ABSTRACT
BACKGROUND: Cyanobacterial carbohydrates, such as sucrose, have been considered as potential renewable feedstock to support the production of fuels and chemicals. However, the separation and purification processes of these carbohydrates will increase the production cost of chemicals. Co-culture fermentation has been proposed as an efficient and economical way to utilize these cyanobacterial carbohydrates. However, studies on the application of co-culture systems to achieve green biosynthesis of platform chemicals are still rare. RESULTS: In this study, we successfully achieved one-step conversion of sucrose derived from cyanobacteria to fine chemicals by constructing a microbial consortium consisting of the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 and Escherichia coli to sequentially produce sucrose and then the platform chemical 3-hydroxypropionic acid (3-HP) from CO2 under photoautotrophic growth conditions. First, efforts were made to overexpress the sucrose permease-coding gene cscB under the strong promoter P cpc560 in S. elongatus UTEX 2973 for efficient sucrose secretion. Second, the sucrose catabolic pathway and malonyl-CoA-dependent 3-HP biosynthetic pathway were introduced into E. coli BL21 (DE3) for heterologous biosynthesis of 3-HP from sucrose. By optimizing the cultivation temperature from 37 to 30 °C, a stable artificial consortium system was constructed with the capability of producing 3-HP at up to 68.29 mg/L directly from CO2. In addition, cell growth of S. elongatus UTEX 2973 in the consortium was enhanced, probably due to the quick quenching of reactive oxygen species (ROS) in the system by E. coli, which in turn improved the photosynthesis of cyanobacteria. CONCLUSION: The study demonstrated the feasibility of the one-step conversion of sucrose to fine chemicals using an artificial consortium system. The study also confirmed that heterotrophic bacteria could promote the cell growth of cyanobacteria by relieving oxidative stress in this microbial consortium, which further suggests the potential value of this system for future industrial applications.
ABSTRACT
Two-component signal transduction systems are still poorly functionally characterized in the model cyanobacterium Synechocystis sp. PCC 6803. To address the issue, a GC-MS based comparative metabolomic analysis was conducted on a library of 44 knockout mutants for the response regulators (RRs) in Synechocystis. The metabolomic profiling analysis showed that 7 RRs mutants, namely Δslr1909, Δsll1291, Δslr6040, Δsll1330, Δslr2024, Δslr1584, and Δslr1693, were significantly different at metabolomic level, although their growth patterns are similar to the wild type under the normal autotrophic growth condition, suggesting regulatory diversity of RRs at metabolite level in Synechocystis. Additionally, a detailed metabolomic analysis coupled with RT-PCR verification led to useful clues for possible function of these 7 RRs, which were found involved in regulation of multiple aspects of cellular metabolisms in Synechocystis. Moreover, an integrative metabolomic and evolutionary analysis of all RR showed that four groups of RR genes clustered together in both metabolomic and evolutionary trees, suggesting of possible functional conservation of these RRs during the evolutionary process. Meanwhile, six groups of RRs with close evolutionary origin were found with different metabolomic profiles, suggesting possible functional changes during evolution. In contrast, more than 10 groups of RR genes with different clustering patterns in the evolutionary tree were found clustered together in metabolomics-based tree, suggesting possible functional convergences during the evolution. This study provided a metabolomic view of RR function, and the most needed functional clues for further characterization of these regulatory proteins in Synechocystis.
ABSTRACT
Dietary omega-3 long-chain polyunsaturated fatty acids docosahexaenoic acid (DHA, C22:6) can be synthesized in microalgae Crypthecodinium cohnii; however, its productivity is still low. Here, we established a new protocol termed as "chemical modulator based adaptive laboratory evolution" (CM-ALE) to enhance lipid and DHA productivity in C. cohnii. First, ACCase inhibitor sethoxydim based CM-ALE was applied to redirect carbon equivalents from starch to lipid. Second, CM-ALE using growth modulator sesamol as selection pressure was conducted to relive negative effects of sesamol on lipid biosynthesis in C. cohnii, which allows enhancement of biomass productivity by 30% without decreasing lipid content when sesamol was added. After two-step CM-ALE, the lipid and DHA productivity in C. cohnii was respectively doubled to a level of 0.046â¯g/L/h and 0.025â¯g/L/h in culture with addition of 1â¯mM sesamol, demonstrating that this two-step CM-ALE could be a valuable approach to maximize the properties of microalgae.
