Brettanomyces: Friend, Foe or… Funk?

by RC

In 2014 alone, there were over 196 trillion liters of beer and 28 billion liters of wine were produced globally (1). These two beverages make up a significant portion of the global economy with many people consuming alcohol on a regular basis. The origins of alcoholic beverages like beer and wine date back to the early Neolithic period over 8,000 years ago, when some sort of plant or fruit (possibly grapes) was left to spontaneously ferment during storage until someone tried this “rotten” fruit, and the rest is history (2).

The spontaneous fermentation encountered by ancient humans is clearly much different from the production of alcohol today. Modern beer and wine production requires lots of care, and often employ very well defined single strain starter cultures of yeast. These single strain cultures have been carefully designed to do exactly what modern brewers require of them and prevent the spoilage of the beverage with other microbes. There are hundreds or even thousands of possible yeast strains that can be used to get the job done, each with slightly unique characteristics.  Modern starter cultures commonly consist of either a Saccharomyces cerevisiae strain or a closely related species (3). If a single strain starter culture is used, there is a high likelihood that the flavor profile of the alcohol will be narrow, while a multi strain culture can have numerous distinct and complex notes. Recently, more unconventional yeasts are being added to fermentations to add a unique flavor profile of the finished product.

One of these unique alternative fermenters is Brettanomyces. Originally isolated in the early twentieth century by Niels Hjelte Claussen at the Carlsberg brewery in Copenhagen during an investigation into the spoilage of English style ales, this yeast was also found to be responsible for the secondary fermentation and the unique flavors found in these ales (4). In addition to being found in English ales during its initial discovery, it has become an indispensible component of modern lambics and gueuze beers. These types of beers have a unique depth of flavors and are often quite sour. Many microbreweries have begun to promote their own versions of Brettanomyces fermented beers as special “funk” style beers, with numerous unique and complex potential flavor profiles. There is, however, a fine balance between the desirable characteristics imparted by the presence of Brettanomyces in mixed culture fermentation and the unpleasant spoilage often seen in wines and beers. In many cases, a high concentration of Brettanomyces can give a beer or wine an unpleasant smoky, sweaty, or even horsy odor and taste. These extreme additions to a beer or wine have led to the idea that Brettanomyces is one of the worst spoilage microbes and great care is taken by many breweries or wineries to avoid contamination, particularly in long term fermentations (5).

Members of the Brettanomyces, while somewhat controversial in the beverage industry, may have additional applications in the production of bioethanol for use in biofuels. They are often found growing spontaneously in traditional bioethanol production sites due to their high pH and stress tolerances, as well as their highly efficient metabolism (3). Most yeast are only able to produce ethanol through fermentation in the absence of oxygen, but some like Brettanomyces are able to produce ethanol even if oxygen is present. This is due to a unique adaptation called the Crabtree effect, where the yeast produces ethanol rather than biochemical energy known as ATP. This does require specific nutrition provided to the fermentation cultures, and Brettanomyces seems to thrive when high concentrations of sugar are present (6). The Custer effect is also seen in large cultures of Brettanomyces, where the production of ethanol decreases as the environment becomes more anaerobic As production of bioethanol has increased in recent decades in attempts to find alternative fuel sources, the need to aerate large cultures requiring oxygen has significantly raised costs, and in this case S. cerevisiae may appear more favorable. It is known that S. cerevisiae can only obtain its required nitrogen from ammonium ions, while Brettanomyces is able to obtain nitrogen from lignocellulose in the medium. Lignocellulose is dry plant biomass, and is the most abundant raw material available on Earth. This high availability and the ability of Brettanomyces to utilize it as a source of nutrition may give it an advantage over other commonly used fermenters (3).

