Recent progress in metabolic engineering has led to autotrophic production of ethanol in various cyanobacterial hosts. However, cyanobacteria are known to be sensitive to ethanol, which restricts further efforts to increase ethanol production levels in these renewable host systems. To understand the mechanisms of ethanol tolerance so that engineering more robust cyanobacterial hosts can be possible, in this study, the responses of model cyanobacterial Synechocystis sp. PCC 6803 to ethanol were determined using a quantitative proteomics approach with iTRAQ LC–MS/MS technologies. The resulting high-quality proteomic data set consisted of 24 887 unique peptides corresponding to 1509 identified proteins, a coverage of approximately 42% of the predicted proteins in the Synechocystis genome. Using a cutoff of 1.5-fold change and a p-value less than 0.05, 135 and 293 unique proteins with differential abundance levels were identified between control and ethanol-treated samples at 24 and 48 h, respectively. Functional analysis showed that the Synechocystis cells employed a combination of induced common stress response, modifications of cell membrane and envelope, and induction of multiple transporters and cell mobility-related proteins as protection mechanisms against ethanol toxicity. Interestingly, our proteomic analysis revealed that proteins related to multiple aspects of photosynthesis were up-regulated in the ethanol-treated Synechocystis cells, consistent with increased chlorophyll a concentration in the cells upon ethanol exposure. The study provided the first comprehensive view of the complicated molecular mechanisms against ethanol stress and also provided a list of potential gene targets for further engineering ethanol tolerance in Synechocystis PCC 6803.Keywords: ethanol; iTRAQ proteomics; Synechocystis; tolerance;

•First de novo transcriptomic analysis for DHA-producing Crypthecodinium cohnii during fed-batch fermentation.•A total of 82,106 unigenes identified from Crypthecodinium cohnii.•Most up-regulated transcripts enriched in fatty acid and PUFA biosynthesis during late stage of the fermentation.•Transcripts potentially related to lipid and DHA biosynthesis investigated.The heterotrophic microalga Crypthecodinium cohnii accumulates lipids with a high fraction of docosahexaenoic acid (DHA). However, lack of genomic information limits the understanding of its physiological metabolism for better DHA production. In this study, de novo assembly of the C. cohnii transcriptome from three growth stages (i.e., fast growth, fatty acid accumulation and DHA conversion stages) was conducted, leading to identification of a total of 82,106 unigenes with an N50 of 1822 bp, among which 64.7% were annotated based on sequence similarity to known genes in the database. In addition, pathway enrichment analysis showed that transcripts related to fatty acid biosynthesis, starch and sucrose metabolism as well as biosynthesis of unsaturated fatty acids were significantly up-regulated during late-stage fermentation. Interestingly, several polyketide synthases (PKSs) and multiple fatty acid desaturases potentially involved in DHA biosynthesis were identified in the C. cohnii transcriptome, most of which were significantly up-regulated during lipid and DHA accumulation, implying that C. cohnii might utilize a combination of PKS systems and desaturase steps for DHA biosynthesis. The results were further confirmed by qRT-PCR and GC–MS-based metabolomic analyses. Overall, integrative analysis of de novo transcriptomic and metabolomic data provided important functional genomic information necessary for not only a better understanding of C. cohnii growth and DHA biosynthesis but also future genetic engineering of industry-important C. cohnii.Download high-res image (212KB)Download full-size image

The unicellular model cyanobacterium Synechocystis sp. PCC 6803 is considered a promising microbial chassis for biofuel production. However, its low tolerance to biofuel toxicity limits its potential application. Although recent studies showed that bacterial small RNAs (sRNAs) play important roles in regulating cellular processes in response to various stresses, the role of sRNAs in resisting exogenous biofuels is yet to be determined.Based on genome-wide sRNA sequencing combined with systematic analysis of previous transcriptomic and proteomic data under the same biofuel or environmental perturbations, we report the identification of 133 trans-encoded sRNA transcripts with high-resolution mapping of sRNAs in Synechocystis, including 23 novel sRNAs identified for the first time. In addition, according to quantitative expression analysis and sRNA regulatory network prediction, sRNAs potentially involved in biofuel tolerance were identified and functionally confirmed by constructing sRNA overexpression or suppression strains of Synechocystis. Notably, overexpression of sRNA Nc117 revealed an improved tolerance to ethanol and butanol, while suppression of Nc117 led to increased sensitivity.The study provided the first comprehensive responses to exogenous biofuels at the sRNA level in Synechocystis and opens an avenue to engineering sRNA regulatory elements for improved biofuel tolerance in the cyanobacterium Synechocystis.

