Microbial chassis as the platform for production of dihydroxy xanthophyll-based carotenoids: an overview of recent advances in biomanufacturing.

Physiological and environmental cues prompt microbes to synthesize diverse carotenoids, including dihydroxy xanthophylls, facilitating their adaptation and survival. Lutein and its isomeric counterpart, zeaxanthin, are notable dihydroxy xanthophylls with bioactive properties such as antioxidative, anti-inflammatory, anticancer, and neuroprotective effects, particularly beneficial for human ocular health. However, global natural resources for co-producing lutein and zeaxanthin are scarce, with zeaxanthin lacking commercial sources, unlike lutein sourced from marigold plants and microalgae. Traditionally, dihydroxy xanthophyll production primarily relies on petrochemical synthetic routes, with limited biological sourcing reported. Nonetheless, microbiological synthesis presents promising avenues as a commercial source, albeit challenged by low dihydroxy xanthophyll yield at high cell density. Strategies involving optimization of physical and chemical parameters are essential to achieve high-quality dihydroxy xanthophyll products. This overview briefly discusses dihydroxy xanthophyll biosynthesis and highlights recent advancements, discoveries, and industrial benefits of lutein and zeaxanthin production from microorganisms as alternative biofactories.

Outdoor cultivation and metabolomics exploration of Chlamydomonas engineered for bisabolene production.

Demonstrating outdoor cultivation of engineered microalgae at considerable scales is essential for their prospective large-scale deployment. Hence, this study focuses on the outdoor cultivation of an engineered Chlamydomonas reinhardtii strain, 3XAgBs-SQs, for bisabolene production under natural dynamic conditions of light and temperature. Our preliminary outdoor experiments showed improved growth, but frequent culture collapses in conventional Tris-acetate-phosphate medium. In contrast, modified high-salt medium (HSM) supported prolonged cell survival, outdoor. However, their subsequent outdoor scale-up from 250 mL to 5 L in HSM was effective with 10 g/L bicarbonate supplementation. Pulse amplitude modulation fluorometry and metabolomic analysis further validated their improved photosynthesis and uncompromised metabolic fluxes towards the biomass and the products (natural carotenoids and engineered bisabolene). These strains could produce 906 mg/L bisabolene and 54 mg/L carotenoids, demonstrating the first successful outdoor photoautotrophic cultivation of engineeredC. reinhardtii,establishing it as a one-cell two-wells biorefinery.

Dynamic metabolomic crosstalk between Chlorella saccharophila and its new symbiotic bacteria enhances lutein production in microalga without compromising its biomass.

The microalgae Chlorella saccharophila UTEX247 was co-cultured with its symbiotic indigenous isolated bacterial strain, Exiguobacterium sp., to determine the possible effects of bacteria on microalgae growth and lutein productivity. Under optimal conditions, the lutein productivity of co-culture was 298.97 µg L-1 d-1, which was nearly 1.45-fold higher compared to monocultures i.e., 103.3 µg L-1 d-1. The highest lutein productivities were obtained in co-cultures, accompanied by a significant increase in cell biomass up to 0.84-fold. These conditions were analyzed using an untargeted metabolomics approach to identify metabolites enhancing valuable renewables, i.e., lutein, without compromising growth. Our qualitative metabolomic analysis identified nearly 30 (microalgae alone), 41 (bacteria alone), and 75 (co-cultures) metabolites, respectively. Among these, 46 metabolites were unique in the co-culture alone. The co-culture interactions significantly altered the role of metabolites such as thiamine precursors, reactive sugar anomers like furanose and branched-chain amino acids (BCAA). Nevertheless, the central metabolism cycle upregulation depicted increased availability of carbon skeletons, leading to increased cell biomass and pigments. In conclusion, the co-cultures induce the production of relevant metabolites which regulate growth and lutein simultaneously in C. saccharophila UTEX247, which paves the way for a new perspective in microalgal biorefineries.

Media engineering in marine diatom Phaeodactylum tricornutum employing cost-effective substrates for sustainable production of high-value renewables.

