This post is supposed to be a video, but because there has some interest about my “work” in this area, I’m writing up a more technical post now for those who’ve expressed an interest. For those looking for something less technical, a lay-audience targeted video should follow sometime around Christmas. This post surrounds a couple of controversial moves to try and patent the entire genus of Lachancea, and/or species/strains within the genus, for the brewing of beer [patent 1 | patent 2].
These patents created a fair amount of anger among the Milk the Funk crew, and I imagine, among many other brewers as well. The idea that someone would patent a genus (or even species) of naturally existing yeast for brewing beer seems to run contrary to both what patents should protect, and to brewing culture. To make matters worse, using Lachancea in brewing is hardly new. Lachancea has a long history of use in wine making, with commercial wine yeast blends containing this yeast being widely available. Lachancea is also present in kombucha, cider, fermented foods, cachaça, olive oil, kefir, coconut water, and quite likely in homebrewed beer as well.
So how could possibly patent any Lachancea for beer brewing? I’m not a patent expert, so I don’t know – but they’ve tried. Both patents also have “fall back” positions of specific strains they’d like to patent if the attempt to patent the whole species or genus fails.
In this post I’m only tackling one of these patents – the one submitted by the University of the Sciences in Philadelphia, with the supposedly new species of Lachancea this patent application is aiming to patent currently available to brewers as the Philly Sour strain from Lallemand.
This patent seems to be built upon a very specific foundation, specifically:
Claims 1-6 basically set out the attempts to patent the genus for beer brewing (claims 1-5), and claim 6 is the specific “species” they claim to have found. This species, the patent claims, is “a new strain of yeast, dubbed GY7B, which is related to, but genetically and phenotypically distinct from, Lachancea thermotolerans”.
But is it?
At this point the more “lay” description of what I’m doing ends, and we’re going to dive deep into yeast phylogenetics and biochemistry without my usual attempts to provide simplified explanations of what I did and of the data…those descriptions will need to wait for the video.
Brief Technical Introduction
Lachancea is a genus of yeast with the unusual property that it produces a mixture of ethanol and lactic acid as end-products of fermentation. These yeasts are closely related to the Saccharomyces genus, and belong to the same Saccharomycetales order of yeasts. As a consequence of their similarities with other members of the Saccharomycetales, when first described yeasts of the Lachancea genus were incorrectly assigned to the Kluyveromyces, Zygosaccharomyces, Debaryomyces, Torulaspora, and Saccharomyces genera. This was corrected in 2003, when Cletus P. Kurtzman reclassified these mis-identified yeasts into their own genera, which he named after Marc-Andre Lachance, a biologist (and colleague of mine) who made many contribution to yeast phylogenetics. Yeasts of the Lachancea genus are commonly found in wild fermentations, including in kombucha, cider, fermented foods, cachaça, olive oil, kefir, and coconut water. Additionally, Lachancea are produced commercially for wine making, and have been tentatively identified in home brewed beer via an inadvertent fungemia of an immunocompromised homebrewer.
In 2017 Mathew Farber of the University of the Sciences in Philadelphia submitted a patent attempting to patent the use of Lachancea for beer brewing, or at a minimum, a “new strain of yeast, dubbed GY7B, which is related to, but genetically and phenotypically distinct from, Lachancea thermotolerans” for brewing beer. As part of this patent application the “inventors” of GY7B submitted 3 DNA sequences (ribosomal D1-D2 and ITS regions, plus a portion of actin), which were compared to L. thermotolerans strain NRRL Y8284 (AKA CBS 6340), which at that time was the only strain of L. thermotolerans for which we have a full genome sequence. Both GY7B and CBS 6340 were purportedly isolated from tree bark, with GY7B isolated from Philadelphia (USA) and CBS 6340 from Russia.
While GY7B showed no variation from CBS 6340 in the D1-D2 region, there were 2 base-pairs difference in the ITS sequence (0.3% difference across 580 bp of sequence) and 7 base-pairs difference in the actin sequence (0.6% difference across 1128 bp of sequence). In addition, a carbon oxidation/assimilation assay is provided, showing that GY7B and CBS 6340 have different carbon utilization across 6 carbon sources, and in addition, that GY7B oxidizes a range of organic compounds not oxidized by CBS 6340. Based on these findings, Farber et al conclude that their strain is unique from Lachancea thermotolerans and therefore is potentially patentable. This claim runs contrary to the convention in fungal biology of using a 3% cutoff in the ITS sequence as the threshold between two species. Moreover, these comparisons are only made using a single comparator strain from a distant geographical location. Therefore, whether GY7B is truly a unique species from Lachancea thermotolerans cannot be determined from the data provided in the patent, raising questions regarding whether it is patentable as a unique strain/species.
Lachancia ITS Sequences: The ITS sequences, and where available, either the geographical location or associated genera, for all known Lachancea species were downloaded from the UNITE fungal ITS database (https://unite.ut.ee/) and imported into Excel. For sequences with an associated genera but not location, the location was estimated based on the geographical range of the associated genera. The ITS sequence of GY7B was downloaded from patent application WO2019018803A1, and confirmed in-house by ITS sequencing using ITS1 and ITS4 primers as described previously. The resulting Excel data was exported as FASTA-formatted files containing all Lachancea sequences, or all geographically-defined Lachancea thermotolerans species.
|Supplemental File 1: Excel file of all available Lachancea ITS sequences and associated metadata.|
|Supplemental File 2: FASTA-formatted text file of all available Lachancea ITS sequences.|
|Supplemental File 3: FASTA-formatted text file of all geographically defined Lachancea thermotolerans ITS sequences.|
ITS Alignment and Phylogenic Tree: All phylogenetic analyses were performed in MEGA X (https://www.megasoftware.net/). ITS sequences were aligned using ClustalW with gap opening penalties of 15 and gap extension penalties of 6.66. Phylogenic trees were built using maximum-likelihood approach using the default settings in MegaX, and then presented as a circular tree.
