Hik28-dependent and Hik28-independent ABC transporters were revealed by proteome-wide analysis of ΔHik28 under combined stress.

Synechocystis histidine kinase, Sll0474: Hik28, a signal protein in a two-component signal transduction system, plays a critical role in responding to a decrease in growth temperature and is also involved in nitrogen metabolism. In the present study, under combined stress from non-optimal growth temperature and nitrogen depletion, a comparative proteomic analysis of the wild type (WT) and a deletion mutant (MT) of Synechocystis histidine kinase, Sll0474: Hik28, in a two-component signal transduction system identified the specific groups of ABC transporters that were Hik28-dependent, e.g., the iron transporter, and Hik28-independent, e.g., the phosphate transporter. The iron transporter, AfuA, was found to be upregulated only in the WT strain grown under the combined stress of high temperature and nitrogen depletion. Whereas, the expression level of the phosphate transporter, PstS, was increased in both the WT and MT strains. Moreover, the location in the genome of the genes encoding Hik28 and ABC transporters in Synechocystis sp. PCC6803 were analyzed in parallel with the comparative proteomic data. The results suggested the regulation of the ABC transporters by the gene in a two-component system located in an adjacent location in the genome.

Ensemble-AHTPpred: A Robust Ensemble Machine Learning Model Integrated With a New Composite Feature for Identifying Antihypertensive Peptides.

Hypertension or elevated blood pressure is a serious medical condition that significantly increases the risks of cardiovascular disease, heart disease, diabetes, stroke, kidney disease, and other health problems, that affect people worldwide. Thus, hypertension is one of the major global causes of premature death. Regarding the prevention and treatment of hypertension with no or few side effects, antihypertensive peptides (AHTPs) obtained from natural sources might be useful as nutraceuticals. Therefore, the search for alternative/novel AHTPs in food or natural sources has received much attention, as AHTPs may be functional agents for human health. AHTPs have been observed in diverse organisms, although many of them remain underinvestigated. The identification of peptides with antihypertensive activity in the laboratory is time- and resource-consuming. Alternatively, computational methods based on robust machine learning can identify or screen potential AHTP candidates prior to experimental verification. In this paper, we propose Ensemble-AHTPpred, an ensemble machine learning algorithm composed of a random forest (RF), a support vector machine (SVM), and extreme gradient boosting (XGB), with the aim of integrating diverse heterogeneous algorithms to enhance the robustness of the final predictive model. The selected feature set includes various computed features, such as various physicochemical properties, amino acid compositions (AACs), transitions, n-grams, and secondary structure-related information; these features are able to learn more information in terms of analyzing or explaining the characteristics of the predicted peptide. In addition, the tool is integrated with a newly proposed composite feature (generated based on a logistic regression function) that combines various feature aspects to enable improved AHTP characterization. Our tool, Ensemble-AHTPpred, achieved an overall accuracy above 90% on independent test data. Additionally, the approach was applied to novel experimentally validated AHTPs, obtained from recent studies, which did not overlap with the training and test datasets, and the tool could precisely predict these AHTPs.

Ensemble of Multiple Classifiers for Multilabel Classification of Plant Protein Subcellular Localization.

The accurate prediction of protein localization is a critical step in any functional genome annotation process. This paper proposes an improved strategy for protein subcellular localization prediction in plants based on multiple classifiers, to improve prediction results in terms of both accuracy and reliability. The prediction of plant protein subcellular localization is challenging because the underlying problem is not only a multiclass, but also a multilabel problem. Generally, plant proteins can be found in 10-14 locations/compartments. The number of proteins in some compartments (nucleus, cytoplasm, and mitochondria) is generally much greater than that in other compartments (vacuole, peroxisome, Golgi, and cell wall). Therefore, the problem of imbalanced data usually arises. Therefore, we propose an ensemble machine learning method based on average voting among heterogeneous classifiers. We first extracted various types of features suitable for each type of protein localization to form a total of 479 feature spaces. Then, feature selection methods were used to reduce the dimensions of the features into smaller informative feature subsets. This reduced feature subset was then used to train/build three different individual models. In the process of combining the three distinct classifier models, we used an average voting approach to combine the results of these three different classifiers that we constructed to return the final probability prediction. The method could predict subcellular localizations in both single- and multilabel locations, based on the voting probability. Experimental results indicated that the proposed ensemble method could achieve correct classification with an overall accuracy of 84.58% for 11 compartments, on the basis of the testing dataset.

