin, thiamine Biotin, thiamine Biotin, thiamine

Oki et al. (1972) Yokote et al. (1974) Yokote et al. (1974) Asai et al. (1976)

“Number of strains reported in paper. bNot essential for but stimulative to growth.

References

Levine and Cooney (1973) Asai et al. (1976)

K. Kato et al. (1974)

Henninger and Windisch (1975) Minaini et al. (1978)

TABLE IV OXIDATION OF METHANOL Step CH,OH

+

HCHO

Enzymes (1) Alcohol oxidase cohctor; FAD

(2) Alcohol dehydrogenase electron acceptor; NAD: glutathione requiring (3) Catalase (peroxidative) HCHO -+ HCOOH

(4) Formaldehyde dehydrogenase electron acceptor; NAD: glutathione dependent (HCHO + HCO-glutathione)

(5) Alcohol oxidase substrate; hydrated formaldehyde

HCOOH + CO,

(6) Catalase (peroxidative) (7) Formate dehydrogenase electron acceptor; NAD (HCO-glutathione -+ CO, or HCO-glutathione + H,O 4 HCOOH + CO,) (8) Catalase (peroxidative)

IN

YEASTS

Strains

Kloeckera sp. 2201 Candida N-16 Candida boidinii Hansenula polymorpha Pichia pinus, Kloeckeru sp. 2201, Candida boidinii Hansenula polymwpha Candida boidinii Candida N-16 Hansenula polymorpha Candida N-16 Kloeckera sp. 2201 Candida boidinii Hansenula polymorpha Candida N-16 Candida boidinii Hansenula polymorpha Kloeckera sp. 2201 Hansenula polymwpha Candida N-16 Candida boidinii Kloeckera sp. 2201 Hansenula polymorpha Hansenula polymorpha

References Tani et al. (1972a,b) Fujii and Tonomura (1972) Sahm and Wagner (1973a) Kato et al. (1976) Mehta (1975a,b) Dudina et al. (1977) Roggenkamp et al. (1974) Fujii and Tonomura (1975b) van Dijken et al. (1972) Fujii and Tonomura (1972) Kato et al. (1972) Sahm and Wagner (1973b), Shiitte et al. (1976) van Dijken et al. (1976a) Fujii and Tonomura (197513) Sahm (1975) Kato et al. (1976) van Dijken et al. (1975b) Fujii and Tonomura (1972) Sahm and Wagner (1973b), Schiitte et al. (1976) N. Kato et al. (197413) van Dijken et al. (1976a) van Dijken et ul. (197513)

UTILIZATION OF METHANOL BY YEASTS

173

1 . Oxidation of Methanol to Formaldehyde

A unique characteristic of the methanol oxidation system of yeasts appears

in the first step, methanol to formaldehyde. All yeasts examined have an

alcohol oxidase (EC 1.1.3.13) which catalyzes the following reaction using molecular oxygen as the electron acceptor: CH,OH

