Cloning and Expression of an A-Acetolactate Decarboxylase Gene from Streptococcus Lactis Subsp

Home , Acetoin

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1988, p. 1889-1891 Vol. 54, No. 7 0099-2240/88/071889-03$02.00/0 Copyright X) 1988, American Society for Microbiology

Cloning and Expression of an a-Acetolactate Decarboxylase Gene from Streptococcus lactis subsp. diacetylactis in Escherichia coli DETLEF GOELLING AND ULF STAHL* Technische Universitat Berlin and Institut fur Gaerungsgewerbe, Abteilung Mikrobiologie, Seestrasse 13, D-1000 Berlin 65, Federal Republic of Germany Received 28 January 1988/Accepted 19 April 1988

The Streptococcus lactis gene coding for a-acetolactate decarboxylase (ADC) was cloned in Escherichia coli. Subsequent subcloning in E. coli showed that the ADC gene was located within a 1.3-kilobase DNA fragment. The ADC gene was controlled by its own promotor. Gas chromatography showed that S. lactis and the transformed E. coli strains produced the two optical isomers of acetoin in different ratios.

Diacetyl (2,3-butanedione), one of the major compounds carried out (14). DNA from S. lactis was restricted with causing off-flavor in beer, is formed by a nonenzymatic Sau3A and then hybridized with pADC1 and pBR322. The reaction from o-acetolactate, an intermediate compound of vector pADC1 hybridized to a 4.5-kilobase (kb) fragment, the isoleucine-valine-pathway (Fig. 1). During beer matura- whereas pBR322 showed no homology to the DNA of S. tion, which may require several weeks, diacetyl is enzymat- lactis (Fig. 2). ically reduced by yeast to acetoin and subsequently to pADC1 carries a 4.5-kb DNA fragment of S. lactis, 2,3-butandiole, which, in contrast to diacetyl, does not enabling E. coli to produce the same amount of acetoin (16 influence beer flavor. ,ug/ml) as the source strain (Table 1). By subsequent dele- The direct conversion of a-acetolactate into acetoin is tions characterized by restriction analysis, this fragment catalyzed by a-acetolactate decarboxylase (ADC). This en- could be shortened to about 1.3 kb (isolate p31-H1) without zyme is found in various procaryotes, for example, Strepto- affecting the quantity of acetoin produced. Deletions which coccus lactis subsp. diacetylactis (6) and Enterobacter aero- included this whole area (like pADC4) or part thereof genes (13, 16) but is not present in yeasts or other eucaryotes (pADC3 and pADC5) led to a total loss of acetoin produc- (9). of to or Addition ADC the fermenting wort to freshly tion, whereas deletions beyond this area had no effect brewed beer (young beer) can dramatically reduce the for- (pADC2 and p31-3S). It can therefore be concluded that the mation of diacetyl, allowing a shorter lagering time (7, 8, 10). whole ADC gene must be located on An alternative method for using ADC in beer maturation this subfragment. Furthermore, as the orientation of this subfragment relative could be the utilization of a yeast brewing genetically altered to the adjacent vector DNA did not influence acetoin forma- to produce ADC. In order to comply with current food tion, it seems plausible to conclude that the ADC gene is legislation, the donor of the ADC gene must be absolutely under the control of its own promotor. harmless and suitable for food production. In contrast to As described above, to screen for acetoin-forming trans- Enterobacter spp., which are putrefying bacteria, S. lactis is formants the VP reaction was used. Since the VP reaction is already used in the cheese-making industry. In addition, positive not only with acetoin but also with diacetyl (19) ADC from S. lactis has proved to be much more active in reducing the formation of diacetyl than the Enterobacter (Fig. 1), it was necessary to confirm whether the positive VP reaction of the transformants was in fact caused by the enzyme when added to fermenting wort (5). presence of acetoin. For its identification, the vicinal dike- The initial stage in producing such a yeast was to clone the tones were extracted with diethyl ether from the culture ADC gene of S. lactis and demonstrate its expression in Escherichia coli. Total cell DNA of S. lactis subsp. diacety- filtrate of SF8(pADC2), dried with sodium sulfate, concen- trated, and separated by gas chromatography (Carbowax 20 lactis DSM 20384 (11) was with partially digested Sau3A, M, 70°C/4°C). According to data ligated into the BamHI site of pBR322, and transferred to E. the chromatography (not shown), only acetoin was present; no traces of diacetyl were coli SF 8 (for a description of the host strain, see reference found. 17). All cloning steps were carried out as described previ- To clarify whether the formation of acetoin was in fact ously (4, 14). Plasmid-carrying E. coli clones were selected with ampicillin. catalyzed directly by ADC or indirectly by diacetyl reduc- As shown by the Voges-Proskauer reaction E. tase (Fig. 1), the ability of the transformants to convert (VP) (12), diacetyl (added instead of pyruvate to the substrate) to coli cannot form acetoin or diacetyl when grown on pyruvate acetoin was examined. In all cases, acetoin could not be as the sole carbon source (Table 1). Among more than 600 E. detected. coli transformants 1 In addition, other workers (2, 3, 18) have shown carrying pieces of the S. lactis genome, that procaryotes, in contrast to eucaryotes, do not contain was found that gave a positive VP reaction. As this clone, the pyruvate dehydrogenase complex, which can combine SF8(pADC1), seemed to carry the ADC gene of S. lactis, its acetaldehyde and pyruvate or two molecules of acetal- extrachromosomal DNA was further characterized and its dehyde (head to head) to form acetoin. It is therefore ability to form acetoin was verified by gas chromatography. obvious that the cloned of S. lactis must In order to demonstrate that the cloned DNA fragment fragment contain the ADC gene. From investigations with Bacillus polymyxa, from S. were originated lactis, hybridization experiments it is known that the proportion of the two optical isomers of acetoin [(R)- and (S)-acetoin] formed by ADC is strain * Corresponding author. specific (18). It is not known whether this specificity is solely 1889 1890 NOTES APPL. ENVIRON. MICROBIOL.

