Synthesis of 2-Methoxy-3-alkylpyrazines by

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Downloaded by UNIV LAVAL on May 5, 2016 | http://pubs.acs.org Publication Date: August 25, 1986 | doi: 10.1021/bk-1986-0317.ch021

Synthesis of 2-Methoxy-3-alkylpyrazines by Pseudomonas perolens R. C. McIver1 and G. A. Reineccius Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108 The influence of media components on the production of 2-methoxy-3-isopropyl and 2-methoxy-3-secbutyl pyrazines by Pseudomonas perolens (ATCC 10757) and selected mutants of this culture was studied. Pyrazine production was observed only during the stationary growth phase. The parent culture produced a maximum of 42 ng/mL pyrazines while selected mutants were found to produce a maximum of 15,760 ng/mL. The parent and mutant strains were found to exhibit similar responses to nutrient sources. Maximum pyrazine production was observed using pyruvate (1%), lactate (1%) or nutrient broth as carbon source. Nitrogen source had no influence on pyrazine production. Maximum pyrazine formation was observed when phosphate level ranged from 0.4 mM to 1.2 mM in the media. There are many reports of Pseudomonas cultures producing musty, earthy, and potato-like odors (1-6). The work of Morgan et al. (7) established 2-methoxy-3-isopropyl pyrazine to be partially responsible for these odors. Subsequently, 2-methyoxy-3-isopropyl pyrazine was found in bell peppers (8), a similar compound 2-methoxy-3-secbutyl pyrazine was identified in galbanum oil (9), and several 2-methoxy-3-alkyl pyrazines were identified in various raw botanicals (10). The odor threshold exhibited by 2-methoxy-3-isobutyl pyrazine (1 part in 10 1 2 ,8) indicates flavor significance for these compounds even at the exceptionally low concentrations in which they occur in foods and other natural products. Producing these compounds from microbial fermentations could be an economical source of flavor for the food industry. The work of MacDonald (11, 12) on the synthesis of aspergillic acid from valine and isoleucine suggests that 2-methoxy-3-alkyl pyrazine, as one of a group of substituted pyrazines and diketopiCurrent address: Sunkist Growers Inc., 760 Sunkist St., Ontario, CA 91761

1

0097-6156/ 86/ 0317-0266506.00/ 0 © 1986 American Chemical Society Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

21.

Synthesis of

MCI V E R A N D R E I N E C C I U S

2-Methoxy-3-alkylpyrazines

267

pyrazines, is formed metabolically by microorganisms. Furthermore, the odor intensity and character of Pseudomonas perolens cultures has been shown to be influenced by media composition (4, _5)· Because the potential for producing "natural" 2-methoxy-3-alkyl pyrazines v i a fermentation might be improved by optimizing media composition, we studied the influence of media components on the synthesis 2-methoxy-3-isopropyl pyrazine by cultures of Pseudomonas perolens and selected mutant strains. Materials and Methods Microbial. Pseudomonas perolens subsp gdansk ATCC 10757 was obtained from the American Type Culture C o l l e c t i o n (Rockville, MD) and revived according to the procedure recommended by the supplier. The parent s t r a i n and subsequently isolated mutants (obtained by treatment with N-methyl-N -nitro-N-nitrosoguanidine, Γ3) were main tained by combining equal volumes of an early logarithmic phase nutrient broth culture with an 80% glycerol solution in small v i a l s and storing at -20°C. Cultures were routinely grown in minimal salt media containing per l i t e r : Na2HP04*7H20 8.2g, KH2PO4 2.7g, NH4CI l.Og, FeS04'7H 0 2.0mg, MgS04«7H20 O.lg, Ca(N03>2 5mg at pH 7.2. The carbon sources were f i l t e r s t e r i l i z e d and added immediately before use. For investigations of d i f f e r e n t media components, a culture from frozen stock was inoculated into an 0.5% glucoseminimal salts culture and grown overnight. The c e l l s were c e n t r i fuged and washed twice with minimal media (no carbon or nitrogen source) then inoculated into fresh media at a l e v e l of approximately 10^ cfu/mL. A l l cultures were grown at ambient temperature ( 22°C) with rotary shaking at 180 rpm. At appropriate i n t e r v a l s , samples were taken for c e l l counts, pH and pyrazine analysis.


