Search

The hydrogen isotope ratios (d2HAA values) of amino acids in all organisms are substantially fractionated relative to growth water. In addition, they exhibit large variations within microbial biomass, animals, and human tissues, hinting at rich biochemical information encoded in such signals. In lipids, such d2H variations are thought to primarily reflect NADPH metabolism. Analogous biochemical controls for amino acids remain largely unknown, but must be elucidated to inform the interpretation of these measurements. Here, we measured the d2H values of amino acids from five aerobic, heterotrophic microbes grown on different carbon substrates, as well as five Escherichia coli mutant organisms with perturbed NADPH metabolisms. We observed similar d2HAA patterns across all organisms and growth conditions, which-consistent with previous hypotheses-suggests a first-order control by biosynthetic pathways. Moreover, d2HAA values varied systematically with the catabolic pathways activated for substrate degradation, with variations explainable by the isotopic compositions of important cellular metabolites, including pyruvate and NADPH, during growth on each substrate. As such, amino acid d2H valuesmay be useful for interrogating organismal physiology and metabolism in the environment, provided we can further elucidate the mechanisms underpinning these signals. [ABSTRACT FROM AUTHOR]

The hydrogen isotope ratios (δ2HAA values) of amino acids in all organisms are substantially fractionated relative to growth water. In addition, they exhibit large variations within microbial biomass, animals, and human tissues, hinting at rich biochemical information encoded in such signals. In lipids, such δ2H variations are thought to primarily reflect NADPH metabolism. Analogous biochemical controls for amino acids remain largely unknown, but must be elucidated to inform the interpretation of these measurements. Here, we measured the δ2H values of amino acids from five aerobic, heterotrophic microbes grown on different carbon substrates, as well as five Escherichia coli mutant organisms with perturbed NADPH metabolisms. We observed similar δ2HAA patterns across all organisms and growth conditions, which–consistent with previous hypotheses–suggests a first-order control by biosynthetic pathways. Moreover, δ2HAA values varied systematically with the catabolic pathways activated for substrate degradation, with variations explainable by the isotopic compositions of important cellular metabolites, including pyruvate and NADPH, during growth on each substrate. As such, amino acid δ2H values may be useful for interrogating organismal physiology and metabolism in the environment, provided we can further elucidate the mechanisms underpinning these signals.

Glucose is a crucial carbon source for the growth of Staphylococcus aureus, but an excess of glucose is detrimental and even leads to cell death. Pyruvate, the central metabolite of glycolysis, has been shown to have anti-inflammatory and antioxidant properties. This study aimed to investigate the protective effect of pyruvate on S. aureus under high glucose conditions. Sodium pyruvate greatly increased the cytotoxicity of S. aureus strain BAA-1717 to human erythrocytes and neutrophils in vitro. However, the cytotoxicity and survival of S. aureus were significantly reduced by high glucose, which was restored to normal levels by the addition of sodium pyruvate. The expression of hlg and lukS in S. aureus was higher in the LB-GP cultures than that in LB-G cultures, but there was no significant difference in cytotoxicity between LB-GP and LB-G cultures. Furthermore, the hemolytic activity of S. aureus supernatants could be inhibited by the cellfree culture medium (CFCM) of LB-G cultures, suggesting that high levels of extracellular proteases were presence in the CFCM of LB-G cultures, resulting in degradation of the hemolytic factors. The expression of sarA, which negatively regulates extracellular protease secretion, was higher in LB-GP cultures than that in LB-G cultures. Additionally, sodium pyruvate increased acetate production in S. aureus, which helps maintain cell viability under acidic environment. In conclusion, pyruvate plays an important role in the survival and cytotoxicity of S. aureus under high glucose conditions. This finding may aid in the development of effective treatments for diabetic foot infections. [ABSTRACT FROM AUTHOR]