+ Oz+

HCHO

+ H202

This type of enzyme has been found only in Basidiomycetes (Farmer et al., 1960; Janssen et al., 1965; Fukuda and Branron, 1971). The electron from methanol in this reaction is transferred to molecular oxygen to form HzOz. In comparison with the bacterial enzyme, this first step of methanol oxidation in yeasts is disadvantageous to ATP regeneration. The enzyme has been purified from the cell-free extract of a methanolutilizing yeast, Kloeckera sp. 2201, by a procedure which includes ammonium sulfate fractionation and DEAE-cellulose and Sephadex G-200 column chromatographies (Tani et d., 1972a). The crystals obtained had a specific activity about 12-fold that of the crude cell-free extract. The content of the enzyme in the cells was estimated to be about 8% of the total soluble protein. The ease of the preparation makes possible the use of the enzyme in the determination of alcohols (Guilbault, 1970). Physicochemical and enzymological properties of alcohol oxidases from different yeasts are similar (Tani et al., 1972a,b; Kato et al., 1976; Fujii and Tonomura, 1972; Sahm and Wagner, 1973a; van Dijken, 1976) (Table V). The enzyme of Hansenula polymorpha, a thermotolerant yeast, differs in its behavior to temperature. The enzyme is composed of eight subunits, each of which contains one coenzyme, FAD. Each subunit is arranged in an octad aggregate composed of two tetragons face to face based on electron microscopical observations (Kato et al., 1976) (Fig. 3). The enzyme can be induced when the yeast is grown on a methanol medium (Tani et al., 1972a). This enzyme should be called an alcohol oxidase because it is almost equally active for ethanol and methanol: To a lesser extent it is active for several primary alcohols (Tani et al., 1972b). The oxidation of methanol by alcohol oxidase is accompanied by the formation of a toxic compound, HzOz. Catalase is also induced when the yeast is grown on a methanol medium (N. Kato et al., 197413; Fujii and Tonomura, 1975a; Yasuhara et al., 1976). Table VI shows the simultaneous formation of alcohol oxidase and catalase in methanol-utilizing yeasts. Catalase also functions like peroxidase to oxidize methanol (Roggenkamp et al., 1974; Fujii and Tonomura, 1975b; van Dijken et al., 1975b), formaldehyde, and formate (van Dijken et al., 197513) in the presence of excess HzOz.

PROPERTIES OF

TABLE V ALCOHOLOXIDASES

OF YEASTS

Relative activityn for Optimum temperature

K , for methanol (mM)

Molecular weight

Molecular weight of subunit

FAD content (moles/mole)

83,000

8.4

-

ec,

Ethanol

Kloeckera sp. 2201

35

106

79

0.44

673,000

Candida N-16 Candida boidinii Hansenuh polymorpha DL-1 Hansenulu polymorpha CBS 4732

35

93 75

75 25

210,000

6o0,OOo

50

44

2.1 2.0 0.23

669,000

74,000 83,000

7.4

Tani et al. (1972a,b) Kato et al. (1976) Fujii and Tonomura (1972) Sahm and Wagner (1973a) Kato et al. (1976)

78

60.5

1.3

616,000

77,000

8

van Dijken (1976)

Origin of enzyme

30 45

50

n-Propanol

"Relative activity is expressed as 100 for the activity against methanol.

References

UTILIZATION OF METHANOL BY YEASTS

175

FIG. 3. Electron micrograph of alcohol oxidase. The crystalline enzyme of Hansenuh polymorpha was negatively stained with sodium phosphotungstate at pH 7.2.

Subcellular localization of enzymes involved in methanol oxidation is an interesting feature of methanol-utilizing yeasts. A specific organelle called the microbody (Fukui et al., 1975a; Sahm et al., 1975)or the peroxisome (van Dijken et al., 1975a), which is surrounded by a single-unit membrane, has been found in methanol-grown cells. This fine structure also occurs in other methanol-utilizing yeasts (Tsubouchi et al., 1976). The organelle has been isolated using density gradient centrifugation (Roggenkamp et al., 1975; Fukui et a l . , 1975b). Alcohol oxidase and catalase are found in this particle, but formaldehyde dehydrogenase and formate dehydrogenase are not (Fukui et d.,1975b). The localization of alcohol oxidase was confirmed using a cytochemical staining technique (Veenhuis et al., 1976). This organelle may be responsible for the first step in methanol oxidation. Immobilization of the organelle has been studied using photocrosslinkable resins (Tanaka et al., 1977). Immobilized microbodies may be useful as a multifunctional biocatalyst. The presence of an enzyme other than alcohol oxidase in the oxidation of methanol to formaldehyde has been reported. Activity of NAD-dependent alcohol dehydrogenase was observed in extracts of all the methanol-utilizing yeasts tested (N. Kato et al., 1974~).Sahm and Wagner (1973a), in a study

