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State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaKey Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science & Technology, Ministry of Education, Tianjin 300457, P.R. ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaTianjin Key Laboratory of Industrial Microbiology, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, P.R. China
State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaKey Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science & Technology, Ministry of Education, Tianjin 300457, P.R. ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaTianjin Key Laboratory of Industrial Microbiology, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, P.R. China
State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaKey Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science & Technology, Ministry of Education, Tianjin 300457, P.R. ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaTianjin Key Laboratory of Industrial Microbiology, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, P.R. China
State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaKey Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science & Technology, Ministry of Education, Tianjin 300457, P.R. ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaTianjin Key Laboratory of Industrial Microbiology, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, P.R. China
State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaKey Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science & Technology, Ministry of Education, Tianjin 300457, P.R. ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaTianjin Key Laboratory of Industrial Microbiology, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, P.R. China
State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaKey Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science & Technology, Ministry of Education, Tianjin 300457, P.R. ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaTianjin Key Laboratory of Industrial Microbiology, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, P.R. China
State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaKey Laboratory of Industrial Fermentation Microbiology, Tianjin University of Science & Technology, Ministry of Education, Tianjin 300457, P.R. ChinaNational Engineering Laboratory for Industrial Enzymes, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaTianjin Key Laboratory of Industrial Microbiology, Tianjin University of Science & Technology, Tianjin 300457, P.R. ChinaCollege of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, P.R. China
A combined proteomic and metabonomic approach was used to investigate the metabolism of Lactococcus lactis ssp. lactis subjected to glucose stress treatment. A proteomic method was used to determine 1,427 altered proteins, including 278 proteins with increased expression and 255 proteins with decreased expression. A metabonomic approach was adopted to identify 98 altered metabolites, including 62 metabolites with increased expression and 26 metabolites with decreased expression. The integrated analysis indicated that the RNA and DNA mismatch repair process and energy metabolism were enhanced in response to high-glucose stress in L. lactis. Lactococcus lactis responded to glucose stress by up-regulating oxidoreductase activity, which acted on glycosyl bonds, hydrolase activity, and organic acid transmembrane transporter activity. This led to an improvement in the metabolic flux from glucose to pyruvate, lactate, acetate, and maltose. Down-regulation of amino acid transmembrane transporter, aminoacyl-transfer RNA ligase, hydroxymethyl-, formyl-, and related transferase activities resulted in a decrease in the nitrogen metabolism-associated metabolic pathway, which might be related to inhibition of the production of biogenic amines. Overall, we highlight the response of metabolism to glucose stress and provide potential possibilities for the reduced formation of biogenic amines in improved level of sugar in the dairy fermentation industry. Moreover, according to the demand for industrial production, sugar concentration in fermented foods should be higher, or lower, than a set value that is dependent on bacterial strain and biogenic amine yield.
Lactococcuslactis strains are widely used in food preservation and the dairy fermentation industry, especially in fermented milk products such as yogurt, kefir, and cheese, and are economically considered the most important lactic acid bacteria (
). Moreover, L. lactis is considered a traditional food fermentation microorganism, and it is recognized as having generally regarded as safe (GRAS) status. Based on industrial interests, the L. lactis group includes 3 phenotypes: lactis (L. lactis ssp. lactis), cremoris (L. lactis ssp. cremoris), and diacetylactis (L. lactis ssp. hordniae) phenotypes (
). Lactococcus lactis ssp. lactis can adapt easily to environments that contain milk or dairy products, and hence quickly and easily metabolize monosaccharides (glucose) and disaccharides (lactose;
During the initiation of the culture and industrial fermentation process, L. lactis encounters various environmental stress factors. Several studies have focused on the molecular adaptation mechanisms of L. lactis to various stresses such as acids (
). Recent results have shown that higher levels of up to 1 g per 100 g of glucose or lactose can reduce the yield of putrescine by repressing gene transcription involved in the agmatine deiminase pathway in L. lactis ssp. lactis and L. lactis ssp. cremoris (
) showed that 10 g/L of glucose or lactose concentration can decrease the yield of putrescine, indicating the sugar content in fermented dairy food should be higher than 1%. Therefore, improving the glucose concentration might be a potential way to reduce the toxicity of substances such as biogenic amines in fermented foods and beverages. Moreover, according to the demand imposed by industrial production, the sugar concentration in fermented food should be higher than a set value or not lower than a set value depending on strain and biogenic amine yield: however, the molecular mechanisms underlying the proteomic and metabolic changes remain unclear. Furthermore, in the industrial fermentation environment, L. lactis might encounter local high sugar contents. In this study, proteomics analysis was performed by tandem mass tag (TMT) labeling technology combined with liquid chromatography–tandem mass spectrometry (LC-MS/MS). A metabonomic analysis in L. lactis ssp. lactis was carried out after treatment in a glucose stress environment. The results will advance our understanding of the mechanisms underlying the metabolic adaptation of L. lactis used in fermented foods and beverages to higher levels of glucose and aim at establishing guidelines around the use of biogenic amine-producing strains and their proper application in the fermentation.