N-acetylneuraminic acid (Neu5Ac) possesses the ability to promote mental health and enhance immunity and is widely used in both medicine and food fields as a supplement. Enzymatic production of Neu5Ac using N-acetyl-D-glucosamine (GlcNAc) as substrate was significant. However, the high-cost GlcNAc limited its development. In this study, an in vitro multi-enzyme catalysis was built to produce Neu5Ac using affordable chitin as substrate. Firstly, exochitinase SmChiA from Serratia proteamaculans and N-acetylglucosaminosidase CmNAGase from Chitinolyticbacter meiyuanensis SYBC-H1 were screened and combined to produce GlcNAc, effectively. Then, the chitinase was cascaded with N-acetylglucosamine-2-epimerase (AGE) and N-neuraminic acid aldolase (NanA) to produce Neu5Ac; the optimal conditions of the multi-enzyme catalysis system were 37°C and pH 8.5, the ratio of AGE to NanA (1:4) and addition of pyruvate (70 mM), respectively. Finally, 9.2 g/L Neu5Ac could be obtained from 20 g/L chitin within 24 h along with two supplementations with pyruvate. This work will lay a good foundation for the production of Neu5Ac from cheap chitin resources. [ABSTRACT FROM AUTHOR]

N-acetylneuraminic acid (Neu5Ac) possesses the ability to promote mental health and enhance immunity and is widely used in both medicine and food fields as a supplement. Enzymatic production of Neu5Ac using N-acetyl-D-glucosamine (GlcNAc) as substrate was significant. However, the high-cost GlcNAc limited its development. In this study, an in vitro multi-enzyme catalysis was built to produce Neu5Ac using affordable chitin as substrate. Firstly, exochitinase SmChiA from Serratia proteamaculans and N-acetylglucosaminosidase CmNAGase from Chitinolyticbacter meiyuanensis SYBC-H1 were screened and combined to produce GlcNAc, effectively. Then, the chitinase was cascaded with N-acetylglucosamine-2-epimerase (AGE) and N-neuraminic acid aldolase (NanA) to produce Neu5Ac; the optimal conditions of the multi-enzyme catalysis system were 37°C and pH 8.5, the ratio of AGE to NanA (1:4) and addition of pyruvate (70 mM), respectively. Finally, 9.2 g/L Neu5Ac could be obtained from 20 g/L chitin within 24 h along with two supplementations with pyruvate. This work will lay a good foundation for the production of Neu5Ac from cheap chitin resources.

The hydrogen isotope ratios (δ 2 H AA values) of amino acids in all organisms are substantially fractionated relative to growth water. In addition, they exhibit large variations within microbial biomass, animals, and human tissues, hinting at rich biochemical information encoded in such signals. In lipids, such δ 2 H variations are thought to primarily reflect NADPH metabolism. Analogous biochemical controls for amino acids remain largely unknown, but must be elucidated to inform the interpretation of these measurements. Here, we measured the δ 2 H values of amino acids from five aerobic, heterotrophic microbes grown on different carbon substrates, as well as five Escherichia coli mutant organisms with perturbed NADPH metabolisms. We observed similar δ 2 H AA patterns across all organisms and growth conditions, which-consistent with previous hypotheses-suggests a first-order control by biosynthetic pathways. Moreover, δ 2 H AA values varied systematically with the catabolic pathways activated for substrate degradation, with variations explainable by the isotopic compositions of important cellular metabolites, including pyruvate and NADPH, during growth on each substrate. As such, amino acid δ 2 H values may be useful for interrogating organismal physiology and metabolism in the environment, provided we can further elucidate the mechanisms underpinning these signals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Silverman, Wijker and Sessions.)

