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.

Evolving perspectives on lutein production from microalgae - A focus on productivity and heterotrophic culture.

Increased consumer awareness for healthier and more sustainable products has driven the search for naturally sourced compounds as substitutes for chemically synthesized counterparts. Research on pigments of natural origin, such as carotenoids, particularly lutein, has been increasing for over three decades. Lutein is recognized for its antioxidant and photoprotective activity. Its ability to cross the blood-brain barrier allows it to act at the eye and brain level and has been linked to benefits for vision, cognitive function and other conditions. While marigold flower is positioned as the only crop from which lutein is extracted from and commercialized, microalgae are proposed as an alternative with several advantages over this terrestrial crop. The main barrier to scaling up lutein production from microalgae to the commercial level is the low productivity compared to the high costs. This review explores strategies to enhance lutein production in microalgae by emphasizing the overall productivity over lutein content alone. Evaluation of how culture parameters, such as light quality, nitrogen sufficiency, temperature and even stress factors, affect lutein content and biomass development in batch phototrophic cultures was performed. Overall, the total lutein production remains low under this metabolic regime due to the low biomass productivity of photosynthetic batch cultures. For this reason, we describe findings on microalgal cultures grown under different metabolic regimes and culture protocols (fed-batch, pulse-feed, semi-batch, semi-continuous, continuous). After a careful literature examination, two-step heterotrophic or mixotrophic cultivation strategies are suggested to surpass the lutein productivity achieved in single-step photosynthetic cultures. Furthermore, this review highlights the urgent need to develop technical feasibility studies at a pilot scale for these cultivation strategies, which will strengthen the necessary techno-economic analyses to drive their commercial production.

Developing a Chromochloris zofingiensis Mutant for Enhanced Production of Lutein under CO2 Aeration.

Microalgae are competitive and commercial sources for health-benefit carotenoids. In this study, a Chromochloris zofingiensis mutant (Cz-pkg), which does not shut off its photosystem and stays green upon glucose treatment, was generated and characterized. Cz-pkg was developed by treating the algal cells with a chemical mutagen as N-methyl-N'-nitro-N-nitrosoguanidine and followed by a color-based colony screening approach. Cz-pkg was found to contain a dysfunctional cGMP-dependent protein kinase (PKG). By cultivated with CO2 aeration under mixotrophy, the mutant accumulated lutein up to 31.93 ± 1.91 mg L-1 with a productivity of 10.57 ± 0.73 mg L-1 day-1, which were about 2.5- and 8.5-fold of its mother strain. Besides, the lutein content of Cz-pkg could reach 7.73 ± 0.52 mg g-1 of dry weight, which is much higher than that of marigold flower, the most common commercial source of lutein. Transcriptomic analysis revealed that in the mutant Cz-pkg, most of the genes involved in the biosynthesis of lutein and chlorophylls were not down-regulated upon glucose addition, suggesting that PKG may regulate the metabolisms of photosynthetic pigments. This study demonstrated that Cz-pkg could serve as a promising strain for both lutein production and glucose sensing study.

AgZDS, a gene encoding ζ-carotene desaturase, increases lutein and ß-carotene contents in transgenic Arabidopsis and celery.

