Algal Research

Light is an essential key player in biomass and metabolite production in microalgae and the green lineage system. Light-sensing proteins (photoreceptors) can influence photosynthesis fitness, thereby regulating biomass and various metabolic activities. However, genetic engineering is mainly employed for enhancing astaxanthin (an important therapeutic bioactive metabolite) production in C. reinhardtii. In the present study, for the first time, we report opto-biomaufacturing of astaxanthin under blue light illumination in C. reinhardtii without any genetic modification. Moreover, we identified light-regulated biosynthetic gene cluster mediated metabolite production in green lineage. We employed data-driven systems biology-based analysis, array of biochemical and molecular techniques to investigate the imperative effect of different illumination conditions on metabolite production. The results indicated that blue light substantially enhances the biomanufacturing of astaxanthin and pigments via the photoreceptor. Thus, it suggests that fine-tuned illumination conditions modulate molecular components in specific metabolite production. Moreover, crosstalk between photoreceptors with the protein family of carotenoid metabolic pathway, cell signalling, and identified biosynthetic gene clusters (BGCs) in green lineage opens a new avenue in opto-biotechnology approaches for bioproducts generation from the green lineage system and other organisms simply by illumination. HighlightsO_LIChlamydomonas reinhardtii is a green biofactory for valuable metabolite production. C_LIO_LIOpto-biomanufacturing of astaxanthin in C. reinhardtii is achieved simply by illumination. C_LIO_LIBiosynthetic gene clusters of metabolite production are also controlled by light. C_LIO_LIPhotoreceptor based opto-biomanufacturing valuable bioactive is established. C_LIO_LIOpto-biomanufacturing opens avenues for bioactive production in various organisms. C_LI

Determining seaweed protein concentration and the associated phenotype is critical for food industries that require precise tools to moderate concentration fluctuations and attenuate risks. Algal protein extraction and profiling have been widely investigated, but content determination involves a costly, time-consuming, and high-energy, laboratory-based fractionation technique. The present study examines the potential of field spectroscopy technology as a precise, high-throughput, non-destructive tool for on-site detection of red seaweed protein concentration. By using information from a large dataset of 144 Gracilaria sp. specimens, studied in a land-based cultivation set-up, under six treatment regimes during two cultivation seasons, and an artificial neural network, machine learning algorithm and diffuse visible-near infrared reflectance spectroscopy, predicted protein concentrations in the algae were obtained. The prediction results were highly accurate (R2 = 0.95; RMSE = 0.84), exhibiting a high correlation with the analytically determined values. External validation of the model derived from a separate trial, exhibited even better results (R2 = 0.99; RMSE = 0.45). This model, trained to convert phenotypic spectral measurements and pigment intensity into accurate protein content predictions, can be adapted to include diversified algae species and usages. HighlightNon-destructive determination of protein content in the edible red seaweed Gracilaria sp. by in-situ, VIS-NIR spectroscopy and a machine learning algorithm.

Our increasing global population combined with the UN Sustainable Development Goals of zero hunger and good health require greater protein intake per capita and higher protein production. Consequently, sustainable food alternatives such as cultivated meat (CM) are urgently required. However, large-scale CM cell-systems face key challenges, particularly high media costs driven by amino acids and the need for ethically-sourced growth factors. Microalgae offer promising solutions, producing high protein yields with all essential amino acids simply from light, CO2, water and nutrients or spent CM media. Here we present Chlorella BDH-1 grown in spent CM media waste as a substitute-source for reduced amino acids and fetal bovine serum in cell culture media, enabling a circular strategy through beneficial mammalian cell-algae co-cultivation. We identified optimal algal growth conditions for maximum protein yield and demonstrated that two recycling rounds using industry-derived spent CM media maximize microalgal biomass yield per unit volume of waste media. We obtained algal lysate, determined thermal processing as the most cost-effective and mammalian cell-beneficial approach, and identified consumed lysate components. Compared to standard media, our lysate increased mammalian cell proliferation over 2-fold in reduced serum and amino acid conditions, replacing costly cell media components. We finally closed the loop by demonstrating a synergistic effect of the algal lysate with our co-cultivation - which co-produces algal biomass. The combination boosted mammalian cell proliferation 1.45-fold, conservatively estimating a media cost reduction by [~]66%. These findings establish parameters to advance the field towards cost-effective sustainable circular cell culture systems with applications in CM production and other biotechnology fields requiring large-scale tissue culture. Technology Readiness:

