The industrial cultivation of microalgae for high-value products faces significant challenges, particularly in maintaining long-term, cost-effective operations. Semi-continuous cultivation presents a promising solution to this problem. In this study, the green alga Chlorella sorokiniana IPPAS C-1 was cultivated in a flat-panel 5 L photobioreactor under optimized conditions, with three biological replicates. We evaluated batch mode against three semi-continuous dilution fractions (50%, 75%, and 87.5%). The 75% dilution fraction demonstrated superior performance, achieving the highest biomass productivity with an average specific productivity of 1.36 g DW L−1 day−1 over seven harvest cycles. Furthermore, this regime ensured stable biochemical composition—including proteins, lipids, carbohydrates, and pigments—as well as a consistent lipid profile and sustained photosynthetic activity throughout the cultivation. These findings are useful for the development of scalable and efficient technological protocols for the industrial production of Chlorella in flat-panel photobioreactors.

This dataset comprises 400 gas chromatography-mass spectrometry (GC-MS) chromatograms, recorded in total ion current mode, of a test mixture containing 38 fatty acid methyl esters (FAMEs). The mixture contains saturated (8:0–24:0), monounsaturated (14:1–24:1), di- and polyunsaturated (18:2–22:6) fatty acids, including positional isomers of the double bonds. Chromatograms were acquired on a highly polar HP-88 capillary column under both isothermal and temperature-programmed (linear heating) conditions, utilizing two carrier gas control modes: constant helium flow (1 mL/min) and constant pressure (30 psi). This dataset facilitates the selection of optimal parameters for the efficient separation of FAME mixtures. Furthermore, it can be utilized to develop analytical methods for identifying individual fatty acids using instrumentation lacking a mass spectrometric detector, and to construct models describing the relationships between the chromatographic properties of FAMEs and their molecular structures—specifically, acyl chain length and the number and position of double bonds.

The positional distribution of fatty acids (FAs) in Vischeria punctata strain IPPAS H-242 during nitrogen starvation was analysed, and 22 individual FAs were identified in total lipids, TAGs, and native sn-1,2-diacylglycerols, comprising both saturated and polyunsaturated FAs. The predominant FAs were 14:0, 16:0, 16:1n-7, 18:1n-9, and 20:5n-3, accounting for more than 98.5% of the total FAs. TAGs demonstrated unique regioselectivity, with stearic, oleic, and eicosapentaenoic acids acylating the molecules at sn-1 and sn-3 positions. Instead, the sn-2 position was acylated with myristic, palmitic (8.4% and 40.1%, respectively) and palmitoleic (50.6%) acids, resulting in a total contribution of 99.1% from these three FAs. Palmitic, palmitoleic, and oleic acids made up 7.5, 73.9, and 13.1% of the sn-1(3) positions, respectively, or 94.5% in total. The molecular types of TAGs that accumulated in the cells of the microalga Vischeria punctata were as follows: UUU, 41.3%, and USU, 39.2%; TAGs of the UUS and USS types accounted for 9.4% and 9.0%, respectively. Tripalmitoleoyl-triacylglycerol, sn-1,3-dipalmitoleoyl-2-palmitoyl-glycerol, rac-1-oleoyl-2,3-dipalmitoleoyl-glycerol, and rac-1-oleoyl-2-palmitoyl-3-palmitoleoyl-glycerol were found as the most common molecular species of TAGs. Thus, Vischeria punctata strain IPPAS H-242 may be considered as a potential producer of structured TAGs with palmitic acid at the sn-2 position and palmitoleic acid at the sn-1 and sn-3 positions. The subsequent hydrolysis of such TAGs by pancreatic lipase produces palmitoleic acid and sn-2-palmitoylglycerol that have significant nutraceutical value and serve as a source of digestible palmitic acid for infant nutrition.

