Metabolic engineering of ketocarotenoids biosynthetic pathway in Chlamydomonas reinhardtii strain CC-4102.

In Chlamydomonas reinhardtii, ketocarotenoid biosynthesis is limited to the diploid zygospore stage. In this study, we attempted to engineer the ketocarotenoid pathway into Chlamydomonas haploid vegetative green cells by overexpressing the key enzyme ß-carotene ketolase (CrBKT). We chose strain CC-4102 for the approach; competitive pathways, α-carotene biosynthesis and xanthophyll cycle are silenced in this strain. Driven by the strong constitutive HSP70/RBCS2 promoter CrBKT overexpression resulted in the production of canthaxanthin, the ketolation product from ß-carotene as well as a drastic reduction in the chlorophyll concentration. Intriguingly, these phenotypes could only be detected from lines transformed and grown heterotrophically in the dark. Once exposed to light, these transformants lost the aforementioned phenotypes as well as their antibiotic resistance. This phenomenon is in agreement with the fact that we were unable to recover any canthaxanthin-producing line among light-selected transformants.

Improved production of echinenone and canthaxanthin in transgenic Nostoc sp. PCC 7120 overexpressing a heterologous crtO gene from Nostoc flagelliforme.

Echinenone and canthaxanthin are important carotenoid pigments with food and industrial applications. Biosynthesis of echinenone and/or canthaxanthin is catalyzed by ß-carotene ketolase (CrtO), with ß-carotene as the substrate. In this study, we generated transgenic Nostoc sp. PCC 7120 overexpressing a heterologous crtO gene from Nostoc flagelliforme and evaluated the productivity of both pigments. Normal (BG11 medium, 30 °C) and osmotic stress (BG11 medium supplemented with 0.4 M mannitol, 30 °C) conditions were used for cultivation. As compared to control strain, production of echinenone and canthaxanthin in transgenic strain were respectively increased by more than 16 % and 80 %, under either normal or osmotic stress conditions. Especially upon the stress condition, higher proportion of echinenone and canthaxanthin in total pigments was achieved, which should be beneficial for downstream separation and purification. In addition, transgenic strain showed drought tolerance and could revive from desiccation treatment after rewetting. Thus, this study provided technical clues for production of both pigments in engineered cyanobacteria as well as for cyanobacterial anhydrobiotic engineering.

Transfer of Meiothermus chliarophilus (Tenreiro et al.1995) Nobre et al. 1996, Meiothermus roseus Ming et al. 2016, Meiothermus terrae Yu et al. 2014 and Meiothermus timidus Pires et al. 2005, to Calidithermus gen. nov., as Calidithermus chliarophilus comb. nov., Calidithermus roseus comb. nov., Calidithermus terrae comb. nov. and Calidithermus timidus comb. nov., respectively, and emended description of the genus Meiothermus.

Chemotaxonomic parameters, phylogenetic analysis of the 16S rRNA gene, phylogenetic analysis of 90 housekeeping genes and 855 core genes, amino acid identity (AAI), average nucleotide identity (ANI) and genomic characteristics were used to examine the 13 species of the genus Meiothermuswith validly published names to reclassify this genus. The results indicate that the species of the genus Meiothermus can be divided into three lineages on the basis of the results of the phylogenetic analysis, AAI, the guanine+cytosine (G+C) mole ratio, the ability to synthesize the red-pigmented carotenoid canthaxanthin and the colony colour, as well as other genomic characteristics. The results presented in this study circumscribe the genus Meiothermus to the species Meithermus ruber, Meiothermus cateniformans, Meiothermus taiwanensis, Meiothermus cerbereus, Meiothermus hypogaeus, Meiothermus luteus, Meiothermus rufus and Meiothermus granaticius, for which it is necessary to emend the genus Meiothermus. The species Meiothermus silvanus, which clearly represents a separate genus level lineage was not reclassified in this study for lack of any distinctive phenotypic or genotypic characteristics. The results of this study led us to reclassify the species Meiothermus chliarophilus, Meiothermus timidus, Meiothermus roseus and Meiothermus terrae as species of a novel genus for which we propose the epithet Calidithermus gen. nov.

From Golden Rice to aSTARice: Bioengineering Astaxanthin Biosynthesis in Rice Endosperm.

