Biosynthesis of -Nonalactone in Yeast - American Chemical Society

Chapter 14

Biosynthesis of γ-Nonalactone in Yeast L.-A. Garbe, H. Lange, and R. Tressl

Downloaded by UNIV OF PITTSBURGH on April 7, 2017 | http://pubs.acs.org Publication Date: August 14, 2001 | doi: 10.1021/bk-2001-0794.ch014

Institut für Biotechnologie, Technische Universität Berlin, Seestrasse 13, D-13353 Berlin, Germany γ-Nonalactone is known as aroma active compound in fermented products such as beer. In a series of labeling experiments with deuterated linoleic acid and [ O ]-13- and 9hydroxyoctadecadienoic acid, respectively, two biosynthetic routes to γ-nonalactone in yeast were elucidated: (i) 13lipoxygenation / reduction and (β-oxidation followed by one α -oxidationstep results in (S)-γ-nonalactone (~ 60 % e.e.); (ii) 9lipoxygenation / reduction and Baeyer-Villiger oxidation yields azelaic acid and 2E,4E-nonadien-1-ol which is further trans formed into (R)-y-nonalactone (~ 46 % e.e.). 18

1

Introduction y-Lactones are widely distributed important flavor compounds occurring in fruits as well as in fermented foods such as beer. The biosynthesis of y-decalactone and y-dodecalactone has been investigated in fruits and microorganisms (1-3). The common initial step is the introduction of oxygen into the carbon chain. In Sporobolomyces odorus metabolization of oleic acid proceeds via (R)12-hydroxylation and subsequent degradation of the resultant ricinoleic acid by Poxidation yielding (/?)-y-decalactone (2). Epoxygenation of oleic or palmitoleic acid has been characterized as additional pathway operative in S. odorus (3). Metabolization of the intermediate epoxy fatty acid leads to y-dodecalactone and y-decalactone, respectively. The stereochemical course of the microbial degradation of the epoxy- and their corresponding dihydroxy fatty acids is currently under investigation (4). Degradation of these oxygenated fatty acids by P-oxidation yields lactones with an even number of carbon atoms. The formation of lactones with odd numbered chain lengths, e.g. y-nonalactone, cannot be explained by this path way. Furthermore, y-nonalactone obtained by yeast-catalyzed reactions exhibits low optical purity compared to y- or 8-decalactone (Table I).

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© 2001 American Chemical Society Takeoka et al.; Aroma Active Compounds in Foods ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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Experimental 2

Synthesis of [9,10,12,13-H]-9,12-octadecadienoic acid and its lipoxygenase products (hydroperoxyoctadecadienoic acid, (HPOD) and hydroxyoctadecadienoic acid (HOD)) were performed as described previously (5). [9- or 4

18

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13- 0 ]-9- or 13-HPOD and [9- or 13- 0,]-9- or 13-HOD were synthesized by enzymatic lipoxygenation of linoleic acid with tomato or soy bean lipoxygenase under [ 0 ]-oxygen gas (HPOD) and subsequent reduction with NaBH (HOD), respectively. Incubation experiments (150 or 250 mg/L of substrate) were performed in 1000 mL shake flasks with 200 ml yeast cultures. Saccharomyces cerevisiae (IfG 06136, RH-strain) was purchased from the IfGB Berlin, Germany. Sporobolomyces odorus (CBS 2636) was obtained from the CBS, The Netherlands. 2

l8


2

4

Table I. Configuration and Optical Purity of Naturally Occurring yNonalactone Source

Configuration

S odorus ATCC 24259 S. odorus KTCC 26697

milk Cheddar cheese

Enantiomeric purity %e.e.

(R) (R) (R)

44 66 80

(S)

13

Consequently, we investigated the biosynthesis of y-nonalactone in Saccharomyces cerevisiae and Sporobolomyces odorus in order to elucidate the

enzymatic steps involved in the formation of lactones with odd numbered carbon chains starting from linoleic acid.

Results and Discussion In a series of labeling experiments two different pathways were shown to be involved in the biosynthesis of y-nonalactone in yeast (Figure 1). Pathway I is based on a 13-lipoxygenase / reductase catalyzed route involving subsequent P-oxidation of 13-HOD into (5)-5-decalactone. In isotopic labeling experiments [13- 0 ]-(S)-13-H(P)OD as well as [9,10,12,13- H ]-(S)13-H(P)OD and [13- H!]-(±)-13-HOD were transformed into labeled (5)-8decalactone (~ 60-100% e.e.). 18

2

1(2)

2

Takeoka et al.; Aroma Active Compounds in Foods ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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(5)-8-Decalactone and (S)-5-hydroxydecanoic acid are further metabolized in S. cerevisiae by a-oxidation, a new metabolic route shown to be very effective for metabolization of (S)-configurated 5- and 3-hydroxy fatty acids in yeasts. cc-Oxidation pathways are known in plants and were recently found to be involved in the metabolization of 3-methyl branched fatty acids in mammalian liver tissue. The yeast S. cerevisiae obviously uses an enzymatic a-oxidation step to transform (S)-5-hydroxydecanoic acid into (S)-4-hydroxynonanoic acid and (5)-y-nonalactone (Figure 2). The origin of y-nonalactone by this pathway could be demonstrated by labeling experiments with H - , 0 - and C- precursors. y-Nonalactone and 4-hydroxynonanoic acid are further metabolized in S. cerevisiae by a-oxidation. The resultant 3-hydroxyoctanoic acid shows (S)configuration with high enantiomeric purity (~ 96 % e.e.). This is in accordance with a metabolization of (i?)-3-hydroxyoctanoic acid by the multifunctional protein (MFP) of yeast possessing (/?)-stereospecificity (6). (S)-3-hydroxyoctanoic acid is transformed to hexanoic acid by two a-oxidation steps.