MATERIALS AND METHODS
Lactic Acid Bacterial Strain and Growth Conditions
The L. lactis ssp. lactis CICC 23200 used in this study was obtained from China Center of Industrial Culture Collection. Lactococcus lactis 23200 was first grown in 10 mL of M17 medium overnight, diluted in 100 mL of M17 medium, and inoculated during the logarithmic phase (optical density at 600 nm ≈ 1.0). For glucose treatments, 10 mL of cells were transferred into a 250-mL anaerobic bottle up to the final volume of 250 mL medium containing 5, 10, 15, 30, 60, and 120 mM glucose, respectively. Next, the cells were grown at 30°C for 6 h and then collected for further analysis.
Protein Extraction and Trypsin Digestion
For protein extractions, samples were lysed (8 M urea, 1% protease inhibitor cocktail, sonication treatment). The supernatant was collected by centrifugation (12,000 × g, 4°C, 10 min) and the protein concentration was detected using a BCA kit. Protein digestion was conducted according to the method described in the previous work (
The LC-MS/MS analysis was conducted using MS/MS in Q Exactive Plus (Thermo Fisher Scientific) coupled online to the EASY-nLC 1000 UPLC system (Thermo Fisher Scientific) with a 300Extend-C18 column (5 μm particles, 4.6 mm i.d., 250 mm length, Agilent Technologies, Santa Clara, CA). The labeled peptides were first separated into 60 fractions (a gradient of 8% to 32% acetonitrile, pH 9.0, 60 min). Then, they were combined into 18 fractions and dried by vacuum centrifugation. The applied electrospray voltage was 2.0 kV, and the scan range was set to a full scan ranging from 350 to 1,800 m/z. Labeled peptides were detected in the Orbitrap (Thermo Fisher Scientific) at a resolution of 70,000 and selected for MS/MS using an normalized collision energy setting of 28. Fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure was applied that alternated between one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. The automatic gain control was set at 5E4. The fixed first mass was set at 100 m/z.
Statistical Analysis
The obtained MS/MS data were processed using the MaxQuant search engine (v.1.5.2.8; https://www.maxquant.org/). Tandem mass spectra were searched against the MASCOT 2.2 server database (www.matrixscience.com/) concatenated to the reverse decoy database. Protein sequences, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were analyzed using the UniProt protein database (https://www.uniprot.org/) and KEGG database (https://www.kegg.jp/). The GO and KEGG enrichment analysis was undertaken using the hypergeometric test method with R language (false discovery rate <1% and peptide minimum score >40 were considered significant).
Metabolite Extraction and GC-MS Analysis
Metabolite extraction and GC-MS analyses were performed using the method previously described (
Non-targeted metabolomic reveals the effect of salt stress on global metabolite of halotolerant yeast Candida versatilis and principal component analysis.
J. Ind. Microbiol. Biotechnol.2014; 41 (25085740): 1553-1562
). Metabolites were extracted in pure methanol and investigated by GC-MS after oximation and silylation derivatization. The GC-MS analyses were conducted with a Varian 4000 (Varian, Palo Alto, CA) GC/MS coupled to an HP 5973 quadrupole mass selective detector (Agilent Technologies) with an electron ionization source operated at 70 eV. The capillary column used for all analyses was a VF-5MS column (Agilent Technologies; 30 × 0.25 µm × 0.25 mm). Each compound was identified by reference to the NIST05a library (www.nist.gov/). Principal component analysis (PCA) was conducted with SPSS Statistics 18.0 package software (SPSS Inc., Chicago, IL). Metabolic pathways were investigated based on the KEGG database.
RESULTS AND DISCUSSION
Effect of Glucose Treatment on Growth of L. lactis and pH
Growth of L. lactis and the pH of the medium exposed to glucose stress were detected (Figures 1A-1B). The results showed a significant increase in the biomass of L. lactis with increasing glucose; however, the growth of L. lactis was slightly inhibited when the content of glucose reached 120 mM. Lactococcus lactis entered the stationary phase at 6 h. The growth of L. lactis led to the acidification of the medium from pH 6.7 to approximately pH 6.2 after 6 h of fermentation in cultures supplemented with 30 mM. Changes from pH 6.5 to approximately pH 4.5 occurred in 60 and 120 mM glucose. Therefore, through the combined analysis of growth and pH, the metabonomic-proteomic analysis samples were obtained at 6 h.