Carotenoids are important phytonutrients with antioxidant properties, and are widely used in foods and feedstuffs as supplements. Astaxanthin, a red-colored ketocarotenoid, has strong antioxidant activity and thus can benefit human health. However, astaxanthin is not produced in most higher plants. Here we report the bioengineering of astaxanthin biosynthesis in rice endosperm by introducing four synthetic genes, sZmPSY1, sPaCrtI, sCrBKT, and sHpBHY, which encode the enzymes phytoene synthase, phytoene desaturase, ß-carotene ketolase, and ß-carotene hydroxylase, respectively. Transgneic overexpression of two (sZmPSY1 and sPaCrtI), three (sZmPSY1, sPaCrtI and sCrBKT), and all these four genes driven by rice endosperm-specific promoters established the carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways in the endosperm and thus resulted in various types of germplasm, from the yellow-grained ß-carotene-enriched Golden Rice to orange-red-grained Canthaxanthin Rice and Astaxanthin Rice, respectively. Grains of Astaxanthin Rice were enriched with astaxanthin in the endosperm and had higher antioxidant activity. These results proved that introduction of a minimal set of four transgenes enables de novo biosynthesis of astaxanthin in the rice endosperm. This work provides a successful example for synthetic biology in plants and biofortification in crops; the biofortified rice products generated by this study could be consumed as health-promoting foods and processed to produce dietary supplements.

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.

Synthesis of Carotenoids of Industrial Interest in the Photosynthetic Bacterium Rhodopseudomonas palustris : Bioengineering and Growth Conditions.

Rhodopseudomonas palustris is a purple photosynthetic bacterium that accumulates in the inner membrane the photosynthetic pigment spirilloxanthin, formed from lycopene. Here, we describe the procedures used to successfully engineer Rps. palustris strains to reroute the production of lycopene toward the synthesis of ß-carotene or canthaxanthin. The crtCD genes specifically involved in spirilloxanthin were replaced by crtY and crtW genes from Bradyrhizobium ORS278 to synthesize ß-carotene and (or) canthaxanthin, two pigments of industrial interest. Since the synthesis of canthaxanthin depends on the presence of oxygen, the procedure to optimize their production is also proposed. By modulating the light and oxygen during the growth process, a single species of photosynthetic bacteria, with an efficient growth rate, produces various carotenoids of economical interest.

Isolation of High Carotenoid-producing Aurantiochytrium sp. Mutants and Improvement of Astaxanthin Productivity Using Metabolic Information.

The marine eukaryotic microheterotroph thraustochytrid genus Aurantiochytrium is a known producer of polyunsaturated fatty acids, carotenoids, and squalene. We previously constructed a lipid fermentation system for Aurantiochytrium sp. strains using underutilized biomass, such as canned syrup and brown macroalgae. To improve the productivity, in this study, Aurantiochytrium sp. RH-7A and RH-7A-7 that produced high levels of carotenoids, such as astaxanthin and canthaxanthin, were isolated through chemical mutagenesis. Moreover, metabolomic analysis of the strain RH-7A revealed that oxidative stress impacts carotenoid accumulation. Accordingly, the addition of ferrous ion (Fe2+), as an oxidative stress compound, to the culture medium significantly enhanced the production of astaxanthin by the mutants. These approaches improved the productivity of astaxanthin up to 9.5 mg/L/day at the flask scale using not only glucose but also fructose which is the main carbon source in fermentation systems with syrup and brown algae as the raw materials.

Control of light-dependent keto carotenoid biosynthesis in Nostoc 7120 by the transcription factor NtcA.

In Nostoc PCC 7120, two different ketolases, CrtW and CrtO are involved in the formation of keto carotenoids from ß-carotene. In contrast to other cyanobacteria, CrtW catalyzes the formation of monoketo echinenone whereas CrtO is the only enzyme for the synthesis of diketo canthaxanthin. This is the major photo protective carotenoid in this cyanobacterium. Under high-light conditions, basic canthaxanthin formation was transcriptionally up-regulated. Upon transfer to high light, the transcript levels of all investigated carotenogenic genes including those coding for phytoene synthase, phytoene desaturase and both ketolases were increased. These transcription changes proceeded via binding of the transcription factor NtcA to the promoter regions of the carotenogenic genes. The binding was absolutely dependent on the presence of reductants and oxo-glutarate. Light-stimulated transcript formation was inhibited by DCMU. Therefore, photosynthetic electron transport is proposed as the sensor for high-light and a changing redox state as a signal for NtcA binding.