The alcohol business is an enormous part of the global economy, both for consumption and the use in biofuels. The potential for unusual yeasts like Brettanomyces for fermentation has not been seriously looked at until very recently. It is commonly found as a serious contaminate of wine and beer fermentations and imparting unpleasant tastes. These same tastes are often valued in certain circles for the uniqueness they bring to the table that is unable to be found in traditional sources. Even in much larger scale production of bioethanol for the use in biofuels, these non-conventional yeasts are beginning to emerge as potential candidates for alternative solutions due to their unique fermentation abilities. While much more work is done on the vast potential of Brettanomyces in bioethanol production, it is very likely that they will become a highly prominent yeast in the near future.

Brettanomyces flavor wheel

1.          Beer Industry - Statistics & Facts | http://www.statista.com.

2.         Chambers PJ, Pretorius IS. 2010. Fermenting knowledge: the history of winemaking, science and yeast research. EMBO Rep 11:914–920.

3.         Steensels J, Daenen L, Malcorps P, Derdelinckx G, Verachtert H, Verstrepen KJ. 2015. Brettanomyces yeasts — From spoilage organisms to valuable contributors to industrial fermentations. Int J Food Microbiol 206:24–38.

4.         Claussen NH. 1904. On a Method for the Application of Hansen’s Pure Yeast System in the Manufacturing of Well-Conditioned English Stock Beers. J Inst Brew 10:308–331.

5.         Wedral D, Shewfelt R, Frank J. 2010. The challenge of Brettanomyces in wine. LWT - Food Sci Technol 43:1474–1479.

6.         Schifferdecker AJ, Dashko S, Ishchuk OP, Piškur J. 2014. The wine and beer yeast Dekkera bruxellensis. Yeast Chichester Engl 31:323–332.

Guide RNAs: A Uniquely Kinetoplastid Way of Editing Mitochondrial mRNA

by Paul Hoffman

            Mitochondria are found nearly all eukaryotic cells on Earth, generating most of the energy for eukaryotic life. These organelles have their own genome, independent from the nuclear genome that controls a majority of a cell’s functions. Much like nuclear DNA, mitochondrial DNA, or mtDNA, follows the central dogma of molecular biology: DNA gets transcribed to messenger RNA, or mRNA, which gets translated to protein or other products. The roles of the mitochondrial genome vary by organism, with some species utilizing their mtDNA more than others. As such, expression of mtDNA varies wildly across eukaryotic life. The kinetoplastids, for example, are a class of protozoans known for having large mitochondria with a massive network of mtDNA, containing multiple duplicates of the entire mitochondrial genome. Kinteoplasts are organized into circular fragments, which vary in size and function. Unlike most mtDNA, or nearly all DNA for that matter, kinetoplast DNA, or kDNA, is encrypted and mRNA produced from it needs to be decoded before it can be translated into useable products. A unique editing process is required for the mRNA to be functional, and this is what makes the kinetoplastids so unique.

Guide RNAs, or gRNAs, are encoded by certain fragments of the kDNA. Kinetoplast fragments can be organized into two classes: maxicircles, approximately twenty thousand to forty thousand base-pairs in length, and minicircles, approximately five hundred to one thousand base pairs in length. Maxicircles encode protein products, and are transcribed into mRNA transcripts. These transcripts are called cryptogenes; the message that is normally readable in regular mRNA is hidden due to highly conserved mutations within kDNA. In order for the message to be read by the cellular machinery, the mRNA transcripts need to be decoded into mRNA, and that is where gRNAs come into play. gRNAs come from the minicircles, vary from species to species (1), and range in number and class between species (2).