Lack of LrgAB renders cariogenic Streptococcus mutans more sensitive to oxidative stress, as well as limits the capacity of this organism to re-uptake pyruvate upon starvation. This study was aimed at investigating the ecological and metabolic contribution of LrgAB to competitive fitness, using S. mutans strains, that either lack or overexpress lrgAB. These experiments revealed that impaired aerobic growth of the ΔlrgAB mutant can be effectively restored by supplementation of pyruvate, and that perturbated expression of lrgAB significantly affects pyruvate flux and the conversion of pyruvate to acetyl-CoA by the Pdh pathway, verifying that LrgAB is closely linked to pyruvate catabolism. In vitro competition assays revealed that LrgAB plays an important role in S. mutans competition with H2O2-producing S. gordonii, an interaction which can also be modulated by external pyruvate. However, no obvious competitive disadvantage was observed against S. gordonii by either the S. mutans lrgAB mutant or lrgAB overexpression strain in vivo using a mouse caries model. Organic acid analysis of mouse dental biofilms revealed that metabolites produced by the host and/or dental plaque microbiota could complement the deficiency of a lrgAB mutant, and favored S. mutans establishment compared to S. gordonii. Collectively, these results reinforce the importance of the oral microbiota and the metabolic environment in the oral cavity battleground, and highlight that pyruvate uptake through LrgAB may be crucial for interspecies competition that drives niche occupancy.

Fluctuating environments force bacteria to constantly adapt and optimize the uptake of substrates to maintain cellular and nutritional homeostasis. Our recent findings revealed that LrgAB functions as a pyruvate uptake system in Streptococcus mutans, and its activity is modulated in response to glucose and oxygen levels. Here, we show that the composition of the growth medium dramatically influences the magnitude and pattern of lrgAB activation. Specifically, tryptone (T) medium does not provide a preferred environment for stationary phase lrgAB activation, which is independent of external pyruvate concentration. The addition of pyruvate to T medium can elicit PlrgA activation during exponential growth, enabling the cell to utilize external pyruvate for improvement of cell growth. Through comparison of the medium composition and a series of GFP quantification assays for measurement of PlrgA activation, we found that acetate and potassium (K+) play important roles in eliciting PlrgA activation at stationary phase. Of note, supplementation of pooled human saliva to T medium induced lrgAB expression at stationary phase and in response to pyruvate, suggesting that LrgAB is likely functional in the oral cavity. High concentrations of acetate inhibit cell growth, while high concentrations of K+ negatively regulate lrgAB activation. qPCR analysis also revealed that growth in T medium (acetate/K+ limited) significantly affects the expression of genes related to the catabolic pathways of pyruvate, including the Pta/AckA pathway (acetate metabolism). Lastly, stationary phase lrgAB expression is not activated when S. mutans is cultured in T medium, even in a strain that overexpresses lytST. Taken together, these data suggest that lrgAB activation and pyruvate uptake in S. mutans are connected to acetate metabolism and potassium uptake systems, important for cellular and energy homeostasis. They also suggest that these factors need to be implemented when planning metabolic experiments and analyzing data in S. mutans studies that may be sensitive to stationary growth phase.

Capnophilic lactic fermentation (CLF) is a novel anaplerotic pathway able to convert sugars to lactic acid (LA) and hydrogen using CO2 as carbon enhancer in the hyperthermophilic bacterium Thermotoga neapolitana. In order to give further insights into CLF metabolic networks, we investigated the transcriptional modification induced by CO2 using a RNA-seq approach. Transcriptomic analysis revealed 1601 differentially expressed genes (DEGs) in an enriched CO2 atmosphere over a total of 1938 genes of the T. neapolitana genome. Transcription of PFOR and LDH genes belonging to the CLF pathway was up-regulated by CO2 together with 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydratase (EDD) of the Entner–Doudoroff (ED) pathway. The transcriptomic study also revealed up-regulation of genes coding for the flavin-based enzymes NADH-dependent reduced ferredoxin:NADP oxidoreductase (NFN) and NAD-ferredoxin oxidoreductase (RNF) that control supply of reduced ferredoxin and NADH and allow energy conservation-based sodium translocation through the cell membrane. These results support the hypothesis that CO2 induces rearrangement of the central carbon metabolism together with activation of mechanisms that increase availability of the reducing equivalents that are necessary to sustain CLF. In this view, this study reports a first rationale of the molecular basis of CLF in T. neapolitana and provides a list of target genes for the biotechnological implementation of this process.