Fatty acid desaturases (FADs) belong to of the oxygenase superfamily. They play important roles in metabolic pathways and adaption mechanisms in a wide range of organisms, including bacteria and humans. These enzymes dehydrogenate a single bond in the acyl chain of fatty acids (FAs), forming a double bond. Multiple parameters influence the precise position of double bond formation and acyl chain docking in the catalytic pocket of various FADs, such as the length of an acyl chain, the position of previously generated double bonds, the location of the enzyme's metal catalytic site, and so on. The "counting" mode differs from one FAD to another. The cyanobacterium Synechocystis sp. strain PCC 6803 has four FADs (D9, D12, D6, and D15 or w3) that synthesize mono-, di-, tri-, and tetraenoic FAs. The substrate preferences and "counting" modes for the first three FADs have been identified, but the substrate specificity for the terminal w3-FAD remains uncertain. We used molecular cloning, heterologous expression with exogenous FAs, and molecular docking to explore the substrate selectivity and counting mode of w3-FAD. Our results show that w3-FAD "counts" from the carboxyl (D) end, introduces a double bond between 15 and 16 atoms, and requires a specific acyl substrate configuration with two pre-existing double bonds at D9 and D12 positions. (c) 2025 Elsevier B.V. and Soci & eacute;t & eacute; Fran & ccedil;aise de Biochimie et Biologie Mol & eacute;culaire (SFBBM). All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Fatty acid desaturases (FADs) represent a class of oxygen-dependent enzymes that dehydrogenate C-C bonds in fatty acids (FAs) producing unsaturated C=C bonds that markedly change the properties of lipid membranes, which is critical for protein and lipid diffusion, perception and transduction of environmental signals, cell division, etc . Membrane-located FADs (acyl-lipid FADs of plants and bacteria, as well as animal acyl-coenzyme A (CoA) FADs) are highly conserved from viruses to human. These enzymes are highly specific towards their acyl substrates, as well as towards the position and geometric configuration of the newly introduced cis double bonds. The regiospecificity of soluble Acyl-Carrier-Protein (ACP) Δ9-FADs was determined relative to the carboxyl end of the FA. Similar regiospecificity was suggested for acyl-lipid and acyl-CoA FADs Δ9-FADs. It was previously thought that acyl-lipid Δ12-FADs (also known as ω6-FADs) also count from the carboxyl terminus of an FA. However, heterologous expression and supplementation of model yeast or cyanobacterial strains with exogenous monounsaturated odd- and even-chain FAs revealed that plant and cyanobacterial acyl-lipid Δ12-FADs use neither end of the FA (Δ or ω) as a counting reference point; but count three carbons toward the methyl end from an existing double bond in the monoene precursors irrespective of FA chain length. Δ6-FADs appeared to 'count' from the carboxyl terminus. ω3-FADs that contribute to unsaturation of C16-C18 FAs of membrane lipids, in fact, are Δ15-FADs that also 'count' from the C-terminus of acyl chains. The exact knowledge of counting order is crucial for understanding the modes of FADs activities and for further construction of biochemical pathways for biosynthesis of polyunsaturated FAs.

Protein phosphorylation is a pivotal mechanism for signal transduction, regulation of biochemical processes essential for reproduction, growth, and adaptation of organisms to changing conditions. Bacteria, which emerged more than 3.5 billion years ago, faced the need to adapt to a variety of ecological niches from the very beginning of their existence. It is not surprising that they developed a wide range of different types of kinases and target amino acid residues for phosphorylation. To date, many examples of phosphorylation of serine, threonine, tyrosine, histidine, arginine, lysine, aspartate, and cysteine have been discovered. Bacterial histidine kinases as part of two-component systems have been studied in most detail. More recently eukaryotic type serine-threonine and tyrosine kinases based on the conserved catalytic domain have been described in the genomes of many bacteria. The term “eukaryotic” is misleading, since evolutionary origin of these enzymes goes back to the last common universal ancestor – LUCA. Bioinformatics, molecular genetics, omics, and biochemical strategies combined provide new tools for researchers to establish relationship between the kinase abundance/activity and proteome changes, including studying of the kinase signaling network (kinome) within the cell. This manuscript presents several approaches to investigation of the serine-threonine protein kinases of cyanobacteria, as well as their combination, which allow to suggest new hypotheses and strategies for researchers.

Fatty acid desaturases (FADs) belong to of the oxygenase superfamily. They play important roles in metabolic pathways and adaption mechanisms in a wide range of organisms, including bacteria and humans. These enzymes dehydrogenate a single bond in the acyl chain of fatty acids (FAs), forming a double bond. Multiple parameters influence the precise position of double bond formation and acyl chain docking in the catalytic pocket of various FADs, such as the length of an acyl chain, the position of previously generated double bonds, the location of the enzyme's metal catalytic site, and so on. The "counting" mode differs from one FAD to another. The cyanobacterium Synechocystis sp. strain PCC 6803 has four FADs (Δ9, Δ12, Δ6, and Δ15 or ω3) that synthesize mono-, di-, tri-, and tetraenoic FAs. The substrate preferences and "counting" modes for the first three FADs have been identified, but the substrate specificity for the terminal ω3-FAD remains uncertain. We used molecular cloning, heterologous expression with exogenous FAs, and molecular docking to explore the substrate selectivity and counting mode of ω3-FAD. Our results show that ω3-FAD "counts" from the carboxyl (Δ) end, introduces a double bond between 15 and 16 atoms, and requires a specific acyl substrate configuration with two pre-existing double bonds at Δ9 and Δ12 positions.

