Genome-Scale Metabolic Modeling Combined with Transcriptome Profiling Provides Mechanistic Understanding of Streptococcus thermophilus CH8 Metabolism

Abstract

Streptococcus thermophilus is a lactic acid bacterium adapted toward growth in milk and is a vital component of starter cultures for milk fermentation. Here, we combine genome-scale metabolic modeling and transcriptome profiling to obtain novel metabolic insights into this bacterium. Notably, a refined genome-scale metabolic model (GEM) accurately representing S. thermophilus CH8 metabolism was developed. Modeling the utilization of casein as a nitrogen source revealed an imbalance in amino acid supply and demand, resulting in growth limitation due to the scarcity of specific amino acids, in particular sulfur amino acids. Growth experiments in milk corroborated this finding. A subtle interdependency of the redox balance and the secretion levels of the key metabolites lactate, formate, acetoin, and acetaldehyde was furthermore identified with the modeling approach, providing a mechanistic understanding of the factors governing the secretion product profile. As a potential effect of high expression of arginine biosynthesis genes, a moderate secretion of ornithine was observed experimentally, augmenting the proposed hypothesis of ornithine/putrescine exchange as part of the protocooperative interaction between S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in yogurt. This study provides a foundation for future community modeling of food fermentations and rational development of starter strains with improved functionality. IMPORTANCE Streptococcus thermophilus is one the main organisms involved in the fermentation of milk and, increasingly, also in the fermentation of plant-based foods. The construction of a functional high-quality genome-scale metabolic model, in conjunction with in-depth transcriptome profiling with a focus on metabolism, provides a valuable resource for the improved understanding of S. thermophilus physiology. An example is the model-based prediction of the most significant route of synthesis for the characteristic yogurt flavor compound acetaldehyde and identification of metabolic principles governing the synthesis of other flavor compounds. Moreover, the systematic assessment of amino acid supply and demand during growth in milk provides insights into the key challenges related to nitrogen metabolism that is imposed on S. thermophilus and any other organism associated with the milk niche.

Keywords: RNA-Seq; Streptococcus thermophilus; food fermentation; genome-scale metabolic modeling; microbial biotechnology; microbial metabolism.

FIG 1

Maximum likelihood phylogeny of CH8 and certain S. thermophilus strains with publicly available genome sequences, as inferred from an alignment of 534 core genes. The location of S. salivarius strain NCTC8618 as an outgroup is indicated by a dotted line, which is not to scale.

FIG 2

(A) Growth and acidification curves of CH8 in CDM (left) and milk (right). Arrows indicate OD₆₀₀ and pH values of RNA sample harvest points. (B) Principal-component analysis plot of sample gene expression profiles. (C) Fold change distributions and quantity of differentially expressed genes, defined as having a q value below 0.05 and log₂ fold change above |±1|. Expression changes are defined as values of the first condition in relation to values of the second condition. MI, milk; CD, CDM; ML, mid-log growth phase; LL, late-log growth phase.

FIG 3

Map of CH8 metabolism overlaid with transcriptome data. The darker brown color signifies higher log₂ fold expression changes between milk mid-log versus CDM mid-log conditions, while “nd” denotes reactions associated with genes displaying no statistically significant differential expression. Insert: transcription level of corresponding enzyme-encoding genes, as estimated from read counts. The darker color signifies higher read counts. For each gene, the highest gene count among conditions is selected.

FIG 4

Predicted secretion product profile as a function of three factors, namely, growth rate, ratio of lactate secretion/lactose uptake, and biosynthetic load; biosynthetic load is represented by GAPN (NADP-dependent glyceraldehyde-3-phosphate dehydrogenase) flux. (A) Exchange fluxes as a function of growth rate, with a constant experimentally determined lactate/lactose exchange ratio of 1.625 (as defined by the lactate secretion flux and lactose uptake flux). (B) Exchange fluxes and growth rate as a function of lactate/lactose exchange ratio. Lactose uptake remains constant. (C) Exchange fluxes and growth rate as a function of biosynthetic load with a constant lactate/lactose exchange ratio (1.625). Solid lines indicate pFBA flux predictions. Dotted vertical lines indicate the predicted values with rates determined experimentally (Table 1).

FIG 5

Casein amino acid composition and effect on growth. (A) Comparison of casein amino acid composition ratio and amino acid composition ratio of S. thermophilus total protein content. (B) Effect of casein uptake and free amino acid availability on growth. Graphs represent growth with casein, either without any free amino acids available (w/o AA), with individual free amino acids available (Asp, Met, Ala, Trp, and Cys), or with all amino acids freely available (w/AA) except methionine and cysteine. The interval of casein uptake rate in which sulfur-amino acids are growth limiting is marked in a transparent gray shade. (C) Experimental acidification curves of CH8 milk fermentation, with or without 40 mg/L free methionine supplementation.