Subject(s)
Dinoflagellida/metabolism , Directed Molecular Evolution/methods , Lipids/biosynthesis , Metabolic Networks and Pathways/drug effects , Metabolic Networks and Pathways/genetics , Acetyl-CoA Carboxylase/metabolism , Benzodioxoles/pharmacology , Biomass , Carbon/metabolism , Cyclohexanones/pharmacology , Docosahexaenoic Acids/biosynthesis , Malonyl Coenzyme A/metabolism , Phenols/pharmacology , Reactive Oxygen Species/metabolism , Starch/metabolismABSTRACT
It is well-known that high-nitrogen content inhibits cell growth and docosahexaenoic acid (DHA) biosynthesis in heterotrophic microalgae Crypthecodinium cohnii. In this study, two nitrogen feeding strategies, pulse-feeding and continuous-feeding, were evaluated to alleviate high-nitrogen inhibition effects on C. cohnii. The results showed that continuous-feeding with a medium solution containing 50% ( w/v) yeast extract at 2.1 mL/h during 12-96 h was the optimal nitrogen feeding strategy for the fermentation process, when glucose concentration was maintained at 15-27 g/L during the same period. With the optimized strategy, 71.2 g/L of dry cell weight and DHA productivity of 57.1 mg/L/h were achieved. In addition, metabolomic analysis was applied to determine the metabolic changes during different nitrogen feeding conditions, and the changes in amino acids, polysaccharides, purines, and pentose phosphate pathway were observed, providing valuable metabolite-level information for exploring the mechanism of the high-nitrogen inhibition effect and further improving DHA productivity in C. cohnii.
Subject(s)
Dinoflagellida/metabolism , Docosahexaenoic Acids/biosynthesis , Microalgae/metabolism , Nitrogen/metabolism , Biomass , Culture Media/chemistry , Culture Media/metabolism , Dinoflagellida/chemistry , Dinoflagellida/growth & development , Fermentation , Metabolomics , Microalgae/chemistry , Microalgae/growth & developmentABSTRACT
The heterotrophic microalga Crypthecodinium cohnii has attracted considerable attention due to its capability of accumulating lipids with a high fraction of docosahexaenoic acid (DHA). In our previous study, ethanolamine (ETA) was identified as an effective chemical modulator for lipid accumulation in C. cohnii. In this study, to gain a better understanding of the lipid metabolism and mechanism for the positive effects of modulator ETA, metabolic flux analysis was performed using 13C-labeled glucose with and without 1 mM ETA modulator. The analysis of flux distribution showed that with the addition of ETA, flux in glycolysis pathway and citrate pyruvate cycle was strengthened while flux in pentose phosphate pathway was decreased. In addition, flux in TCA cycle was slightly decreased compared with the control without ETA. The enzyme activity of malic enzyme (ME) was significantly increased, suggesting that NADP+-dependent ME might be the major source of NADPH for lipid accumulation. The flux information obtained by this study could be valuable for the further efforts in improving lipid accumulation and DHA production in C. cohnii.