176

YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA TABLE VI ACTIVITYOF ALCOHOLOXIDASE AND

CATALASE IN YEASTSa

Activity in methanol-grown

cells

Activity in glucose-grown cells

Strains

Alcohol oxidaseb

Catalaseb

Alcohol oxidaseb

Catalaseb

Kloeckera sp. 2201 Candida rnethanolica Torulopsis pinus Torulopsis methanolovescens Hansenuh capsulata Pichia pinus Pichia trehalophila

0.13 0.40 0.16 0.08 0.07 0.24

12.9 10.7 6.2 9.4 5.7 6.9 3.2

-e -

0.64 0.20 0.18 0.18 0.09 0.35 0.20

0.48

0.002

-

0.002

aAdapted from N. Kato et al. (1974~). %pecific activity: alcohol oxidase, pmoles of H,Oz/min/mg protein; catalase, AE,Jmin/mg protein. Nondetectable.

using Candidu boidinii, showed that the alcohol dehydrogenase was inactive for methanol, that it was not induced by methanol, and that a mutant which could not grow on methanol had this enzyme activity. This suggests that the enzyme has no physiological significance. Some researchers, however, assume that there is positive participation of the alcohol dehydrogenase in methanol oxidation. Glutathione-dependent dehydrogenation has been shown in cell-free extracts of several yeasts (Mehta, 1975a,b). In this case, cooperative action of contaminating enzymes, such as alcohol oxidase and glutathione-dependent formaldehyde dehydrogenase, may indicate that a dehydrogenase activity reduces NAD. However, methanol oxidation has recently been detected also under anaerobic conditions in which alcohol oxidase should be inactive (Dudina et al., 1977). Therefore, whether methanol dehydrogenase is active in vivo, which would be advantageous for ATP-regeneration and would result in better cell yields, is still debatable. 2 . Oxidation of Formaldehyde to Furmute A glutathione-dependent dehydrogenase which uses NAD as the electron acceptor is known to catalyze formaldehyde oxidation in various organisms, including methanol-utilizing bacteria. Formaldehyde dehydrogenase (EC 1.2.1.1) has also been found in methanol-utilizing yeasts (Fujii and Tonomura, 1972; Kato et al., 1972; Sahm and Wagner, 1973b; Schutte et al., 1976; van Dijken et al., 1976a).

UTILIZATION OF METHANOL BY YEASTS HCHO

177

+ NAD + H,O -+ HCOOH + NADH,

The enzyme can be induced when the yeast is g r o w on a methanol medium. Its content in the cells is about 0.8% as calculated from the specific activity of a highly purified preparation (Schutte et al., 1976). The enzyme is highly specific for formaldehyde. The reaction mechanism is: HCHO -t GSH + H,O + HOCH,-S-G NADH, HOCHZ-S-G + NAD + HCO-S-G GSH HCO-S-G + HZ0 -+ HCOOH

+ +

In this sequence, the oxidation of formaldehyde occurs after the nonenzymatic formation of hemimercaptal from formaldehyde and glutathione. The reaction product, S -formylglutathione, is then hydrolyzed to formate by a hydrolase. Induction of the hydrolase by methanol has also been reported (Schutte et al., 1976). The electron of formaldehyde is introduced into a respiratory chain through the reduction of NAD. It appears that, in contrast to the first step, the second step in methanol oxidation is responsible for energy production. Oxidation offormaldehyde by alcohol oxidase has been found (Sahm, 1975; Fujii and Tonomura, 197513; Kato et al., 1976). This reaction occurs because more than 99% of the formaldehyde is hydrated to form an alcoholic compound in aqueous solution. The apparent K , value of alcohol oxidase for formaldehyde (2.40 mM) is much higher than that of formaldehyde dehydrogenase for formaldehyde (0.29 mM) and that of alcohol oxidase for methanol (0.44 mM) (Katoet al., 1976). A mutant ofCandida boidinii, which lacks alcohol oxidase, oxidizes formaldehyde as well as the parent strain does (Sahm, 1975).These data support the hypothesis that there is no physiological significance in the oxidation of formaldehyde by alcohol oxidase in vim. Formaldehyde oxidation by dehydrogenase is favorable since it provides electrons for the respiratory chain to produce ATP. Another possible oxidation system for formaldehyde is catalase-catalyzed peroxidation (van Dijken et al., 1975b). $3. Oxidation of Formute to C o g

