Improvement of Beer Brewing by Using Genetically Modified Yeast

Chapter 14

Improvement of Beer Brewing by Using Genetically Modified Yeast 1

2

1

J. Vogel , K. Wackerbauer , and U. Stahl 1

Fachgebiet für Mikrobiologie und Genetik, Technische Universität Berlin, Gustav-Meyer-Allee 25, D-13355 Berlin, Germany Forschungsinstitut für Brauerei und Mälzerei der Versuchs- und Lehranstalt für Brauerei in Berlin, Seestrasse 13, D-13353 Berlin, Germany

Downloaded by UNIV OF ARIZONA on September 7, 2015 | http://pubs.acs.org Publication Date: October 5, 1995 | doi: 10.1021/bk-1995-0605.ch014

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During fermentation of beer, yeast cells produce ethanol as well as diacetyl, a substance with a low taste threshold of about 0.2 ppm (1). This unpleasant butter-like flavor can be removed by means of a separate maturation period of 2-6 weeks during which yeast cells degrade the diacetyl. Recombinant DNA techniques have made it possible to reduce the amount of α-acetolactate, the precursor molecule of diacetyl, in yeast. The α-acetolactatedecarboxylase (ALDC) gene from Acetobacter pasteurianus, which decarboxylates α-acetolactate directly into acetoin without forming diacetyl, was isolated and transferred into brewer`s yeast. Test fermentation with recombinant yeast having A L D C activity showed no differences in fermentation properties. At the end of the fermentation process hardly any diacetyl was measurable; thus the subsequent maturation period was unnecessary. Microorganisms play a significant role in food biotechnology. They are used in brewing, baking, dairy products as well as for the production of desired flavors. Lactic acid bacteria are used to produce products such as yogurt, cottage cheese and butter milk. They are also used as starter cultures, not only for lactic acid production, but also for the production of diacetyl to give a "genuine" butter flavor. Soy products as an alternative to meat have become more popular over the last few years. Anyone who has eaten such a "meat substitute" will testify that it tastes like meat. This is due to the Rhizopus and Mucor species which possess proteolytic activity and therefore degrade the soy protein into peptides, which then confers the final product a meat-like flavor. Some fungi species are able to produce familiar, or pleasant odor, one example being a smell reminiscent of coconut which is often emitted when Trichoderma species have been grown on non-defined media. It is thus not surprising that efforts have been made to use microorganisms as producers of odor compounds for the food industry. Lactones are widely used in the flavoring industry and are characterized by being generally pleasing in odor and flavor. They are described as being fruity, coconut-like, buttery, nut-like or sweet. A variety of yeast and filamentous fungi such as Pénicillium notatum produce lactones when incubated with keto acids. To produce this odor or flavor by chemical means requires seven successive steps, so the advantage of using 0097-6156/95/0605-0160$12.00/0 © 1995 American Chemical Society

In Genetically Modified Foods; Engel, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1995.