1

2

Pyrazine Quantitation: Culture samples were analyzed for pyrazine content using a commercial dynamic headspace apparatus (Hewlett Packard, Purge and Trap Headspace Concentrator, model 7675A). This apparatus used Tenax as the adsorbent and desorbed v i a heating (180°C). Purging of the cultures was done with helium (100 mL/min) for 10 min at ambient temperature. Analysis was performed on a Hewlett Packard model 5880 gas chromatograph using a nitrogen speci f i c detector (NPD)· A 15 m χ 0.32 mm DB-5 (0.25 pm film thickness) column was used for separation (J & W S c i e n t i f i c , Rancho Cordova, CA). Isobutyl pyrazine was used as the internal standard. Identity of the pyrazines from microbial cultures was confirmed by GC-MS using authentic reference compounds. Results and Discussion E f f e c t of Carbon Source. The growth of Pseudomonas perolens as well as culture levels of 2-methoxy-3-isopropyl pyrazine in minimal salts plus 1% pyruvate are shown in Figure 1. High c e l l density was obtained but the presence of 2-methoxy-3-isopropyl pyrazine was not detected u n t i l after c e l l numbers were no longer increasing. At this point, the amount of pyrazine increased rapidly reaching a maximum on the f i f t h day. The growth and pyrazine synthesis with other carbon sources showed similar behavior. The results are pre sented in Table I, and plotted for selected carbon sources at 1%

Parliment and Croteau; Biogeneration of Aromas ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

BIOGENERATION OF AROMAS

268

l e v e l in Figure 2. No growth was observed when sucrose or lactose was the sole carbon source, nor when any carbon source was present at 5% or greater concentration.

Table I.


Carbon Source Glucose

The influence of carbon source on the production of 2-methyl-3-isopropyl pyrazine by P.perolens*

%

(w/v) 0.2 0.5 1.0 2.0

2-Methoxy 3-isoPropyl Pyrazine (ng/mL) 36 25 18 9

% vs. 1% Lactate 75 52 38 19

Fructose

0.2 0.5 1.0 2.0

25 19 20 10

52 40 42 21

Pyruvate

0.2 0.5 1.0 2.0

14 14 42 31

29 29 88 65

Lactate

0.2 0.5 1.0 2.0

15 14 48 37

31 29 (100) 77

Na Acetate

1.0

20

—

Nutrient broth

—

46

96

With the exception of sodium acetate containing cultures, viable c e l l counts were similar in the range of lO^-lO^O cfu/mL. In a l l cultures where growth was observed, a strong musty-green potato odor, c h a r a c t e r i s t i c of 2-methoxy-3-isopropyl pyrazine was present. Although 2-methoxy-3-secbutyl pyrazine was not detected by GC, a d e f i n i t e green-pea pod l i k e odor was noted at the retention time corresponding to this compound when the effluent from the gc column was s n i f f e d , suggesting culture concentrations below the l i m i t of detection. The reported threshold of this pyrazine (14) is except i o n a l l y low and apparently below the detection l i m i t of the GC. As can be seen from Table I, pyruvate and lactate at 1% resulted in the highest y i e l d of 2-methoxy-3-isopropyl pyrazine. At the 2% level these substrates resulted in a decrease of approximately 30% in the amount of pyrazine with a much greater drop at 0.2% and 0.5% l e v e l s . This is in contrast to glucose and fructose as carbon source where the amount of pyrazine synthesized was inversely related to the i n i t i a l concentration of sugar in the media. Sodium acetate yielded about 20 ng/mL which is impressive considering that the c e l l numbers were 3-4 orders of magnitude lower than that found



21. MclVER AND REINECCIUS

Synthesis of 2-Methoxy-3-alkylpyrazines

Figure 1. Relationship between the growth of ?AmdomoncU> pQJwtzvib ( ) d the production of 2-methoxy-3-isopropyl pyrazine ( )· a n

2-Me-3-lsop(ng/ml)

°

1

2

TIMEfDAYS)

3

5

6

7

8

Figure 2. The influence of carbon source on the production of 2-methoxy-3-isopropyl pyrazine (ng/mL). Pyruvate ( ), lactate ( ), Glucose ( ).