ζ-Carotene desaturase (ZDS) is one of the key enzymes regulating carotenoids biosynthesis and accumulation. Celery transgenic efficiency is low and it is difficult to obtain transgenic plants. The study on ZDS was limited in celery. Here, the AgZDS gene was cloned from celery and overexpressed in Arabidopsis thaliana and celery to verify its function. The AgZDS has typical characteristic of ZDS protein and is highly conserved in higher plants. Phylogenetic analysis showed that AgZDS has the closest evolutionary relationship with ZDSs from Solanum lycopersicum, Capsicum annuum and Tagetes erecta. Overexpression of AgZDS gene in A. thaliana and celery resulted in increased accumulations of lutein and ß-carotene and up-regulated the expression levels of the genes involved in carotenoids biosynthesis. The contents of lutein and ß-carotene in two lines, AtL1 and AgL5, were the highest in transgenic A. thaliana and celery, respectively. The relative expression levels of 5 genes (AtPDS, AtZISO, AtZEP, AtNCED3, and AtCCD4) were up-regulated compared to the wild type plants. The relative expression levels of most genes in carotenoids biosynthesis pathway, such as AgPDS, AgCRTISO1, and AgZISO, were up-regulated in transgenic celery plants. The antioxidant capacity of A. thaliana and photosynthetic capacity of celery were also enhanced. This research is the first report on the function of structure gene related to carotenoid biosynthesis in transgenic celery plants. The findings in this study demonstrated the roles of AgZDS in regulating carotenoids metabolism of celery, which laid a potential foundation for quality improvement of celery.

Structural basis for plant lutein biosynthesis from α-carotene.

Two cytochrome P450 enzymes, CYP97A3 and CYP97C1, catalyze hydroxylations of the ß- and ε-rings of α-carotene to produce lutein. Chirality is introduced at the C-3 atom of both rings, and the reactions are both pro-3R-stereospecific. We determined the crystal structures of CYP97A3 in substrate-free and complex forms with a nonnatural substrate and the structure of CYP97C1 in a detergent-bound form. The structures of CYP97A3 in different states show the substrate channel and the structure of CYP97C1 bound with octylthioglucoside confirms the binding site for the carotenoid substrate. Biochemical assays confirm that the ferredoxin-NADP+ reductase (FNR)-ferredoxin pair is used as the redox partner. Details of the pro-3R stereospecificity are revealed in the retinal-bound CYP97A3 structure. Further analysis indicates that the CYP97B clan bears similarity to the ß-ring-specific CYP97A clan. Overall, our research describes the molecular basis for the last steps of lutein biosynthesis.

Functional Identification of Two Types of Carotene Hydroxylases from the Green Alga Dunaliella bardawil Rich in Lutein.

The salt-tolerant unicellular alga Dunaliella bardawil FACHB-847 can accumulate large amounts of lutein, but the underlying cause of massive accumulation of lutein is still unknown. In this study, genes encoding two types of carotene hydroxylases, i.e., ß-carotene hydroxylase (DbBCH) and cytochrome P450 carotenoid hydroxylase (DbCYP97s; DbCYP97A, DbCYP97B, and DbCYP97C), were cloned from D. bardawil. Their substrate specificities and enzyme activities were tested through functional complementation assays in Escherichia coli. It was showed that DbBCH could catalyze the hydroxylation of the ß-rings of both ß- and α-carotene, and displayed a low level of ε-hydroxylase. Unlike CYP97A from higher plants, DbCYP97A could not hydroxylate ß-carotene. DbCYP97A and DbCYP97C showed high hydroxylase activity toward the ß-ring and ε-ring of α-carotene, respectively. DbCYP97B displayed minor activity toward the ß-ring of α-carotene. The high accumulation of lutein in D. bardawil may be due to the multiple pathways for lutein biosynthesis generated from α-carotene with zeinoxanthin or α-cryptoxanthin as intermediates by DbBCH and DbCYP97s. Taken together, this study provides insights for understanding the underlying reason for high production of lutein in the halophilic green alga D. bardawil FACHB-847.

Metabolic Engineering of Chlamydomonas reinhardtii for Enhanced ß-Carotene and Lutein Production.

The metabolic engineering of Chlamydomonas reinhardtii, one of the fastest-growing microalgae, is a potential alternative for enhanced carotenoid productivity. CrtYB (phytoene-ß-carotene synthase - PBS) gene from red yeast Xanthophyllomyces dendrorhous encodes for a bifunctional enzyme that harbours both phytoene synthase (psy) and lycopene cyclization (lcyb) activities. Heterologous expression of this bifunctional PBS gene led to 38% enhancement in ß-carotene along with 60% increase in the lutein yields under low light conditions of 75 µmol photons m-2 s-1. Short Duration-High Light induction strategy led to overall 72% and 83% increase in ß-carotene and lutein yield reaching up to 22.8 mg g-1 and 8.9 mg g-1, respectively. This is the first report of expression of heterologous bifunctional PBS gene resulting in simultaneous enhancement in ß-carotene and lutein content in phototrophic engineered cells. Graphical Abstract.