Nitrogen is an important element for all living organisms. Photoautotrophic organisms need to assimilate nitrogen from the environment, therefore changes in nitrogen availability have a strong influence on their growth and metabolism. Many microalgae have been known to contain crystalline inclusions, and recently, it has been shown that many of these consist of purines like guanine and thus must be linked to the cellular nitrogen metabolisms. The alveolate alga Chromera velia contains such guanine crystals, and during its lifecycle, the alga is thought to be subjected to strong changes in external nitrogen availability. Here, we investigated the formation or decline of crystalline guanine in dependence of the availability of inorganic nitrogen in the growth medium. Cells were examined using polarised light microscopy, Raman micro-spectroscopy, chromatography (HPLC), transmission and scanning electron microscopy. The cellular guanine crystal content decreased during nitrogen starvation and increased upon transfer of the cells back to standard growth medium containing nitrate. Raman micro-spectroscopy showed that the crystals were composed of anhydrous guanine in beta-polytype. They appear in unspecific positions throughout the cell, and staining with the green dye Lysotracker DND-26 suggests that they are within vacuoles. Stacks of crystals could be observed in cells via freeze fracture and freeze etching electron microscopy, which unambiguously showed a membrane around the crystal aggregates, in a similar arrangement as has been shown for guanine storage vacuoles (GSV) in Chlamydomonas reinhardtii. We developed a method to isolate the guanine crystals from whole cells, and were able to obtain crystals which retained their flat, plate-like structure, matching the electron microscopic observations from whole cells. The isolated crystals were shown to consist of nitrogen rich compounds via energy-dispersive X-ray (EDX) analysis, and Raman micro-spectroscopy confirmed that they consist of guanine.

Mammalian cell culture technologies are crucial for recombinant protein production, organoid generation, medical applications, and the generation of in vitro cultivated meat. However, they are limited by high costs, vascular O2 provision, and the resultant inhibition of 3D tissue formation. Effective media usage along with oxygenation and waste management to extend culture health and longevity are key to improving all three. Microalgae, utilizing organic or inorganic CO2, produce O2 from light which complements oxygen-consuming and CO2-respiring mammalian cells and tissue culture. However, common microalgal cultivation conditions differ in temperature and salinity from mammalian cell cultivation environments, making co-cultivation short-lived and challenging. We screened several different microalgae species to identify locally isolated Chlorella BDH-1 as candidate that has high growth rates in mammalian culture conditions while, unlike other Chlorella species, does not compete for glucose as an energy source. In mammalian cell co-culture, BDH1 reduces cellular waste products, stabilizes pH, doubles culture longevity, increases growth performance up to 80%, and reduces expensive and ethically challenging foetal bovine serum requirements. Chlorella BDH-1 was also non-inflammatory and tolerant of clinical antibiotics. Collectively, mammalian cell/BDH1 co-cultivation improves tissue culture health and reduces costs, paving the path for applications in the biotechnology and medical sectors.