Figure 1(A) The growth curves of Lactococcus lactis after treatment with 5, 10, 15, 30, 60, and 120 mM glucose; (B) pH of medium exposed to different glucose stresses; (C) Pearson's R2 showing the correlation of the differentially expressed proteins (log2 ratio) between 3 biological replicates; (D) protein sequence coverage (%); and (E) subcellular location of differentially expressed proteins in L. lactis. OD600 = optical density at 600 nm.
Identification of Proteins Using TMT Labeling Technology Combined with LC-MS/MS
The TMT correlation between the biological replicates was analyzed and the results are illustrated in Figure 1C. A total of 1,427 proteins were identified using TMT labeling technology combined with LC-MS/MS, and the protein mass in the majority of the altered proteins ranged from 20 to 100 kDa (Figure 1D). A protein was considered to be significantly expressed if it indicated at least a 1.2-fold change. Therefore, 278 proteins were up-regulated and 255 proteins were down-regulated in our data (Supplemental Table S1; https://doi.org/10.3168/jds.2019-17810). The subcellular location of the altered proteins was classified as cytoplasmic (72%), membrane (20%), and extracellular (8%), respectively (Figure 1E). The considerable proportion of down-regulated membrane proteins (26% down-regulated proteins, 67 down-regulated proteins involved; 15% up-regulated proteins, 41 up-regulated proteins involved) in response to glucose stress indicated that these essential metabolic enzymes may be involved in the decrease of some metabolic pathways.
Functional Classification and Annotation Analysis of Differentially Expressed Proteins
Differentially expressed proteins of L. lactis were functionally classified by GO analysis, as shown in Figure 2. Differentially expressed proteins related to molecular functions were classified as follows: catalytic activity (55%), binding (33%), transporter activity (6%), nucleic acid binding transcription factor activity (2%), structural molecule activity (2%), antioxidant activity, electron carrier activity, molecular transducer activity, and signal transducer activity (2% each; Figure 2A).
Figure 2Gene Ontology categories for differentially expressed proteins and characteristics of Lactococcus lactis treated with glucose stress. (A) Functional classification of differentially expressed proteins in the molecular functions in response to glucose treatment, and (B) functional classification of differentially expressed proteins in the biological processes in response to glucose treatment.
Expressed proteins in L. lactis related to biological processes were classified as follows: metabolic processes (33%), cellular processes (26%), single-organism processes (23%), localization (8%), biological regulation (5%), response to stimulus (3%), cellular component organization or biogenesis (1%), and signaling and biological adhesion (1%; Figure 2B). As differentially expressed proteins focused on catalytic activity, metabolic processes are the most likely adaptable strategies to relieve the pressure of glucose stress.
Functional Enrichment Analysis of Differentially Expressed Proteins
Gene Ontology enrichment of differentially expressed proteins of L. lactis treated with glucose stress is demonstrated in Figure 3. Differentially expressed proteins related to molecular functions were enriched as follows: hydrolase activity, acting on glycosyl bonds, hydrolase activity, hydrolyzing O-glycosyl compounds, amino acid transmembrane transporter activity, hydroxymethyl-, formyl- and related transferase activity, oxidoreductase activity, acting on the CH-OH group of donors, pyridoxal phosphate binding, and organic acid transmembrane transporter activity (Figure 3A). Differentially expressed proteins related to biological process were enriched as follows: pyrimidine-containing compound metabolic process, de novo inosine monophosphate (IMP) biosynthetic process, IMP metabolic process, IMP biosynthetic process, nucleobase metabolic process, pyrimidine-containing compound biosynthetic process, nitrogen compound transport, amide transport, peptide transport, and organophosphate catabolic process (Figure 3A).
Figure 3Histogram displaying Gene Ontology enrichment of differentially expressed proteins of Lactococcus lactis treated with glucose stress according to Fisher's exact test P-value. (A) Molecular functions and biological process enrichment of differentially expressed proteins in 60 versus 30 mM glucose treatment; (B) protein domain enrichment of differentially expressed proteins 60 versus 30 mM glucose treatment; (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of up-regulated proteins 60 versus 30 mM glucose treatment; and (D) KEGG enrichment of down-regulated proteins 60 versus 30 mM glucose treatment, respectively. Each related protein number is shown to the right of the bars. TCA = tricarboxylic acid; tRNA = transfer RNA; IMP = inosine monophosphate.
Figure 3Histogram displaying Gene Ontology enrichment of differentially expressed proteins of Lactococcus lactis treated with glucose stress according to Fisher's exact test P-value. (A) Molecular functions and biological process enrichment of differentially expressed proteins in 60 versus 30 mM glucose treatment; (B) protein domain enrichment of differentially expressed proteins 60 versus 30 mM glucose treatment; (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of up-regulated proteins 60 versus 30 mM glucose treatment; and (D) KEGG enrichment of down-regulated proteins 60 versus 30 mM glucose treatment, respectively. Each related protein number is shown to the right of the bars. TCA = tricarboxylic acid; tRNA = transfer RNA; IMP = inosine monophosphate.