ABSTRACT
BACKGROUND: Photosynthetic cyanobacteria have attracted a significant attention as promising chassis to produce renewable fuels and chemicals due to their capability to utilizing solar energy and CO2. Notably, the enhancing supply of key precursors like malonyl-CoA would benefit the production of many bio-compounds. Nevertheless, the lacking of genetic tools in cyanobacteria, especially the knockdown strategies for essential pathways, has seriously restricted the attempts to re-direct carbon flux from the central carbohydrate metabolism to the synthesis of bioproducts. RESULTS: Aiming at developing new genetic tools, two small RNA regulatory tools are reported for the model cyanobacterium Synechocystis sp. PCC6803, based on paired termini RNAs as well as the exogenous Hfq chaperone and MicC scaffold (Hfq-MicC) previously developed in Escherichia coli. Both regulatory tools functioned well in regulating exogenous reporter gene lacZ and endogenous glgC gene in Synechocystis sp. PCC6803, achieving a downregulation of gene expression up to 90% compared with wildtype. In addition, the Hfq-MicC tool was developed to simultaneously regulate multiple genes related to essential fatty acids biosynthesis, which led to decreased fatty acids content by 11%. Furthermore, aiming to re-direct the carbon flux, the Hfq-MicC tool was utilized to interfere the competing pathway of malonyl-CoA, achieving an increased intracellular malonyl-CoA abundance up to 41% (~ 698.3 pg/mL/OD730 nm) compared to the wildtype. Finally, the Hfq-MicC system was further modified into an inducible system based on the theophylline-inducible riboswitch. CONCLUSIONS: In this study, two small RNA regulatory tools for manipulating essential metabolic pathways and re-directing carbon flux are reported for Synechocystis sp. PCC6803. The work introduces efficient and valuable metabolic regulatory strategies for photosynthetic cyanobacteria.
ABSTRACT
Although numerous studies have demonstrated that social support affects a range of life experiences, few have examined its moderating and mediating effects. In the current study, 479 Chinese parents of children with ASD (aged 3-18 years) completed the surveys assessing parenting stress, social support and life satisfaction. Results indicated that parenting stress, social support and life satisfaction were significantly related. Moreover, social support both mediated and moderated the influence of parenting stress on life satisfaction. These findings imply that parenting stress and social support are critical indicators of life satisfaction and can serve as basic intervention strategies that promote life satisfaction among Chinese parents of children with ASD.
Subject(s)
Autism Spectrum Disorder/psychology , Parenting/psychology , Parents/psychology , Personal Satisfaction , Social Support , Stress, Psychological/psychology , Adolescent , Adult , Autism Spectrum Disorder/epidemiology , Child , Child, Preschool , Female , Humans , Male , Stress, Psychological/epidemiologyABSTRACT
Functions of transcriptional regulators (TRs) are still poorly understood in the model cyanobacterium Synechocystis sp. PCC 6803. To address the issue, we constructed knockout mutants for 32 putative TR-encoding genes of Synechocystis, and comparatively analyzed their phenotypes under autotrophic growth condition and metabolic profiles using liquid chromatography-mass spectrometry-based metabolomics. The results showed that only four mutants of TR genes, sll1872 (lytR), slr0741 (phoU), slr0395 (ntcB), and slr1871 (pirR), showed differential growth patterns in BG11 medium when compared with the wild type; however, in spite of no growth difference observed for the remaining TR mutants, metabolomic profiling showed that they were different at the metabolite level, suggesting significant functional diversity of TRs in Synechocystis. In addition, an integrative metabolomic and gene families' analysis of all TR mutants led to the identification of five pairs of TR genes that each shared close relationship in both gene families and metabolomic clustering trees, suggesting possible conserved functions of these TRs during evolution. Moreover, more than a dozen pairs of TR genes with different origin and evolution were found with similar metabolomic profiles, suggesting a possible functional convergence of the TRs during genome evolution. Finally, a protein-protein network analysis was performed to predict regulatory targets of TRs, allowing inference of possible regulatory gene targets for 4 out of five pairs of TRs. This study provided new insights into the regulatory functions and evolution of TR genes in Synechocystis.