Ensemble-AMPPred: Robust AMP Prediction and Recognition Using the Ensemble Learning Method with a New Hybrid Feature for Differentiating AMPs.

Antimicrobial peptides (AMPs) are natural peptides possessing antimicrobial activities. These peptides are important components of the innate immune system. They are found in various organisms. AMP screening and identification by experimental techniques are laborious and time-consuming tasks. Alternatively, computational methods based on machine learning have been developed to screen potential AMP candidates prior to experimental verification. Although various AMP prediction programs are available, there is still a need for improvement to reduce false positives (FPs) and to increase the predictive accuracy. In this work, several well-known single and ensemble machine learning approaches have been explored and evaluated based on balanced training datasets and two large testing datasets. We have demonstrated that the developed program with various predictive models has high performance in differentiating between AMPs and non-AMPs. Thus, we describe the development of a program for the prediction and recognition of AMPs using MaxProbVote, which is an ensemble model. Moreover, to increase prediction efficiency, the ensemble model was integrated with a new hybrid feature based on logistic regression. The ensemble model integrated with the hybrid feature can effectively increase the prediction sensitivity of the developed program called Ensemble-AMPPred, resulting in overall improvements in terms of both sensitivity and specificity compared to those of currently available programs.

Spirulina-in Silico-Mutations and Their Comparative Analyses in the Metabolomics Scale by Using Proteome-Based Flux Balance Analysis.

This study used an in silico metabolic engineering strategy for modifying the metabolic capabilities of Spirulina under specific conditions as an approach to modifying culture conditions in order to generate the intended outputs. In metabolic models, the basic metabolic fluxes in steady-state metabolic networks have generally been controlled by stoichiometric reactions; however, this approach does not consider the regulatory mechanism of the proteins responsible for the metabolic reactions. The protein regulatory network plays a critical role in the response to stresses, including environmental stress, encountered by an organism. Thus, the integration of the response mechanism of Spirulina to growth temperature stresses was investigated via simulation of a proteome-based GSMM, in which the boundaries were established by using protein expression levels obtained from quantitative proteomic analysis. The proteome-based flux balance analysis (FBA) under an optimal growth temperature (35 °C), a low growth temperature (22 °C) and a high growth temperature (40 °C) showed biomass yields that closely fit the experimental data obtained in previous research. Moreover, the response mechanism was analyzed by the integration of the proteome and protein-protein interaction (PPI) network, and those data were used to support in silico knockout/overexpression of selected proteins involved in the PPI network. The Spirulina, wild-type, proteome fluxes under different growth temperatures and those of mutants were compared, and the proteins/enzymes catalyzing the different flux levels were mapped onto their designated pathways for biological interpretation.

Revealing the key point of the temperature stress response of Arthrospira platensis C1 at the interconnection of C- and N- metabolism by proteome analyses and PPI networking.