270


f o r the 3- and 6-carbon s u b s t r a t e s . The complex growth media sup p o r t e d dense growth and s u b s t a n t i a l amounts of p y r a z i n e s y n t h e s i s .


E f f e c t of N i t r o g e n s o u r c e . N i t r o g e n source and c o n c e n t r a t i o n e x e r t e d a minor i n f l u e n c e on the amount of 2 - m e t h o x y - 3 - i s o p r o p y l p y r a z i n e s y n t h e s i z e d in c u l t u r e s . I t has been r e p o r t e d by a number o f i n v e s t i g a t o r s t h a t ammonium s a l t s as the source of n i t r o g e n r e s u l t s in good growth but show n e g a t i v e e f f e c t s on m e t a b o l i t e p r o d u c t i o n (15-19). The i n f l u e n c e of i n c r e a s i n g c o n c e n t r a t i o n s of ammonium c h l o r i d e as the s o l e n i t r o g e n source and the e f f e c t s of d i f f e r i n g c o n c e n t r a t i o n s of o t h e r sources of n i t r o g e n on p y r a z i n e p r o d u c t i o n is shown in Table I I . I n a l l c a s e s , the growth c u r v e s and pH p r o f i l e s were s i m i l a r . A l s o , appearance of 2 - m e t h o x y - 3 - i s o p r o p y l p y r a z i n e c o i n c i d e d w i t h the c e s s a t i o n of the

Table I I .

The i n f l u e n c e of n i t r o g e n source and c o n c e n t r a t i o n on the p r o d u c t i o n of 2 - m e t h o x y - 3 - i s o p r o p y l p y r a z i n e by P. perolens. 2-Methoxy-3-isopropyl

Ν

source

NH4CL

L-valine L-proline L-of-amino--η-butyric a c i d

p y r a z i n e (ng/mL) Ν concentration 50 15 30 25 31 31 33 35 27 29

mM 100 15 29 36 30

growth phase. The o n l y i n s t a n c e where the r e s u l t s d i f f e r e d s i g n i f i c a n t l y was when ammonium c h l o r i d e was p r e s e n t at an i n i t i a l con c e n t r a t i o n of 100 mM. The f a c t t h a t L - p r o l i n e , L - v a l i n e and L-ot-amino η-butyric a c i d gave s i m i l a r r e s u l t s suggests t h a t exoge nous a d d i t i o n of v a l i n e , the p r o b a b l e p r e c u r s o r of 2 - m e t h o x y - 3 - i s o p r o p y l p y r a z i n e , does not i n c r e a s e the amount of the p y r a z i n e made by the c e l l . S i m i l a r r e s u l t s have been r e p o r t e d by Morgan ( 2 0 ) . E f f e c t of Phosphate. I n c o n t r a s t to the l i m i t e d e f f e c t s of c a r b o n and n i t r o g e n source and c o n c e n t r a t i o n , the amount of 2- m e t h o x y - 3 - i s o p r o p y l p y r a z i n e s y n t h e s i z e d by Pseudomonas perolens was found to be s e n s i t i v e to the l e v e l of phosphate in the media ( T a b l e I I I ) . The u s u a l media c o n t a i n e d 50 mM phosphate as b o t h a n u t r i e n t and to p r o v i d e b u f f e r i n g c a p a c i t y . I n o r d e r to b u f f e r the media at lowered amounts of phosphate, 0.2 M 3- ( N - m o r p h o l i n o ) - p r o p a n e s u l f o n i c a c i d (MOPS) was added ( 2 1 ) . A l t h o u g h c l e a r d i f f e r e n c e s were observed in the amounts of 2 - m e t h o x y - 3 - i s o p r o p y l p y r a z i n e produced, the data were more s t r i k i n g when p o p u l a t i o n d e n s i t y was c o n s i d e r e d . When compared on the b a s i s o f p y r a z i n e s y n t h e s i z e d per 10*^ cfu/mL, the c u l t u r e grown in media c o n t a i n i n g 0.2 mM phosphate produced almost 50 f o l d more p y r a z i n e than the one grown in 50 mM phosphate. The i m p o r t a n t r o l e of phosphate in the c o n t r o l of m e t a b o l i t e s y n t h e s i s has been r e c o g n i z e d f o r a number of y e a r s as reviewed by Weinberg ( 2 2 ) . As w i t h the cases of c a r b o n and n i t r o g e n s o u r c e s , s e n s i t i v i t y to c o n t r o l by these s u b s t r a t e s depends on the organism and the m e t a b o l i t e . R e c e n t l y , Young e t a l . (19) overcame the


21.