Under standard laboratory conditions, Synechococcus elongatus PCC 7942 lacks EcaASyn, a periplasmic carbonic anhydrase (CA). In this study, a S. elongatus transformant was created that expressed the homologous EcaACya from Cyanothece sp. ATCC 51142. This additional external CA had no discernible effect on the adaptive responses and physiology of cells exposed to changes similar to those found in S. elongatus natural habitats, such as fluctuating CO2 and HCO3− concentrations and ratios, oxidative or light stress, and high CO2. The transformant had a disadvantage over wild-type cells under certain conditions (Na+ depletion, a reduction in CO2). S. elongatus cells lacked their own EcaASyn in all experimental conditions. The results suggest the presence in S. elongatus of mechanisms that limit the appearance of EcaASyn in the periplasm. For the first time, we offer data on the expression pattern of CCM-associated genes during S. elongatus adaptation to CO2 replacement with HCO3−, as well as cell transfer to high CO2 levels (up to 100%). An increase in CO2 concentration coincides with the suppression of the NDH-14 system, which was previously thought to function constitutively.

The PAS (Per-ARNT-Sim) domain is a sensory protein regulatory module found in archaea, prokaryotes, and eukaryotes. Histidine and serine/threonine protein kinases, chemo- and photoreceptors, circadian rhythm regulators, ion channels, phosphodiesterases, and other cellular response regulators are among these proteins. Hik33 is a multifunctional sensory histidine kinase that is implicated in cyanobacterial responses to cold, salt, hyperosmotic, and oxidative stressors. The functional roles of individual Hik33 domains in signal transduction were investigated in this study. Synechocystis Hik33 deletion variants were developed, in which either both or a portion of the transmembrane domains and/or the PAS domain were deleted. Cold stress was applied to the mutant strains either under illumination or in the dark. The findings show that the transmembrane domains govern temperature responses, whereas PAS domain may be involved in regulation of downstream gene expression in light-dependent manner.

The cyanobacterial strain Cyanobacterium sp. IPPAS B-1200 isolated from Lake Balkhash is characterized by high relative amounts of myristic (30%) and myristoleic (10%) acids. The remaining fatty acids (FAs) are represented mainly by palmitic (20%) and palmitoleic (40%) acids. We expressed the genes for lysophosphatidic acid acyltransferase (LPAAT; EC 2.3.1.51) and Δ9 fatty acid desaturase (FAD; EC 1.14.19.1) from Cyanobacterium sp. IPPAS B-1200 in Synechococcus elongatus PCC 7942, which synthesizes myristic and myristoleic acids at the level of 0.5–1% and produces mainly palmitic (~60%) and palmitoleic (35%) acids. S. elongatus cells that expressed foreign LPAAT synthesized myristic acid at 26%, but did not produce myristoleic acid, suggesting that Δ9-FAD of S. elongatus cannot desaturate FAs with chain lengths less than C16. Synechococcus cells that co-expressed LPAAT and Δ9-FAD of Cyanobacterium synthesized up to 45% palmitoleic and 9% myristoleic acid, suggesting that Δ9-FAD of Cyanobacterium is capable of desaturating saturated acyl chains of any length.

This mini review focuses on the phenomenon of homeoviscous adaptation (HVA). The concept, which dominated for decades, had a significant impact on membrane and lipid research. It includes the functional characterization of biological membranes and their domains, the role of lipids and fatty acids in cell metabolic control, and the characterization of fatty acid desaturases and their roles in membrane properties modulation. This hypothesis led to the discovery of a feed-back manner of desaturase expression and membrane-associated temperature sensors in bacteria.