Fluorescent reporters cover a wide range of applications in both basic and applied research. Whether a study involves microscopic imaging to study (co)-localization of proteins, FRET, biosensing, or quantifying gene expression, fluorophores are attractive reporter candidates due to their relatively straightforward in vivo readout. For microbiological applications, a wide variety of fluorescent proteins with varying excitation and emission wavelengths, brightness levels, and maturation times are available. Careful consideration is required when selecting from this large suite of proteins, especially when choosing multiple fluorophores. This is further complicated in phototrophic organisms, which exhibit strong autofluorescence, especially towards the red part of the spectrum, effectively eliminating common candidates such as mCherry. In this study, the specific properties and performance of a selection of fluorescent proteins are systematically evaluated against the background of photosynthetic pigment-derived autofluorescence in the cyanobacterium Synechocystis sp. PCC 6803. Specific readouts of different combinations of fluorescent proteins are also analyzed using high-throughput methods, namely plate reader fluorescent scans and single-cell flow cytometry to quantify fluorescence. The ultimate goal is to assess each fluorescent protein with regard to: 1.) Its ability to be discerned from cyanobacterial autofluorescence. 2.) Its compatibility with other fluorophores in this context. 3.) Its overall suitability in cyanobacterial research. Several highly suitable fluorescent proteins for use in cyanobacteria are identified, including mTagBFP2, mNeonGreen and mScarlet-I and suitable combinations, covering nearly the whole spectrum of visible light. This study expands the knowledge and toolset for current and future researchers and uncovers a whole spectrum of possibilities for fluorescent protein selection in cyanobacterial cell biology.

The polyextremophilic Cyanidiales are eukaryotic red microalgae with promising biotechnological properties arising from their low pH and elevated temperature requirements which can minimize culture contamination at scale. Cyanidioschyzon merolae 10D is a cell wall deficient species with a fully sequenced genome that is amenable to nuclear transgene integration by targeted homologous recombination. C. merolae maintains a minimal carotenoid profile and here, we sought to determine its capacity for ketocarotenoid accumulation mediated by heterologous expression of a green algal {beta}-carotene ketolase (BKT) and hydroxylase (CHYB). To achieve this, a synthetic transgene expression cassette system was built to integrate and express Chlamydomonas reinhardtii (Cr) sourced enzymes by fusing native C. merolae transcription, translation and chloroplast targeting signals to codon-optimized coding sequences. Chloramphenicol resistance was used to select for the integration of synthetic linear DNAs into a neutral site within the host genome. CrBKT expression caused accumulation of canthaxanthin and adonirubin as major carotenoids while co-expression of CrBKT with CrCHYB generated astaxanthin as the major carotenoid in C. merolae. Unlike green algae and plants, ketocarotenoid accumulation in C. merolae did not reduce total carotenoid contents, but chlorophyll a reduction was observed. Light intensity affected global ratios of all pigments but not individual pigment compositions and phycocyanin contents were not markedly different between parental strain and transformants. Continuous illumination was found to encourage biomass accumulation and all strains could be cultivated in simulated summer conditions from two different extreme desert environments. Our findings present the first example of carotenoid metabolic engineering in a red eukaryotic microalga and open the possibility for use of C. merolae 10D for simultaneous production of phycocyanin and ketocarotenoid pigments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/530181v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@dd010dorg.highwire.dtl.DTLVardef@1700e5corg.highwire.dtl.DTLVardef@1bee8eaorg.highwire.dtl.DTLVardef@ad67da_HPS_FORMAT_FIGEXP M_FIG C_FIG

Achieving enhanced lipid yield without compromising biomass is one of the long-standing challenges in our quest to produce algal biofuel sustainably. Multiple factors, including temperature, nutrients and light conditions impact lipid production, however such lipid-enhancing strategies often lead to reduced biomass, thereby offsetting the total volume of lipid recovered. Hydrodynamic cues remain poorly studied, specifically in the context of lipid production in motile algae, concurrently with biomass generation and photo-physiology, a key fitness parameter. By imposing hydrodynamic cues to biophysically stress distinct strains of raphidophyte Heterosigma akashiwo at specific time points along the growth stages (indicating different nutritional states), we quantify the lipid production, alongside algal biomass and photo-physiology. Early induction (hydrodynamic cues implemented during the lag phase) and delayed induction (hydrodynamic cues implemented during the exponential phase) were studied. Delayed induction of hydrodynamic cues suppressed growth and photo-physiology without significant enhancement of lipid production, however, early induction allowed to significantly increase lipid content, up to 300%, without observable changes in biomass and photo-physiology. Based on this, we propose a hydrodynamic strategy for enhanced lipid production with sustained biomass and physiological fitness. This work presents hydrodynamic perturbation and its onset timing as tunable parameters to advance lipid production technologies across diverse motile species.