The growth and lutein accumulation in heterotrophic Chlorella protothecoides provoked by waste Monascus fermentation broth feeding.

Although the potential of heterotrophic microalgae served as a sustainable source for lutein, it was still crucial to formulate a suitable medium to offset the cost involved in algal biomass cultivation while improve inherent lutein productivity. The objective of this study was to investigate the feasibilities of waste Monascus fermentation broth medium (MFBM) toward heterotrophic Chlorella protothecoides-enriched lutein. The results indicated that C. protothecoides subjected to MFBM batch feeding achieved 7.1 g/L biomass and 7.27 mg/g lutein. The resulting lutein productivity (7.34 mg/L/day) represented 1.54-fold more than that of frequently used Basal medium. Concurrently, the effective metabolism and absorption of carbon, nitrogen, and phosphorus in MFBM by C. subellipsoidea cultivation make it easily complied with the permissible dischargeable limits for fermentation broth. When response to fed-batch culture mode, the biomass and lutein productivity peaked 20.4 g/L and 9.11 mg/L/day with concentrated MFBM feeding. Transcriptomics data hinted that MFBM feeding manipulated lutein biosynthesis key checkpoints (e.g., lycopene ß-cyclase and lycopene ε-cyclase) while accelerated energy pathways (e.g., glycolysis and TCA cycle) to contribute such high lutein productivity in C. protothecoides. These encouraging findings not only provided indications in applying nutrient-rich fermentation broth for affordable microalgae cultivation but also presented possibilities in linking algal high value-added products like lutein with high-efficient biological nutrition removal from industrial fermentation processing.

Enhancing production of lutein by a mixotrophic cultivation system using microalga Scenedesmus obliquus CWL-1.

This work studies a series of strategies in the production of lutein by Scenedesmus obliquus CWL-1 under mixotrophic cultivation. Our experimental results revealed that the optimal conditions associated with light-related strategies were 12 h light period followed by a 12 h dark period and blue to red light under mixotrophic cultivation. Under such conditions, the biomass, lutein content and lutein productivity were maximized to 9.88 (g/L), 1.78 (mg/g) and 1.43 (mg/L/day), respectively. Moreover, the assimilation of 4.5 g/L of calcium nitrate into S. obliquus CWL-1 increased the maximal biomass (12.73 g/L) and the highest maximal lutein productivity (3.06 mg/L/day), while the assimilation of 1.5 g/L of calcium nitrate yielded the highest maximal lutein content of 2.45 mg/g. The highest maximal lutein productivity of 4.96 (mg/L/day) was obtained when fed-batch fermentation was conducted, and this value was approximately 11-folds that obtained using the batch system.

Exploring biostimulation of plant hormones and nitrate supplement to effectively enhance biomass growth and lutein production with thermo-tolerant Desmodesmus sp. F51.

In this study, the interactive effect of plant hormone-salicylic acid and succinic acid on biomass growth, lutein content, and productivity of Desmodesmus sp. F51 were investigated. The results demonstrated that the synergistic action of salicylic acid and succinic acid could effectively enhance the assimilation of nitrate and significantly improve lutein production. The maximal lutein content 7.01 mg/g and productivity 5.11 mg/L/d could be obtained with a supplement of 100 µM salicylic acid and 2.5 mM succinic acid in batch culture. Furthermore, operation strategy of nitrate fed-batch coupled with supplementation for succinic acid and salicylic acid resulted in further enhancement of lutein content and productivity by 7.50 mg/g and 5.78 mg/L/d, respectively. The performance is better than most of the previously reported values.