Microbial small RNAs (sRNAs) have been proposed as valuable regulatory elements for optimizing cellular metabolism for industrial purposes. However, little information is currently available on functional relevance of sRNAs to biofuels tolerance in cyanobacteria.Here, we described the identification and functional characterization of a novel 124 nt sRNA Ncl1460 involved in tolerance to biofuel 1-butanol in Synechocystis sp. PCC 6803. The expression of Ncl1460 was verified by blotting assay and its length was determined through 3′ RACE. Further analysis showed that Ncl1460 was a negative regulator of slr0847 (coaD) and slr0848 operon responsible for coenzyme A (CoA) synthesis possibly via promoter-directed transcriptional silencing mechanisms which has been widely discovered in eukaryote; thus Ncl1460 was designated as CoaR (CoA Biosynthesis Regulatory sRNA). The possible interaction between CoaR and target genes was suggested by CoA quantification and green fluorescent protein assays. Finally, a quantitative proteomics analysis showed that CoaR regulated tolerance to 1-butanol possibly by down-regulating CoA biosynthesis, resulting in a decrease of fatty acid metabolism and energy metabolism.As the first reported sRNA involved CoA synthesis and 1-butanol tolerance in cyanobacteria, this study provides not only novel insights in regulating mechanisms of essential pathways in cyanobacteria, but also valuable target for biofuels tolerance and productivity modifications.

Gene-expression heterogeneity among individual cells determines the fate of a bacterial population. Here we report the first bacterial single-cell RNA sequencing (RNA-seq), BaSiC RNA-seq, a method integrating RNA isolation, cDNA synthesis and amplification, and RNA-seq analysis of the whole transcriptome of single cyanobacterium Synechocystis sp. PCC 6803 cells which typically contain approximately 5–7 femtogram total RNA per cell. We applied the method to 3 Synechocystis single cells at 24 h and 3 single cells at 72 h after nitrogen-starvation stress treatment, as well as their bulk-cell controls under the same conditions, to determine the heterogeneity upon environmental stress. With 82–98% and 31–48% of all putative Synechocystis genes identified in single cells of 24 and 72 h, respectively, the results demonstrated that the method could achieve good identification of the transcripts in single bacterial cells. In addition, the preliminary results from nitrogen-starved cells also showed a possible increasing gene-expression heterogeneity from 24 h to 72 h after nitrogen starvation stress. Moreover, preliminary analysis of single-cell transcriptomic datasets revealed that genes from the “Mobile elements” functional category have the most significant increase of gene-expression heterogeneity upon stress, which was further confirmed by single-cell RT-qPCR analysis of gene expression in 24 randomly selected cells.

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.

•Acid-response network mediated by Slr1909 characterized in Synechocystis.•Signal pathway mediated by Slr1909 independent from that by SphS–SphR.•Synechocystis Δslr1909 mutant was more sensitive to low-pH.•24 and 10 proteins were up- and down-regulated upon slr1909 deletion.•A dozen metabolites for discrimination of Δslr1909 and wild type were identified.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 pH 6.2–6.5 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 significanceLow 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.