BACKGROUND: Growth-temperature stress causes biochemical changes in the cells and reduction of biomass yield. Quantitative proteome of Arthrospira platensis C1 in response to low- and high temperature stresses was previously analysed to elucidate the stress response mechanism. The data highlighted the linkage of signaling proteins and proteins involved in nitrogen and ammonia assimilation, photosynthesis and oxidative stress. RESULTS: After phosphoproteome analysis was carried out in this study, the tentative temperature response cascade of A. platensis C1 was drawn based on data integration of quantitative proteome and phosphoproteome analysis and protein-protein interaction (PPI) networks. The integration revealed 31 proteins regulated at the protein-expression and post-translational levels; thus, this group of proteins was designated bi-level regulated proteins. PPI networks were then constructed based on A. platensis C1 gene inference from publicly available interaction data. The key two-component system (TCS) proteins, SPLC1_S082010 and SPLC1_S230960, were identified as bi-level regulated proteins and were linked to SPLC1_S270380 or glutamate synthase, an important enzyme in nitrogen assimilation that synthesizes glutamate from 2-oxoglutarate, which is known as the signal compound that regulates the carbon/nitrogen (C/N) balance of cells. Moreover, the role of the p-site in the PPIs of some phosphoproteins of interest was determined using site-directed mutagenesis and a yeast two-hybrid system. Evidence showing the critical role of the p-site in the PPI was observed for the multi-sensor histidine kinase SPLC1_S041070 (Hik28) and glutamate synthase. PPI subnetwork also showed that the Hik28 involved with the enzymes in fatty acid desaturation and nitrogen metabolism. The effect of Hik28-deletion was validated by fatty acid analysis and measurement of photosynthetic activity under nitrogen depletion. CONCLUSIONS: Taken together, the data clearly represents (i) the multi-level regulation of proteins involved in the stress response mechanism and (ii) the key point of the temperature stress response at the interconnection of C- and N- metabolism.

Natural ACE inhibitory peptides discovery from Spirulina (Arthrospira platensis) strain C1.

Bioactive peptides from natural sources are utilized as food supplements for disease prevention and are increasingly becoming targets for drug discovery due to their specificity, efficacy and the absence of undesirable side effects, among others. Hence, the 'SpirPep' platform was developed to facilitate the in silico-based bioactive peptide discovery of these highly sought-after biomolecules from Spirulina(Arthrospira platensis) and to select the protease (thermolysin) used for in vitro digestion. Analysis of the predicted and experimentally-derived peptides suggested that they were mainly involved in ACE inhibition; thus, an ACEi assay was used to study the ACE inhibitory activity of five candidate peptides (SpirPep1-5), chosen from common peptides with multifunctional bioactivity and 100% bioactive peptide coverage, originating from phycobiliproteins. Results showed that SpirPep1 inhibited the activity of ACE with IC50 of 1.748 mM and was non-toxic to fibroblasts of African green monkey kidney and human dermal skin. The molecular docking and MD simulation analysis revealed SpirPep1 had significantly lower binding scores than others and showed greater specificity to ACE. The non-bonded interaction energy of SpirPep1 and ACE was -883 kJ/mol. The SpirPep1 indirectly bound to ACE via the ACE substrate binding sites residues (D121, E123, S516, and S517) found in natural ACE inhibitory peptides (angiotensin II and bradykinin potentiating peptides). In addition, two unreported substrate binding sites including R124 and S219 were found. These results indicate that 'SpirPep' platform could increase the success rate for natural bioactive peptide discovery.

SpirPep: an in silico digestion-based platform to assist bioactive peptides discovery from a genome-wide database.

BACKGROUND: Bioactive peptides, including biological sources-derived peptides with different biological activities, are protein fragments that influence the functions or conditions of organisms, in particular humans and animals. Conventional methods of identifying bioactive peptides are time-consuming and costly. To quicken the processes, several bioinformatics tools are recently used to facilitate screening of the potential peptides prior their activity assessment in vitro and/or in vivo. In this study, we developed an efficient computational method, SpirPep, which offers many advantages over the currently available tools. RESULTS: The SpirPep web application tool is a one-stop analysis and visualization facility to assist bioactive peptide discovery. The tool is equipped with 15 customized enzymes and 1-3 miscleavage options, which allows in silico digestion of protein sequences encoded by protein-coding genes from single, multiple, or genome-wide scaling, and then directly classifies the peptides by bioactivity using an in-house database that contains bioactive peptides collected from 13 public databases. With this tool, the resulting peptides are categorized by each selected enzyme, and shown in a tabular format where the peptide sequences can be tracked back to their original proteins. The developed tool and webpages are coded in PHP and HTML with CSS/JavaScript. Moreover, the tool allows protein-peptide alignment visualization by Generic Genome Browser (GBrowse) to display the region and details of the proteins and peptides within each parameter, while considering digestion design for the desirable bioactivity. SpirPep is efficient; it takes less than 20 min to digest 3000 proteins (751,860 amino acids) with 15 enzymes and three miscleavages for each enzyme, and only a few seconds for single enzyme digestion. Obviously, the tool identified more bioactive peptides than that of the benchmarked tool; an example of validated pentapeptide (FLPIL) from LC-MS/MS was demonstrated. The web and database server are available at http://spirpepapp.sbi.kmutt.ac.th . CONCLUSION: SpirPep, a web-based bioactive peptide discovery application, is an in silico-based tool with an overview of the results. The platform is a one-stop analysis and visualization facility; and offers advantages over the currently available tools. This tool may be useful for further bioactivity analysis and the quantitative discovery of desirable peptides.

