It’s time for another long due update. I’m very excited to share our latest manuscript on diastatic brewing yeast (or “Saccharomyces diastaticus” as it was formerly known as)! Keep in mind that this is a preprint and the manuscript is currently under peer-review. Below I’ll summarize the main findings of our small project (that ended up having quite significant results).

Edit: The manuscript has now been published in Applied Microbiology and Biotechnology: https://link.springer.com/article/10.1007/s00253-019-10021-y

First of all, here is the link to the preprint: https://www.biorxiv.org/content/10.1101/654681v1

So as most of you are aware, the presence or absence of the STA1 gene is commonly used as a marker for diastatic yeast. However, as recent work by e.g. the Hutzler lab and Escarpment Laboratories have shown, not all strains carrying the STA1 gene are problematic (i.e. not all STA1+ strains can super-attenuate beer or ferment starch). This is of course a problem. While the PCR-based method for detecting diastatic yeast with the SD-5A/SD-6B primers is rapid, it can’t differentiate between the highly and poorly diastatic strains. I set out to find out the reasons behind the variation in diastatic ability that we have observed in STA1+ strains.

We started out by screening 15 strains of S. cerevisiae carrying the STA1 gene (thanks to Mathias Hutzler and Richard Preiss for sharing strains). We tested the diastatic ability of the strains using three different tests: 1) the ability to grow on starch agar, 2) the ability to grow in beer (wort force-fermented with a lager yeast), and 3) the ability to grow on dextrin as a sole carbon source. In all three tests, the 15 strains grouped into two distinct groups: the highly diastatic, and the poorly diastatic. This was already promising.

Next, we sequenced the open reading frame and the upstream region of the STA1 gene in all 15 strains. In simpler terms, we looked at the sequence coding the actual gene and the sequence in front of the gene (which usually controls how much of the gene is expressed into the protein it codes for). Here we observed something fascinating: all 10 of the poorly diastatic strains had a large 1162 bp deletion in the promoter of STA1 (i.e. in the upstream region / the sequence in front of the gene). Within this region that was deleted, is a transcription factor binding site (transcription factors are proteins that control the expression of the gene). We hypothesised that this deletion in the STA1 promoter decreases the expression of STA1 (i.e. less of the glucoamylase enzyme is produced), which in turn results in poor diastatic ability. It seemed very obvious at this point already that this deletion was the cause of the variable diastatic ability, but we still needed to confirm this.

To confirm the effect of the deletion, we performed CRISPR/Cas9-aided reverse engineering. Wyeast 3711 was the most diastatic strain out of all 15 that we tested (in all three tests). So we used CRISPR/Cas9 to replicate this same 1162 bp deletion in the promoter of STA1 in WY3711. We then repeated the three diastatic tests. We compared the original WY3711, the WY3711 variant with the deletion in the STA1 promoter, and WLP570 that naturally has the deletion in the STA1 promoter. The deletion resulted in decreased ability to grow in beer and on dextrin (WLP570 and the WY3711 variant with the deletion performed similarly poorly), confirming that it is indeed this 1162 bp deletion in the STA1 promoter which causes the poor diastatic ability in some of the STA1+ strains. To test our hypothesis that STA1 expression is affected, we measured STA1 mRNA levels with RT-qPCR in the same three strains as above, and witnessed 150-200 fold higher levels of STA1 mRNA in WY3711 compared to WLP570 and the WY3711 variant.

So what can we do in practice with this information? We designed a new set of PCR and qPCR primers that bind within the deleted region in the promoter. So these primers will only give a signal (produce an amplicon) if the strain has the full STA1 promoter (i.e. the strain is highly diastatic). These new primers can therefore distinguish between very active (full promoter) and slow (deletion in the promoter) diastatic strains. They can also be multiplexed with the commonly used SD-5A/6B primers! So this means that you can either use these new primers on their own, which will produce a signal for very active diastatic strains, and no signal for both poorly diastatic strains and strains lacking STA1 completely. Or, you can use these new primers in the same PCR reaction together with the SD-5A/6B primers, which will produce two bands for highly diastatic strains, one band for poorly diastatic strains, and no bands for strains lacking STA1 completely. If this sounds confusing, you can check out the image below (and two above) for some examples. Now we can use PCR to distinguish the highly diastatic and poorly diastatic strains! We tested the primers on some lager yeast cultures that we contaminated with various ratios of WY3711, and we could reliably detect WY3711 down to a concentration of 10^-4 with regular PCR and 10^-5 with qPCR using these new primers.