Phaeodactylum tricornutum is a marine diatom, rich in omega-3 polyunsaturated fatty acids especially eicosapentaenoic acid (EPA) and brown pigment, that is, fucoxanthin. These high-value renewables (HVRs) have a high commercial and nutritional relevance. In this study, our focus was to enhance the productivities of such renewables by employing media engineering strategy via., photoautotrophic (P1, P2, P3) and mixotrophic (M1, M2, M3, M4) modes of cultivation with varying substrate combinations of carbon (glycerol: 0.1 m) and nitrogen (urea: 441 mm and/or sodium nitrate: 882 mm). Our results demonstrate that mixotrophic [M4] condition supplemented with glycerol (0.1 m) and urea (441 mm) feed enhanced productivities (mg L-1  day-1 ) as follows: biomass (770.0), total proteins (36.0), total lipids (22.0), total carbohydrates (23.0) with fatty acid methyl esters (9.6), EPA (2.7), and fucoxanthin (1.1), respectively. The overall yield of EPA represents 28% of total fatty acids in the mixotrophic [M4] condition. In conclusion, our improved strategy of feeding urea to a glycerol-supplemented medium defines a new efficient biomass valorization paradigm with cost-effective substrates for the production of HVRs in oleaginous diatoms P. tricornutum.

Multi-Fold Enhancement of Tocopherol Yields Employing High CO2 Supplementation and Nitrate Limitation in Native Isolate Monoraphidium sp.

Tocopherols are the highly active form of the antioxidant molecules involved in scavenging of free radicals and protect the cell membranes from reactive oxygen species (ROS). In the present study, we focused on employing carbon supplementation with varying nitrate concentrations to enhance the total tocopherol yields in the native isolate Monoraphidium sp. CABeR41. The total tocopherol productivity of NRHC (Nitrate replete + 3% CO2) supplemented was (306.14 µg·L-1 d-1) which was nearly 2.5-fold higher compared to NRVLC (Nitrate replete + 0.03% CO2) (60.35 µg·L-1 d-1). The best tocopherol productivities were obtained in the NLHC (Nitrate limited + 3% CO2) supplemented cells (734.38 µg·L-1 d-1) accompanied by a significant increase in cell biomass (2.65-fold) and total lipids (6.25-fold). Further, global metabolomics using gas chromatography-mass spectrometry (GC-MS) was done in the defined conditions to elucidate the molecular mechanism during tocopherol accumulation. In the present study, the Monoraphidium sp. responded to nitrogen limitation by increase in nitrogen assimilation, with significant upregulation in gamma-Aminobutyric acid (GABA). Moreover, the tricarboxylic acid (TCA) cycle upregulation depicted increased availability of carbon skeletons and reducing power, which is leading to increased biomass yields along with the other biocommodities. In conclusion, our study depicts valorization of carbon dioxide as a cost-effective alternative for the enhancement of biomass along with tocopherols and other concomitant products like lipids and carotenoids in the indigenous strain Monoraphidium sp., as an industrial potential strain with relevance in nutraceuticals and pharmaceuticals.

Channeling of Carbon Flux Towards Carotenogenesis in Botryococcus braunii: A Media Engineering Perspective.

Microalgae, due to their unique properties, gained attention for producing promising feedstocks having high contents of proteins, antioxidants, carotenoids, and terpenoids for applications in nutraceutical and pharmaceutical industries. Optimizing production of the high-value renewables (HVRs) in microalgae requires an in-depth understanding of their functional relationship of the genes involved in these metabolic pathways. In the present study, bioinformatic tools were employed for characterization of the protein-encoding genes of methyl erythritol phosphate (MEP) pathway involved in carotenoid and squalene biosynthesis based upon their conserved motif/domain organization. Our analysis demonstrates nearly 200 putative genes showing a conservation pattern within divergent microalgal lineages. Furthermore, phylogenomic studies confirm the close evolutionary proximity among these microalgal strains in the carotenoid and squalene biosynthetic pathways. Further analysis employing STRING predicts interactions among two rate-limiting genes, i.e., phytoene synthase (PSY) and farnesyl diphosphate farnesyl synthase (FPPS), which are specifically involved in the synthesis of carotenoids and squalene. Experimentally, to understand the carbon flux of these rate-limiting genes involved in carotenogenesis, an industrial potential strain, namely, Botryococcus braunii, was selected in this study for improved biomass productivity (i.e., 100 mg L-1 D-1) along with enhanced carotenoid content [0.18% dry cell weight (DCW)] when subjected to carbon supplementation. In conclusion, our approach of media engineering demonstrates that the channeling of carbon flux favors carotenogenesis rather than squalene synthesis. Henceforth, employing omics perspectives will further provide us with new insights for engineering regulatory networks for enhanced production of high-value carbon biorenewables without compromising growth.