Protein domains are high-level parts of proteins that exhibit evolutionary conservation, and many protein domains perform the specific functions of their proteins (
). Differentially expressed proteins related to protein domains were enriched as follows: the amino acid permease/SLC12A domain, glycoside hydrolase, catalytic domain, oligopeptide/dipeptide ABC transporter, C-terminal, 6-phosphogluconate dehydrogenase, domain 2, aminotransferase, class I/class II, glycoside hydrolase superfamily, 6-phosphogluconate dehydrogenase, NADP-binding, biotin/lipoyl attachment, single hybrid motif, nitroreductase-like, dihydrofolate reductase-like domain, nitroreductase, and transfer RNA (tRNA)-binding arm (Figure 3B).
The KEGG pathways of differentially expressed proteins were analyzed (Figures 3C-3D). As shown in the figure, carbon metabolism (starch and sucrose metabolism, pyruvate metabolism, citrate cycle (tricarboxylic acid) and glycolysis/gluconeogenesis) could be enriched with increased protein expression (Figure 3C). Purine metabolism and nitrogen metabolism (valine, leucine and isoleucine degradation, phenylalanine metabolism, tryptophan metabolism, alanine, aspartate and glutamate metabolism, histidine metabolism, aminoacyl-tRNA biosynthesis) were enriched with proteins with both increased and decreased expression (Figure 3D). In addition, 9 aminoacyl-tRNA ligases (thrS, pheT, pheS, tyrS, glyQ, lysS, ileS, valS, and serS) were found to have decreased expression in this study. Our data confirmed the key role of metabolic alterations in the response to glucose stress in L. lactis.
Functional Enrichment Cluster Analysis of Differentially Expressed Proteins
The functional enrichment cluster analysis of differentially expressed proteins of L. lactis treated with glucose stress is demonstrated in Figure 4. Unsupervised hierarchical clustering of differentially expressed proteins in 60 mM versus 30 mM glucose treatment in the 4 groups included: quantitative category 1 (0 < ratio ≤ 1/1.3 and P < 0.05), quantitative category 2 (1/1.3 < ratio ≤ 1/1.2 and P < 0.05), quantitative category 3 (1.2 < ratio ≤ 1.3 and P < 0.05), and quantitative category 4 (ratio > 1.3 and P < 0.05). The results indicated that down-regulated proteins (Q1, ratio ≤ 1/1.3) were most strongly associated with histidine metabolism, nitrogen metabolism, biosynthesis of amino acids, cysteine and methionine metabolism, alanine, aspartate and glutamate metabolism, quorum sensing, beta-lactam resistance, sulfur metabolism, purine metabolism, ABC transporters, and one carbon pool by folate. Up-regulated proteins (quantitative category 4, ratio > 1.3) were most strongly associated with pyruvate metabolism, the citrate cycle, starch and sucrose metabolism, glycolysis/gluconeogenesis, and propanoate metabolism.
Figure 4Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of differentially expressed proteins of Lactococcus lactis treated with glucose stress. Unsupervised hierarchical clustering of differentially expressed proteins 60 versus 30 mM glucose treatment in 4 groups included: quantitative category 1 (Q1; 0 < ratio ≤ 1/1.3 and P < 0.05), quantitative category 2 (Q2; 1/1.3 < ratio ≤ 1/1.2 and P < 0.05), quantitative category 3 (Q3; 1.2 < ratio ≤1.3 and P < 0.05), and quantitative category 4 (Q4; ratio > 1.3 and P < 0.05). tRNA = transfer RNA.
Metabonomic Analysis of L. lactis Treated with Different Concentrations of Glucose
Metabonomic analysis of L. lactis after treatment with 5, 10, 15, 30, 60, and 120 mM glucose, followed by metabolomic profiling, is illustrated in Figure 5. A total of 98 metabolites were detected, which were categorized as 28.57% acids (28), 23.46% amino acids (23), 17.35% sugars (17), 15.31% derivatives of ammonia or amine (15), 12.24% heterocycles of ketone and phenol (12), and 3.06% alcohols (3) (Supplemental Table S2 and Figure S1A; https://doi.org/10.3168/jds.2019-17810).
Figure 5Heat map of unsupervised hierarchical clustering of all metabolites of Lactococcus lactis treated with glucose stress. Green and red indicate decreased and increased expression of metabolites, respectively. Values are scaled to the mean and SD of the metabolite level row (see color scale).
