Abstract
Pulque is a culturally important 4,000-year-old traditional Mexican fermented drink. Pulque is produced by adding fresh aguamiel (agave sap) to mature pulque, resulting in a mixture of microbial communities and chemical compositions. We performed shotgun metagenomic sequencing of five stages of pulque fermentation to characterize organismal and functional diversity. We identified 6 genera (Acinetobacter, Lactobacillus, Lactococcus, Leuconostoc, Saccharomyces and Zymomonas) and 10 species (Acinetobacter boissieri, Acinetobacter nectaris, Lactobacillus sanfranciscensis, Lactococcus lactis, Lactococcus piscium, Lactococcus plantarum, Leuconostoc citreum, Leuconostoc gelidum, Zymomonas mobilis and Saccharomyces cerevisiae) that were present ≥ 1% in at least one stage of pulque fermentation. The abundance of genera and species changed during fermentation and was associated with a decrease in sucrose and increases in ethanol and lactic acid, suggesting that resource competition shapes organismal diversity. We also predicted functional profiles, based on organismal gene content, for each fermentation stage and identified an abundance of genes associated with the biosynthesis of folate, an essential B-vitamin. Additionally, we investigated the evolutionary relationships of S. cerevisiae and Z. mobilis, two of the major microbial species found in pulque. For S. cerevisiae, we used a metagenomics assembly approach to identify S. cerevisiae scaffolds from pulque, and performed phylogenetic analysis of these sequences along with a collection of 158 S. cerevisiae strains. This analysis suggests that S. cerevisiae from pulque is most closely related to Asian strains isolated from sake and bioethanol. Lastly, we isolated and sequenced the whole-genomes of three strains of Z. mobilis from pulque and compared their relationship to seven previously sequenced isolates. Our results suggest pulque strains may represent a distinct lineage of Z. mobilis.
Highlights
Pulque is a culturally important 4,000-year-old traditional Mexican fermented drink
Lactic acid bacteria and S. cerevisiae have been isolated from a guamiel[17,18,19], which likely represent the major source of microorganisms for pulque fermentation
As a quality control step to demonstrate the integrity of metagenomic DNA extraction, we successfully amplified the 16S rDNA (V3–V4) locus in each of the technical replicates (Supplementary Figure S2)
Summary
Pulque is produced by adding fresh aguamiel (agave sap) to mature pulque, resulting in a mixture of microbial communities and chemical compositions. For S. cerevisiae, we used a metagenomics assembly approach to identify S. cerevisiae scaffolds from pulque, and performed phylogenetic analysis of these sequences along with a collection of 158 S. cerevisiae strains This analysis suggests that S. cerevisiae from pulque is most closely related to Asian strains isolated from sake and bioethanol. Species from the lactic acid bacteria genus Leuconostoc produce extracellular polysaccharides (EPS) resulting in pulque’s distinctive v iscosity[16]. Lactic acid bacteria and S. cerevisiae have been isolated from a guamiel[17,18,19], which likely represent the major source of microorganisms for pulque fermentation. The identification of Z. mobilis from honey b ees[22] and the practice of filtering aguamiel to remove insects and other debris suggests a possible insect transmission[11]
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