The enzyme which catalyzes the final step of methanol oxidation in yeasts is formate dehydrogenase (EC 1.2.1.2)(Fujii and Tonomura, 1972; Sahm and Wagner, 1973b; N. Kato et al., 197413; Schutte et al., 1976; van Dijken et al., 1976a). It has also been found in bacterial methylotrophs. HCOOH

+ NAD + CO, + NADH,

178

YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA

The enzyme can be induced when the yeast is grown on a methanol medium. Its content in the cell have been calculated to be about 3% for Kloeckeru sp. 2201 (Kato et al., 1974b) and about 5% for Candida boidinii (Schutte et al., 1976). The enzyme may be useful for determining formate since it is highly specific for that compound. The high K , value of the enzyme for formate, 22 mM for the enzyme of Kloeckeru sp. 2201 (Kato et al., 1974b), has been debated. This places doubt on the physiological significance of the enzyme in an energy-giving system, although it is advantageous for the supply of formaldehyde to the assimilation pathway. Recently, van Dijken et al. (1976a) reported that the true substrate of the enzyme is S -formylglutathione, not formate: HCO-S-G

+ NAD + H,O

--f

COP

+ NADH, + GSH

When S-formylglutathione is used as substrate, the K , value of formate dehydrogenase for it is about 1mM. Direct oxidation of S-formylglutathione to COz, without hydrolysis to formate, is possibly the best way to complete the oxidation of methanol. Induction of S -formylglutathione hydrolase by methanol has been reported (Schutte et al., 1976). Further studies on the final step of methanol oxidation, however, are necessary. Results of studies of the oxidation system of methanol are as follows (Fig. 4): In methanol-utilizing yeasts, methanol is first oxidized to formaldehyde

FIG.4. Schematic representation of the localization and the mechanism of oxidation of formaldehyde nonenzymatic, methanol by yeasts: alcohol oxidase, formate dehydrogenase, dehydrogenase, S-formylglutathione dehydrogenase.

0@

@

@

@

UTILIZATION OF METHANOL BY YEASTS

179

by alcohol oxidase and by catalase in the manner of peroxidase. These reactions occur in a specific organelle, the microbody (peroxisome). Subsequently formaldehyde, which is hydrated in aqueous solution, is oxidized to S-formylglutathione by cytoplasmic formaldehyde dehydrogenase after the nonenzymatic formation of hemimercaptal. Then, S-formylglutathione is oxidized to C 0 2 by formate dehydrogenase alone or in cooperation with hydrolase. In addition, the oxidation of methanol to formate through formaldehyde by the catalysis of alcohol oxidase is also possible, but the physiological significance of this reaction is questionable.