Target of rapamycin (TOR) is a conserved protein kinase that regulates the balance between catabolic and anabolic processes in response to nutrient availability. Although the central role of TOR kinase in nutrient stress responses is well-recognized, little is known about the molecular basis of TOR signaling in ecologically important secondary algae with plastids of red algal origin, such as diatoms, as assessing in vivo TOR kinase activity is a difficult task. To assess TOR kinase activity, the phosphorylation status of downstream components, such as ribosomal protein S6 (RPS6), must be measured. Unlike for model organisms, an antibody that detects phosphorylated (P-) RPS6 in diatoms is not commercially available. Therefore, we developed a convenient method in which P-RPS6 and non-P-RPS6 were detected via Phos-tag affinity electrophoresis and immunoblotting with a commercial antibody that cross-reacts with RPS6 (both P- and non-P-RPS6) in the diatom, Phaeodactylum tricornutum. Using this Phos-tag-based method, we observed a dose-dependent decrease in the P-RPS6/total RPS6 ratio in P. tricornutum cells treated with the TOR kinase inhibitor, AZD-8055. We also observed a reduction in the P-RPS6/total RPS6 ratio during the nitrogen-deficient culture of P. tricornutum, which indicated the inactivation of TOR kinase in response to nitrogen deficiency. Finally, we demonstrated the potential application of the Phos-tag-based method to other ecologically, evolutionarily, and industrially important secondary algae, such as Nannochloropsis oceanica (Stramenopiles), the haptophyte Tisochrysis lutea, and Euglena gracilis (Euglenid). As all experimental materials are commercially available, the Phos-tag-based method can be used to promote studies on TOR in diverse algae in different contexts.

The increasing world population necessitates new sustainable nutrient sources, making microalgae like Chlorella sorokiniana interesting due to its rich nutrient profile and sustainable cultivation methods. With genetic optimization tools like CRISPR/Cas9, microalgae as a nutrient source can be improved even further. However, degradation of the rigid cell wall of microalgae, and thereby developing protoplasts, is often necessary prior to transformation, but monitoring protoplast development in spherical, single-celled organisms like C. sorokiniana is challenging using bright-field microscopy. Carbotrace 480 and 630 were tested as fluorescent markers of the cell wall of a C. sorokiniana mutant for protoplast detection, and Carbotrace 480 was successfully used to distinguish protoplast from normal cells in a cell suspension. The enzymes Driselase, Glucanex, Snailase, and Saczyme were tested in different combinations to degrade the cell wall of the mutant, with Snailase as the most effective yielding ~60 % protoplasts. This study provides a quick and easy tool for monitoring protoplast development in the microalgae C. sorokiniana, the first step to improve C. sorokiniana as a sustainable nutrient source using genetic optimization tools like CRISPR/Cas9.

Cyanate (OCN-) is potentially an important organic nitrogen source in aquatic environments given the prevalence and activity of cyanate lyase genes in microalgae. However, the conditions under which these genes are expressed and the actual capacity of microalgae to assimilate cyanate remain underexplored. Here, we studied the nitrogen metabolism of the cosmopolitan picoalga Pelagomonas calceolata (Pelagophyceae, Stramenopiles) in environmental metatranscriptomes and transcriptomes from culture experiments under different nitrogen sources and concentrations. We observed that cyanate lyase is over-expressed in nitrate-poor oceanic regions, suggesting that cyanate is an important molecule contributing to the persistence of P. calceolata in oligotrophic environments. In the laboratory, we confirmed that this gene is over-expressed in low-nitrate medium together with several genes involved in nitrate recycling from endogenous molecules. Non-axenic cultures of P. calceolata were capable of growing on various nitrogen sources, including nitrate, urea and cyanate, but not ammonium. RNA sequencing of these cultures revealed that cyanate lyase was under-expressed in the presence of cyanate, indicating that this gene in not involved in the catabolism of extracellular cyanate to ammonia. Conversely, axenic P. calceolata cultures were not able to grow on cyanate, suggesting that the bacterial community consumes cyanate and provides an available form of nitrogen for growth of the alga. Based on environmental datasets and laboratory experiments, we propose that cyanate lyase is important in nitrate-poor environments to reduce the toxicity of intracellular cyanate produced by endogenous nitrogenous compound recycling, rather than being used to metabolise imported extracellular cyanate as an alternative nitrogen source.