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fungi to produce lactones is quite obvious. Another advantage of using biological flavors is, that biotransformation reactions are stereoselective so only one enantiomer is formed and no contaminations of other unpleasant substances are present. In addition, in Germany a "natural" flavor compound is more likely to be accepted by the food administration authorities and the public than a chemical one. Some fungi produce flavor compounds which are unpleasant and not wanted in the final product. They are regarded as contamination and might even be toxic. It is therefore necessary to remove these undesired compounds. Technological improvements such as the optimizing of temperature or the supplementing of nutrients may be one method of solving the problem. However, as the metabolism is ultimately dependent on its genetic constitution, a genetic approach could also be a solution. One possibility could be to interrupt the pathway in which an unpleasant flavor compound is synthesized by using mutagenesis methods. This would lead to the accumulation of an intermediate product which must be flavorless and non toxic for the cell in higher concentrations. Another possibility is the conversion of an unpleasant compound by the cells into a neutral substance, which must of course also be flavorless and nontoxic for the cell. For this approach a gene transfer is necessary, which is only possible by genetic engineering techniques. Diacetyl, an Off-Flavor Compound Produced by Yeast During Fer mentation of Beer Yeast cells used in the brewing process affect the flavor produced. Although beer making is a long-established, traditional process, difficulties occasionally occur in obtaining a product of steady quality. One reason for this is that during fermentation yeast cells, apart from producing ethanol, also produce "off flavor compounds" such as dimethylsulfide (reminiscent of boiled vegetables), hydroxy-2-butenolide ("Maggi"like flavor) and diacetyl which arises from α-acetolactate, an intermediate of the valineisoleucine-pathway. In the main fermentation α-acetolactate leaks out of the yeast cells into the wort, where it is spontaneously oxidatively decarboxylated to diacetyl. Since this compound can be used by the cell as an electron-acceptor, it is taken up again by the yeast cells and reduced via acetoin to 2,3-butanediol. The non-enzymatic conversion of α-acetolactate to diacetyl is a slow reaction, so by the end of the main fermentation α-acetolactate remains in the wort and therefore a maturation must follow. During the maturation α-acetolactate is converted to diacetyl, which is then incorporated into the cells and reduced to acetoin (Figure 1). Because of the low temperature and reduced yeast cell numbers during the maturation the conversion of aacetolactate to diacetyl and the incorporation of diacetyl into the cells takes time, but is necessary during the production of lager beer. Diacetyl has a butter-like flavor with a low taste threshold of about 0.2 ppm. To remove this compound a maturation period of 2-6 weeks is required in order to allow the diacetyl to be reabsorbed into the yeast cells. This makes the brewing process costly. Strategies to Reduce the Diacetyl Content in Beer A way to reduce lagering time would be by simply adding purified bacterial A L D C to the fermenting wort. This enzyme which decarboxylates α-acetolactate directly to acetoin without forming diacetyl has been found in several gram-negative and grampositive bacteria such as Lactobacillus lactis, Acetobacter pasteurianus, Bacillus brevis and Enterobacter aerogenes (2). Beer fermented in the presence of A L D C purified from Enterobacter aerogenes showed no analytical differences to normal beer, except that the


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Figure 1 : Pathway of the isoleucine- and valine-synthesis and the formation of viccinal diketones by yeast.



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diacetyl concentration was very low and therefore lagering time could be reduced from some weeks to several hours (3). Although this procedure is advantageous, two points must be considered. Firstly, commercially prepared A L D C has to be added which makes the process more costly and secondly, the addition of any enzymes during the brewing process is prohibited in Germany and would contravene the German beerbrewing purity laws ("Deutsches Reinheitsgebot"). Since it is not possible to decrease the spontaneous conversion rate from a acetolactate to diacetyl, one has to consider whether it may be possible to decrease the α-acetolactate pool in the yeast itself by strain improvement. With respect to the isoleucine-valine pathway, different strategies are possible which could reduce the aacetolactate pool in yeast. One could decrease the activity of the acetolactate synthase so that less α-acetolactate is formed (4). The alternative is the molecular cloning of an additional ILV5 gene coding for reductoisomerase, into yeast, which results in an increased reductoisomerase activity (5). Both methods interfere with the amino acid pathway of valine, leucine and isoleucine and could have negative influences on growth. Another strategy for reducing the a-acetolactate-pool in the yeast or the acetolactate-content in beer is the isolation and transfer of the ALDC-gene from an appropriate organism to yeast. In 1988 the ALDC-gene was firstly isolated from Enterobacter aerogenes (6), followed by a corresponding gene of Klebsiella(J). Both genes could be expressed in yeast and, as expected, the diacetyl-content was reduced. Since both, Enterobacter and Klebsiella belong to the Enterobacteriaceae, using a G R A S (general recommended as safe) organism would increase the chances of obtaining approval for the transformed yeast by authorities, especially since Acetobacter pasteurianus, a bacterium widely used in food industry to produce vinegar, could be chosen as the source of the ALDC-gene. Cloning and Analysis of the Acetobacter pasteurianus