Lack of LrgAB renders cariogenic Streptococcus mutans more sensitive to oxidative stress, as well as limits the capacity of this organism to re-uptake pyruvate upon starvation. This study was aimed at investigating the ecological and metabolic contribution of LrgAB to competitive fitness, using S. mutans strains, that either lack or overexpress lrgAB. These experiments revealed that impaired aerobic growth of the Δ lrgAB mutant can be effectively restored by supplementation of pyruvate, and that perturbated expression of lrgAB significantly affects pyruvate flux and the conversion of pyruvate to acetyl-CoA by the Pdh pathway, verifying that LrgAB is closely linked to pyruvate catabolism. In vitro competition assays revealed that LrgAB plays an important role in S. mutans competition with H2O2-producing S. gordonii , an interaction which can also be modulated by external pyruvate. However, no obvious competitive disadvantage was observed against S. gordonii by either the S. mutans lrgAB mutant or lrgAB overexpression strain in vivo using a mouse caries model. Organic acid analysis of mouse dental biofilms revealed that metabolites produced by the host and/or dental plaque microbiota could complement the deficiency of a lrgAB mutant, and favored S. mutans establishment compared to S. gordonii. Collectively, these results reinforce the importance of the oral microbiota and the metabolic environment in the oral cavity battleground, and highlight that pyruvate uptake through LrgAB may be crucial for interspecies competition that drives niche occupancy. [ABSTRACT FROM AUTHOR]

Fluctuating environments force bacteria to constantly adapt and optimize the uptake of substrates to maintain cellular and nutritional homeostasis. Our recent findings revealed that LrgAB functions as a pyruvate uptake system in Streptococcus mutans , and its activity is modulated in response to glucose and oxygen levels. Here, we show that the composition of the growth medium dramatically influences the magnitude and pattern of lrgAB activation. Specifically, tryptone (T) medium does not provide a preferred environment for stationary phase lrgAB activation, which is independent of external pyruvate concentration. The addition of pyruvate to T medium can elicit P lrgA activation during exponential growth, enabling the cell to utilize external pyruvate for improvement of cell growth. Through comparison of the medium composition and a series of GFP quantification assays for measurement of P lrgA activation, we found that acetate and potassium (K+) play important roles in eliciting P lrgA activation at stationary phase. Of note, supplementation of pooled human saliva to T medium induced lrgAB expression at stationary phase and in response to pyruvate, suggesting that LrgAB is likely functional in the oral cavity. High concentrations of acetate inhibit cell growth, while high concentrations of K+ negatively regulate lrgAB activation. qPCR analysis also revealed that growth in T medium (acetate/K+ limited) significantly affects the expression of genes related to the catabolic pathways of pyruvate, including the Pta/AckA pathway (acetate metabolism). Lastly, stationary phase lrgAB expression is not activated when S. mutans is cultured in T medium, even in a strain that overexpresses lytST. Taken together, these data suggest that lrgAB activation and pyruvate uptake in S. mutans are connected to acetate metabolism and potassium uptake systems, important for cellular and energy homeostasis. They also suggest that these factors need to be implemented when planning metabolic experiments and analyzing data in S. mutans studies that may be sensitive to stationary growth phase. [ABSTRACT FROM AUTHOR]

Capnophilic lactic fermentation (CLF) is a novel anaplerotic pathway able to convert sugars to lactic acid (LA) and hydrogen using CO2 as carbon enhancer in the hyperthermophilic bacterium Thermotoga neapolitana. In order to give further insights into CLF metabolic networks, we investigated the transcriptional modification induced by CO2 using a RNA-seq approach. Transcriptomic analysis revealed 1601 differentially expressed genes (DEGs) in an enriched CO2 atmosphere over a total of 1938 genes of the T. neapolitana genome. Transcription of PFOR and LDH genes belonging to the CLF pathway was up-regulated by CO2 together with 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydratase (EDD) of the Entner–Doudoroff (ED) pathway. The transcriptomic study also revealed up-regulation of genes coding for the flavin-based enzymes NADH-dependent reduced ferredoxin:NADP oxidoreductase (NFN) and NAD-ferredoxin oxidoreductase (RNF) that control supply of reduced ferredoxin and NADH and allow energy conservation-based sodium translocation through the cell membrane. These results support the hypothesis that CO2 induces rearrangement of the central carbon metabolism together with activation of mechanisms that increase availability of the reducing equivalents that are necessary to sustain CLF. In this view, this study reports a first rationale of the molecular basis of CLF in T. neapolitana and provides a list of target genes for the biotechnological implementation of this process. [ABSTRACT FROM AUTHOR]