There are two major subgroups within the kinetoplastids, the trypanosomatids and the bononids, with major differences to gRNAs and kinetoplast structure between, and even within, these groups (3). Leishmania species fall under the former group, and form a clade, or close relationship, with Crithidia species within the trypanosomatids, with the Trypanosoma species forming another (4). This clade only has one gRNA coded per minicircle. Some trypanosomes contain multiple genes per minicircle (5), with three gRNAs per minicircle being coded for in some species of Trypanosoma (6). RNA editing in the Trypanosoma clade is performed at both ends of each editing domain while the  Leishmania-Crithidia clade is at the 5’ prime end of a domain (3). The bononids are often seen as more primitive than their cousins. Unlike the trypanosomatids, the bononids have unconcatenated ktDNA, meaning that it is not tightly bound, but rather loose and free-floating. Furthermore, the bononids have two gRNAs encoded per minicircle, unlike the Leishmania-Crithidia clade’s one or the Trypanosoma’s three to four (3). These differences make the kinetoplasts and gRNAs unique between the subdivisions of the kinetoplastids and incompatible between one another, despite sharing a common lineage. Of these groups, the trypanosomatids are far better studied, and seen as the model for studying gRNAs.

            Of the two clades that exist in the trypanosomatids, the Leishmania-Crithidia clade is often less studied, but just as important as these are the organisms with the most amount of classification to gRNAs. In some species of Leishmania, there can be upwards of sixty different classes of minicircles, each of which can encode multiple gRNAs. Each class is a unique minicircle sequence that codes for a group of gRNAs that act on a certain mRNA sequence (7). Minicircles all share a conserved sequence and differentiate based on variable sequences (8). In Leishmania species, the coding region for gRNAs is around one hundred fifty base-pairs from the conserved region; each minicircle codes for a single specific gRNA (9). Certain Leishmania species contain gRNAs with duplicate functions, with some having upwards of nineteen redundant gRNAs (10). These redundant gRNAs vary in expression level, with some of the redundant gRNAs being completely non-functional (5). Some of this redundancy may allow for minicircles to be lost and gained without any major change in function to the cell. Leishmania species have a wide range of minicircle numbers, even between strains of the same species, yet are able to function normally and have all the required gRNAs needed for survival (3).

            gRNA function can be boiled down to adding or removing uracil-residues from mRNA transcripts (11). All genetic material is coded for with nucleotides: DNA uses adenine, guanine, cytosine, and thymine; while RNAs swap out thymine for uracil. gRNAs decode mRNA transcripts by shifting the coding region of these transcripts by the addition and deletion of uracils in the transcript. The gRNAs have precise points within the transcripts where they perform their editing: gRNAs bind to a block of bases, ranging from one to ten nucleotides, within the transcript and signal a cleavage reaction in these blocks (12). Multiple blocks are strung together to create an editing domain. It is within these domains that the vast majority of mRNA transcript editing occurs. Different combinations of gRNA blocks can be formed on the same transcript to create different domains, yielding different mRNA products from the same transcript. Should a transcript be misedited by the wrong gRNA, other gRNAs can re-edit the transcript to form a correct sequence (12).

            A big question is how gRNAs, and the kinetoplast structure as a whole, arose from the standard mitochondrion. Evidence for early divergence of  the kinetoplastids from the euglenoids comes from ribosomal RNA sequences (13). The kinetoplastids are highly divergent from other euglenoids and, unlike many other groups, are monophyletic (14). The first models on the origin of the kinetoplast and gRNAs is a three step process: the ability to edit mRNA is acquired from enzymatic activity, then mutations occur at certain sites within the mRNA, finally editing mRNA becomes essential for growth (15). Expansions to this model propose that cryptogenes could then code for multiple proteins with different editing procedures unlocking different protein products from the mRNA. Another model suggests that RNA editing with gRNAs arose to combat mutations. kDNA, like other mtDNA, is highly unstable when not in use. Leishmania species have complex lifecycles, and different genes within the kDNA are needed at different times. When selective pressure is relived from kDNA and mtDNA, mutation rates tend to skyrocket within the sequences, leading to a vastly different kinetoplast genome than before. RNA editing with gRNAs could combat these mutations, restoring lost functionality by returning the mRNA transcripts to a working state. Furthermore, the kinetoplastids is that they do not use the universal genetic code (3). In most organisms, the codon UGA signals and end to reading an mRNA sequence when translating from nucleic acids to protein. Kinetoplastids, however, use UGA to code for tryptophan. However, the kinetoplasts themselves do not code for tRNA, or transfer RNA, that carries tryptophan to the mRNA sequence in the process of translating to protein. Because these tRNAs are imported into the kinetoplast, they rely on the genetic code of the cell rather than that of the kinetoplast. The differences between the cell’s code and the kinetoplast’s code may warrant RNA editing to be preserved and selected for in the kinetoplastids.