Ketocarotenoid Production in Soybean Seeds through Metabolic Engineering.

The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. Because soybean is already an important component in animal feed, production of these carotenoids in soybean could be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv. Jack was transformed with the crtB gene from Pantoea ananatis, which codes for phytoene synthase, an enzyme which catalyzes the first committed step in the carotenoid pathway. The crtB gene was engineered together in combinations with ketolase genes (crtW from Brevundimonas sp. strain SD212 and bkt1 from Haematococcus pluvialis) to produce ketocarotenoids; all genes were placed under the control of seed-specific promoters. HPLC results showed that canthaxanthin is present in the transgenic seeds at levels up to 52 µg/g dry weight. Transgenic seeds also accumulated other compounds in the carotenoid pathway, such as astaxanthin, lutein, ß-carotene, phytoene, α-carotene, lycopene, and ß-cryptoxanthin, whereas lutein was the only one of these detected in non-transgenic seeds. The accumulation of astaxanthin, which requires a ß-carotene hydroxylase in addition to a ß-carotene ketolase, in the transgenic seeds suggests that an endogenous soybean enzyme is able to work in combination with the ketolase transgene. Soybean seeds that accumulate ketocarotenoids could potentially be used in animal feed to reduce or eliminate the need for the costly addition of these compounds.

Regulation of astaxanthin and its intermediates through cloning and genetic transformation of ß-carotene ketolase in Haematococcus pluvialis.

Astaxanthin, a high-value ketocarotenoid used in the pharmaceutical and nutraceutical industries is mainly produced from green alga, Haematococcus pluvialis. It is biosynthesized by the action of key enzyme, ß-carotene ketolase (BKT) on ß-carotene through intermediates echinenone and canthaxanthin. In this study, the ß-carotene ketolase (bkt) gene was isolated from H. pluvialis and cloned in a vector pRT100 and further mobilized to a binary vector pCAMBIA 1304. The T-DNA of pCAMBIA 1304, which consists of cloned bkt, was successfully transformed to H. pluvialis through Agrobacterium mediation. The cloning and transformation of bkt in H. pluvialis was confirmed by Southern blotting and also by PCR analysis. Total carotenoids and astaxanthin content in the transformed cells were found to be 2-3-fold higher, while the intermediates like echinenone and canthaxanthin were found to be 8-10-fold higher than in the control cells. The expression level of carotenogenic genes like phytoene synthase (psy), phytoene desaturase (pds), lycopene cyclase (lcy), bkt, and ß-carotene hydroxylase (bkh) were found to be higher in transformed cells compared to the non-transformed (NT) H. pluvialis.

Canthaxanthin biosynthesis by Dietzia natronolimnaea HS-1: effects of inoculation and aeration rate.

The interest in production of natural colorants by microbial fermentation has been currently increased. The effects of D-glucose concentration (3.18-36.82 g/L), inoculum size (12.5 × 10(9)-49.5 × 10(9) cfu cells/mL) and air-flow rate (1.95-12.05 L/L min) on the biomass, total carotenoid and canthaxanthin (CTX) accumulation of Dietzia natronolimnaea HS-1 in a batch bioreactor was scrutinized using a response surface methodology-central composite rotatable design (RSM-CCRD). Second-order polynomial models with high R (2) values ranging from 0.978 to 0.990 were developed for the studied responses using multiple linear regression analysis. The models showed the maximum cumulative amounts of biomass (7.85 g/L), total carotenoid (5.48 mg/L) and CTX (4.99 mg/L) could be achieved at 23.38 g/L of D-glucose, 31.2 × 10(9) cfu cells/mL of inoculation intensity and air-flow rate of 7.85 L/L min. The predicted values for optimum conditions were in good agreement with experimental data.