•∆sll0649 mutant was more sensitive to Cd2 + and other metal ions in Synechocystis.•156 and 151 proteins were down- and up-regulated upon sll0649 deletion.•Two direct regulatory targets of Sll0649 were identified by EMSAs.•Three mutants of Sll0649 downstream genes were also more sensitive to Cd2 +.Photosynthetic cyanobacteria are sensitive to toxicity of metal cadmium (Cd2 +). Although metabolic responses against Cd2 + exposure have been described, the related regulatory mechanism is still unclear in cyanobacteria. In this study, we identified in Synechocystis sp. PCC 6803 a response regulator (RR)-encoding gene sll0649, whose mutant was more sensitive to Cd2 + stress. Further phenotypic analysis revealed that ∆sll0649 becomes more sensitive to Cu2 +, Fe2 +, Mn2 + and Zn2 + stress as well. Using a quantitative iTRAQ-LC–MS/MS proteomics approach, we showed that a total of 156 and 151 unique proteins were down- and up-regulated for at least 2-fold in the ∆sll0649 mutant grown under Cd2 + stress, respectively. In addition, electrophoretic mobility shift assays showed that Sll0649 was able to bind directly to the upstream regions of sll1598 and slr0798, which encode an Mn2 + transporter MntC and a Zn2 + transporting P-type ATPases ZiaA, respectively, suggesting that Sll0649 was involved in Cd2 + tolerance by regulating and maintaining intracellular metal homeostasis. The involvement of sll1598 and slr0798 genes in Cd2 + tolerance was also verified by comparative mutant analyses. The study provided a proteomic description of the Cd2 + response network mediated by the response regulator Sll0649, and revealed novel insights on the metal-tolerance mechanism in Synechocystis.Biological significanceAs a major pollutant on earth, Cd2 + is toxic to both prokaryotic and eukaryotic organisms. It is thus important to obtain a better understanding of cellular response to Cd2 + and the related regulatory mechanism. In this study, by screening 44 gene knockout mutants of putative RR-encoding genes in Synechocystis for their sensitivity change to Cd2 + stress, we identified the orphan RR, Slr0649, involved in Cd2 + tolerance in Synechocystis. The ∆sll0649 mutant was also found to be more sensitive to high-concentration Cu2 +, Fe2 +, Mn2 + and Zn2 +, when compared with the wild type. Using an iTRAQ-LC–MSMS based quantitative proteomic analysis coupled with EMSAs, we found that, in addition to its positive regulation on genes directly related to Cd2 + utilization, Sll0649 can also functions as a key positive regulator either directly or indirectly on expression of multiple genes related to transporting and utilization of several other metal ions. The study provided a proteomic description of the Cd2 + response network mediated by the response regulator Sll0649, and revealed novel insights on the metal-tolerance mechanism in Synechocystis.

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.

We report here the characterization of a novel orphan response regulator Slr1588 directly involved in the synthesis and transport of compatible solutes against salt stress. In the Δslr1588 mutant, salt tolerance was found to be decreased by 2–3-fold. Using a high performance Q-EXACTIVE hybrid quadrupole-Orbitrap mass spectrometer, we found that proteins involved in the synthesis and transport of glucosylglycerol, a key compatible solute, were up-regulated in the Δslr1588 mutant grown in 4.0% NaCl, suggesting that Slr1588 might function as a repressor for glucosylglycerol metabolism. The functional assignment was further confirmed using an electrophoretic mobility shift assay (EMSA) showing that the purified his-tagged Slr1588 could bind in vitro directly to the upstream regions of sll1566 (ggpS) genes required for glucosylglycerol biosynthesis. In addition, quantitative proteomic analysis showed that the biosynthesis of another key compatible solute in Synechocystis, sucrose, was also up-regulated in the Δslr1588 mutant under 4.0% NaCl, and EMSA showed that the purified his-tagged Slr1588 bound in vitro directly to the upstream regions of sll0045 (spsA) gene required for sucrose biosynthesis. Moreover, proteomic analysis showed that 113 and 127 unique proteins were and up- and down-regulated in the Δslr1588 mutant grown under 4.0% NaCl, respectively. Notably, a dozen transporter genes were down-regulated in the Δslr1588 mutant under salt stress. The study revealed a novel salt-tolerant regulatory mechanism mediated by Slr1588, and also provided a proteomic description of the possible Slr1588 regulon in Synechocystis.

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.