ABSTRACT
BACKGROUND: Microalgae have been recognized as a good food source of natural biologically active ingredients. Among them, the green microalga Euglena is a very promising food and nutritional supplements, providing high value-added poly-unsaturated fatty acids, paramylon and proteins. Different culture conditions could affect the chemical composition and food quality of microalgal cells. However, little information is available for distinguishing the different cellular changes especially the active ingredients including poly-saturated fatty acids and other metabolites under different culture conditions, such as light and dark. RESULTS: In this study, together with fatty acid profiling, we applied a gas chromatography-mass spectrometry (GC-MS)-based metabolomics to differentiate hetrotrophic and mixotrophic culture conditions. CONCLUSIONS: This study suggests metabolomics can shed light on understanding metabolomic changes under different culture conditions and provides a theoretical basis for industrial applications of microalgae, as food with better high-quality active ingredients.
Subject(s)
Bioreactors/microbiology , Dietary Supplements/microbiology , Euglena/metabolism , Fatty Acids/metabolism , Metabolome/physiology , Microalgae/metabolism , Cell Culture Techniques/methods , Culture Media/metabolism , Euglena/classification , Metabolic Flux Analysis/methods , Microalgae/classification , Species SpecificityABSTRACT
3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. So far large-scale production of 3-HP has been mainly through petroleum-based chemical processes, whose sustainability and environmental issues have attracted widespread attention. With the ability to fix CO2 directly, cyanobacteria have been engineered as an autotrophic microbial cell factory to produce fuels and chemicals. In this study, we constructed the biosynthetic pathway of 3-HP in cyanobacterium Synechocystis sp. PCC 6803, and then optimized the system through the following approaches: i) increasing expression of malonyl-CoA reductase (MCR) gene using different promoters and cultivation conditions; ii) enhancing supply of the precursor malonyl-CoA by overexpressing acetyl-CoA carboxylase and biotinilase; iii) improving NADPH supply by overexpressing the NAD(P) transhydrogenase gene; iv) directing more carbon flux into 3-HP by inactivating the competing pathways of PHA and acetate biosynthesis. Together, the efforts led to a production of 837.18 mg L(-1) (348.8 mg/g dry cell weight) 3-HP directly from CO2 in Synechocystis after 6 days cultivation, demonstrating the feasibility photosynthetic production of 3-HP directly from sunlight and CO2 in cyanobacteria. In addition, the results showed that overexpression of the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) gene from Anabaena sp. PCC 7120 and Synechococcus sp. PCC 7942 led to no increase of 3-HP production, suggesting CO2 fixation may not be a rate-limiting step for 3-HP biosynthesis in Synechocystis.
Subject(s)
Carbon Dioxide/metabolism , Lactic Acid/analogs & derivatives , Metabolic Engineering/methods , NADP/metabolism , Oxidoreductases/metabolism , Synechocystis/metabolism , Lactic Acid/biosynthesis , Lactic Acid/isolation & purification , Light , Metabolic Networks and Pathways/physiology , NADP/genetics , Oxidoreductases/genetics , Photosynthesis/physiology , Photosynthesis/radiation effects , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Synechocystis/genetics , Synechocystis/radiation effectsABSTRACT
Cyanobacteria have been engineered to produce ethanol through recent synthetic biology efforts. However, one major challenge to the cyanobacterial systems for high-efficiency ethanol production is their low tolerance to the ethanol toxicity. With a major goal to identify novel transporters involved in ethanol tolerance, we constructed gene knockout mutants for 58 transporter-encoding genes of Synechocystis sp. PCC 6803 and screened their tolerance change under ethanol stress. The efforts allowed discovery of a mutant of slr0982 gene encoding an ATP-binding cassette transporter which grew poorly in BG11 medium supplemented with 1.5% (v/v) ethanol when compared with the wild type, and the growth loss could be recovered by complementing slr0982 in the Δslr0982 mutant, suggesting that slr0982 is involved in ethanol tolerance in Synechocystis. To decipher the tolerance mechanism involved, a comparative metabolomic and network-based analysis of the wild type and the ethanol-sensitive Δslr0982 mutant was performed. The analysis allowed the identification of four metabolic modules related to slr0982 deletion in the Δslr0982 mutant, among which metabolites like sucrose and L-pyroglutamic acid which might be involved in ethanol tolerance, were found important for slr0982 deletion in the Δslr0982 mutant. This study reports on the first transporter related to ethanol tolerance in Synechocystis, which could be a useful target for further tolerance engineering. In addition, metabolomic and network analysis provides important findings for better understanding of the tolerance mechanism to ethanol stress in Synechocystis.