SpirPro: A Spirulina proteome database and web-based tools for the analysis of protein-protein interactions at the metabolic level in Spirulina (Arthrospira) platensis C1.

BACKGROUND: Spirulina (Arthrospira) platensis is the only cyanobacterium that in addition to being studied at the molecular level and subjected to gene manipulation, can also be mass cultivated in outdoor ponds for commercial use as a food supplement. Thus, encountering environmental changes, including temperature stresses, is common during the mass production of Spirulina. The use of cyanobacteria as an experimental platform, especially for photosynthetic gene manipulation in plants and bacteria, is becoming increasingly important. Understanding the mechanisms and protein-protein interaction networks that underlie low- and high-temperature responses is relevant to Spirulina mass production. To accomplish this goal, high-throughput techniques such as OMICs analyses are used. Thus, large datasets must be collected, managed and subjected to information extraction. Therefore, databases including (i) proteomic analysis and protein-protein interaction (PPI) data and (ii) domain/motif visualization tools are required for potential use in temperature response models for plant chloroplasts and photosynthetic bacteria. DESCRIPTIONS: A web-based repository was developed including an embedded database, SpirPro, and tools for network visualization. Proteome data were analyzed integrated with protein-protein interactions and/or metabolic pathways from KEGG. The repository provides various information, ranging from raw data (2D-gel images) to associated results, such as data from interaction and/or pathway analyses. This integration allows in silico analyses of protein-protein interactions affected at the metabolic level and, particularly, analyses of interactions between and within the affected metabolic pathways under temperature stresses for comparative proteomic analysis. The developed tool, which is coded in HTML with CSS/JavaScript and depicted in Scalable Vector Graphics (SVG), is designed for interactive analysis and exploration of the constructed network. SpirPro is publicly available on the web at http://spirpro.sbi.kmutt.ac.th . CONCLUSIONS: SpirPro is an analysis platform containing an integrated proteome and PPI database that provides the most comprehensive data on this cyanobacterium at the systematic level. As an integrated database, SpirPro can be applied in various analyses, such as temperature stress response networking analysis in cyanobacterial models and interacting domain-domain analysis between proteins of interest.

Identification of regulatory regions and regulatory protein complexes of the Spirulina desD gene under temperature stress conditions: role of thioredoxin as an inactivator of a transcriptional repressor GntR under low-temperature stress.

In the present study, electrophoretic mobility shift assays were used to identify temperature responsive elements in the 5' upstream region (5' UTR) of the Spirulina desD gene. Overlapping, synthetic oligonucleotides of both sense and anti-sense strands that spanned the entire 5' UTR of the gene were analyzed. The responsive DNA-binding protein complexes were identified using liquid chromatography-tandem mass spectrometry. The results indicated that the cold-responsive elements were located at -453 to -247, -197 to -151, -105 to -76, and -50 to -1, whereas the low-temperature specific regulatory regions were located at -372 to -352. Moreover, the heat-responsive elements were located at -347 to -243, -197 to -151, and -124 to -1, whereas the high-temperature specific elements were located between -130 to -101 and -30 to -1. In terms of regulatory protein complexes under the two stress conditions, Trx was only detected in the low-temperature responsive protein complex, and divalent cations were essential for the binding of the protein complex to the regulatory elements. Furthermore, Trx was shown to play a critical role as a reducing agent that inactivates the Spirulina desD repressor, GntR. Consequently, the desD gene expression is induced under the low-temperature condition.