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

The west coast of Saudi Arabia borders the Red Sea, which maintains high average temperatures and increased salinity compared to other seas or oceans. Summer conditions in the Arabian Peninsula may exceed the temperature tolerance of most currently cultivated microalgae. The Cyanidiales are polyextremophilic red algae whose native habitats are at the edges of acidic hot springs. Cyanidioschyzon merolae 10D has recently emerged as an interesting model organism capable of high-cell density cultivation on pure CO2 with optimal growth at 42 {degrees}C and low pH between 0.5-2. C. merolae biomass has an interesting macromolecular composition, is protein rich, and contains valuable bio-products like heat-stable phycocyanin, carotenoids, {beta}-glucan, and starch. Here, photobioreactors were used to model C. merolae 10D growth performance in simulated environmental conditions of the mid-Red Sea coast across four seasons, it was then grown at various scales outdoors in Thuwal, Saudi Arabia during the Summer of 2022. We show that C. merolae 10D is amenable to cultivation with industrial-grade nutrient and CO2 inputs outdoors in this location and that its biomass is relatively constant in biochemical composition across culture conditions. We also show the adaptation of C. merolae 10D to high salinity levels of those found in Red Sea waters and conducted further modeled cultivations in nutrient enriched local sea water. It was determined that salt-water adapted C. merolae 10D could be cultivated with reduced nutrient inputs in local conditions. The results presented here indicate this may be a promising alternative species for algal bioprocesses in outdoor conditions in extreme desert summer environments.

Living cells of brown algae are difficult to observe in 3D because pigments such as fucoxanthin and chlorophyll diffract light. Furthermore, at the beginning of their life, brown algae develop slowly in seawater. To gain insight into the 3D shape and size of brown algal cells during embryogenesis, we designed a fluorescence probe that efficiently and selectively labels the plasma membrane. Styryl benzoindoleninium sulfonate (SBIS) is a bright orange fluorogenic probe that is soluble and virtually non-emissive in seawater and is activated upon binding to the plasma membrane. Unlike Calcofluor White, SBIS enables observation of cells at thicknesses of up to 25 {micro}m. More importantly, SBIS allows three-dimensional observation of the cells in the growing uniseriate filaments of Ectocarpus sp., the polystichous filaments of Sphacelaria rigidula and the cellular monolayered lamina of Saccharina latissima over periods of up to seven days. Altogether, these properties allow visualization of the entire cell contours in living brown algae, making the study of early development at the cellular level in 4D now possible in these marine organisms.

BackgroundPolyphosphates (polyP) and ATP are phosphate-containing metabolites present in prokaryotes and eukaryotes. PolyP has a wide variety of functions including phosphate and cation storage. ATP is a central metabolite in cellular bioenergetics and the phosphate providing substrate of polyP. In the green microalga Chlamydomonas reinhardtii, polyP synthesis is suggested to buffer ATP concentration, and the role of polyP in energetic metabolism requires further investigation. In this aim, relative quantification of both metabolites is needed. Because ATP and polyP half-lives differ greatly, harvesting generates biases in this relative quantification in current methods. For this reason, we present here a joint protocol optimised to compromise between maximal yield and specific constraints of both assays. MethodsThe optimised method quantifies ATP and polyP from the same C. reinhardtii cell extract after neutral phenol-chloroform extraction. Cells are directly pipetted from the culture to the phenol-chloroform- EDTA extraction mix. After a second chloroform extraction, ATP is quantified directly from the extract, while polyP measurement requires purification by ethanol precipitation. We used one-way analysis of variance or Kruskall-Wallis testing and appropriate post hoc testing to evaluate statistical effects in our results. ResultsWe show that he polyP/ATP ratio of the reference strain CC-4533 in exponential mixotrophic growth is around 65. While the optimised protocol performs as well as specific protocols for either ATP or polyP, the dispersion of the polyP/ATP ratio is twice better than for the separate metabolites. Direct sampling from the culture works better than centrifugation and filtration to maintain physiological conditions and high polyP yield. Using spiking with ATP and polyP, we show that ATP and longer chain polyP are fully recovered but not very short chain polyP. Finally, we show that the polyP chain length distribution extracted from CC-4533 is very broad, reaching up to several thousand P with a mean around 200 P. DiscussionOur protocol improves the precision of relative quantification of ATP and polyP by using neutral phenol-chloroform extraction and allows polyP/ATP ratio calculation from low-density samples and without normalisation. It can be applied to other microorganisms or cells, in a variety of physiological and stress conditions.