Functional Analysis of the Marigold (Tagetes erecta) Lycopene ε-cyclase (TeLCYe) Promoter in Transgenic Tobacco.

Lycopene ε-cyclases (LCYEs) are key enzymes in carotenoid biosynthesis converting red lycopene to downstream lutein. The flowers of marigold (Tagetes erecta) have been superior sources to supply lutein. However, the transcriptional regulatory mechanisms of LCYe in lutein synthesis are still unclear in marigold. In this work, the expression pattern of the TeLCYe gene in marigold indicated that TeLCYe mainly expressed in floral organs. To gain a better understanding of the expression and regulatory mechanism of TeLCYe gene, the TeLCYe promoter was isolated, sequenced, and analyzed through bioinformatics tools. The results suggested that the sequence of TeLCYe promoter contained various putative cis-acting elements responsive to exogenous and endogenous factors. The full-length TeLCYe promoter and three 5'-deletion fragments were fused to the GUS reporter gene and transferred into tobacco to test the promoter activities. A strong GUS activity was observed in stems of seedlings, leaves of seedlings, middle stems, top leaves, petals, stamens, and stigmas in transgenic tobacco containing full-length TeLCYe promoter LP0-2086. Deletion of - 910 to - 429 bp 5' to ATG significantly increased the GUS activity in chloroplast-rich tissues and floral organs, while deletion occurring from 1170 to 910 bp upstream ATG decreased the TeLCYe promoter strength in stems of seedlings, leaves of seedlings, top leaves and sepals. Functional characterization of the full-length TeLCYe promoter and its' deletion fragments in stable transgenic tobacco indicated that the LP0-2086 contains some specific cis-acting elements, which might result in the high-level expression of in floral organs, and LP2-910 might contain some specific cis-acting elements which improved GUS activities in vegetable tissues.

Effect of light quality on growth rate, carbohydrate accumulation, fatty acid profile and lutein biosynthesis of Chlorella sp. AE10.

Microalgae are feedstocks for multiple product development based on algal biorefinery concept. The effects of light quality (white, red and blue light emitting diodes) and macro-element starvations on Chlorella sp. AE10 were investigated under 20% CO2 and 850 µmol m-2 d-1. Nitrogen and phosphorus starvations had negative effects on its growth rate. The biomass productivities were decreased from day 1 and the highest one was 1.90 g L-1 d-1 under white light conditions. Phosphorus starvation promoted carbohydrate accumulation under three LED light sources conditions and the highest carbohydrate content was 75.9% using red light. Blue light increased lutein content to 9.58 mg g-1. The content of saturated fatty acids was significantly increased from 37.51% under blue light and full culture medium conditions to 77.44% under blue light and nitrogen starvation conditions. Chlorella sp. AE10 was a good candidate for carbohydrate and lutein productions.

Enhancing lutein production with mixotrophic cultivation of Chlorella sorokiniana MB-1-M12 using different bioprocess operation strategies.

A lutein-enriched mutant, Chlorella sorokiniana MB-1-M12 was grown mixotrophically for lutein production. The lutein production efficiency of the strain was enhanced via optimizing the operating strategies. The results show that using semi-continuous cultivation with a medium replacement ratio of 75% resulted in a higher lutein productivity and lutein concentration of 6.24 mg/L/d and 50.6 mg/L, respectively, which were markedly higher than those obtained from batch and fed-batch cultivation. Cultivation under simulated outdoor cultivation conditions (i.e., temperature of 35 °C/25 °C for a 12 h/12 h light/dark cycle) could achieve the highest lutein productivity and lutein concentration of 3.34 mg/L/d and 30.8 mg/L, respectively. Lutein production via outdoor cultivation of MB-1-M12 strain with a 60-L tubular photobioreactor was performed using semi-continuous operation. With a medium replacement ratio of 75%, a good lutein productivity (4.46 mg/L/d) and concentration (27.4 mg/L) was obtained, indicating the feasibility of producing lutein under outdoor cultivation of the microalgal strain.