ABSTRACT
Low ethanol tolerance is a crucial factor that restricts the feasibility of bioethanol production in renewable cyanobacterial systems. Our previous studies showed that several transcriptional regulators were differentially regulated by exogenous ethanol in Synechocystis. In this study, by constructing knockout mutants of 34 Synechocystis putative transcriptional regulator-encoding genes and analyzing their phenotypes under ethanol stress, we found that three mutants of regulatory gene sll1392, sll1712 and slr1860 grew poorly in the BG11 medium supplemented with ethanol when compared with the wild type in the same medium, suggesting that the genes may be involved in the regulation of ethanol tolerance. To decipher the regulatory mechanism, targeted LC-MS and untargeted GC-MS approaches were employed to determine metabolic profiles of the three mutants and the wild type under both normal and ethanol stress conditions. The results were then subjected to PCA and WGCNA analyses to determine the responsive metabolites and metabolic modules related to ethanol tolerance. Interestingly, the results showed that there was a significant overlapping of the responsive metabolites and metabolic modules between three regulatory proteins, suggesting that a possible crosstalk between various regulatory proteins may be involved in combating against ethanol toxicity in Synechocystis. The study provided new insights into ethanol-tolerance regulation and knowledge important to rational tolerance engineering in Synechocystis.
Subject(s)
Adaptation, Biological , Bacterial Proteins/metabolism , Cyanobacteria/metabolism , Ethanol/metabolism , Metabolomics , Cluster Analysis , Cyanobacteria/genetics , Gene Expression Regulation, Bacterial , Gene Knockout Techniques , Metabolic Networks and Pathways , Metabolome , Metabolomics/methods , Mutation , Transcription, GeneticABSTRACT
The heterotrophic dinoflagellate alga Crypthecodinium cohnii is known to accumulate lipids with a high fraction of docosahexaenoic acid (DHA). In this study, we first evaluated two antioxidant compounds, butylated hydroxyanisole (BHA) and propyl gallate (PG), for their effects on lipid accumulation in C. cohnii. The results showed that antioxidant BHA could increase lipid accumulation in C. cohnii by 8.80% at a final concentration of 30 µM, while PG had no obvious effect on lipid accumulation at the tested concentrations. To decipher the molecular mechanism responsible for the increased lipid accumulation by BHA, we employed an integrated GC-MS and LC-MS metabolomic approach to determine the time-series metabolic profiles with or without BHA, and then subjected the metabolomic data to a principal component analysis (PCA) and a weighted gene coexpression network analysis (WGCNA) network analyses to identify the key metabolic modules and metabolites possibly relevant to the increased lipid accumulation. LC-MS analysis showed that several metabolites, including NADPH, could be important for the stimulation role of BHA on lipid accumulation. Meanwhile GC-MS and network analyses allowed identification of eight metabolic modules and nine hub metabolites possibly relevant to the stimulation role of BHA in C. cohnii. The study provided a metabolomics view of the BHA mode of action on lipid accumulation in C. cohnii, and the information could be valuable for a better understanding of antioxidant effects on lipid accumulation in other microalgae as well.