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a;*o-O1hydroxy AOC DRn 23.1 9.4 8.5 a-Ksto- Aoetoin 4.3 lsoislete jA TAI AR 2.3 2.0 Valin- 2.3-Eutendlol FIG. 1. Main steps of the valine-biosynthetic pathway (1, 15). Dependent on the organism, acetoin may be formed directly from a-acetolactate or via diacetyl which arises spontaneously. Abbrevi- ations for the enzymes catalyzing the individual steps: AS, acetolactate synthase; RI, reductoisomerase; DH, dehydrase; TA, tran- saminase; DR, diacetyl reductase; AR, acetoin reductase. dependent on the ADC gene or can also be influenced by the FIG. 2. Hybridization of Sau3A-digested S. lactis DNA with host strain. To examine these two alternatives, the amount pADC1 and pBR322. Lanes 1 and 3, S. lactis DNA digested with of (R)- and (S)-acetoin was determined in culture filtrates of Sau3A; lane 2, S. lactis DNA hybridized with pADC1; lane 4, S. S. lactis and E. coli carrying pADC2 by gas chromatography lactis DNA hybridized with pBR322; lane 5, X DNA digested with under the following conditions: acetoin was extracted with HindIll as size markers. diethyl ether, derivated with (R)-(+)-a-methoxy-a-tri- fluoromethyl-phenylacetic acid-chloride and separated on a known in eucaryotes. Subsequent fermentation tests will column (DB210, 140NC isotherm). The data obtained showed show whether the maturation time of young beer can be that S. lactis produced about 91% (R)- and 9% (S)-acetoin reduced by using a genetically engineered yeast strain in the (which is in good agreement with the values found in B. same way as adding the appropriate enzyme (ADC) to polymyxa), whereas surprisingly, the transformed E. coli fermenting wort. strain showed a proportion of 52 and 48, respectively. As a chemical racemerization can be excluded (the culture me- We thank J. Heidlas for the gas chromatography. We are also dium was buffered at pH 7.0), one can suppose either that indebted to K. Lehmann for technical assistance and to E. Quaite the ADC expressed by E. coli had lost its original stereospe- for critical reading of the manuscript. cificity (and eventually, by posttranslational modification, This research was supported by the Arbeitsgemeinschaft Indu- became different in the two organisms) or that the host strain strieller Forschung. contains a racemase which is by chance specific for acetoin. LITERATURE CITED At present it is not possible to determine which of these 1. Adler-Nilsen, J. 1987. Newer uses of microbial enzymes in food alternatives is correct. processing. Trends Biotechnol. 5:170-174. After having cloned and identified the ADC gene from S. 2. Alkonyi, I., E. Bolygo, L. Gyocsi, and D. Szabo. 1976. Studies on lactis, the next step towards a diacetylless brewing yeast will the formation of acetoin from acetaldehyde by the pyruvate be expression of the ADC gene in Saccharomyces cerevi- dehydrogenase complex and its regulation. Eur. J. Biochem. siae, although as yet, no naturally occurring ADC activity is 66:551-557. 3. Baggetto, L. G., and A. L. Lehninger. 1987. Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits TABLE 1. Acetoin production by E. coli transformants contain- pyruvate oxidation as well as other aldehydes. Biochem. ing pADC1 or various subfragments of pADC1 by shotgun cloning Biophys. Res. Commun. 145:153-159. from S. lactis genomic DNAa 4. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513-1523. Brau. 1111trains Charatericzation of the 5. Dellweg, H. 1985. Diacetyl und Bierreifung. Monatsschr. Z Stre*ptococal DNA Acetoin [ppm) 6:262-266. Clones Cloned In E.coll 6. Godtfredsen, S. E., H. Lorck, and P. Sigsgaard. 1983. On the occurrence of a-acetolactate decarboxylases among microorga- S.ueoti ODM 20384 Source Strain 1t nisms. Carlsberg Res. Commun. 48:239-247. 111ool*Fape Cloningati raln not detesctble 7. Godtfredsen, S. E., and M. Ottesen. 1982. Maturation of beer U PM upUPR *FS pAOC1 4.5kb 10 with a-acetolactate decarboxylase. Carlsberg Res. Commun. UPS' pAOC2 le 47:93-102. UpS' p31-3U 16 8. S. M. P. K. T. UPS/p 31-M, 16 Godtfredsen, E., Ottesen, Sigsgaard, Erdal, Up5epADC 3 not deteotable Mathiasen, and B. Ahrenst-Larsen. 1983. Use of a-acetolactate Sp/pADOC4 - not dete-ctble decarboxylase for accelerated maturation of beer. EBC Con- *Sp/ pAOC 5 not detectable gress Lecture No. 17, p. 161-168. 9. Godtfredsen, S. E., A. M. Rasmussen, M. Ottesen, T. Mathiasen, a For comparison, the acetoin data for the cloning and source strains are and B. Ahrenst-Larsen. 1984. Application of the a-acetolactate also given. Acetoin was determined spectrophotometrically (at 540 nm) by the decarboxylase from Lactobacillus casei for accelerated matura- VP reaction in culture filtrates (culturing time, 24 h). The limit of detection 49:69-74. was 3 ppm of acetoin. The bars in the middle column indicate the cloned tion of beer. Carlsberg Res. Commun. streptococcal DNA areas out of the 4.5-kb large DNA fragment shown which 10. Godtfredsen, S. E., A. M. Rasmusen, M. Ottesen, P. Rafn, and was gained from the streptococcal genomic library. Some restriction sites are N. Peitersen. 1984. Occurrence of a-acetolactate decarboxylases given: S, Sau3A; P, PvuII; H, HindIII; E, EcoRV; R, RsaI. among lactic acid bacteria and their utilization for maturation of VOL. 54, 1988 NOTES 1891