Photosynthetic Carbon Partitioning and Metabolic Regulation in Response to Very-Low and High CO2 in Microchloropsis gaditana NIES 2587.

Photosynthetic organisms fix inorganic carbon through carbon capture machinery (CCM) that regulates the assimilation and accumulation of carbon around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). However, few constraints that govern the central carbon metabolism are regulated by the carbon capture and partitioning machinery. In order to divert the cellular metabolism toward lipids and/or biorenewables it is important to investigate and understand the molecular mechanisms of the CO2-driven carbon partitioning. In this context, strategies for enhancement of CO2 fixation which will increase the overall biomass and lipid yields, can provide clues on understanding the carbon assimilation pathway, and may lead to new targets for genetic engineering in microalgae. In the present study, we have focused on the physiological and metabolomic response occurring within marine oleaginous microalgae Microchloropsis gaditana NIES 2587, under the influence of very-low CO2 (VLC; 300 ppm, or 0.03%) and high CO2 (HC; 30,000 ppm, or 3% v/v). Our results demonstrate that HC supplementation in M. gaditana channelizes the carbon flux toward the production of long chain polyunsaturated fatty acids (LC-PUFAs) and also increases the overall biomass productivities (up to 2.0 fold). Also, the qualitative metabolomics has identified nearly 31 essential metabolites, among which there is a significant fold change observed in accumulation of sugars and alcohols such as galactose and phytol in VLC as compared to HC. In conclusion, our focus is to understand the entire carbon partitioning and metabolic regulation within these photosynthetic cell factories, which will be further evaluated through multiomics approach for enhanced productivities of biomass, biofuels, and bioproducts (B3).

Multifaceted applications of isolated microalgae Chlamydomonas sp. TRC-1 in wastewater remediation, lipid production and bioelectricity generation.

Green microalga, Chlamydomonas sp. TRC-1 (C. TRC-1), isolated from the outlet of effluent treatment plant of textile dyeing mill, was investigated for its competence towards bioremediation. Algal biomass obtained after remediation (ABAR) was implied for bioelectricity and biofuel production. C. TRC-1 could completely decolorize the effluent in 7 days. Significant reduction in pollution-indicating parameters was observed. Chronoamperometric studies were carried out using cyclic voltammetry and electrochemical impedance spectroscopy (EIS). Maximum current density, power and power density of 3.6 A m-2, 4.13 × 10-4 W and 1.83 W m-2, respectively were generated in ABAR. EIS studies showed a decrease in resistance of ABAR, supporting better electron transfer as compared to algal biomass before remediation (ABBR). Its candidature for biofuel production was assessed by estimating the total lipid content. Results revealed enhancement in lipid content from 46.85% (ABBR) to 79.1% (ABAR). Current study advocates versatile potential of isolated C. TRC-1 for bioremediation of wastewater, bioelectricity production and biofuel generation.

Molecular profiling of an oleaginous trebouxiophycean alga Parachlorella kessleri subjected to nutrient deprivation for enhanced biofuel production.