The cidAB and lrgAB operons of Streptococcus mutans encode proteins that are structurally similar to the bacteriophage lambda family of holin-antiholin proteins, which are believed to facilitate cell death in other bacterial species. Although their precise function is not known, cidAB and lrgAB are linked to multiple virulence traits of S. mutans , including oxidative stress tolerance, biofilm formation, and autolysis. Here we investigate the regulation of lrgAB which in S. mutans shows a complex dependence on growth conditions that is not fully understood. By combining single-cell imaging of a fluorescent gene reporter with microfluidic control of the extracellular environment, we identify specific environmental cues that trigger lrgA expression and characterize cell-to-cell heterogeneity in lrgA activity. We find that the very abrupt activation of lrgA at stationary phase is tightly synchronized across the population. This activation is controlled by a small number of inputs that are sensitive to growth phase: extracellular pyruvate, glucose, and molecular oxygen. Activation of lrgA appears to be self-limiting, so that strong expression of lrgA is confined to a short interval of time. lrgA is programmed to switch on briefly at the end of exponential growth, as glucose and molecular oxygen are exhausted and extracellular pyruvate is available. Our findings are consistent with studies of other bacteria showing that homologs of lrgAB participate, with input from lytST , in the reimport of pyruvate for anaerobic fermentative growth. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii (Ab) is one of the most treacherous pathogens among those causing hospital-acquired pneumonia (HAP). A. baumannii possesses an adaptable physiology, seen not only in its antibiotic resistance and virulence phenotypes but also in its metabolic versatility. In this study, we observed that A. baumannii undergoes global transcriptional changes in response to human pleural fluid (PF), a key host-derived environmental signal. Differential gene expression analyses combined with experimental approaches revealed changes in A. baumannii metabolism, affecting cytotoxicity, persistence, bacterial killing, and chemotaxis. Over 1,220 genes representing 55% of the differentially expressed transcriptomic data corresponded to metabolic processes, including the upregulation of glutamate, short chain fatty acid, and styrene metabolism. We observed an upregulation by 1.83- and 2.61-fold of the pyruvate dehydrogenase complex subunits E3 and E2, respectively. We also found that pyruvate (PYR), in conjunction with PF, triggers an A. baumannii pathogenic behavior that adversely impacts human epithelial cell viability. Interestingly, PF also amplified A. baumannii cytotoxicity against murine macrophages, suggesting an immune evasion strategy implemented by A. baumannii. Moreover, we uncovered opposing metabolic strategies dependent on the degree of pathogenicity of the strains, where less pathogenic strains demonstrated greater utilization of PYR to promote persister formation in the presence of PF. Additionally, our transcriptomic analysis and growth studies of A. baumannii suggest the existence of an alternative phenylalanine (PA) catabolic route independent of the phenylacetic acid pathway, which converts PA to phenylpyruvate (PP) and shuttles intermediates into styrene metabolism. This alternative route promoted a neutrophil-evasive state, as PF-induced degradation of PP significantly reduced overall human neutrophil chemotaxis in ex vivo chemotactic assays. Taken together, these data highlight A. baumannii pathoadaptabililty in response to host signals and provide further insight into the role of bacterial metabolism in virulence traits, antibiotic persistence strategies, and host innate immune evasion.