B. ASSIMILATIONOF METHANOL It is essential to determine the pathway that assimilates methanol in order to make use of methanol-utilizing yeasts. Bacterial methylotrophs assimilate reduced C compounds through the ribulose monophosphate or the serine pathway. Therefore, there have been several attempts to determine the pathway in yeasts. However, sufficient data to explain the assimilation pathway of yeasts have not yet been accumulated. The first step, itself, in the incorporation of C, compounds (possiblyformaldehyde and/or formate) is not yet clear. A system different from that in bacteria may occur in yeasts as seen in the oxidation pathway. Sugar phosphates have been postulated to be early intermediates in the assimilation pathway in yeasts. The incorporation of I4C-labeled C, compounds to phosphate esters of hexose, e.g., fructose and glucose, was first reported by Fujii and Tonomura (1973). This suggests the presence of a pathway similar to the ribulose monophosphate pathway and different from the serine pathway. The absence of hydroxypyruvate reductase, a key enzyme in the serine pathway, in cell-free extracts of Kloeckera sp. 2201 (Diel et al., 1974) supports this suggestion. Hexulose phosphate synthase is therefore thought to catalyze the first condensation step as in the bacterial pathway. The special feature in the reaction of yeasts is the ATP requirement (Fujii et a l . , 1974; Fujii and Tonomura, 1974), which is not known for any bacterial system. Measurement of the hexulose phosphate synthase activity is only possible using the radioactivity of a 14C-labeledC, compound incorporated into isolated hexose phosphates. The activity of this enzyme seems inadequate for the first-step reaction of the assimilation pathway (Diel et al., 1974; Fujii and Tonomura, 1974; Sahm and Wagner, 1974; Trotsenko et al., 1976), although the enzyme has been induced during adaptation from a glucose to a methanol medium (Sahm, 1977). No phosphohexulose isomerase activity, which is known to catalyze the isomerization of ~arabino-3-hexulose 6-phosphate to fructose 6-phosphate after the condensation reaction of for-

,

180

YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA

maldehyde to ribulose 5-phosphate in the bacterial pathway, could be detected in the cell-free extract of Kloeckwu sp. 2201 (Kato et al., 197713). A positive change in enzyme activities related to the ribulose monophosphate pathway has been found when a yeast is grown on a methanol medium (Sahm, 1977). Properties of hexulose phosphate synthase also have been reported (Bykovskaya and Voronkov, 1977; Sahm, 1977). The enzyme responsible for the first reaction in the assimilation pathway of methanol in yeast may still be unknown as studies so far are not definitive. Identifications of the substrate and of the product of “synthase” are the most needed studies, at present, in the research on methanol-utilizing yeasts. IV. Cell Yield and the Metabolic Pathway The first aim of the microbial utilization of methanol has been to produce single-cell protein. Knowledge of methanol metabolism may be sufficient to enable us to produce a theoretical cell yield value. Pathways to dissimilate and assimilate methanol have been summarized in terms of cell yield (Fig. 5) where ATP is the principal compound. The oxidation of methanol to CO, functions as an ATP-producing system. The pathway leading to the cell material described as C4H,0,N, from the C1 compound through 3-phosphoglycerate, a common intermediate in the biosynthesis of cell materials, is represented to be an ATP-consuming system (van Dijken and Harder, 1975). Assuming that the oxidative phosphorylation system is also used in methylotrophs, then the type of electron acceptor should directly determine the amount of ATP formed by the oxidation of methanol. The electron transferred to NAD(P) by the oxidation of one molecule of a C1 compound gives three ATPs but its transfer to molecular oxygen, as in the alcohol oxidase reaction in yeasts, produces no ATP. Phenazine methosulfate-dependent primary alcohol dehydrogenase is distributed in almost all bacterial methylotrophs. The electron acceptor of the enzyme in uiuo is thought to be S e r m pathway or Rtbulose mMophosprate pathway

A

X CH3OH

XHz

Y

YHz

HCHO -HCOOH

Z

ZH2 CO2

FIG. 5. Pathways of dissimilation and assimilation of methanol. Adapted from van Dijken and Harder (1975).