The investigation of metabolites was subjected to PCA. The PCA score plots show that each group was clearly discriminated from the others (Supplemental Figure S1B). The biplot of metabolites analyzed for the 5 principal components is shown in Supplemental Figure S1C and Table S3 (https://doi.org/10.3168/jds.2019-17810). Principal components 1 and 2 accounted for 73.34% of the accumulative variance. As summarized in Supplemental Table S3, principal components 1 and 2 possessed 55 and 24 metabolites, respectively.
The PCA loading plots (Supplemental Figure S1C; https://doi.org/10.3168/jds.2019-17810) reveal that acetamide (metabolite 4), lactate (metabolite 10), proline (metabolite 17), glycerol (metabolite 23), pentanedioic acid (metabolite 43), ornithine (metabolite 50), lysine (metabolite 55), phosphoric acid (metabolite 56), fructofuranose (metabolite 58), glucose (metabolite 59), fructopyranose (metabolite 62), fructose (metabolite 67), sorbose (metabolite 68), mannopyranose (metabolite 70), galactose (metabolite 71), and ascorbic acid (metabolite 75) were clearly distinguishable from the other samples.
Acetamide is widely present in milk, beef, eggs, and roasted coffee beans, and is produced during food processing (
). Acetamide is formed from thermal breakdown of N-acetylated metabolites (monosaccharides N-acetylglucosamine, N-acetylcysteine) and glycans during vaporization of GC-MS analysis (
). Therefore, acetamide can be involved in carbohydrate metabolism and considered to have harmful components. However, the acute toxicity of acetamide is very low and its lethal dose 50% value is in the grams per kilogram range (
). Amino acids such as l-proline, ornithine, and l-lysine are involved in amino acid metabolism and widely used in food additives and the feed industry (
Modular pathway engineering of Corynebacterium glutamicum for production of the glutamate-derived compounds ornithine, proline, putrescine, citrulline, and arginine.
). Proline is generated from glutamate, through catalysis by glutamate 5-kinase, glutamate-5-semialdehyde dehydrogenase, delta-1-pyrroline-5-carboxylate synthetase, and pyrroline-5-carboxylate reductase. The glutamate 5-kinase was down-regulated 0.64-fold at 60 mM versus 30 mM. Ornithine is generated from glutamate, via catalysis by amino acid N-acetyltransferase, acetylglutamate kinase, acetylornithine deacetylase, and aminoacylase. Lysine is generated from aspartate, via catalysis by aspartate kinase, aspartate-semialdehyde dehydrogenase, succinyl-diaminopimelate desuccinylase, and diaminopimelate decarboxylase. Pentanedioic acid is a natural product in the catabolism of l-lysine via the aminovalerate pathway in bacteria (
). Fructose, fructofuranose, fructopyranose, galactose, mannopyranose, sorbose, and glycerol are involved in fructose, mannose, and galactose metabolism. Proline, glycerol, and sorbose can be classified as a compatible solute or osmoprotectant that plays important roles in response to various stresses (
). In water solution, beta-d-fructopyranose is the only crystalline form of d-fructose; in addition, there exists an interconversion between pyranose and furanose, and glucopyranose could be isomerized to fructofuranose (
). Ascorbic acid is produced from glucose-6P (plants) or glucose-1P (animals). Some studies showed that yeast cells can synthesize ascorbic acid via the pathway naturally used for d-erythroascorbic acid biosynthesis (
Metabolic pathways were enriched in differential metabolites, and the results showed an enrichment of 9 metabolic pathways (Figure 6). Up-regulated metabolites were enriched in fatty acid, fructose, mannose, galactose, propanoate, valine, leucine, and isoleucine metabolism. Moreover, down-regulated metabolites were enriched in nitrogen metabolism, which was in line with the proteomic results (Figure 3D).
Figure 6Kyoto Encyclopedia of Genes and Genomes enrichment of the metabolites of Lactococcus lactis after treatment under a glucose stress. Green and red highlighting represent those metabolites with up-regulated and down-regulated expression, respectively.
Effect of Glucose Stress on DNA Repair Process of L. lactis
Mismatch repair pathway-related proteins, such DNA polymerase III (BN927_01049, 1.208-fold, BN927_02385, 1.275-fold), DNA-directed DNA polymerase (BN927_01802, 1.203), single-stranded DNA-binding protein (BN927_02507, 1.403, BN927_00106, 1.472), and priA were up-regulated. Ribosome-related proteins, DNA-directed RNA polymerase (rpoB, 0.813, rpoC, 0.805, rpoZ, 0.785), and ribonucleotide reductase (BN927_01203, 0.629) were down-regulated in L. lactis under high-glucose stress. Previous studies have shown that single-stranded DNA-binding protein genes were highly expressed under acid or erythromycin stress (
). Therefore, we inferred that RNA- and DNA-related regulatory mechanisms may also participate in the response to stress.