Canthaxanthin biosynthesis by Dietzia natronolimnaea HS-1: effects of inoculation and aeration rate

The interest in production of natural colorants by microbial fermentation has been currently increased. The effects of D-glucose concentration (3.18-36.82 g/L), inoculum size (12.5 x 10(9)-49.5 x 10(9) cfu cells/mL) and air-flow rate (1.95-12.05 L/L min) on the biomass, total carotenoid and canthaxanthin (CTX) accumulation of Dietzia natronolimnaea HS-1 in a batch bioreactor was scrutinized using a response surface methodology-central composite rotatable design (RSM-CCRD). Second-order polynomial models with high R² values ranging from 0.978 to 0.990 were developed for the studied responses using multiple linear regression analysis. The models showed the maximum cumulative amounts of biomass (7.85 g/L), total carotenoid (5.48 mg/L) and CTX (4.99 mg/L) could be achieved at 23.38 g/L of D-glucose, 31.2 x 10(9) cfu cells/mL of inoculation intensity and air-flow rate of 7.85 L/L min. The predicted values for optimum conditions were in good agreement with experimental data.

Biotechnological production of carotenoids by yeasts: an overview.

Nowadays, carotenoids are valuable molecules in different industries such as chemical, pharmaceutical, poultry, food and cosmetics. These pigments not only can act as vitamin A precursors, but also they have coloring and antioxidant properties, which have attracted the attention of the industries and researchers. The carotenoid production through chemical synthesis or extraction from plants is limited by low yields that results in high production costs. This leads to research of microbial production of carotenoids, as an alternative that has shown better yields than other aforementioned. In addition, the microbial production of carotenoids could be a better option about costs, looking for alternatives like the use of low-cost substrates as agro-industrials wastes. Yeasts have demonstrated to be carotenoid producer showing an important growing capacity in several agro-industrial wastes producing high levels of carotenoids. Agro-industrial wastes provide carbon and nitrogen source necessary, and others elements to carry out the microbial metabolism diminishing the production costs and avoiding pollution from these agro-industrial wastes to the environmental. Herein, we discuss the general and applied concepts regarding yeasts carotenoid production and the factors influencing carotenogenesis using agro-industrial wastes as low-cost substrates.

Feeding strategies for the improved biosynthesis of canthaxanthin from enzymatic hydrolyzed molasses in the fed-batch fermentation of Dietzia natronolimnaea HS-1.

The effect of two enzymatic hydrolyzed molasses (EHM)-feeding strategies including constant-(CFR) and exponential-(EFR) feeding rate on canthaxanthin (CTX) biosynthesis by Dietzia natronolimnaea HS-1 fed-batch fermentation was studied. The results showed that the CFR of 7 ml/h with an EHM content of 45 g/l led to the highest values of specific growth rate (0.127 h(-1)), biomass dry weight (17.66 g/l), total carotenoid (16.31 mg/l) and CTX (14.67 mg/l). A significant decrease in the kinetic growth and production parameters by the increasing EHM concentration from 30 to 60 g/l during EFR fed-batch bioprocess was observed (p<0.01). This study concluded that EHM alone can displace glucose-based medium towards improved CTX biosynthesis from D. natronolimnaea HS-1 using a CFR strategy during fed-batch culture.

Induction of canthaxanthin production in a Dactylococcus microalga isolated from the Algerian Sahara.

Secondary carotenoids are high-valued anti-oxidants which can be produced by some algae when exposed to an environmental stress (e.g. nutrient deprivation, high light intensities). To this end, we characterized the stress-induced carotenoid production of a new microalgal strain, Dactylococcus dissociatus MT1, which was isolated from the Sahara Desert of Algeria. Nitrate starvation, oxidative stress and varying light intensities were applied to determine the effect of illumination on carotenogenesis. Canthaxanthin was the main secondary carotenoid and light intensity had an important influence on the rate of its accumulation. The addition of NaCl also enhanced canthaxanthin production while nitrate depletion had more of an effect on lipid production. However, these two stresses in combination synergistically increased the production of both. Our results represent a step toward the development of strains suitable for secondary carotenoid production at the industrial scale.

Anti-oxidative, anti-tumor-promoting, and anti-carcinogenic activities of adonirubin and adonixanthin.

Anti-oxidative, anti-tumor-promoting, and anti-carcinogenic activities of adonirubin and adonixanthin, which are biosynthetic intermediates from ß-carotene to astaxanthin, were investigated. Both adonirubin and adonixanthin showed almost the same activities for inhibition of lipid peroxidation and quenching of singlet oxygen as those of astaxanthin. Furthermore, adonirubin and adonixanthin exhibited an inhibitory effect on Epstein-Barr virus early antigen activation in Raji cells and carcinogensis of mouse skin tumors initiated by 7,12-dimethylbenz[a]anthracene and promoted by 12-O-tetradecanoylphorbol-13-acetate.