Subject(s)
Antioxidants/metabolism , Butylated Hydroxyanisole/metabolism , Dinoflagellida/metabolism , Lipid Metabolism , Dinoflagellida/chemistry , Dinoflagellida/genetics , Gas Chromatography-Mass Spectrometry , Gene Regulatory Networks , Metabolomics , Propyl Gallate/metabolismABSTRACT
BACKGROUND: Recent efforts demonstrated the potential application of cyanobacteria as a "microbial cell factory" to produce butanol directly from CO2. However, cyanobacteria have very low tolerance to the toxic butanol, which limits the economic viability of this renewable system. RESULTS: Through a long-term experimental evolution process, we achieved a 150% increase of the butanol tolerance in a model cyanobacterium Synechocystis sp. PCC 6803 after a continuous 94 passages for 395 days in BG11 media amended with gradually increased butanol concentration from 0.2% to 0.5% (v/v). To decipher the molecular mechanism responsible for the tolerance increase, we employed an integrated GC-MS and LC-MS approach to determine metabolomic profiles of the butanol-tolerant Synechocystis strains isolated from several stages of the evolution, and then applied PCA and WGCNA network analyses to identify the key metabolites and metabolic modules related to the increased tolerance. The results showed that unstable metabolites of 3-phosphoglyceric acid (3PG), D-fructose 6-phosphate (F6P), D-glucose 6-phosphate (G6P), NADPH, phosphoenolpyruvic acid (PEP), D-ribose 5-phosphate (R5P), and stable metabolites of glycerol, L-serine and stearic acid were differentially regulated during the evolution process, which could be related to tolerance increase to butanol in Synechocystis. CONCLUSIONS: The study provided the first time-series description of the metabolomic changes related to the gradual increase of butanol tolerance, and revealed a metabolomic basis important for rational tolerance engineering in Synechocystis.
Subject(s)
Butanols/metabolism , Directed Molecular Evolution/methods , Synechocystis , Synechocystis/genetics , Synechocystis/metabolismABSTRACT
Various combinations of acetate (Ac), Fe(2+) and high light (HL) stress conditions were evaluated to maximize astaxanthin accumulation and biomass production in Haematococcus pluvialis, and then GC-MS and LC-MS based metabolomics were applied to determine molecular mechanisms responsible for enhancing astaxanthin accumulation under the stress conditions. With the optimized analytical protocols, the GC-MS and LC-MS analyses allowed identification of 93 stable and 24 unstable intracellular metabolites from H. pluvialis, respectively. In addition, a metabolic network was constructed based on GC-MS metabolomic datasets using a weighted correlation network analysis (WGCNA) approach. The network analysis uncovered 2, 1 and 1 distinguished metabolic modules highly associated with HL, Fe(2+) & HL, and Ac & Fe(2+) & HL conditions, respectively. Finally, LC-MS analysis found that AKG, Glu and R5P may be metabolites associated with the Fe(2+) & HL condition. The study provided the first metabolomic view of cell growth and astaxanthin accumulation in H. pluvialis.
Subject(s)
Chlorophyta/physiology , Metabolic Networks and Pathways/physiology , Metabolome/physiology , Metabolomics/methods , Stress, Physiological/physiology , Chlorophyta/metabolism , Chromatography, Liquid/methods , Gas Chromatography-Mass Spectrometry/methods , Metabolic Networks and Pathways/genetics , Metabolome/genetics , Xanthophylls/biosynthesis , Xanthophylls/genetics , Xanthophylls/metabolismABSTRACT
Low pH is recognized as a major environmental stress to cyanobacteria that play a pivotal role in the global carbon cycling. Although several cellular mechanisms in response to acid stress were proposed, the regulatory mechanism related to acid stress has not been fully elucidated. By screening gene knockout mutants for all 44 putative response regulator (RR)-encoding genes of Synechocystis sp. PCC 6803 grown under acid stress, we found that a mutant of slr1909 (previously known as rre9), which encoded an orphan RR, grew poorly in BG11 medium at pH6.2-6.5 when compared with the wild type. Using a quantitative iTRAQ-LC-MS/MS proteomics approach coupled with GC-MS based metabolomics and quantitative real-time reverse transcription-PCR (RT-qPCR), we further determined the possible acid response network mediated by Slr1909. The results showed that the signal transduction pathway mediated by Slr1909 may be independent from that mediated by SphS-SphR previously discovered, as none of the proteins and their coding genes regulated by SphS-SphR were differentially