UTILIZATION OF METHANOL BY YEASTS

181

cytochrome c (Anthony, 1975; Netrusov et al., 1977). In this case, one molecule of ATP is available for the oxidation of one molecule of methanol to formaldehyde through the respiratory chain. NAD(P) is effective as the electron acceptor for ATP production but it is usually inactive for the first step of methanol oxidation. This may be because the oxidation-reduction potential in this oxidation is not sufficient to reduce NAD(P). The ATP balance of the assimilation pathways of methanol is shown in Fig. 6. The serine and ribulose monophosphate pathways have several reactions related to the consumption or production of ATP up to the synthesis of a common intermediate, 3-phosphoglycerate. The regeneration system of glyoxylate and glycine in the serine pathway is considered here. The serine pathway consumes four molecules of ATP to form one molecule of 3-phosphoglycerate, while the ribulose monophosphate pathway gives three molecules of ATP. Possibly, the later pathway in yeasts consumes one molecule of ATP in the first step to incorporate formaldehyde. Thus, an organism having the ribulose monophosphate pathway is more advantageous for cell yield than is one having the serine pathway. Cyclic oxidation of formaldehyde through 6-phosphogluconate (Strflm et al., 1974; Colby and Zatman, 1975a,b; Ben-Bassat and Goldberg, 1977) produces a greater amount of ATP for the ribulose monophosphate pathway. Van Dijken and Harder (1975) reported yields of microorganisms grown on methanol, based on a , Y of 10.5. Methanol-utilizing yeasts have values of O,3, and 3, for X, Y and 2, respectively, in the dissimilation pathway (Fig. Serine pathway 2 HCHO COP 4 ATP + 3-phosphoglycerate + 4 ADP + 4 Pi Hydroxypyruvate NADH,+glycerate NAD Glycerate ATP -+ 3-phosphoglycerate ADP + Pi Malate ATP CoA + acetyl-CoA + glyoxylate + ADP + Pi Succinate flavoprotein 4 malate flavoprotein.H2 Malate NAD + oxaloacetate NADH,

+

+

+ + + + +

+

+ +

+

Ribulose monophosphate pathway 3 HCHO 3 ADP 3 Pi + 3-phosphoglycerate + 3 ATP (3 HCHO + 3-phosphoglycerate) Fructose 6-phosphate ATP+fructose 1,g-diphosphate ADP Pi Dihydroxyacetone phosphate + NAD + ADP + Pi + 3-phosphoglycerate NADH, ATP (HCHO pentose phosphate ATP + hexose phosphate + ADP + Pi)

+

+

+

+

+

+ +

+

+

FIG. 6. Overall reactions leading to the synthesis of 3-phosphoglyceratefrom methanol. The reactions within parentheses may be possible in yeasts.

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YOSHIKI TANI, NOBUO KATO, AND HIDEAKI YAMADA

5) and possibly have the ribulose monophosphate pathway. Obligate methanol-utilizing bacteria have values of 1, 3, and 3 for X, Y, and 2 and possess the ribulose monophosphate pathway. Calculations based on these values showed a higher cell yield for the methanol-utilizing bacteria (0.63 gm per gram methanol) than for the methanol-utilizing yeasts (0.54 gm per gram methanol). Van Dijken et al. (1976b) also reported a low yield for a yeast (0.38 gm per gram methanol) due to alcohol oxidase. Primary alcohol dehydrogenase also has been reported to be responsible for the oxidation of formaldehyde (Heptinstall and Quayle, 1970). Th’IS enzyme may transfer the electron of formaldehyde to cytochromec . Therefore, the cell yield of the bacterium should be reduced. However, methanol oxidation by a dehydrogenase in yeasts may increase cell yield.