BLAST: NCBI Blast was used for all nucleotide searches. BLAST was performed using the default settings, and the search performed against the NCBI nucleotide collection.
Comparative Biochemical Qualities: The carbon utilization of GY7B was compared to the formal species definition for Lachancea thermotolerans as reported in The Yeasts (5th edition, 2011).
ITS Sequence of Lachancea GY7B
To ensure that the DNA sequence provided in the patent was accurate, a heat-extraction of DNA from Philly Sour (GY7B) was performed and sequenced in-house. This produced 418 base-pairs of sequence which was aligned with the 580 bp sequence provided in the patent (Figure 1). The two sequences were 100% identical, confirming the validity of the patent sequence.
GY7B’s Location In the Lachancea Genus
A phylogenic reconstruction of all available Lachancea ITS sequences using a maximum likelihood approach produced a phylogenic tree similar to that reported previously (Figure 2). Previously identified species formed distinct non-overlapping clades. Lachancea GY7B was embedded deep within the L. thermotolerans clade, within a major sub-clade of closely related strains.
To further assess the location of GY7B within the Lachancea thermotolerans species, the GY7B ITS sequence from the patent was searched against the NCBI nucleotide collection using a nucleotide BLAST. GY7B was found to have a 100% match to Lachancea thermotolerans strain LL12-057 (Figure 3), as well as 100% match to two additional Lachancea thermotolerans partial ITS sequences, clearly identifying GY7B as a member of the Lachancea thermotolerans species.
Geographic Analysis of GY7B Phylogeny
Given the strong clustering of GY7B with a sub-clade of Lachancea thermotolerans (Figure 2), a second phylogenic analysis was performed using ITS sequences from strains of Lachancea thermotolerans from known geographical locations (Figure 4). GY7B clustered with a large clade of North American isolates, mostly found on trees of the Quercus and Fagus (oak and beech) genera, but was part of a genetically distinct clade from the CBS 6340 type strain used as a comparator in the patent. Indeed, these two strains fall onto two distinct clades which separate at the root of the phylogenetic tree.
Comparative Biochemical Analysis
The patent’s authors provide a table of growth in medium containing a range of carbon sources, along with some additional readouts of GY7B’s oxidizing capabilities. These are compared to the type strain CBS 6340, a Russian strain that is phylogenetically distant to GY7B (Figure 4). While many differences in substrate oxidation were identified between BY7B and CBS 6340, no biochemical basis was identified and the degree of oxidation tended to be weak. Given the broad range of substrates oxidized by GY7B, this most likely represents a non-specific oxidation, likely from metabolically-produced oxidants aggravated by Lachancea thermotolerans poor anti-oxidant capabilities.
The more conventional carbon utilization assay identified a small number of differences between GY7B and CBS 6340, which I then compared to the reported rates of these features across a broad range of L. thermotolerans strains (Table 2). Of the 6 carbon sources showing differential usage between GY7B and CBS 6340, four are known to be variably processed by L. thermotolerans strains, with adonitol (ribotol) utilization unique to GY7B compared CBS 6340 (but is untested in other L. thermotolerans strains). Unexpectedly, CBS6 340 metabolized a-d lactose plus-xylose, contrary to the previous report describing this strains carbon utilization profile [Lachance and Kurtzman (2011)].
|Carbon Source||GY7B||CBS 6340||L. thermotolerans|
|2-keyto-d-gluconic acid||–||+||Variable (as gluconate)|
|d-gluconic acid||–||+||Variable (as gluconate)|
|a-d lactose plus-xylose||–||+||–|
Contrary to the claims of the patent, D1-D2 and ITS sequences clearly place GY7B as a member of the Lachancea thermotolerans species, and show that GY7B is deeply embedded in – and minimally different from – a clade of Lachancea thermotolerans native to eastern North America. The degree of ITS sequence diversity between GY7B and CBS 6340 falls well within the reported 1.5% ITS diversity of Lachancea thermotolerans species. While the actin gene did show a greater degree of difference from the type strain CBS 6340 than did the ITS sequence, the degree of difference (0.6%) was minimal and well within the reported intraspecies differences within the closely related Saccharomyces cerevisiae (0.87%, data not shown). Furthermore, GY7B’s carbon utilization falls within the reported norms for Lachancea thermotolerans.
In conclusion, GY7B is clearly a strain within the Lachancea thermotolerans species, and may be identical to a previously reported strain LL12-057. The choice of CBS 6340 as a comparator strain – especially given that over 50 ITS sequences for Lachancea thermotolerans were available at the time of patent submission – may have been a deliberate choice to maximize the apparent differences between GY7B and Lachancea thermotolerans. A more detailed analysis using data drawn from multiple strains clearly demonstrates that GY7B falls entirely within the phylogenetic and biochemical range of the Lachancea thermotolerans species, and moreover, that GY7B is deeply rooted within a highly similar clade of Lachancea thermotolerans that is geographically located to eastern North America – the same region where GY7B itself was isolated.