Draft genome sequence of Arthrospira platensis C1 (PCC9438).

Arthrospira platensis is a cyanobacterium that is extensively cultivated outdoors on a large commercial scale for consumption as a food for humans and animals. It can be grown in monoculture under highly alkaline conditions, making it attractive for industrial production. Here we describe the complete genome sequence of A. platensis C1 strain and its annotation. The A. platensis C1 genome contains 6,089,210 bp including 6,108 protein-coding genes and 45 RNA genes, and no plasmids. The genome information has been used for further comparative analysis, particularly of metabolic pathways, photosynthetic efficiency and barriers to gene transfer.

Comparative analysis of the Spirulina platensis subcellular proteome in response to low- and high-temperature stresses: uncovering cross-talk of signaling components.

The present study focused on comparative proteome analyses of low- and high-temperature stresses and potential protein-protein interaction networks, constructed by using a bioinformatics approach, in response to both stress conditions.The data revealed two important points: first, the results indicate that low-temperature stress is tightly linked with oxidative stress as well as photosynthesis; however, no specific mechanism is revealed in the case of the high-temperature stress response. Second, temperature stress was revealed to be linked with nitrogen and ammonia assimilation. Moreover, the data also highlighted the cross-talk of signaling pathways. Some of the detected signaling proteins, e.g., Hik14, Hik26 and Hik28, have potential interactions with differentially expressed proteins identified in both temperature stress conditions. Some differentially expressed proteins found in the Spirulina protein-protein interaction network were also examined for their physical interactions by a yeast two hybrid system (Y2H). The Y2H results obtained in this study suggests that the potential PPI network gives quite reliable potential interactions for Spirulina. Therefore, the bioinformatics approach employed in this study helps in the analysis of phenomena where proteome analyses of knockout mutants have not been carried out to directly examine for specificity or cross-talk of signaling components.

Identification of a heat shock-responsive cis-acting DNA sequence and its transcriptional regulator: Their roles in the expression of the Spirulina-desD gene in response to heat stress.

This study addresses the importance of a heat-shock-responsive cis-acting DNA element and its transcriptional regulator, which play key roles in the regulation of the Spirulina-desD gene on exposure to high temperatures. Temperature response analysis studies showed that the AT-rich region that is located between nt -98 to -80 of the Spirulina-desD gene promoter serves as a binding site for its transcriptional regulator. LC-MS/MS analysis of the DNA-binding protein complex revealed that the amino acid sequences of the bound proteins were homologous to those of several proteins, including a DNA-binding protein, heat shock protein-90 (Hsp90 or HtpG), GroEL and various protein kinases. In addition, western blot analysis indicated that the chaperones GroEL and Hsp90 and a dephosphorylation reaction played a role in the response to elevated temperatures. We conclude that the regulatory DNA segments and the corresponding regulatory binding proteins are distinct for each particular stress condition. This is true, irrespective of whether the regulatory mechanisms that govern the expression of the cold- and heat-regulated desD gene depend on similar phosphorylation- and dephosphorylation-dependent conformational changes that modulate the association of the co-chaperone.

Subcellular proteomic characterization of the high-temperature stress response of the cyanobacterium Spirulina platensis.