Microalgae is currently gaining attention as an alternative source for food production. The market is currently demanding colorless algae with high protein and lutein content as an alternative to currently available commodities. This research was aimed at performing random UV mutagenesis on Chlorella vulgaris to obtain mutants with enhanced growth rates and increased growth characteristics. A total of seven mutants were selected to be analyzed after the random mutagenesis. Small (40 mL) and larger-scale (1,000 mL) reactors were used to analyze the production of C. vulgaris mutant biomass, focusing on the dry matter, starch, chlorophyll, protein, fatty acids, and lutein contents. Results implied that mutants showed a higher specific growth rate (2.1-2.5-fold higher) as compared to the wild type. The three mutants (MT 1, 2, and 3) that exhibited a yellow color were subsequently chosen for further scalability. In larger-scale reactors, all mutants exhibited higher protein contents while displaying lower carbohydrate and chlorophyll contents in comparison to the wild type. Moreover, MT 1 exhibited the highest concentration of lutein (0.37%-0.38%) and the lowest concentration of chlorophyll (0.1-0.14%), both of which are of significance for potential applications in the food industry.

Recirculating aquaculture system (RAS) effluents contain substantial nitrate and phosphate loads, posing a significant eutrophication risk to aquatic ecosystems if left untreated. While microalgal bioremediation is a promising strategy, expanding opportunities for valorisation is crucial for its successful implementation. This study couples the cultivation of nitrate-utilizing Chlamydomonas reinhardtii strain CC-1690 in RAS effluent with photobiological hydrogen (H2) production to achieve nutrient removal, bioenergy production, and high-quality biomass. Within 70 h, CC-1690 achieved near-complete depletion of nitrate and phosphate from the effluent. Subsequently, H2 production was induced via carbon limitation and anoxia, yielding a cumulative 49 {micro}mol H2 mg-1 Chl over 72 h. Although decreased chlorophyll content and Fv/Fm indicated physiological stress, key photosynthetic subunits (PsbA, PSBO, CP47, PetB and PsaA) remained largely stable. Importantly, the process preserved biomass quality; total lipid content increased slightly, enriched in palmitic (16:0) and -linolenic (18:3{omega}-3) fatty acids while protein content was unaffected. These results demonstrate that integrating H2 production with RAS effluent remediation offers a robust circular economy approach, valorising wasted nutrients into bioenergy and high-quality biomass with potential downstream applications.