Acetolactatedecarboxylase-Gene from

In order to clone the ALDC-gene the total D N A of A. pasteurianus was isolated, restricted with Hindlll and randomly integrated into the LacZ-gene of the E. coli vector pUC19. After transfer into the E. coli recipient NM522, approximately 2000 colonies were assayed using the Voges-Proskauer reaction, for their ability to form acetoin which was not present in untransformed cells. In analyzing one of the acetoin-positive clones it was found that it carried an Acetobacter-fragment of about 3 kb in size. As it can be assumed that only a part of the fragment is responsible for the formation of acetoin, it was sequentially truncated and found that an EcoRV/Hindlll-fragment of only lkb in length was necessary for A L D C expression. Sequencing experiments revealed a 879bp ORF on the EcoRV/Hindlll fragment which seems to code for the A L D C (Figure 2). Expression of the Acetolactatedecarboxylase-Gene in Laboratory Yeast Strains In order to render the ALDC-gene from Acetobacter pasteurianus expressible in yeast it was integrated into an expression-cassette consisting of the alcoholdehydrogenase I(ADHl)-promoter and the tryptophan I(trp)-terminator. In addition, the plasmid contains the G418 resistance-gene for the selection of plasmid-carrying yeast cells and a 2μπι origin which is necessary for autonomous replication in yeast (Figure 3). As might be expected in transformed yeast cells, only a slight ALDC-activity could be detected because four start codons are located at the putative beginning of the ORF.



164


CCCCGGCCAG

CCCCAGCCGG

ATATGTCCCA

GCTCACCCCG

GGCCAGACCG

TGGGCGGTCA

60

CCAGAAACTG

GGCCTGCAGG

TCCAGAATGG

TGCGGGCGCG

GGGCAGGAGG

GTCGCGCCAA

120

TTTCGGTCAG

CTTCACACCG

CGCGTCTGCC

GTTCAAACAG

CGACGCCAGT

TTTTGCTCCA

180

GCTGCTTGAT

CTGCTGGCTG

AGGGGCGGCT

GTTCCATCCC

TAGCTCTTCC

GCAGCCCGGG

240

TAATACTGCC

GTGGTCTGCC

ACGGTGACAA

AGTAACGCAG

300 EcoRV

ATATTTATAG

GATATGGATA

ATGCGTTCAA

TATATATTGG

AAGACTAGAT

CCCGCTGTAA

360

TAACGTCTGT

GTCATTGAGA

TCAGCAACAA

TGAGTTTGGA

ACAGTGCGCA

CqATQAAGAA

420

CAGCCCGGTG

GCAGACATGG

ACGTTCGGTC

TTCTGCACTC

GGAAACGGTG

TGGGTAAGAA

480

ACCCGTCGCC

AACCGTCTTT

ATCAGACCTC

CACCATGGCC

GCTCTGCTGG

ATGCCGTGTA

540

CGATGGCGAA

ACCACGCTGG

ACGAAGTGCT

GCACCACGGC

AATTTCGGCC

TTGGCACGTT

600

TAACGCGCTG

GATGGCGAAA

TGATTGTGAC

CGATGGTGTC

GCACGCCAGT

TCCGTGCGGA

660

AGGGCAGGCT

GCCGAGGTTC

CCGGTTCTCT

CAAAACGCCT

TTTGCCTGCG

TGACATATTT

720

TGAGCCGGAA

AAAACGCTCA

ATATTGATAC

ACCGCAGACA

AAAGAAACAT

TTGAAGCACT

780

GGTCGACCAG

TTGGTGGGTA

ATCCCAACCT

GTTTGGTGCC

GTTCGCTTTA

CCGGGCAGTT

840

TGAGCGGGTG

GATACGCGCA

CGGTGTTCTG

CCAGTGCAAG

CCCTATCCGC

ACATGCTGGA

900

TGTTGTGAAA

AAGCAGCCCA

CTCTGACCAT

GGAATCCGTG

ACCGGCACCA

TGATCGGCTT

960

CCGCACCCCG

GTTTATATGC

AGGGTGTGAA

CGTGGCGGGT

TATCATCTGC

ACTTCCTGAC

1020

GGAAGACCAG

AAACGCGGTG

GGCACGTGAC

GGAATACCGG

CTGGTGCGTG

GCCAGCTTGA

1080

GGTTGCCGTG

ATCTCCGATC

TTGAAATTCA

GCTGCCGCGC

ACAGAGCAGT

TTGCAAAAGC

1140

AAACCTTAAT

CCTGAGCATC

TGAGTGAAGC

CATTCGGATT

CGGCAAGGCG

GCTGAAGCTT

1200

HindlH

Figure 2: The complete nucleotide sequence of the ALDC-gene and its 5'flanking sequence.



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Amp

Acetobacter D N A

E.coli ori

ADH1 Promoter

2um ori

tip Terminator

KanVG418

Figure 3: Physical map of the plasmid pPTK-EB51, containing a lkb bacter pasteurianus DNA fragment.