The present study examined the changes in protein expression in Spirulina platensis upon exposure to high temperature, with the changes in expression analyzed at the subcellular level. In addition, the transcriptional expression level of some differentially expressed proteins, the expression pattern clustering, and the protein-protein interaction network were analyzed. The results obtained from differential expression analysis revealed up-regulation of proteins involved in two-component response systems, DNA damage and repair systems, molecular chaperones, known stress-related proteins, and proteins involved in other biological processes, such as capsule formation and unsaturated fatty acid biosynthesis. The clustering of all differentially expressed proteins in the three cellular compartments showed: (i) the majority of the proteins in all fractions were sustained tolerance proteins, suggesting the roles of these proteins in the tolerance to high temperature stress, (ii) the level of resistance proteins in the photosynthetic membrane was 2-fold higher than the level in two other fractions, correlating with the rapid inactivation of the photosynthetic system in response to high temperature. Subcellular communication among the three cellular compartments via protein-protein interactions was clearly shown by the PPI network analysis. Furthermore, this analysis also showed a connection between temperature stress and nitrogen and ammonia assimilation.

Proteome analysis at the subcellular level of the cyanobacterium Spirulina platensis in response to low-temperature stress conditions.

The present study addresses the differential expression of Spirulina platensis proteins detected during cold-induced stress, analyzed at the subcellular level. In performing differential expression analysis, the results revealed upregulated proteins in every subcellular fraction, including two-component response systems, DNA repair, molecular chaperones, stress-induced proteins and proteins involved in other biological processes such as secretion systems and nitrogen assimilation. The chlorophyll biosynthetic proteins, protochlorophyllide oxidoreductase and ChlI, had unique expression patterns as detected in the thylakoid membrane; the levels of these proteins immediately decreased during the first 45 min of low-temperature exposure. In contrast, their expression levels significantly increased after low-temperature exposure, indicating the relevance of the chlorophyll biosynthesis in Spirulina in response to low-temperature stress in the light condition. In addition, this is the first report in which genome-based protein identification in S. platensis by peptide mass fingerprinting was performed using the database derived from the unpublished Spirulina genome sequence.

A combined stress response analysis of Spirulina platensis in terms of global differentially expressed proteins, and mRNA levels and stability of fatty acid biosynthesis genes.

Changes in gene expression play a critical role in enhancing the ability of cyanobacteria to survive under cold conditions. In the present study, Spirulina platensis cultures were grown at the optimal growth temperature, in the light, before being transferred to dark conditions at 22 degrees C. Two dimensional-differential gel electrophoresis was then performed to separate differentially expressed proteins that were subsequently identified by MS. Among all differentiated proteins identified, a protein involved in fatty acid biosynthesis, (3R)-hydroxymyristoyl-[acyl-carrier-protein]-dehydratase encoded by fabZ, was the most up-regulated protein. However, the fatty-acid desaturation proteins were not significantly differentiated. This raised the question of how the unsaturated fatty acid, especially gamma-linolenic acid, content in the cells in the cold-dark shift remained stable compared with that of the cold shift. Thus, a study at the transcriptional level of these desaturase genes, desC, desA and desD, and also of the fabZ gene was conducted. The results indicated that in the dark, where energy is limited, mRNA stability was enhanced by exposure to low temperatures. The data demonstrate that when the cells encounter cold stress with energy limitation, they can maintain their homeoviscous adaptation ability via mRNA stability.

Truncation mutants highlight a critical role for the N- and C-termini of the Spirulina Delta(6) desaturase in determining regioselectivity.

The results of our previous study on heterologous expression in Escherichia coli of the gene desD, which encodes Spirulina Delta(6) desaturase, showed that co-expression with an immediate electron donor-either cytochrome b ( 5 ) or ferredoxin-was required for the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6) desaturase. Since a system for stable transformation of Spirulina is not available, studies concerning Spirulina-enzyme characterization have been carried out in heterologous hosts. In this present study, the focus is on the role of the enzyme's N- and C-termini, which are possibly located in the cytoplasmic phase. Truncated enzymes were expressed in E. coli by employing the pTrcHisA expression system. The truncation of the N- and C-terminus by 10 (N10 and C10) and 30 (N30 and C30) amino acids, respectively, altered the enzyme's regioselective mode from one that measures from a preexisting double bond to that measuring from the methyl end of the substrate.