Effects of Glucose Stress on Energy and Carbohydrate Metabolism of L. lactis
Proteins with up-regulated expression were involved in energy metabolism such as starch and sucrose metabolism (12 proteins involved), pyruvate metabolism (11), glycolysis/gluconeogenesis (11), amino sugar and nucleotide sugar metabolism (8), citrate cycle (5), pentose phosphate pathway (5), and galactose metabolism (4). The increased expression of these energy generation-related metabolism suggested that metabolic activity was enhanced in response to high-glucose condition.
The most efficient sugar uptake system in bacteria is the phosphotransferase (PTS) system, which includes histidine-containing phosphocarrier protein (HPr) plus enzyme I and enzyme II (
Genetic characterization of the CcpA-dependent, cellobiose-specific PTS system comprising CelB, PtcB and PtcA that transports lactose in Lactococcus lactis IL1403.
Int. J. Food Microbiol.2011; 145 (21262549): 186-194
). Enzyme II have 3 domains, IIA, IIB and IIC, and IIA and IIB, involved in phosphoryl group transfer, whereas domain IIC could form the translocation channel. The higher expression of proteins involved in PTS are beta-glucoside-specific (BN927_02039, 3.562-fold), 6-phospho-beta-glucosidase (BN927_02404, 1.294), cellobiose-specific IIB component (BN927_01839, 1.538), cellobiose-specific IIA component (BN927_01838, 1.715), lactose-specific IIA (BN927_02727, 1.204) maltose phosphorylase/trehalose phosphorylase (BN927_02410, 1.217), sucrose-specific PTS enzyme IIABC (BN927_01824, 1.228), mannose-specific IID (BN927_02452, 0.774), and trehalose-specific IIB (BN927_01823, 0.813). Moreover, beta-glucosidase (BN927_02040, 2.843-fold, BN927_02010, 1.265-fold) could hydrolyze glucosidic linkages in aryl-, amino-, or alkyl-β-d-glucosides, cyanogenic glucosides, and oligo- or disaccharides (
). Maltose produced from glycogen by catalysis of alpha-amylase (BN927_02411, 1.3 fold), cytoplasmic alpha-amylase (BN927_00812, 1.431), and neopullulanase (BN927_02673, 1.526) is an extracellular alpha-amylase (
). Some sugar utilization systems consist of operon structures without PTS. These operons contain genes involved in both sugar transport and metabolism (
Cloning and biochemical analysis of β-glucoside utilization (bgl) operon without phosphotransferase system in Pectobacterium carotovorum subsp. carotovorum LY34.
reported activation of glucose uptake by a cellobiose-specific PTS system in L. lactis in response to acid, ethanol, heat, and oxidative stress. Our data indicated that cellobioses, lactose, mannose, and sucrose also played important roles in response to high-glucose and glucose-limited stress (
Genomic resequencing combined with quantitative proteomic analyses elucidate the survival mechanisms of Lactobacillus plantarum P-8 in a long-term glucose-limited experiment.
), which suggested that lactose transport is different in L. lactis in response to various stresses. Sucrose and maltose can protect membrane structures against fusion and leakage (
A comparative study of the influence of sugars sucrose, trehalose, and maltose on the hydration and diffusion of DMPC lipid bilayer at complete hydration: investigation of structural and spectroscopic aspect of lipid-sugar interaction.
demonstrated that maltose functions as a sugar protectant in Candida oleophila against heat stress. Therefore, the results indicated that the disaccharide maltose, derived from glucose for glycan biosynthesis, might serve as a potential osmoprotectant and potential carbon storage source in L. lactis.
Pyruvate metabolism was enhanced under glucose stress, and the expression of phosphoglycerate kinase (pgk, 1.261-fold), lactate dehydrogenase (BN927_01433, 1.317), aldehyde-alcohol dehydrogenase (BN927_02274, 1.435), acetate kinase (ackA, 1.411), and acylphosphatase (BN927_02165, 1.398) was markedly up-regulated. The levels of lactate, ethanol, and acetate increased in L. lactis in response to glucose, which indicated metabolic flux from pyruvate to lactate and acetate, leading to a decrease in the pH of the culture medium. Moreover, abundant acetyl-CoA was generated from pyruvate-flavodoxin oxidoreductase (BN927_01829, 1.465), dihydrolipoamide acetyltransferase (BN927_00448, 1.633), acetoin dehydrogenase (BN927_00449, 1.457, BN927_00450, 1.453), dihydrolipoyl dehydrogenase (BN927_00447, 1.54), phenylalanine [6-aminohexanoate-cyclic-dimer hydrolase (BN927_01315, 1.266)], tryptophan [3-ketoacyl-CoA thiolase @ acetyl-CoA acetyltransferase (BN927_01919, 1.786)], 6-aminohexanoate-cyclic-dimer hydrolase (BN927_01315, 1.266), histidinol-phosphate aminotransferase (hisC, 1.213), lysine, valine, leucine, isoleucine [dihydrolipoyl dehydrogenase (BN927_00447, 1.54)], 3-ketoacyl-CoA thiolase @ acetyl-CoA acetyltransferase (BN927_01919, 1.786), 2-hydroxy-3-oxopropionate reductase (BN927_00152, 1.27), branched-chain amino acid aminotransferase (BN927_00772, 1.224), and acetyl-CoA flux to the citrate cycle [dihydrolipoyl dehydrogenase (BN927_00447, 1.54)]. 3-Ketoacyl-CoA thiolase @ acetyl-CoA acetyltransferase catalyzes the final step of fatty acid oxidation to release acetyl-CoA. Moreover, the levels of acetyl-CoA precursor substances such as phenylalanine, tryptophan, lysine, valine, leucine, and isoleucine are increased in L. lactis in response to glucose.