Aceto-



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In order to try to increase the amount of A L D C transcripted, the first A T G (which is out of frame), the second codon (which is in frame with the fourth one) and the third A T G (which is in frame with the first one) were deleted. Whereas the absence of the first and the second codon had no effect on ALDC-activity, the additional deletion of the third increased the activity six-fold. It is therefore obvious that the last of the four A T G codons is the translation initiation codon of the ALDC-gene which is 780bp in length.


Fermentation Experiments with Laboratory Yeast Strains In order to investigate the ALDC-activity during fermentation, the yeast strains containing the A L D C were compared with its parental strain in 500ml scale fermentations at 20°C for 8 days. As shown in Figure 4, there was no substantial difference in fermentation performance. The pH value in both strains decreased from 4.6 in the first day to 4.1 by the fourth day of fermentation, and remained thereafter constant. The progress of the apparent extract was as expected - a decrease until the fourth day and then a constant level. Ethanol was produced by both strains in the same manner. The low ethanol concentration of 0.6 % at the end of fermentation was unexpected. This behavior seems to be characteristic for the lab-strain used and is of course useless for practical purposes. However, during main fermentation (until the fourth day) strong A L D C activity could be detected in comparison with the untransformed strain. The amount of A L D C was obviously sufficient for keeping the concentration of diacetyl and of ccacetolactate in the medium low. The amount of diacetyl is less than O.Olppm in contrast to 0.13 ppm in untransformed cells. The combined values for diacetyl and ocacetolactate in the transformed and untransformed cells are 0.06 ppm and 0.34 ppm, respectively. The stability of the replicative vector used throughout all experiments was not very high. Under non-selective anaerobic conditions about 40% of the population had lost the ALDC-gene within 5 days which is equivalent to about 3 to 5 generations. For practical purposes such a stability is too low. These results revealed, that it is possible to express the ALDC-gene of Acetobacter in a laboratory yeast strain which was subsequently able to convert cc-acetolactate to acetoin. Hence the diacetyl- and ot-acetolactate-content decreases about 15% of the initial concentration. Expression of the Acetolactatedecarboxylase-Gene in Brewer's Yeast Transformation of brewer's yeast with the mentioned construct was carried out and, unfortunately, all the obtained transformants not only propagated very slowly, they also had to be cultivated in complete medium: they were all auxotrophic for valine, leucine and isoleucine. They were not able to sythesize sufficient amounts of these amino acids probably due to the lack of intracellular α-acetolactate. However, as brewer's yeast takes up these amino acids slowly the transformants show reduced growth. Secretion of the Acetolactatedecarboxylase into the Medium To convert only the excreted, and therefore surplus, α-acetolactate into acetoin, the ALDC-gene was integrated into an expression-secretion-cassette comprising the tryptophan I-terminator and alcoholdehydrogenase I-promoter. This promoter is linked to the secretion signal sequence of the mating pheromone α-factor encoding gene


168



6,00

> X

Time (days)

0

1 2

3 4

Time (days)

5 6

Time (days) transformant (T8) parent strain (RH) Figure 5: Fermentation characteristics of the transformed brewer's yeast strain (T8) and its parental strain (RH).


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(MFal). In addition, the plasmid contains the G418 resistance gene and the 2μηι origin. This approach should have no negative influence on amino acid synthesis. This construct worked well in laboratory yeast strains and, as expected, was also expressible in brewer's yeast. A l l obtained transformants propagate like the untransformed yeast cells and no auxotrophy appeared. In addition, we were able to detect A L D C activity in the medium in which the transformants were cultivated, thus clearing obstacles for carrying out fermentation experiments with these transformants.