Butanol is a promising biofuel, and recent metabolic engineering efforts have demonstrated the use of photosynthetic cyanobacterial hosts for its production. However, cyanobacteria have very low tolerance to butanol, limiting the economic viability of butanol production from these renewable producing systems. The existing knowledge of molecular mechanism involved in butanol tolerance in cyanobacteria is very limited. To build a foundation necessary to engineer robust butanol-producing cyanobacterial hosts, in this study, the responses of Synechocystis PCC 6803 to butanol were investigated using a quantitative proteomics approach with iTRAQ — LC-MS/MS technologies. The resulting high-quality dataset consisted of 25,347 peptides corresponding to 1452 unique proteins, a coverage of approximately 40% of the predicted proteins in Synechocystis. Comparative quantification of protein abundances led to the identification of 303 differentially regulated proteins by butanol. Annotation and GO term enrichment analysis showed that multiple biological processes were regulated, suggesting that Synechocystis probably employed multiple and synergistic resistance mechanisms in dealing with butanol stress. Notably, the analysis revealed the induction of heat-shock protein and transporters, along with modification of cell membrane and envelope were the major protection mechanisms against butanol. A conceptual cellular model of Synechocystis PCC 6803 responses to butanol stress was constructed to illustrate the putative molecular mechanisms employed to defend against butanol stress.The responses of Synechocystis PCC 6803 to butanol were investigated using a quantitative proteomics approach with iTRAQ — LC-MS/MS technologies. The results showed that Synechocystis employed multiple and synergistic resistance mechanisms in dealing with butanol stress.Highlights► A quantitative proteomics used to investigate butanol resistance in Synechocystis. ► Synechocystis employs multiple mechanisms in coping with butanol stress. ► Putative resistance mechanisms against butanol in Synechocystis were proposed. ► A list of potential targets for engineering butanol resistance was provided.

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.

•Bi-colored networks constructed to decipher functions of the Synechocystis HPs.•122 HPs were annotated by applying global propagation algorithm to networks.•More than 70% annotated HPs validation based on operon information•Several HPs were found with key metabolic functions in Synechocystis.Functional inference of hypothetical proteins (HPs) is a significant task in the post-genomic era. We described here a network-based protocol for functional inference of HPs using experimental transcriptomic, proteomic, and protein–protein interaction (PPI) datasets. The protocol includes two steps: i) co-expression networks were constructed using large proteomic or transcriptomic datasets of Synechocystis sp. PCC 6803 under various stress conditions, and then combined with a Synechocystis PPI network to generate bi-colored networks that include both annotated proteins and HPs; ii) a global algorithm was adapted to the bi-colored networks for functional inference of HPs. The algorithm ranked the associations between genes/proteins with known GO functional categories, and assumed that the top one ranked HP for each GO functional category might have a function related to the GO functional category. We applied the protocol to all HPs of the model cyanobacterium Synechocystis, and were able to assign putative functions to 122 HPs that have never been functionally characterized previously. Finally, the functional inference was validated by the known biological information of operon, and results showed that more than 70% HPs could be correctly validated. The study provided a new protocol to integrate different types of OMICS datasets for functional inference of HPs, and could be useful in achieving new insights into the Synechocystis metabolism.

We described here a global detection and functional inference of hypothetical proteins involved in stress response in Synechocystis sp. PCC 6803. In the study, we first applied an iTRAQ-LC–MS/MS based quantitative proteomics to the Synechocystis cells grown under five stress conditions. The analysis detected a total of 807 hypothetical proteins with high confidence. Among them, 480 were differentially regulated. We then applied a Weighted Gene Co-expression Network Analysis approach to construct transcriptional networks for Synechocystis under nutrient limitation and osmotic stress conditions using transcriptome datasets. The analysis showed that 305 and 467 coding genes of hypothetical proteins were functionally relevant to nutrient limitation and osmotic stress, respectively. A comparison of responsive hypothetical proteins to all stress conditions allowed identification of 22 hypothetical proteins commonly responsive to all stresses, suggesting they may be part of the core stress responses in Synechocystis. Finally, functional inference of these core stress responsive proteins using both sequence similarity and non-similarity approaches was conducted. The study provided new insights into the stress response networks in Synechocystis, and also demonstrated that a combination of experimental “OMICS” and bioinformatics methodologies could improve functional annotation for hypothetical proteins.Highlights► We described a global detection and functional inference of hypothetical proteins. ► We detected 807 hypothetical proteins using iTRAQ-LC–MS/MS proteomics. ► We identified hypothetical proteins relevant to stress using networks analysis. ► We found 22 hypothetical proteins commonly responsive to all stresses. ► The study provided new insights into the stress response networks in Synechocystis.