BackgroundMicroalgae are an important feedstock for the production of a wide variety of products, including biodiesel. Biodiesel, composed of fatty acid alkyl esters, is produced through the transesterification reaction of triacylglycerol (TAG). Microalgae store their energy reserves primarily as starch and TAGs. Therefore, several studies have focused on understanding the partitioning of carbon precursors between starch and TAG biosynthetic pathways. In this study, 5 starch mutants of Chlorella vulgaris were developed and cultured on different culture media. ResultsChlorella vulgaris starch mutants were generated through UV-random mutagenesis. Five starch mutants were selected for this study: four low-starch producing mutants (st27, st29, st43 and st54) and one high-starch producing mutant (st80). The starch mutants were cultured on media with different organic carbon sources, and lipid and biomass productivity were measured. Mixotrophic growth on glucose resulted in the highest lipid productivity in all the mutants, including st80, without compromising growth, whereas photoautotrophic growth generally did not result in changes in lipid productivity of the starch mutants. The highest increase in lipid productivity was observed for st27, with a 3.8-fold higher lipid productivity than wildtype. ConclusionsAll starch mutants increased their lipid productivities when grown mixotrophically on glucose, suggesting the overflow hypothesis could explain the partitioning of carbon between starch and TAGs. Out of the mutants generated in this work, st27 resulted in the highest increases in lipid productivities, reaching an increase of 380% when grown mixotrophically on glucose, without compromising growth. The high-starch producing mutant st80 provides insight into a possibility to develop starch- and TAG-rich microalgal biomass.

Phycobiliproteins (PBPs) play a vital role in light harvesting by cyanobacteria, which enables efficient utilization of photon energy for oxygenic photosynthesis. The PBPs carry phycobilins, open-chain tetrapyrrole chromophores derived from heme. The structure and chromophore composition of PBPs is dependent on the organisms ecological niche. In cyanobacteria, these holo-proteins typically form large, macromolecular antenna complexes called phycobilisomes (PBSs). The PBS of Synechocystis sp. PCC 6803 (hereafter Synechocystis) consists of allophycocyanin (APC) and phycocyanin (PC), which exclusively harbor phycocyanobilin (PCB) as a chromophore. Investigations into heterologous PBP biosynthesis in E. coli have proven limiting with respect to PBP assembly and their functional characterization. Consequently, we wanted to engineer a platform for the investigation of heterologously produced PBPs, focusing on unusual, phycoerythrobilin (PEB)-containing light-harvesting proteins called phycoerythrins (PEs) in Synechocystis. As a first step, a gene encoding for the synthesis of the natural cyanobacterial chromophore, PEB, was introduced into Synechocystis. We provide spectroscopic evidence for heterologous PEB formation and show covalent attachment of PEB to the -subunit of PC, CpcA, by HPLC and LC-MS/MS analyses. Fluorescence microscopy and PBS isolation demonstrate a cellular dispersal of PBPs with modified phycobilin content. However, these modifications have minor effects on physiological responses, as demonstrated by growth rates, oxygen evolution, nutrient accumulation, and PBP content analyses. As a result, Synechocystis demonstrates the capacity to efficiently manage PEB biosynthesis and therefore reflects a promising platform for both biochemical and physiological investigations of foreign and unusual PEs.

Microalgae are well-known for their role as sustainable bio-factories, offering a promising solution to the global food and nutrition crisis. To clarify the potential of Chlorella sorokiniana UTEX 1230 for food applications, particularly as an alternative protein source, the study employed a mixotrophic cultivation mode with sodium acetate (NaAc) as a cost-effective organic carbon (NaAc-C) source. Varying levels of NaAc-C and nitrate-sourced nitrogen were investigated, optimizing the effect of metabolic characteristics of the microalgal growth. The designed heterotrophic cultivation confirmed the ability of C. sorokiniana UTEX 1230 to grow on NaAc-C, and then the mixotrophic cultures, when supported by both NaAc-C and CO2, exhibited superior growth performance, achieving double the biomass concentration compared to the autotrophic control. The addition of nitrogen (750 mg/L NaNO) facilitated the thorough metabolism of NaAc-C and enhanced photosynthetic activity indicated by a 196% increase in pigment levels, which resulted in a maximum biomass concentration of 2.82 g/L in the 150 mM NaAc-C group. A detailed analysis of nitrogen and protein concentrations over time revealed that higher nitrogen availability led to greater protein accumulation which was then degraded to support essential life activities under nitrogen starvation. Therefore, it is suggested that supplementing nitrate on the 3rd day and harvesting on the 4th day could be strategically implemented to increase protein yield from 0.17 g/L/d to 0.34 g/L/d. These findings offer theoretical guidance for further refining this microalgal strain for use as an alternative protein.