beer. Appl. Microbiol. Biotechnol. 20:23-28. genes. Carlsberg Res. Commun. 49:577-584. 11. Klaenhammer, T. R., L. L. McKay, and K. A. Baldwin. 1978. 16. Sone, H., T. Fuji, K. Kondo, and J.-I. Tanaka. 1987. Molecular Improved lysis of group N streptococci for isolation and rapid cloning of the gene encoding a-acetolactate decarboxylase from characterization of plasmid deoxynucleic acid. Appl. Environ. Enterobacter aerogenes. J. Biotechnol. 5:87-91. Microbiol. 35:592-600. 17. Stahl, U., E. Leitner, and K. Esser. 1987. Transformation of 12. Krampitz, L. 0. 1957. Preparation and determination of acetoin, Penicillium chrysogenum by a vector containing a mitochon- diacetyl and acetolactate. Methods Enzymol. 3:271-283. drial origin of replication. App. Microbiol. Biotechnol. 26:237- 13. L0ken, J. P., and F. C. St0rmer. 1970. Acetolactate decarboxy- 241. lase from Aerobacter aerogenes. Eur. J. Biochem. 14:133-137. 18. Ui, S., T. Masuda, H. Masuda, and H. Muraki. 1986. Mecha- 14. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular nism for the formation of 2,3-butandiole stereoisomers in Bacil- cloning: a laboratory manual. Cold Spring Harbor Laboratory, lus polymyxa. J. Ferment. Technol. 64:481-486. Cold Spring Harbor, N.Y. 19. Wainwright, T. 1973. Diacetyl-a review. J. Inst. Brew. 79:451- 15. Polaina, J. 1984. Cloning of Saccharomyces cerevisiae ILV 470.