The microalgae Vischeria sp. IPPAS C-70 produces eicosapentaenoic acid. Several stresses cause the formation of fatty acid peaks that resemble hexadecadienoic acids. We used the integrated technique including TLC, HPLC, and GC–MS to search and determine these fatty acids. Double bond positioning in these fatty acids indicated that they were conjugated dienes and allenes. We identified and described natural nine isomers of C16 polyunsaturated fatty acids, including common methylene-interrupted dienes (Δ6,9-16:2, Δ7,10-16:2, Δ9,12-16:2), and unusual conjugated dienes (Δ6,8-, Δ7,9-, Δ8,10-, Δ9,11-, and Δ10,12-16:2), as well as allenic diene (Δ9,10-16:2). We hypothesize that the formation of conjugated dienes and allenes among fatty acids is the result of oxidative stress caused by H2O2. Hydrogen peroxide also caused an increase in saturated at the expense of unsaturated fatty acids, suggesting inhibition either fatty acid desaturases activities or the corresponding gene expression.

Recent advances in research techniques have enabled rapid progress in the study of spirulina, an ancient edible cyanobacteria. Nowadays, spirulina species are classified into three genera: Spirulina, Arthrospira, and Limnospira. The latter now refers to industrially manufactured spirulina strains. Whole-genome sequencing revealed gene clusters involved in metabolite production, and the physiology of spirulina. Omics technologies demonstrated the absence of hazardous compounds in spirulina cells, confirming the safety of this biomass as a food product. Spirulina is a good source of different chemicals used in food manufacturing, food supplements, and pharmaceuticals. Spirulina’s enrichment with inherent biologically active substances makes it a potential supplier of natural products for dietary and pharmaceutical applications. Spirulina is also a prospective component of both terrestrial and space-based life support systems. Here, we review current breakthroughs in spirulina research and clarify fallacies that can be found in both professional literature and public media.

Twelve genes for the potential serine-threonine protein kinases (STPKs) have been annotated in the genome of Synechocystis sp. PCC 6803. Based on similarities and distinctive domain organization, they were divided into two clusters: serine/threonine-protein N2-like kinases (PKN2-type) and "activity of bc1 complex" kinases (ABC1-type). While the activity of the PKN2-type kinases have been demonstrated, no ABC1-type kinases activity have hitherto been reported. In this study, a recombinant protein previ-ously annotated as a potential STPK of ABC1-type (SpkH, Sll00 05) was expressed and purified to ho-mogeneity. We demonstrated SpkH phosphorylating activity and substrate preference for casein in in vitro assays using [g-32P]ATP. Detailed analyses of activity showed that Mn2+ had the strongest acti-vation effect. The activity of SpkH was significantly inhibited by heparin and spermine, but not by staurosporine. By means of semi-quantitative mass-spectrometric detection of phosphopeptides, we identified a consensus motif recognized by this kinase -X1X2pSX3E. Thus, we first report here that SpkH of Synechocystis represents a true active serine protein kinase, which shares the properties of casein kinases according to its substrate specificity and sensitivity to some activity effectors. & COPY; 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

The intracellular accumulation of inorganic carbon (Ci) by microalgae and cyanobacteria under ambient atmospheric CO2 levels was first documented in the 80s of the 20th Century. Hence, a third variety of the CO2-concentrating mechanism (CCM), acting in aquatic photoautotrophs with the C3 photosynthetic pathway, was revealed in addition to the then-known schemes of CCM, functioning in CAM and C4 higher plants. Despite the low affinity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of microalgae and cyanobacteria for the CO2 substrate and low CO2/O2 specificity, CCM allows them to perform efficient CO2 fixation in the reductive pentose phosphate (RPP) cycle. CCM is based on the coordinated operation of strategically located carbonic anhydrases and CO2/HCO3− uptake systems. This cooperation enables the intracellular accumulation of HCO3−, which is then employed to generate a high concentration of CO2 molecules in the vicinity of Rubisco’s active centers compensating up for the shortcomings of enzyme features. CCM functions as an add-on to the RPP cycle while also acting as an important regulatory link in the interaction of dark and light reactions of photosynthesis. This review summarizes recent advances in the study of CCM molecular and cellular organization in microalgae and cyanobacteria, as well as the fundamental principles of its functioning and regulation.

