In the beverage fermentation industry, especially at the craft or micro level, there is a movement to incorporate as many local ingredients as possible to both capture terroir and stimulate local economies. In the case of craft beer, this has traditionally only encompassed locally sourced barley, hops, and other agricultural adjuncts. The identification and use of novel yeasts in brewing lags behind. We sought to bridge this gap by bio-prospecting for wild yeasts, with a focus on the American Midwest. We isolated 284 different strains from 54 species of yeast and have begun to determine their fermentation characteristics. During this work, we found several isolates of five species that produce lactic acid and ethanol during wort fermentation: Hanseniaspora vineae, Lachancea fermentati, Lachancea thermotolerans, Schizosaccharomyces japonicus, and Wickerhamomyces anomalus. Tested representatives of these species yielded excellent attenuation, lactic acid production, and sensory characteristics, positioning them as viable alternatives to lactic acid bacteria (LAB) for the production of sour beers. Indeed, we suggest a new LAB-free paradigm for sour beer production that we term "primary souring" because the lactic acid production and resultant pH decrease occurs during primary fermentation, as opposed to kettle souring or souring via mixed culture fermentation.
In the beverage fermentation industry, especially at the craft or micro level, there is a movement to incorporate as many local ingredients as possible to both capture terroir and stimulate local economies. In the case of craft beer, this has traditionally only encompassed locally sourced barley, hops, and other agricultural adjuncts. The identification and use of novel yeasts in brewing lags behind. We sought to bridge this gap by bio-prospecting for wild yeasts, with a focus on the American Midwest. We isolated 284 different strains from 54 species of yeast and have begun to determine their fermentation characteristics. During this work, we found several isolates of five species that produce lactic acid and ethanol during wort fermentation: Hanseniaspora vineae, Lachancea fermentati, Lachancea thermotolerans, Schizosaccharomyces japonicus, and Wickerhamomyces anomalus. Tested representatives of these species yielded excellent attenuation, lactic acid production, and sensory characteristics, positioning them as viable alternatives to lactic acid bacteria (LAB) for the production of sour beers. Indeed, we suggest a new LAB-free paradigm for sour beer production that we term “primary souring” because the lactic acid production and resultant pH decrease occurs during primary fermentation, as opposed to kettle souring or souring via mixed culture fermentation.Chemical compounds studied in this article: Lactic acid (PubChem CID: 612); Ethanol (PubChem CID: 702)Abbreviations: ABV, alcohol by volume; DIC, differential interference contrast; EtOH, ethanol; FG, final gravity; gDNA, genomic DNA; IBU, international bittering unit; LAB, lactic acid bacteria; LASSO, lactic acid specific soft-agar overlay; N-J, neighbor-joining; OG, original gravity; WLN, Wallerstein Laboratories nutrient; YPD, yeast extract, peptone, and dextrose
In plants, nuclear multisubunit RNA polymerases IV and V are RNA Polymerase II-related enzymes that synthesize non-coding RNAs for RNA-directed DNA methylation (RdDM) and transcriptional gene silencing. Here, we tested the importance of the C-terminal domain (CTD) of Pol IV’s largest subunit given that the Pol II CTD mediates multiple aspects of Pol II transcription. We show that the CTD is dispensable for Pol IV catalytic activity and Pol IV termination-dependent activation of RNA-DEPENDENT RNA POLYMERASE 2, which partners with Pol IV to generate dsRNA precursors of the 24 nt siRNAs that guide RdDM. However, 24 nt siRNA levels decrease ∼80% when the CTD is deleted. RNA-dependent cytosine methylation is also reduced, but only ∼20%, suggesting that siRNA levels typically exceed the levels needed for methylation of most loci. Pol IV-dependent loci affected by loss of the CTD are primarily located in chromosome arms, similar to loci dependent CLSY1/2 or SHH1, which are proteins implicated in Pol IV recruitment. However, deletion of the CTD does not phenocopy clsy or shh1 mutants, consistent with the CTD affecting post-recruitment aspects of Pol IV activity at target loci.
A subset of tRNA genes in eukaryotes contains an intron. tRNAs introns are short sequences located 1 nt. 3′ of the anticodon. Introns are removed from precursor tRNAs (pre‐tRNA) by the conserved heterotetrameric tRNA splicing endonuclease (SEN), that recognizes pre‐tRNA structure rather than sequence motifs at splice junctions. In vertebrate cells intron removal from pre‐tRNAs occurs in the nucleoplasm; in contrast, pre‐tRNA splicing in budding yeast and fission yeast occurs on the mitochondrial surface (Yoshihisa et al. 2003; Wan & Hopper, 2018); therefore, pre‐tRNA splicing on the mitochondrial surface has been conserved for greater than 500 million years. Free tRNA introns are rarely detected in cells because they are rapidly and efficiently destroyed. We identified at least 5 separate, family‐specific mechanisms that function in tRNA intron destruction (Wu & Hopper, 2014; Bao, Metcalf & Hopper, unpublished). Interestingly, under particular stress conditions, specific tRNA introns accumulate to high levels, possibly indicative of their functions in stress responses (Peltier, Metcalf, & Hopper, unpublished). Moreover, we discovered that tRNA introns possess long stretches of perfect complementarity to particular budding yeast mRNA ORFs (Bao, Nostramo, & Hopper, unpublished). Deletion of the introns from the 2 copies of the genes encoding tRNAIleUAU results in elevated levels of mRNAs with complementarity, but not mRNAs lacking complementarity; further, transformation of the cells lacking the tRNAIleUAU introns with extrachromosomal intron‐containing tRNAIleUAU suppresses the mRNA enhanced levels. These results support the hypothesis that tRNA introns may function as a novel type of noncoding regulatory RNA, perhaps functioning in stress response (Nostramo & Hopper, unpublished).
Plant nuclear multisubunit RNA polymerase IV plays a key role in the RNA-directed DNA methylation (RdDM) pathway for transcriptional silencing of transposons, viruses and specific genes by synthesizing precursors of 24 nt siRNAs that guide the process. The Pol IV largest subunit, NRPD1 is derived from the Pol II largest subunit but has a unique carboxy-terminal domain (CTD) of unknown function. We show that the NRPD1 CTD is critical for transcriptional silencing of target loci and for producing 24 nt siRNAs at high levels. However, the CTD is surprisingly dispensable for near wild-type levels of Pol IV-dependent genomic cytosine methylation. These results suggest that low levels of 24 nt siRNAs, produced at only 20-30% of wild-type levels, are sufficient for full RNA-directed DNA methylation, yet insufficient for silencing, suggesting additional roles for siRNAs beyond DNA methylation. Moreover, at a subset of target loci, neither siRNA levels nor cytosine methylation are impaired upon deletion of the CTD, yet silencing is lost. Collectively, the non-linear relationships between siRNA levels, cytosine methylation and silencing suggest the existence of additional mechanisms of silencing dependent on Pol IV transcription and mediated by the CTD, such as promoter occlusion to inhibit the activities of other polymerases.
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