Enhancing lutein productivity of Chlamydomonas sp. via high-intensity light exposure with corresponding carotenogenic genes expression profiles.

The marine microalga Chlamydomonas sp. JSC4 is a potential lutein source with high light tolerance. In this study, light intensity was manipulated to enhance cell growth and lutein production of this microalga. High lutein productivity (5.08 mg/L/d) was achieved under high light irradiation of 625 µmol/m2/s. Further increase in light intensity to 750 µmol/m2/s enhanced the biomass productivity to 1821.5 mg/L/d, but led to a decrease in lutein content. Under high light conditions, most carotenoids and chlorophyll contents decreased, while zeaxanthin and antheraxanthin contents increased. Inspection of gene expression profile shows that the lut1 and zep genes, responsible for lutein synthesis and flow of zeaxanthin into violaxanthin, respectively, were downregulated, while zeaxanthin biosynthesis gene crtZ was upregulated when the microalga was exposed to a high light intensity. This is consistent with the decrease in lutein content and increase in zeaxanthin content under high light exposure.

Strategies related to light quality and temperature to improve lutein production of marine microalga Chlamydomonas sp.

The marine microalga Chlamydomonas sp. JSC4 was examined for its potential as a lutein producer. Environmental conditions, including light quality, temperature and light wavelength mixing ratio, were individually altered to enhance the cell growth rate and lutein production in strain JSC4. Results showed that optimal cell growth was obtained under white light and a temperature of 35 °C, while the optimal lutein content was obtained under blue light and a lower temperature of 20-25 °C. The best lutein production occurred when using a mixing ratio of 3:1 (white light: blue light). Strategies related to light quality and temperature (namely, temperature-gradient and two-stage strategies) were then used to further improve lutein production. Among them, the two-stage strategy proved to be effective markedly improving lutein content from 2.52 to 4.24 mg/g and resulting in the highest lutein productivity of 3.25 mg/L/day.

Manipulating Nutritional Conditions and Salinity-Gradient Stress for Enhanced Lutein Production in Marine Microalga Chlamydomonas sp.

Marine microalgae has great potential for lutein production with the advantage of saving fresh water resource. Thus, marine microalga Chlamydomonas sp. JSC4 is investigated as a potential lutein producer in this study. The medium types, nitrate-N and sea salt concentration are individually investigated to promote the cell growth rate and lutein production of JSC4. In Modified Bold Basal 3N medium, cell growth and lutein content are optimal at the nitrate-N concentration of 1000 mg L-1 and sea salt concentration of 2%. In addition, an innovative salinity-gradient strategy is operated to dramatically enhance biomass productivity (560 mg/L/d) and lutein content (3.42 mg g-1 ), resulting in the optimal lutein productivity (1.92 mg/L/d). Overall, this study clearly demonstrates that salinity is a significant inducer of lutein accumulation by strain JSC4 and that lutein production can be successfully optimized using the salinity-gradient strategy, which is beneficial for the outdoor large-scale lutein production in the future.

Photosynthetic Accumulation of Lutein in Auxenochlorella protothecoides after Heterotrophic Growth.