regulated in the ∆slr1909 mutant grown under acid stress. Only 24 and 10 proteins were up- and down-regulated in the ∆slr1909 mutant when compared with the wild type under acid stress condition, respectively. Notably, three proteins, Slr1259, Slr1260 and Slr1261 whose encoding genes seem located in an operon, were down-regulated upon the knockout of the slr1909 gene, suggesting their roles in acid tolerance. In addition, metabolomic analysis allowed identification of a dozen metabolites important for the discrimination of the ∆slr1909 mutant and the wild type under acid stress, including several monosaccharide and fatty acids. The study provided a proteomic and metabolomic characterization of the acid-response network mediated by an orphan regulator Slr1909 in Synechocystis. BIOLOGICAL SIGNIFICANCE: Low pH is recognized as a major environmental stress to cyanobacteria that play a pivotal role in the global carbon cycling. Although several cellular mechanisms in response to acid stress were proposed, the regulatory mechanism related to acid stress is still far from being fully elucidated. In a previous work, one two-component signal transduction system SphS-SphR was found involved in acid stress in Synechocystis. In this work, by screening gene knockout mutants for all 44 putative response regulator (RR)-encoding genes grown under acid stress, we found that a novel two-component response regulator Slr1909 was also involved in acid tolerance in Synechocystis. Moreover, the analysis showed that the signal transduction pathway mediated by Slr1909 may be independent from that mediated by SphS-SphR. Using a quantitative iTRAQ-LC-MS/MS proteomics and coupled with GC-MS based metabolomics and quantitative real-time reverse transcription-PCR (RT-qPCR), we further determined the possible acid response network mediated by Slr1909. The study provided a proteomic and metabolomic characterization of a novel acid-response network mediated by an orphan regulator Slr1909 in Synechocystis, and valuable new insight for better understanding of stress responses to acidity in cyanobacteria.
Subject(s)
Bacterial Proteins/physiology , Metabolome/physiology , Phosphotransferases/physiology , Proteome/biosynthesis , Signal Transduction/physiology , Synechocystis/metabolism , Transcription Factors/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Gene Expression Regulation , Hydrogen-Ion Concentration , Metabolomics/methods , Mutation , Phosphotransferases/genetics , Phosphotransferases/metabolism , Proteome/genetics , Proteomics/methods , Synechocystis/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Early studies in cyanobacteria have found that few genes induced by short-term salt shock (15-60 min) display a stable induction in the long-term (>1 day) salt-acclimated cells; meanwhile, most of the genes responsive to long-term salt stress were different from those by short-term salt shock, suggesting that different regulatory mechanisms may be involved for short-term and long-term salt stress responses. In our previous work using the model cyanobacterium Synechocystis sp. PCC 6803, sll1734 encoding CO2 uptake-related protein (CupA) and three genes encoding hypothetical proteins (i.e., ssr3402, slr1339, and ssr1853) were found induced significantly after a 3-day salt stress, and the corresponding gene knockout mutants were found salt sensitive. To further decipher the mechanisms that these genes may be involved, in this study, we performed a comparative metabolomic analysis of the wild-type Synechocystis and the four salt-sensitive mutants using a gas chromatography-mass spectrometry (GC-MS) approach. A metabolomic data set that consisted of 60 chemically classified metabolites was then subjected to a weighted correlation network analysis (WGCNA) to identify the metabolic modules and hub metabolites specifically related to each of the salt-stressed mutants. The results showed that two, one, zero, and two metabolic modules were identified specifically associated with the knockout events of sll1734, ssr3402, slr1339, and ssr1853, respectively. The mutant-associated modules included metabolites such as lysine and palmitic acid, suggesting that amino acid and fatty acid metabolisms are among the key protection mechanisms against long-term salt stresses in Synechocystis. The metabolomic results were further confirmed by quantitative reverse-transcription PCR analysis, which showed the upregulation of lysine and fatty acid synthesis-related genes. The study provided new insights on metabolic networks involved in long-term salt stress response in Synechocystis.