V. Production of Cells and Metabolites Calculations of cell yields are based only on enzymatic aspects of methanol metabolism. To obtain actual yield data, various factors besides the metabolic pathway must be considered (Cooney and Levine, 1975). One specific drawback to the use of methanol as the sole source of carbon and energy is its toxicity to the growth of the microorganism. Semicontinuous methods of culture (fed-batch culture) have been carried out using methanol-utilizing yeasts and bacteria. In contrast to the usual continuous culture, the fed-batch method keeps the methanol concentration low so that maximum specific growth rate can be obtained during cultivation. Reuss et al. (1975) reported on a fed-batch culture of Candidu boidinii in which the methanol concentration was controlled by measuring the amount of methanol in the exhaust gas. Several methods have been developed using methanol-utilizingbacteria. Yamane et al. (1976) have designed a fed-batch culture which maintains exponential growth with a feed rate programmer. Nishio et al. (1977a)have reported on a culture controlled by pH, in which a methanol-ammonia mixture was fed in response to a direct signal of pH change. Shimizu et al. (1977d) have obtained about 85 gmAiter of Protaminobacter ruber with a fed-batch culture using dissolved oxygen tension as the control indicator. Fermentor design has been improved and allows the use of methanol for the industrial process of single-cell protein. Using methanol-utilizing yeasts, Kuraishi et al. (1977)have cultivated Pichia aganobii in an air-lift fermentor, made as a pilot plant fermentor to obtain high oxygen transfer and miscibility (Kuraishi et. al., 1975). A high cell density culture at a normal dilution rate could be made. Several efforts to obtain metabolites of methylotrophs have been also reported. Accumulation of L-glutamate (6.8 mg/ml) was first reported with

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Methanomonas methylouora (Oki et al., 1973). Other metabolites, e.g., pyruvate, a-ketoglutarate, and p l y saccharide, have also been detected in the culture filtrate. Methylomonas aminofaciens has been isolated as a producer of branched chain amino acids (Ogata et al., 1977). A valine hydroxamate-resistant mutant of the strain accumulated 2.2 mg/ml of Lvaline and 0.8 mg/ml of L-leucine (Izumi et al., 1977). A mutant of Methylomonas methanolophila, resistant to aromatic amino acid analogs, accumulated L-phenylalanine (4 mg/ml), L-tyrosine (1.1 mdml), and L-tryptophan (0.2 mglml) (Suzuki et al., 1977). Methanol-utilizing bacteria also produce vitamin B,, (Tanakaet al., 1974; Nishio et al., 1975a,b, 1977a,b; Toraya et al., 1975; Sato et al., 1977) and polysaccharide (Hagstrom, 1977; Kodama et al., 1977). The production of L-serine by methylotrophs is one use of their unique metabolic pathway, the serine pathway. Serine transhydroxymethylase, which fixes formaldehyde to glycine to form L-serine, is a key enzyme in this pathway. Keune et al. (1976) reported the accumulation of 4.7 mg/ml of L-serine in the culture filtrate of Pseudomonas 3ab. Arthrobacter globtjbrmis, a gram-positive methylotroph, is another L-serine producer. A methionine-requiring mutant of the strain can produce 5.2 mg of L-serine per milliliter (Tani et al., 1978). Methanol-utilizing yeasts have an alcohol oxidase catalyzing the first step of methanol oxidation. The content of the enzyme is 8% of the intracellular soluble protein, and the enzyme has eight molecules of FAD in each molecule (Kato et al., 1976). The increased amount of FAD in the cell and the derepression of FAD pyrophosphorylase, the last enzyme in FAD biosynthesis, have been observed when yeasts are grown on a methanol medium (Shimizu et al., 1977a,b; Eggeling et al., 1977). The induction of FAD biosynthesis by methanol has led to a study of the production of FAD by methanol-utilizing yeasts. Riboflavin or F M N added to a yeast culture on a methanol medium was converted to FAD in a good yield (45.4 pg/ml) (Shimizu et a l . , 1977~). The amounts of metabolites produced by methylotrophs are still low, but the advantage of using methanol as a raw material should promote its utilization not only for single-cell protein production but also for metabolite production. To further develop this field, details of methanol metabolism need to be determined. ACKNOWLEDGMENTS We wish to express our thanks to the late Professor Koichi Ogata, Kyoto University, who was the first to report on a methanol-utilizing yeast at 1969. Due to his leadership and encouragement in the study of rnethylotrophs, this review has been possible.

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