Vibrio parahaemolyticus is a common foodborne pathogen found in seafood, and represents a major threat to human health worldwide. Low-temperature storage is an important seafood processing method, but is not sufficient to completely eliminate the bacteria and avoid foodborne illness. To determine the mechanisms behind such cold tolerance, RNA-seq and iTRAQ analyses were first performed to obtain the global transcriptomic and proteomic patterns of frozen squid and clinical V. parahaemolyticus isolates under cold conditions. The integrated analysis revealed the modulation of multiple pathways such as the co-occurrence of down-regulated pyruvate metabolism and up-regulated fatty acid biosynthesis, which likely contribute to V. parahaemolyticus cold tolerance. Furthermore, we found that increasing concentrations of pyruvate can reduce the fatty acid content to influence V. parahaemolyticus growth in cold conditions. Thus, regulation of pyruvate concentration may be an effective method to control this seafood-borne pathogen.

Acinetobacter baumannii (Ab) is one of the most treacherous pathogens among those causing hospital-acquired pneumonia (HAP). A. baumannii possesses an adaptable physiology, seen not only in its antibiotic resistance and virulence phenotypes but also in its metabolic versatility. In this study, we observed that A. baumannii undergoes global transcriptional changes in response to human pleural fluid (PF), a key host-derived environmental signal. Differential gene expression analyses combined with experimental approaches revealed changes in A. baumannii metabolism, affecting cytotoxicity, persistence, bacterial killing, and chemotaxis. Over 1,220 genes representing 55% of the differentially expressed transcriptomic data corresponded to metabolic processes, including the upregulation of glutamate, short chain fatty acid, and styrene metabolism. We observed an upregulation by 1.83- and 2.61-fold of the pyruvate dehydrogenase complex subunits E3 and E2, respectively. We also found that pyruvate (PYR), in conjunction with PF, triggers an A. baumannii pathogenic behavior that adversely impacts human epithelial cell viability. Interestingly, PF also amplified A. baumannii cytotoxicity against murine macrophages, suggesting an immune evasion strategy implemented by A. baumannii. Moreover, we uncovered opposing metabolic strategies dependent on the degree of pathogenicity of the strains, where less pathogenic strains demonstrated greater utilization of PYR to promote persister formation in the presence of PF. Additionally, our transcriptomic analysis and growth studies of A. baumannii suggest the existence of an alternative phenylalanine (PA) catabolic route independent of the phenylacetic acid pathway, which converts PA to phenylpyruvate (PP) and shuttles intermediates into styrene metabolism. This alternative route promoted a neutrophil-evasive state, as PF-induced degradation of PP significantly reduced overall human neutrophil chemotaxis in ex vivo chemotactic assays. Taken together, these data highlight A. baumannii pathoadaptabililty in response to host signals and provide further insight into the role of bacterial metabolism in virulence traits, antibiotic persistence strategies, and host innate immune evasion. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is a common foodborne pathogen found in seafood, and represents a major threat to human health worldwide. Low-temperature storage is an important seafood processing method, but is not sufficient to completely eliminate the bacteria and avoid foodborne illness. To determine the mechanisms behind such cold tolerance, RNA-seq and iTRAQ analyses were first performed to obtain the global transcriptomic and proteomic patterns of frozen squid and clinical V. parahaemolyticus isolates under cold conditions. The integrated analysis revealed the modulation of multiple pathways such as the co-occurrence of down-regulated pyruvate metabolism and up-regulated fatty acid biosynthesis, which likely contribute to V. parahaemolyticus cold tolerance. Furthermore, we found that increasing concentrations of pyruvate can reduce the fatty acid content to influence V. parahaemolyticus growth in cold conditions. Thus, regulation of pyruvate concentration may be an effective method to control this seafood-borne pathogen. [ABSTRACT FROM AUTHOR]