Effect of Glucose Stress on AA Metabolism of L. lactis
Proteins with altered expression in the amino acid metabolic pathway included aspartate (aspartate aminotransferase (BN927_01322, 1.205-fold), adenylosuccinate synthetase (purA, 0.628), adenylosuccinate lyase (BN927_02371, 0.552), glutamate [glutamate synthase (BN927_00775, 0.391, BN927_00774, 0.403)], histidine [imidazole glycerol phosphate synthase (hisF, 0.58)], histidinol dehydrogenase (hisD, 0.424), imidazole glycerol phosphate dehydratase (hisB, 0.413), ATP phosphoribosyltransferase (hisZ, 0.406), histidinol-phosphate aminotransferase (hisC, 1.213), and arginine [carbamoyl-phosphate synthase (carA, 0.55, carB, 0.512)]. As previously shown, histidinol phosphatase (hisK) was up-regulated in L. lactis ssp. lactis in response to gradient freezing stress (
The level of glutamate increased under glucose stress, which was predicted to be the main factor that acts as a switch for the regulation of cell growth and carbon accumulation. Considering the high levels of glutamate in the cells under glucose stress (
), the down-regulated expression of glutamate synthase (BN927_00775, BN927_00774) and glutamine synthetase (BN927_00137) suggested that the cells readjust nitrogen metabolism by the GS/GOGAT pathway (
Down-regulation of the expression level of protein involved in methionine metabolism, such as methionine biosynthesis and transport regulator MtaR (BN927_01938, 0.704-fold), methionine synthase (BN927_02656, 0.49), and methionine, import ATP-binding protein MetN (metN, 0.472). Previous work showed that the enzymes involved in methionine metabolism increased in response to elevated temperatures in L. lactis, which could directly improve the production of the key cheese flavor compound methanethiol (
It was observed that the expression of serine protease DegP/Htr (BN927_00315, 1.929-fold) was significantly up-regulated. The HtrA is a housekeeping surface protease in L. lactis and is a critical factor for bacterial survival under environmental stress conditions (
). The HtrA proteins are known bifunctional proteins that function as both molecular chaperones and proteases (conserved from bacteria to humans). Their proteolytic activity (in most cases chaperone activity) efficiently counteracted the consequences of stressful conditions (
Effects of Glucose Stress on tRNA Synthetases and Biosynthesis of Biogenic Amine of L. lactis
The levels of many aminoacyl-tRNA biosynthesis-related metabolites such as threonine, phenylalanine, tyrosine, isoleucine, valine, serine, which are the precursors of amino acid-tRNA, were increased. In contrast, aminoacyl-tRNA ligases (thrS, 0.8-fold; pheT, 0.765; pheS, 0.709; tyrS, 0.824; glyQ, 0.818; lysS, 0.795; ileS, 0.789; valS, 0.772; and serS, 0.8) were largely decreased in response to high glucose. The results showed that the activity of aminoacyl-tRNA synthetase was disturbed by glucose stress and that protein biosynthetic processes were suppressed.
Biogenic amines, including cadaverine, tyramine, putrescine, spermine, and spermidine, are derived from the microbial decarboxylation of the corresponding amino acids through substrate-specific decarboxylase enzymes (
Determination of biogenic amines as dansyl derivatives in alcoholic beverages by high-performance liquid chromatography with fluorimetric detection and characterization of the dansylated amines by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.