Multiple ketolases involved in light regulation of canthaxanthin biosynthesis in Nostoc punctiforme PCC 73102.

In the genome of Nostoc punctiforme PCC 73102, three functional ß-carotene ketolase genes exist, one of the crtO and two of the crtW type. They were all expressed and their corresponding enzymes were functional inserting 4-keto groups into ß-carotene as shown by functional pathway complementation in Escherichia coli. They all synthesized canthaxanthin but with different efficiencies. Canthaxanthin is the photoprotective carotenoid of N. punctiforme PCC 73102. Under high-light stress, its synthesis was enhanced. This was caused by up-regulation of the transcripts of two genes in combination. The first crtB-encoding phytoene synthase is the gate way enzyme of carotenogenesis resulting in an increased inflow into the pathway. The second was the ketolase gene crtW148 which in high light takes over ß-carotene conversion into canthaxanthin from the other ketolases. The other ketolases were down-regulated under high-light conditions. CrtW148 was also exclusively responsible for the last step in 4-keto-myxoxanthophyll synthesis.

Isolation and characterization of a lycopene ε-cyclase gene of Chlorella (Chromochloris) zofingiensis. Regulation of the carotenogenic pathway by nitrogen and light.

The isolation and characterization of the lycopene ε-cyclase gene from the green microalga Chlorella (Chromochloris) zofingiensis (Czlcy-e) was performed. This gene is involved in the formation of the carotenoids α-carotene and lutein. Czlcy-e gene encoded a polypeptide of 654 amino acids. A single copy of Czlcy-e was found in C. zofingiensis. Functional analysis by heterologous complementation in Escherichia coli showed the ability of this protein to convert lycopene to δ-carotene. In addition, the regulation of the carotenogenic pathway by light and nitrogen was also studied in C. zofingiensis. High irradiance stress did not increase mRNA levels of neither lycopene ß-cyclase gene (lcy-b) nor lycopene ε-cyclase gene (lcy-e) as compared with low irradiance conditions, whereas the transcript levels of psy, pds, chyB and bkt genes were enhanced, nevertheless triggering the synthesis of the secondary carotenoids astaxanthin, canthaxanthin and zeaxanthin and decreasing the levels of the primary carotenoids α-carotene, lutein, violaxanthin and ß-carotene. Nitrogen starvation per se enhanced mRNA levels of all genes considered, except lcy-e and pds, but did not trigger the synthesis of astaxanthin, canthaxanthin nor zeaxanthin. The combined effect of both high light and nitrogen starvation stresses enhanced significantly the accumulation of these carotenoids as well as the transcript levels of bkt gene, as compared with the effect of only high irradiance stress.

Developing an emulsion model system containing canthaxanthin biosynthesized by Dietzia natronolimnaea HS-1.

An acceptable strategy to incorporate canthaxanthin (CX) as a natural colorant into products is by means of oil-in-water emulsions. The used CX in this study was produced by bacterium Dietzia natronolimnaea HS-1 using a batch bioreactor system. A central composite rotatable design-response surface methodology (CCRD-RSM) consisting of three-factored factorial design with five levels was applied for analysis of the results to obtain the optimal formulation of emulsions. Three independent variables including fenugreek gum (FG, 0.2-0.5%, w/w), coconut oil (CO, 6-10%, w/w), and CO/CX ratio (10:1-50:1) were transformed to coded values and second-order polynomial models was developed to predict the responses (p<0.0001). The studied independent variables were the stability, viscosity and droplet size properties such as volume-weighted mean diameter (D43), specific surface area (S(v)) and polydispersity index (PDI) of emulsions. The 3-D response surface plot derived from the mathematical models was used to determine the optimal conditions. Main emulsion components under the optimum conditions ascertained presently by RSM: 50:1 CO/CX ratio, 0.49% (w/w) FG content and 6.28% (w/w) CO concentration. At this optimum point, stability, viscosity, D43, S(v) and PDI were 90.6%, 0.0118 Pas, 0.595 µm, 12.03 m²/ml and 1.380, respectively.