Fermentation Experiments with Brewer's Yeast These experiments were carried out in EBC columns filled with 2.5 liters of yeastcontaining wort at 10°C for 7 days . As shown in Figure 5, there was no substantial differences in the fermentation performance. The pH value in both strains decreased from 5.9 in the first day to 4.4 by the sixth day of fermentation. They were also no differences in the progress of the apparent extract. During fermentation a strong ALDC-activity could be detected when compared to the untransformed strains. As may be seen, it increased until the fourth day of fermentation, however, at the end of the process activity decreased to a lower level. This is most probably due to pH instability of the enzyme. Other experiments showed that the enzyme is degraded at a pH value of about 4.6 and is constantly stable when potassium phosphate-buffered wort is used for fermentation. However, the amount of the A L D C was sufficient for keeping the concentration of both diacetyl and α-acetolactate in the medium low. The amount of diacetyl was less than 0.01 versus 0.07 ppm in untransformed cells. The data for combined diacetyl and α-acetolactate in transformed and untransformed strains are 0.05 and 0.43 ppm, respectively. The analysis of beer fermented with the control and the transformant are shown in Table I. It is apparent that they hardly differ in characteristics such as alcohol content, color and bitter units, higher alcohols and esters, which are involved in creating the flavor characteristic of the final beer. Table I. Analysis of the young beer fermented with transformed brewer's yeast strain (T8) and its parental strain (RH) ester bitter higher diacetyl diacetyl EtOH color app. (actual) (total) (vol%) attenu (EBC) units alco (ppm) (ppm) (ppm) ation (BE) hols (ppm) (%) RH 87.7 29.0 0.07 23.0 0.43 4.28 16.5 76.9 T8

>0.01

0.05

4.33

77.6

16.5

22.4

88.3

30.3

Because brewer's yeasts are repeatedly used for beer production, A L D C activity has to be stably maintained. In order to observe the stability of the plasmid carrying yeast cells, three successive fermentations were carried out. Although the number of plasmid-carrying yeast cells decreases from 80% during the first fermentation to 69% at completion of the third fermentation, again only 0.06 ppm of total diacetyl was detectable versus 0.31 ppm in control cells. Other experiments have shown that after 30 generations cultivated under non selective conditions, 68% of the yeast cells still contained the plasmid. So, if, as


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mentioned earlier, during one fermentation 3 to 5 generations arise, at least 6 to 10 subsequent fermentation processes are possible, without any diacetyl flavor problems arising. When using such a genetically modified yeast strain in the brewing process the diacetyl content should be much lower than the taste threshold after main fermentation, thus rendering subsequent maturation unnecessary.


Safety

Assessment

Since the ALDC-gene is widely distributed among other Aceto- and Lactic acid bacteria, A L D C can be found in products such as vinegar, yogurt and butter - thus no risks arise in using such a gene for yeast strain improvement. In addition, when released into the environment ALDC-gene-carrying yeast cells have a selective disadvantage when compared to the wild type as they are handicapped in the regeneration of their reduction-equivalents (which are produced by the oxidation of the carbon source). No risks are involved with regards to ecological aspects when using an antibiotic resistance gene as no evidence is available that natural transformation occurs among yeasts and so the direct transfer of genetic material into other organisms is not likely. Transformation of this gene without using any markers, a process which we are at present using in our institute, makes it possible to obtain yeast strains carrying only sequences particular to the yeast and the ALDC-gene from an acetic acid-producing strain. In conclusion, there are no health or environmental risks involved when using such a genetically improved yeast strain, so that production facilities originally designed for fermentation with non-recombinant yeast can be used. Acknowledgments We wish to thank Roslin Bensmann for secreterial assistance. This work was supported by AIF (Arbeitsgemeinschaft Industrieller Forschung). Literature Cited (1) Scherrer, A. Schweizer Βrauerei-Rundschau 1972, 82, 1-23 (2) Godtfredsen, S.E.; Lorck, H.; Sigsgaard, P. Carsberg Res. Commun. 1983a, 48, 239-247 (3) Godtfredsen, S.E.; Ottesen, M.; Sigsgaard, P.; Erdal, K.; Mathiasen, T.; AhrenstLarsen, B. EBC Congress 1983b, Lect. No. 17, 161-168 (4) Ramos-Jeunehomme, C.; Masschelein, C.A. Eur. Brew. Congr. Amsterdam 1977, 267-283 (5) Villanueba, K.D.; Goosens, E.; Masschelein, C. A. ASBC J. 1990, 48, 111-114 (6) Sone, H., T.; Kondo, K.; Shimizu, F.; Tanaka, J.; Inoue, T. Techn. Rep. Kirin 1988, 31, 11-18 (7) Blomquist, K.; Suihko, M.-L.; Knowles, J.; Penttilä, M. Appl. Environ. Microbiol. 1991, 57, 2796-2804 R E C E I V E D July 7, 1995


Improvement of Beer Brewing by Using Genetically Modified Yeast

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