Subject(s)
Amino Acids/metabolism , Bacterial Proteins/genetics , Fatty Acids/metabolism , Gene Expression Regulation, Bacterial , Metabolome/genetics , Sodium Chloride/pharmacology , Synechocystis/drug effects , Bacterial Proteins/metabolism , Gene Knockout Techniques , Gene Regulatory Networks , Metabolic Networks and Pathways , Principal Component Analysis , Salinity , Stress, Physiological , Synechocystis/genetics , Synechocystis/metabolismABSTRACT
Although synthetic biology progress has made it possible to produce various biofuels in more user-friendly hosts, such as Escherichia coli, the large-scale biofuel production in these non-native systems is still challenging, mostly due to the very low tolerance of these non-native hosts to the biofuel toxicity. To address the issues, in this study we determined the metabolic responses of E. coli induced by three major biofuel products, ethanol, butanol, and isobutanol, using a gas chromatography-mass spectrometry (GC-MS) approach. A metabolomic data set of 65 metabolites identified in all samples was then subjected to principal component analysis (PCA) to compare their effects and a weighted correlation network analysis (WGCNA) to identify the metabolic modules specifically responsive to each of the biofuel stresses, respectively. The PCA analysis showed that cellular responses caused by the biofuel stress were in general similar to aging cells at stationary phase, inconsistent with early studies showing a high degree of dissimilarity between metabolite responses during growth cessation as induced through stationary phases or through various environmental stress applications. The WGCNA analysis allowed identification of 2, 4, and 2 metabolic modules specifically associated with ethanol, butanol, and isobutanol treatments, respectively. The biofuel-associated modules included amino acids and osmoprotectants, such as isoleucine, valine, glycine, glutamate, and trehalose, suggesting amino acid metabolism and osmoregulation are among the key protection mechanisms against biofuel stresses in E. coli. Interestingly, no module was found associated with all three biofuel products, suggesting differential effects of each biofuel on E. coli. The findings enhanced our understanding of E. coli responses to exogenous biofuels and also demonstrated the effectiveness of the metabolomic and network analysis in identifying key targets for biofuel tolerance.
Subject(s)
Biofuels/toxicity , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/metabolism , Metabolome/drug effects , Stress, Physiological/drug effects , Amino Acids/metabolism , Butanols , Ethanol , Gas Chromatography-Mass Spectrometry , Metabolomics/methods , Osmoregulation/genetics , Osmoregulation/physiology , Principal Component AnalysisABSTRACT
BACKGROUND: Photosynthetic cyanobacteria have been recently proposed as a 'microbial factory' to produce butanol due to their capability to utilize solar energy and CO2 as the sole energy and carbon sources, respectively. However, to improve the productivity, one key issue needed to be addressed is the low tolerance of the photosynthetic hosts to butanol. RESULTS: In this study, we first applied a quantitative transcriptomics approach with a next-generation RNA sequencing technology to identify gene targets relevant to butanol tolerance in a model cyanobacterium Synechocystis sp. PCC 6803. The results showed that 278 genes were induced by the butanol exposure at all three sampling points through the growth time course. Genes encoding heat-shock proteins, oxidative stress related proteins, transporters and proteins involved in common stress responses, were induced by butanol exposure. We then applied GC-MS based metabolomics analysis to determine the metabolic changes associated with the butanol exposure. The results showed that 46 out of 73 chemically classified metabolites were differentially regulated by butanol treatment. Notably, 3-phosphoglycerate, glycine, serine and urea related to general stress responses were elevated in butanol-treated cells. To validate the potential targets, we constructed gene knockout mutants for three selected gene targets. The comparative phenotypic analysis confirmed that these genes were involved in the butanol tolerance. CONCLUSION: The integrated OMICS analysis provided a comprehensive view of the complicated molecular mechanisms employed by Synechocystis sp. PCC 6803 against butanol stress, and allowed identification of a series of potential gene candidates for tolerance engineering in cyanobacterium Synechocystis sp. PCC 6803.