2

Pathway I

l 8

Pathway II

S. cerevisiae

y-Nonalactone

(S)~40-50 % e.e.

( / ? )

_

4 0

.

5 0

%

e e >

Figure 1. Biosynthetic routes to y-nonalactone in yeast (R = -(CHJe-COOH).


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(3-Oxidation

COOH

v.

HCOOH

Cyclisation

^

a-Oxidation

WTO*

(S)-8-Decalactone

(S)-y-Nonalactone

(5)-100%e.e.

(S)-60 % e.e.

10

300 -(S)-I Oil-13-HOD

23/77

>M

- (S)-( Oi ]-5 -Decalactone

8

-(SH^Oil-y-Nonalactone 6

16

0 /

18

&

Q

2

31 IW 0 50

100 Incubation Time (h)

150

8

200

Figure 2. Metabolization of [13- Oj]-(S)-13'Hydroxy-9Z, 11 E-octadecadienoic acid (150 mg/L) by S, cerevisiae.


180

The (^-configuration of the Ci -hydro(pero)xyde initially formed by lipoxygenation is in accordance with the (^-configuration of the formed 5decalactone (92 % e.e.). y-Nonalactone resulting from this pathway also shows (5)- configuration (50-70 % e.e.). The alternative pathway II to y-nonalactone is based on a 9-lipoxygenase / reductase catalyzed reaction of linoleic acid. Linoleic acid, [9,10,12,13- H ]-(S)9-hydro(pero)xy-10£,12Z-octadecadienoie acid as well as [9- H i ]-(±)-9-hydroxy10£,12Z-octadecadienoic acid are converted to 9-oxo-10£,12Z-octadecadienoic acid, isomerized to 9-oxo-ll£,13£-octadecadienoic acid and oxygenated by a Baeyer-Villiger enzyme. The formed Baeyer-Villiger ester is hydrolyzed to yield azelaic acid and 2£,4£-nonadien-l-ol. 2£,4£-Nonadien-l-ol is further oxidized to 2£,4£-nonadienoic acid, transformed to 3Z-nonenoic acid by acyl-CoA-reductoisomerase and metabolized within the "epoxyde pathway" of yeast to yield (R)-ynonalactone (Figure 3). This "epoxyde pathway" has been intensively studied in the yeast S. odorus (3). Incubation experiments using deuterated 9hydro(pero)xy-10£,12Z-oetadecadienoie acids yielded y-nonalactone (-50 % e.e.). 8

2

4


2

Conclusion The studies revealed two completely different pathways to be operative in the degradation of 13- and 9-hydro(pero)xyoctadecadienoic acids in yeast. A 13lipoxygenase pathway with a-oxidation activity and a 9-lipoxygenase pathway with enzymatic Baeyer-Villiger oxidation activity were characterized in yeast cleaving the carbon chain of oxygenated linoleic acid into C fragments which are further transformed into y-nonalactone and metabolized by peroxysomal Poxidation, respectively. 9

Acknowledgment We are grateful to „Arbeitsgemeinschaft industrieller Forschungsvereinigungen (AiF), Otto von Guericke e. V." for financial support of this study in the course of the research project No. 11428 N . This project has been supported by funds of „Bundesminister fur Wirtschaft (BMWi)".

References 1. 2.

Schöttler, M.; Bohland, W. Helv. Chim. Acta 1996, 79, 1488-1496. Haffner, T.; Tressl, R. J. Agric. Food Chem. 1996, 44, 1218-1223.



181

'COOH

y-Nonalactone

(R)-44 % e.e.

Incubation Time (h)

2

Figure 3. Metabolization of [9J0J2J3' H ]-(S)-9-Hydroxy-10EJ2Z-octadecadienoic acid (250 mg/L) by S. odorus. 4


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3. 4. 5.


6.

Haffner, T.; Tressl, R. Lipids 1998, 33, 47-58. Garbe, L . - A . ; Tressl, R, unpublished Albrecht, W.; Schwarz, M.; Heidlas, J.; Tressl, R. J. Org. Chem. 1992, 57, 1954-1956. Filppula, S.; Sormunen, R. T.; Hertig, A.; Kunau, W. H . ; Hiltunen, K . J. Biol. Chem. 1995, 270, 27453-27457.


Biosynthesis of -Nonalactone in Yeast - American Chemical Society

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