). Lysine decarboxylation results in the formation of cadaverine. Tyrosine decarboxylation occurs to generate tyramine. Transformation of glutamic acid to arginine is found, followed by the formation of putrescine, spermine, and spermidine. The levels of lysine, putrescine, cadaverine, tyramine, spermine, and spermidine are decreased in L. lactis in response to glucose. Decarboxylation of amino acids is the main way in which enhanced lactic acid bacteria survival arises under environmental stress; moreover, the antiporter/decarboxylase system is an indirect proton pump analogous to other mechanisms providing energy and protecting from acidic conditions (
Putrescine production by Lactococcus lactis subsp. cremoris CECT 8666 is reduced by NaCl via a decrease in bacterial growth and the repression of the genes involved in putrescine production.
To further explore the comprehensive influence of proteins and metabolites on the response to glucose treatment, the metabolic network, which consisted of a combination of proteomic and metabonomic data, was established as shown in Figure 7.
Up-regulated metabolic pathways included starch and sucrose metabolic transformation of monosaccharide into polysaccharides, especially enhanced maltose metabolism, glycolysis conversion of glucose into pyruvate and acetic acid, pyruvate oxidation to generate acetyl-CoA, and amino acid metabolic transformation of leucine, valine, and isoleucine into acetyl-CoA connected via fatty acid metabolism. Phenylalanine and tryptophan could also generate acetyl-CoA. The extracellular level of glucose could affect metabolic flux in the stress response of L. lactis (
). Research has shown that the metabolic flux is increased in glycolysis, the citrate cycle, and pyruvate metabolism in L. lactis in response to glucose stress. This result is consistent with a previous study showing that Lactobacillus plantarum activation consists of an increase in the metabolism of carbohydrates (via glycolysis, the citric acid cycle, and pyruvate metabolism) in response to glucose-limited stress (
Genomic resequencing combined with quantitative proteomic analyses elucidate the survival mechanisms of Lactobacillus plantarum P-8 in a long-term glucose-limited experiment.
Among the down-regulated metabolic pathways, metabolic flux was decreased for histidine, lysine, alanine, aspartate, glutamate, and arginine metabolism in L. lactis in response to glucose stress. This led to reduced biogenic amine production, which is in agreement with a previous study indicating that Lactobacillus plantarum activation increases amino acid catabolism (via serine, threonine, and histidine metabolism, lysine degradation, and arginine responses to glucose-limited stress;
Genomic resequencing combined with quantitative proteomic analyses elucidate the survival mechanisms of Lactobacillus plantarum P-8 in a long-term glucose-limited experiment.
). Saccharomyces cerevisiae metabolic pathway regulation of the citrate cycle and alanine, aspartate, and glutamate metabolisms occur in response to ethanol stress (
Lactic acid bacteria could encounter a series of environmental stresses in the manufacture of fermented dairy foods. Consequently, the organism has advantages in terms of high sugar-uptake rates, deamination and decarboxylation of amino acids, and higher acid tolerance allowing survival under harsh stress conditions (
). During acid stress, additional energy is provided by the up-regulation of carbohydrate metabolism, and enhanced regulation of amino acid metabolism and transport to maintain pH homeostasis and ATP generation (
). The levels of glycolytic enzymes (pyruvate oxidase and phosphate acetyltransferase) increase in response to acid, thermal, and osmotic stresses, leading to improved synthesis of glycogen, lysine, and diacetyl/acetoin (
). Under oxidative stress, the levels of alcohol dehydrogenase and pyruvate dehydrogenase complex are reduced, leading to decreased synthesis of lactic acid and ethanol in lactic acid bacteria. Lactococcus lactis possesses a citrate/lactate antiporter, which could metabolize citrate and synthesize acetic acid and ATP under carbohydrate starvation conditions (
). Lactococcus lactis has an advantage in terms of a higher glycine betaine uptake function and membrane-associated proteins (FtsH and HtrA) imparting resistance to osmotic stress (
The response mechanisms underlying the metabolic adaptation of L. lactis to higher levels of glucose were elucidated using a combined proteomic and metabonomic method. The results showed that the RNA and DNA mismatch repair process and energy metabolism were enhanced. Also, the metabolic flux from glucose to pyruvate, lactate, acetate, and maltose was improved. The nitrogen metabolism-associated metabolic pathway decreased, which might be related to the inhibition of the production of biogenic amines. This study constructed a model for global response mechanisms to glucose stress and identified the possibility that reduced formation of biogenic amines improves level of sugar in the dairy fermentation industry. Moreover, according to the demand for industrial production, sugar concentration in fermented foods should be higher than the set value, which is dependent on bacterial strain and biogenic amine yield.
ACKNOWLEDGMENTS
This work was supported by grants from National Natural Science Foundation of China (Beijing; 31501449, 2017YFD0400303, SKLFNS-KF-201912, TD13-5015, and 18JCTPJC56800). The authors have not stated any conflicts of interest.
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Genomic resequencing combined with quantitative proteomic analyses elucidate the survival mechanisms of Lactobacillus plantarum P-8 in a long-term glucose-limited experiment.
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