Twelve genes for the potential serine-threonine protein kinases (STPKs) have been annotated in the genome of Synechocystis sp. PCC 6803. Based on similarities and distinctive domain organization, they were divided into two clusters: serine/threonine-protein N2-like kinases (PKN2-type) and “activity of bc1 complex” kinases (ABC1-type). While the activity of the PKN2-type kinases have been demonstrated, no ABC1-type kinases activity have hitherto been reported. In this study, a recombinant protein previously annotated as a potential STPK of ABC1-type (SpkH, Sll0005) was expressed and purified to homogeneity. We demonstrated SpkH phosphorylating activity and substrate preference for casein in in vitro assays using [γ-32P]ATP. Detailed analyses of activity showed that Mn2+ had the strongest activation effect. The activity of SpkH was significantly inhibited by heparin and spermine, but not by staurosporine. By means of semi-quantitative mass-spectrometric detection of phosphopeptides, we identified a consensus motif recognized by this kinase – X1X2pSX3E. Thus, we first report here that SpkH of Synechocystis represents a true active serine protein kinase, which shares the properties of casein kinases according to its substrate specificity and sensitivity to some activity effectors.

Микроводоросли – одноклеточные фотосинтезирующие организмы, играющие важную роль в образовании кислорода и органических соединений на нашей планете, поэтому их экологическое значение неоценимо. Микроводоросли нашли свое применение в многих сферах деятельности человека. Их потенциальные возможности в продуцировании ценных метаболитов, витаминов и ряда иных органических соединений вызывают к микроводорослям большой интерес. Культивирование микроводорослей в перспективе может создать альтернативу сельскому хозяйству из-за высокой продуктивности производства, отсутствия зависимости от условий окружающей среды и погодных условий, и возможности выращивать микроводоросли в условиях непригодных для высших растений.

Currently, microalgae and cyanobacteria attract the attention of researchers as potential producers of various valuable substances. To increase the profitability of biotechnological processes using these organisms, it is necessary to select highly effective strains and choose the optimal conditions for their growth and maximum productivity. Growth optimization should be carried out, on the one hand, under intensive conditions, as close as possible to large-scale cultivation, and, on the other hand, in small volumes in order to be able to check many different parameters in parallel at minimal cost. In this paper, the authors present a description and characteristics of their laboratory system for intensive cultivation (LSIC—Laboratory System for Intensive Cultivation) with thermo-, light-, and gas regulation and the possibility of cultivation in four repetitions in eight different conditions, differing in light, temperature, and CO 2 concentration. As an example, the results of a number of experiments using the installation are also presented.

Several forms of EcaA protein, correspondent to the extracellular α-class carbonic anhydrase (CA) of cyanobacterium Crocosphaera subtropica ATCC 51142 were expressed in Escherichia coli. The recombinant proteins with no leader peptide (EcaA and its fusion with thioredoxin or glutathione S-transferase) were allocated inside cells in a full-length form; these cells did not display any extracellular CA activity. Soluble proteins (including that of periplasmic space) of E. coli cells that expressed both ЕсаА equipped with its native leader peptide (L-EcaA) as well as L-EcaA fused with thioredoxin or glutathione S-transferase at N-terminus, mainly contained the processed EcaA. The appearance of mature ЕсаА in outer layers of E. coli cells expressed leader peptide-containing forms of recombinant proteins, has been directly confirmed by immunofluorescent microscopy. Those cells also displayed high extracellular CA activity. In addition, the mature EcaA protein was detected in the culture medium. This suggests that cyanobacterial signal peptide is recognized by the secretory machinery and by the leader peptidase of E. coli even as a part of a fusion protein. The efficiency of EcaA leader peptide was comparable to that of PelB and TorA signal peptides, commonly used for biotechnological production of extracellular recombinant proteins in E. coli.

Flat-panel photobioreactors are effective systems for microalgae cultivation. This paper presents the growth characteristics of the microalgae Chlorella sorokiniana IPPAS C-1 as a result of three-stage scale-up cultivation in a specially designed cultivation system. First, C. sorokiniana was grown aseptically in 250 mL glass vessels; then, it was diluted and inoculated into a 5-liter flat-panel horizontal photobioreactor; and, at the last stage, the culture was diluted and inoculated into a 70-liter flat-panel vertical photobioreactor. In the presented cycle, the cultured biomass increased by 326 times in 13 days (from 0.6 to 195.6 g dw), with a final biomass concentration of 2.8 g dw L−1. The modes of semi-continuous cultivation were considered. The biomass harvest and dilution of the suspension were carried out either every day or every 3–4 days. For C. sorokiniana IPPAS C-1, a conversion coefficient of optical density values to dry biomass (g L−1) was refined through a factor of 0.33. The key parameters of the photobioreactors tested in this work are discussed.