Glucose is a crucial carbon source for the growth of Staphylococcus aureus , but an excess of glucose is detrimental and even leads to cell death. Pyruvate, the central metabolite of glycolysis, has been shown to have anti-inflammatory and antioxidant properties. This study aimed to investigate the protective effect of pyruvate on S. aureus under high glucose conditions. Sodium pyruvate greatly increased the cytotoxicity of S. aureus strain BAA-1717 to human erythrocytes and neutrophils in vitro . However, the cytotoxicity and survival of S. aureus were significantly reduced by high glucose, which was restored to normal levels by the addition of sodium pyruvate. The expression of hlg and lukS in S. aureus was higher in the LB-GP cultures than that in LB-G cultures, but there was no significant difference in cytotoxicity between LB-GP and LB-G cultures. Furthermore, the hemolytic activity of S. aureus supernatants could be inhibited by the cell-free culture medium (CFCM) of LB-G cultures, suggesting that high levels of extracellular proteases were presence in the CFCM of LB-G cultures, resulting in degradation of the hemolytic factors. The expression of sarA, which negatively regulates extracellular protease secretion, was higher in LB-GP cultures than that in LB-G cultures. Additionally, sodium pyruvate increased acetate production in S. aureus , which helps maintain cell viability under acidic environment. In conclusion, pyruvate plays an important role in the survival and cytotoxicity of S. aureus under high glucose conditions. This finding may aid in the development of effective treatments for diabetic foot infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen, Xu, Yao and Luo.)

N-acetylneuraminic acid (Neu5Ac) possesses the ability to promote mental health and enhance immunity and is widely used in both medicine and food fields as a supplement. Enzymatic production of Neu5Ac using N-acetyl-D-glucosamine (GlcNAc) as substrate was significant. However, the high-cost GlcNAc limited its development. In this study, an in vitro multi-enzyme catalysis was built to produce Neu5Ac using affordable chitin as substrate. Firstly, exochitinase Sm ChiA from Serratia proteamaculans and N-acetylglucosaminosidase Cm NAGase from Chitinolyticbacter meiyuanensis SYBC-H1 were screened and combined to produce GlcNAc, effectively. Then, the chitinase was cascaded with N-acetylglucosamine-2-epimerase (AGE) and N-neuraminic acid aldolase (NanA) to produce Neu5Ac; the optimal conditions of the multi-enzyme catalysis system were 37°C and pH 8.5, the ratio of AGE to NanA (1:4) and addition of pyruvate (70 mM), respectively. Finally, 9.2 g/L Neu5Ac could be obtained from 20 g/L chitin within 24 h along with two supplementations with pyruvate. This work will lay a good foundation for the production of Neu5Ac from cheap chitin resources., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liu, Wei, Yang, Wang, Chen, Ouyang and Zhang.)

Capnophilic lactic fermentation (CLF) is a novel anaplerotic pathway able to convert sugars to lactic acid (LA) and hydrogen using CO 2 as carbon enhancer in the hyperthermophilic bacterium Thermotoga neapolitana . In order to give further insights into CLF metabolic networks, we investigated the transcriptional modification induced by CO 2 using a RNA-seq approach. Transcriptomic analysis revealed 1601 differentially expressed genes (DEGs) in an enriched CO 2 atmosphere over a total of 1938 genes of the T. neapolitana genome. Transcription of PFOR and LDH genes belonging to the CLF pathway was up-regulated by CO 2 together with 6-phosphogluconolactonase (6PGL) and 6-phosphogluconate dehydratase (EDD) of the Entner-Doudoroff (ED) pathway. The transcriptomic study also revealed up-regulation of genes coding for the flavin-based enzymes NADH-dependent reduced ferredoxin:NADP oxidoreductase (NFN) and NAD-ferredoxin oxidoreductase (RNF) that control supply of reduced ferredoxin and NADH and allow energy conservation-based sodium translocation through the cell membrane. These results support the hypothesis that CO 2 induces rearrangement of the central carbon metabolism together with activation of mechanisms that increase availability of the reducing equivalents that are necessary to sustain CLF. In this view, this study reports a first rationale of the molecular basis of CLF in T. neapolitana and provides a list of target genes for the biotechnological implementation of this process., (Copyright © 2020 d’Ippolito, Landi, Esercizio, Lanzilli, Vastano, Dipasquale, Pradhan and Fontana.)