            Kinetoplasts and gRNAs make up the unique mitochondria system in kinetoplastids. Unlike nearly every other eukaryote, the kinetoplastids have the unique capability to change the expression of their mitochondrial genes by changing their mRNA transcripts using other RNAs as the editor. While the origin of gRNAs are still unclear, their function is well classified and incredibly interesting as no other organism uses this mechanism of RNA editing.

Bibliography

1.         Macina RA, Sanchez DO, Gluschankof DA, Burrone OR, Frasch AC. 1986. Sequence diversity in the kinetoplast DNA minicircles of Trypanosoma cruzi. Mol Biochem Parasitol 21:25–32.

2.         Yurchenko VY, Merzlyak EM, Kolesnikov AA, Martinkina LP, Vengerov YY. 1999. Structure of Leishmania Minicircle Kinetoplast DNA Classes. J Clin Microbiol 37:1656–1657.

3.         Simpson L, Thiemann OH, Savill NJ, Alfonzo JD, Maslov DA. 2000. Evolution of RNA editing in trypanosome mitochondria. Proc Natl Acad Sci U S A 97:6986–6993.

4.         Maslov DA, Avila HA, Lake JA, Simpson L. 1994. Evolution of RNA editing in kinetoplastid protozoa. Nature 368:345–348.

5.         Savill NJ, Higgs PG. 2000. Redundant and non-functional guide RNA genes in Trypanosoma brucei are a consequence of multiple genes per minicircle. Gene 256:245–252.

6.         Pollard VW, Rohrer SP, Michelotti EF, Hancock K, Hajduk SL. 1990. Organization of minicircle genes for guide RNAs in Trypanosoma brucei. Cell 63:783–790.

7.         Fu G, Kolesnikov AA. 1994. Leishmania gymnodactyli and Leishmania infantum minicircles contain the same guide RNA genes as do minicircles of Leishmania tarentolae. Mol Biochem Parasitol 67:171–174.

8.         Ray DS. 1989. Conserved sequence blocks in kinetoplast minicircles from diverse species of trypanosomes. Mol Cell Biol 9:1365–1367.

9.         Sturm NR, Simpson L. 1991. Leishmania tarentolae minicircles of different sequence classes encode single guide RNAs located in the variable region approximately 150 bp from the conserved region. Nucleic Acids Res 19:6277–6281.

10.      Gao G, Kapushoc ST, Simpson AM, Thiemann OH, Simpson L. 2001. Guide RNAs of the recently isolated LEM125 strain of Leishmania tarentolae: an unexpected complexity. RNA N Y N 7:1335–1347.

11.      Frech GC, Bakalara N, Simpson L, Simpson AM. 1995. In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae. EMBO J 14:178–187.

12.      Sciences NA of. 2000. Variation and Evolution in Plants and Microorganisms:: Toward a New Synthesis 50 Years after Stebbins. National Academies Press.

13.      Cavalier-Smith T. 1997. Cell and genome coevolution: facultative anaerobiosis, glycosomes and kinetoplastan RNA editing. Trends Genet 13:6–9.

14.      Lukes J, Jirkû M, Dolezel D, Kral’ová I, Hollar L, Maslov DA. 1997. Analysis of ribosomal RNA genes suggests that trypanosomes are monophyletic. J Mol Evol 44:521–527.

15.      Speijer D. 2006. Is kinetoplastid pan-editing the result of an evolutionary balancing act? IUBMB Life 58:91–96.