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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, L