After we had managed to elucidate the cause behind the variable diastatic ability in STA1+ strains, I still wanted to explore the prevalence and evolutionary role of STA1. Brewers tend to link diastatic yeast with “wild yeast”. This turns out to be incorrect. After screening 1100+ publically available genome assemblies, we show that STA1 is prevalent in the ‘Beer 2’/’Mosaic Beer’ population, and surprisingly a population of yeast isolated from a village in French Guiana (mostly isolates from human feces). So STA1 is clearly linked to domesticated yeast (i.e. no wild strains possessed STA1). The link to ‘Beer 2’ was expected (the saison strains can be found from here), but the French Guiana link was definitely unexpected. The villagers produce and consume a traditional starch-rich beverage called cachiri, so maybe STA1 (allowing starch fermentation) has given the strains a fitness advantage there? Anyways, exploring that was outside the scope of this particular study, but it should definitely be clarified in the future.

If we go back to the ‘Beer 2’ strains, the Gallone et al. 2016 paper has some nice phenotypic data that can be used. I decided to look at the maltotriose use ability of all the STA1+ ‘Beer 2’ strains, and if we divide the strains into two groups based on whether they have the full or partial STA1 promoter, one could see that the strains with the full promoter have significantly higher maltotriose use ability. This hinted that STA1 possibly has some role in enabling maltotriose use in the ‘Beer 2’ strains. The main mechanism for maltotriose use in ‘Beer 1’ brewing strains is through the AGT1/MAL11 transporter. ‘Beer 2’ strains have a non-functional AGT1/MAL11, which means some other unknown mechanism(s) must be responsible. Could STA1 be the unknown mechanism?

I then decided to compare the wort fermentation ability of WY3711 with the WY3711 variant with the deletion in the STA1 promoter. In the first 24 hours of fermentation the strains performed identically. At this time it is mainly glucose that is consumed from the wort. After this time point, the variant with the deletion in the STA1 promoter slowed down significantly. Maltotriose use in particular slowed down. WY3711 had consumed around 80% of the maltotriose after 96 hours, while the variant with the deletion in the STA1 promoter had only consumed 12%. So these initial results already suggested that STA1 has a central role in enabling maltotriose use in STA1+ strains.

To further test this, we next used CRISPR/Cas9 to completely delete STA1 from three of the highly diastatic strains (WY3711 and two TUM strains). We repeated the wort fermentations, and again saw significantly reduced maltotriose use for all three deletion strains. The two TUM strains (TMU PI BA 109 and TUM 71) in particular, barely consumed any maltotriose at all after STA1 had been deleted. This confirmed that STA1 seems to enable efficient maltotriose use through extracellular hydrolysis! We think that the formation and retention of STA1, through the fusion of FLO11 and SGA1, is an alternative evolutionary strategy for efficient utilization of sugars present in brewer’s wort.

The WY3711 deletion strain still showed minimal maltotriose use, so that suggested that while STA1 is the main mechanism for maltotriose use in these strains, it is not the only one. To confirm this, we measured the uptake rate of maltotriose in these strains. This is done with radio-labelled maltotriose (14C). All three strains were able to transport maltotriose into the cell (though at levels lower than what is normally measured for brewing yeast), confirming that while STA1 appears to be the main mechanism for maltotriose use, the strains carry some low affinity maltotriose transporter(s) as well. We performed long-read sequencing of WY3711 using an Oxford Nanopore MinION, and the resulting genome assembly appeared to contain multiple copies of an MTT1-like transporter (which probably explain the maltotriose uptake ability and the low maltotriose use of the STA1 deletion strain). MTT1 is a transporter that has been shown to transport maltotriose.

So to conclude, we saw three major results here in this study. The first is probably of most use to the brewing community: The variable diastatic ability in STA1+ strains seems to be determined by a 1162 bp deletion in the STA1 promoter. Strains with the deletion are not very diastatic. You can use our newly designed PCR primers so differentiate between STA1+ strains with and without the deletion. The second result was that STA1 is not linked to wild yeast, rather it appears to be prevalent only in ‘Beer 2’/’Mosaic Beer’ strains, and surprisingly the ‘French Guiana, human’ strains. STA1 presumably gives a fitness advantage in starch-rich environments (e.g. beer). The third major result was that STA1 enables efficient maltotriose consumption in STA1+ strains. This appears to be the unknown mechanism that has enabled efficient maltotriose use in the ‘Beer 2’ strains (which otherwise have a non-functional AGT1/MAL11 transporter). STA1 therefore seems to be an alternative evolutionary route (‘domestication signature’) to enable efficient utilization of the sugars present in wort. Let me know if you have any questions or feedback, I’d be happy to help! Thanks again to Mathias Hutzler and Richard Preiss for sharing strains!