Comparative Metagenomics Reveals Microbial Communities and Their Associated Functions in Two Types of Fuzhuan Brick Tea

Abstract

Fuzhuan brick tea (FBT) is a unique post-fermented tea product, naturally co-fermented by microorganisms, and has gained global popularity due to its potential health benefits for humans. Considerable efforts have been made toward elucidating the microbial diversity within FBT, but an understanding of the underlying FBT community interactions and functions remains poorly studied. Consequently, the microbial communities of two types of FBT, originating from Hunan and Shaanxi provinces, were investigated using comparative shotgun metagenomic sequencing and functional annotations. Metagenomic analysis indicated that two communities shared similar taxonomic and functional attributes. Two samples shared 486 genera, in which Pseudomonas contributed most to the abundant functions within the two samples. The carbohydrate active enzyme functions of the communities primarily comprised GH (32.92%), GT (26.8%), CEs (20.43%), and AAs (18.04%). Furthermore, the overall metabolic pathways encoded by the metagenomes were largely associated with carbohydrate and amino acid metabolism, with nine metabolic pathways that were differential between two groups including penicillin and cephalosporin biosynthesis. Significantly, a total of 35 potential probiotics were inferred, with Pseudomonas putida being the most abundant inferred probiotic (80%) within the FBT communities. This study provides new insights into FBT microbial communities on their potential functions and roles in FBT characteristics.

Keywords: Fuzhuan brick tea (FBT); metabolic pathway; microbial community; potential function; shotgun metagenomic sequencing.

FIGURE 1

Microbial community composition at the genus level among two FBT groups. (A) The relative abundances of microbial populations. (B) Venn diagram depicting shared taxonomic groups between FBT_H and FBT_S samples. (C) Variation in taxonomic composition among FBT types based on PCA ordination. Different colors indicate different FBT types. The proportion of variation explained by PCA1 and 2 was 63.38 and 34.86%, respectively. FBT_H: H1–H3. FBT_S: S1–S3. (D) ANOSIM analysis of variation based on FBT communities. Inter-group differences are shown by the “Between” box. The “FBT_H and FBT_S” boxes show intra-group differences. (E) STAMP differential analysis of microbial communities between FBT_H and FBT_S samples (error bars indicate Wilcoxon rank-sum tests).

FIGURE 2

LEfSe analysis of (A) bacterial and (B) eukaryal populations that differed between FBT_H and FBT_S samples based on linear discriminant analysis (LDA) scores. Higher LDA scores indicate greater differential effects of samples on species abundances.

FIGURE 3

Contribution of microbial populations to (A) enzyme functions, (B) functional modules, and (C) KEGG pathways. Colors indicate relative abundances of different taxa. The abundance contributed by taxa = (reads of the specific taxa associated with function)/(total reads of all taxa associated with function).

FIGURE 4

Correlation of encoded functions and the most abundant microbial taxa. Cell colors denote correlation coefficient values based on the scales to the right of the heatmaps (*p < 0.05, **p < 0.01, and ***p < 0.001). Profiles are shown for (A) enzyme profiles, (B) module profiles, and (C) KEGG pathway level 3.

FIGURE 5

Distribution of CAZyme database functions. (A) The overall distribution of CAZyme among FBT samples. The length of bars for each FBT on the outer ring represents the percentage of CAZymes within each FBT sample. (B) Comparison of CAZyme functions between FBT_H and FBT_S samples. The data were visualized using STAMP (error bars represent Wilcoxon rank-sum test). (C) The contribution of species to CAZyme functional profiles. AA, auxiliary activities; CBM, carbohydrate-binding modules; CE, carbohydrate esterases; GH, glycoside hydrolases; GT, glycosyl transferases; PL, polysaccharide lyases.

FIGURE 6

Comparison of KEGG pathway level functions between FBT type communities. (A) KEGG pathway level 2 and (B) KEGG pathway level 3.

FIGURE 7

Comparison of ipath metabolic pathways between FBT-type communities. Red and blue lines indicate unique metabolic pathways for FBT_H and FBT_S communities, respectively, while green lines indicate shared metabolic pathways among the two samples. (A) Biosynthesis of secondary metabolites and overall (B) metabolic pathways.

FIGURE 8

Probiotic analysis for FBT communities against probio database. (A) The relative abundances of inferred probiotic in FBT communities. (B) Correlation between probiotic and functional abundances (*p < 0.05, **p < 0.01, and ***p < 0.001). Cell colors indicate the correlation coefficients based on the scale to the right.