BACKGROUND: Decreasing fossil fuels and its impact on global warming have led to an increasing demand for its replacement by sustainable renewable biofuels. Microalgae may offer a potential feedstock for renewable biofuels capable of converting atmospheric CO2 to substantial biomass and valuable biofuels, which is of great importance for the food and energy industries. Parachlorella kessleri, a marine unicellular green alga belonging to class Trebouxiophyceae, accumulates large amount of lipids under nutrient-deprived conditions. The present study aims to understand the metabolic imprints in order to elucidate the physiological mechanisms of lipid accumulations in this microalga under nutrient deprivation. RESULTS: Molecular profiles were obtained using gas chromatography-mass spectrometry (GC-MS) of P. kessleri subjected to nutrient deprivation. Relative quantities of more than 60 metabolites were systematically compared in all the three starvation conditions. Our results demonstrate that in lipid metabolism, the quantities of neutral lipids increased significantly followed by the decrease in other metabolites involved in photosynthesis, and nitrogen assimilation. Nitrogen starvation seems to trigger the triacylglycerol (TAG) accumulation rapidly, while the microalga seems to tolerate phosphorous limitation, hence increasing both biomass and lipid content. The metabolomic and lipidomic profiles have identified a few common metabolites such as citric acid and 2-ketoglutaric acid which play significant role in diverting flux towards acetyl-CoA leading to accumulation of neutral lipids, whereas other molecules such as trehalose involve in cell growth regulation, when subjected to nutrient deprivation. CONCLUSIONS: Understanding the entire system through qualitative (untargeted) metabolome approach in P. kessleri has led to identification of relevant metabolites involved in the biosynthesis and degradation of precursor molecules that may have potential for biofuel production, aiming towards the vision of tomorrow's bioenergy needs.

Isolation, purification and properties of new restriction endonucleases from Bacillus badius and Bacillus lentus.

We tentatively named two enzymes as BbaI and BleI, which were isolated and purified from Gram-positive mesophilic bacteria Bacillus badius 1458 and Bacillus lentus 1689 respectively, by ammonium sulphate precipitation, phosphocellulose and heparin-sepharose column chromatography. SDS-PAGE protein profiles for BbaI and BleI showed denatured molecular weights of 52 and 48 kDa, respectively. BbaI hydrolyzed pUC18 DNA into 1900 and 700 bp, pBR322 DNA into two fragments of 2800 and 1500 bp and Phix174 DNA into 3800 and 1600 bp. BleI hydrolyzed pUC18 DNA into 1800 and 800 bp, pBR322 DNA into two fragments of 2700 and 1600 bp and Phix174 DNA into 3700 and 1700 bp. The effects of temperature, ionic strength, pH and Mg2+ ion concentrations were studied to demonstrate some biochemical properties of BbaI and BleI. Maximum activities of these enzymes were observed at 37 degrees C (pH 8.0) with 100 mM NaCl and 10 mM Mg2+ concentrations.

Bsu2413I and Bfi2411I, two new thermophilic type II restriction endonucleases from Bacillus subtilis and Bacillus firmus: isolation and partial purification. Thermophilic endonucleases from two Bacillus species.

Two new thermophilic type II restriction endonucleases, which we designated as Bsu2413I and Bfi2411I, have been isolated from gram-positive thermophilic bacteria Bacillus subtilis strain 2413 and Bacillus firmus strain 2411 respectively and partially purified. The restriction endonucleases were extracted from cell extracts and purified using single step purification through phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights of 33 and 67 kDa for the Bsu2413I and 39 and 67 kDa for the Bfi2411I. The partially purified Bsu2413I enzyme restricted pBR322 DNA into two fragments of 3250 and 1100 bp whereas Bfi2411I enzyme restricted pBR322 DNA into two fragments of 3500 and 800 bp. The activity of both endonucleases was assayed at 55 degrees C and they required Mg+2 as cofactor like other type II restriction endonucleases.

Isolation and partial purification of a novel type II restriction endonuclease Bsu121 I, from Bacillus subtilis. Bsu121I, a type II restriction endonuclease from Bacillus subtilis.

A new type II restriction endonuclease which we designated as Bsu121I has been isolated from gram-positive bacterium Bacillus subtilis strain 121 and partially purified. The restriction endonuclease was isolated from cell extracts using step-wise purification through ammonium sulfate precipitation, followed by phosphocellulose column chromatography. SDS-PAGE profile showed denatured molecular weights (23 and 67 kDa) of the endonuclease. The partially purified enzyme restricted pBR322 DNA into two fragments of 3200 and 1700 bp. The endonuclease activity required Mg(+2) as cofactor like other type II endonucleases.