Metatranscriptomics Reveals the Functions and Enzyme Profiles of the Microbial Community in Chinese Nong-Flavor Liquor Starter

Abstract

Chinese liquor is one of the world's best-known distilled spirits and is the largest spirit category by sales. The unique and traditional solid-state fermentation technology used to produce Chinese liquor has been in continuous use for several thousand years. The diverse and dynamic microbial community in a liquor starter is the main contributor to liquor brewing. However, little is known about the ecological distribution and functional importance of these community members. In this study, metatranscriptomics was used to comprehensively explore the active microbial community members and key transcripts with significant functions in the liquor starter production process. Fungi were found to be the most abundant and active community members. A total of 932 carbohydrate-active enzymes, including highly expressed auxiliary activity family 9 and 10 proteins, were identified at 62°C under aerobic conditions. Some potential thermostable enzymes were identified at 50, 62, and 25°C (mature stage). Increased content and overexpressed key enzymes involved in glycolysis and starch, pyruvate and ethanol metabolism were detected at 50 and 62°C. The key enzymes of the citrate cycle were up-regulated at 62°C, and their abundant derivatives are crucial for flavor generation. Here, the metabolism and functional enzymes of the active microbial communities in NF liquor starter were studied, which could pave the way to initiate improvements in liquor quality and to discover microbes that produce novel enzymes or high-value added products.

Keywords: Chinese Nong-flavor liquor; Chinese liquor starter; ethanol fermentation; flavor generation; metatranscriptome; saccharification.

Figure 1

The 20 most abundant KEGG pathways in the metatranscriptome of Nong-flavor liquor starter samples (N1, N2, N3, and N4). N1 was sampled at the beginning of liquor starter production, N2 was sampled after 3 days of liquor starter fermentation, N3 was sampled after 9 days of liquor starter fermentation, and N4 was the mature liquor starter. The temperatures of N1, N2, N3, and N4 were 30, 50, 62 and 25°C, respectively.

Figure 2

Key metabolic profiles of scarification, ethanol fermentation and flavor generation in the Nong-flavor liquor starter. In this schematic summary, extracellular and intracellular reactions are separated by the cell membrane, but these reactions are not restricted to one cell. The light-blue color indicates the scarification profile. The pink color indicates the ethanol fermentation profile, and the green color indicates that the derivatives may be related to flavor generation.

Figure 3

Matched numbers of carbohydrate-active enzymes from the Nong-flavor liquor starter samples. CBM, Carbohydrate-Binding Module; GT, Glycosyl Transferase; PL, Polysaccharide Lyase; GH, Glycoside Hydrolase; and CE, Carbohydrate Esterase. N1 was sampled at the beginning of liquor starter production, N2 was sampled after 3 days of liquor starter fermentation, N3 was sampled after 9 days of liquor starter fermentation, and N4 was the mature liquor starter. The temperatures of N1, N2, N3, and N4 were 30, 50, 62, and 25°C, respectively.

Figure 4

Genes related to carbohydrate and energy metabolism were relatively highly expressed in the liquor starter samples (N1, N2, N3, and N4). Four abundant carbohydrate and energy metabolisms were analyzed here, i.e., starch and sucrose metabolism (A), glycolysis (B), pyruvate metabolism (C), and the citrate cycle (D). For each metabolism, relatively high gene expression levels were presented by function, EC number and total RPKM. Relative expression (log₂RPKM)) is shown between the high (red) and low (blue) expression levels. The key enzymes are highlighted with color.