Isolation and functional characterization of Spirulina D6D gene promoter: role of a putative GntR transcription factor in transcriptional regulation of D6D gene expression.

Delta6-Desaturase (D6D) is a key enzyme that catalyzes the synthesis of gamma-linolenic acid (GLA), an essential polyunsaturated fatty acid. We report here the isolation and first functional characterization of the D6D gene promoter from Spirulina platensis C1. Functional analysis of this isolated promoter showed that the Spirulina promoter was functional in Escherichia coli. Site-specific mutation studies demonstrated that the -10 sequence (TATAAT), located at -33bp relative to the translation start site, was essential for D6D promoter function. Temperature responsive deletion analysis studies identified the minimal core promoter within the region -51 to +1, which was sufficient for basal D6D promoter activity, and several cold-shock responsive cis-acting elements with activating and repressing activity. Electrophoretic mobility shift assay and LC-MS/MS studies demonstrated that an 'AT-rich Inverted Repeat' (-192 to -164) served as a target-binding site for a transcriptional regulator (probably a member of the GntR family) from Spirulina. Western blot analysis studies revealed that the DNA-binding transcriptional regulator underwent phosphorylation after a temperature downshift and possibly associated with transcriptional regulation of D6D gene expression. Taken together, our results suggest complex regulation of D6D gene expression in Spirulina.

Revealing differentially expressed proteins in two morphological forms of Spirulina platensis by proteomic analysis.

Spirulina is distinguished from other cyanobacteria by its spiral morphology; however, this cyanobacterium has frequently been observed with a linear morphology in laboratory and industrial conditions. In our laboratory conditions, the simultaneously presence of the linear and spiral forms has also been observed. In the present study, the two forms of S. platensis C1 were separated and grown as axenic cultures in order to study the proteins that were differentially expressed in the soluble and insoluble protein fractions of the spiral and the linear forms. Two dimensional-differential gel electrophoresis (2D-DIGE) was performed to separate differentially expressed proteins that were subsequently identified by mass spectrometry. The differentially expressed proteins suggested two points. First, the morphological change is possibly induced by various environmental stresses such as oxygen level, carbon dioxide level, nutrient availability, and light. Second, the change of cell-shape might be a result of the change in a cell shape determination mechanism. Thus, this study is the first to show evidence at the protein level that may explain this morphological transformation in Spirulina.

Effect of two intermediate electron donors, NADPH and FADH(2), on Spirulina Delta (6)-desaturase co-expressed with two different immediate electron donors, cytochrome b (5) and ferredoxin, in Escherichia coli.

When the gene desD encoding Spirulina Delta(6)-desaturase was heterologously expressed in E. coli, the enzyme was expressed without the ability to function. However, when this enzyme was co-expressed with an immediate electron donor, i.e. the cytochrome b (5) domain from Mucor rouxii, the results showed the production of GLA (gamma-linolenic acid), the product of the reaction catalyzed by Delta(6)-desaturase. The results revealed that in E. coli cells, where cytochrome b (5) is absent and ferredoxin, a natural electron donor of Delta(6)-desaturase, is present at a very low level, the cytochrome b (5) domain can complement for the function of ferredoxin in the host cells. In the present study, the Spirulina-ferredoxin gene was cloned and co-expressed with the Delta(6)-desaturase in E. coli. In comparison to the co-expression of cytochrome b ( 5 ) with the Delta(6)-desaturase, the co-expression with ferredoxin did not cause any differences in the GLA level. Moreover, the cultures containing the Delta(6)-desaturase co-expressed with cytochrome b (5) and ferredoxin were exogenously supplied with the intermediate electron donors, NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) and FADH(2) (flavin adenine dinucleotide, reduced form), respectively. The GLA level in these host cells increased drastically, by approximately 50%, compared to the cells without the intermediate electron donors. The data indicated that besides the level of immediate electron donors, the level of intermediate electron donors is also critical for GLA production. Therefore, if the pools of the immediate and intermediate electron donors in the cells are manipulated, the GLA production in the heterologous host will be affected.