In order to enhance lutein accumulation and to explain the reasons for the difference in lutein accumulation under photoautotrophic and heterotrophic conditions, different culture modes and the associated transcriptome profiles were investigated in Auxenochlorella protothecoides. The heterotrophic-photoautotrophic transition culture mode was investigated for lutein accumulation, changing from organic carbon to increase biomass in dark fermentation to irradiation under nitrogen rich conditions. This strategy increased the lutein content 10 times along with chloroplast regeneration and little biomass loss in 48 h. The highest lutein productivity and production in the heterotrophic-photoautotrophic transition culture reached 12.36 mg/L/day and 34.13 mg/L respectively within seven days. Furthermore, compared to the photoautotrophic conditions, most genes involved in lutein biosynthesis and photosystem generation were down-regulated during heterotrophic growth. By contrast, two ß-ring hydroxylases were transiently upregulated, while violaxanthin de-epoxidase and zeaxanthin epoxidase were mostly downregulated, which explained the extremely low lutein content of heterotrophic cells. Nevertheless, the lutein proportion in total carotenoids reached nearly 100%. This study is the first to our knowledge to report on a comparative transcriptome analysis of lutein biosynthesis, and it provides a promising strategy to boost lutein production in A. protothecoides.

Carotenoids Production: A Healthy and Profitable Industry.

Carotenoids relevance as natural pigments is mainly due to their uses as colorants, feed supplements, nutraceuticals and for medical, cosmetic, and biotechnological purposes. Since they have putative health beneficial effects, the demand and market of carotenoids are growing significantly. There is a diversity of natural and synthetic carotenoids, but only a few of them are commercially produced, including carotenes (ß-carotene and lycopene) and xanthophylls (astaxanthin, canthaxanthin, lutein, zeaxanthin, and capsanthin). Some biotechnological processes for carotenoids production were established some years ago, but new strains and technologies are being developed nowadays for carotenoids widely in demand. This chapter shows a revision of the main carotenoids from a commercial point of view.

Induced High-Yield Production of Zeaxanthin, Lutein, and ß-Carotene by a Mutant of Chlorella zofingiensis.

Natural resources of zeaxanthin are extremely limited. A Chlorella zofingiensis mutant (CZ-bkt1), which could accumulate high amounts of zeaxanthin, was generated and characterized. CZ-bkt1 was achieved by treating the algal cells with a chemical mutagen followed by a color-based colony-screening approach. CZ-bkt1 was found to consist of a dysfunctional carotenoid ketolase, leading to the accumulation of zeaxanthin rather than to its downstream ketocarotenoid astaxanthin. Light irradiation, glucose, NaCl, and nitrogen deficiency all induced CZ-bkt1 to accumulate zeaxanthin. CZ-bkt1 accumulated zeaxanthin up to 7.00 ± 0.82 mg/g when induced by high-light irradiation and nitrogen deficiency and up to 36.79 ± 2.23 mg/L by additional feeding with glucose. Furthermore, in addition to zeaxanthin, CZ-bkt1 also accumulated high amounts of ß-carotene (7.18 ± 0.72 mg/g or 34.64 ± 1.39 mg/L) and lutein (13.81 ± 1.23 mg/g or 33.97 ± 2.61 mg/L). CZ-bkt1 is the sole species up to date with the ability to accumulate high amounts of the three carotenoids that are essential for human health.

Photoautotrophic production of macular pigment in a Chlamydomonas reinhardtii strain generated by using DNA-free CRISPR-Cas9 RNP-mediated mutagenesis.

Lutein and zeaxanthin are dietary carotenoids reported to be protective against age-related macular degeneration. Recently, the green alga Chlamydomonas reinhardtii has received attention as a photosynthetic cell factory, but the potential of this alga for carotenoid production has not yet been evaluated. In this study, we selected the C. reinhardtii CC-4349 strain as the best candidate among seven laboratory strains tested for carotenoid production. A knock-out mutant of the zeaxanthin epoxidase gene induced by preassembled DNA-free CRISPR-Cas9 ribonucleoproteins in the CC-4349 strain had a significantly higher zeaxanthin content (56-fold) and productivity (47-fold) than the wild type without the reduction in lutein level. Furthermore, we produced eggs fortified with lutein (2-fold) and zeaxanthin (2.2-fold) by feeding hens a diet containing the mutant. Our results clearly demonstrate the possibility of cost-effective commercial use of microalgal mutants induced by DNA-free CRISPR-Cas9 ribonucleoproteins in algal biotechnology for the production of high-value products.