MCIVER AND REINECCIUS

Synthesis

of2-Methoxy-3-alkylpyrazines

271

Table I I I . The influence of phosphate concentration on the production of 2-methoxy-3-isopropyl pyrazine by P. perolens . * i n i t i a l PO4 (mM) 0.2 0.4 1.2 5.0 20.0 50.0 100.0

2-Methoxy-3-isopropyl pyrazine (ng/mL) 74 130 133 78 46 37 11

viable c e l l s (cfu/mL) 4xl0 lxlO 4x10 9x10 8xl0 lxlO 9xl0 8

9

9

9

9

1 0

9


grown in minimal salts + 0.2 M MOPS + 1% pyruvate

i n i h i b i t o r y effects of both phosphate and ammonia on lincomycin synthesis by Streptomyces lincolnensis . They added substantial amounts of MgS04 the phosphate containing media which resulted in the p r e c i p i t a t i o n of MgNH4P04. As ammonia and phosphate were removed from the media by the c e l l s , more of the precipitate would dissolve. The e f f e c t was a slow release of the two nutrients. They observed an increase in both the rate of lincomycin synthesis and titer. t 0

Investigation of Mutant Strains. Murray (14) suggested that the amino acids valine, leucine and isoleucine were precursors of 2-methoxy-3-isopropyl, 3-isobutyl and 3-secbutyl pyrazines, respect i v e l y . The effects of mutations in the branched chain amino acid pathways on the synthesis of these compounds was investigated. During the process of i s o l a t i n g mutants, we noticed that one culture produced a very intense musty-green odor. Upon i s o l a t i o n and c u l t u r i n g of the s t r a i n responsible, J. was found to have 2-methoxy-3-isopropyl- and 2-methoxy-3-secbutyl pyrazines at levels of 12,500 and 150 ng/mL, respectively, when grown in nutrient broth culture. The bacterium isolated was not found to have d i f f e r e n t growth requirements from the parent s t r a i n .

Table IV.

The production of 2-methoxy-3-alkyl pyrazines by a mutant of P. perolens*

Carbon Source Glucose

Lactate

Pyruvate

Glycerol Nutrient Broth

Cone. (% w/v) 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0

—

pyrazine (ng/mL) 2-MeO-3-isoPr 2-MeO-3-secB 2030 21 11 960 840 9 68 6280 9890 112 48 4730 55 5150 100 11120 8400 95 3610 86 3170 69 145 12500

ratio isoPr/secB 97 87 92 92 88 99 94 111 88 42 46 86



272

Investigations with d i f f e r e n t carbon sources (Table IV) showed that although the amounts of the two pyrazines varied according to the carbon source used, the r a t i o of the isopropyl to secbutyl pyra zine remained r e l a t i v e l y constant. This suggested that either a v a i l a b i l i t y of precursor followed a set r a t i o , or else enzyme spe c i f i c i t y for the d i f f e r e n t side chains played a role in determining the r e l a t i v e amounts of each compound synthesized. Accordingly, strains auxotrophic for leucine (leu"), isoleucine ( i l u ~ ) , or leucine, valine, isoleucine, and pantothenic acid (leu"> v a l " , i l u " , pant") were isolated from cultures treated with N-methyl-N -nitro-N-nitrosoguanidine. Also, a s t r a i n that grew well on the leucine analog, 4-azaleucine was i s o l a t e d . f


Metabolic Implications of Auxotrophs. The levels and ratios of the 2-methoxy-3-alkyl pyrazines synthesized by these various mutants are contained in Table V. For the auxotrophs, the required nutrients

Table V.

The production of 2-methoxy-3-alkyl pyrazines by selected mutant strains of P. perolens .

Strain

ilu" leu" aza" val",ilu",leu"

C-source (1%) glycerol pyruvate pyruvate pyruvate pyruvate pyruvate pyruvate + t-leu (200 ug/mL)

pyrazine (ng/mL) 2-MeO-3isoPr 2-MeO-3secB 3170 69 100 11120

Synthesis of 2-Methoxy-3-alkylpyrazines by

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