ISOLATION AND CHARACTERIZATION OF THE PHYTOENE DESATURASE GENE AS A POTENTIAL SELECTIVE MARKER FOR GENETIC ENGINEERING OF THE ASTAXANTHIN‐PRODUCING GREEN ALGA CHLORELLA ZOFINGIENSIS (CHLOROPHYTA)1

Phytoene desaturase (PDS) is a rate‐limiting enzyme in carotenoid biosynthesis. Algal PDS is inhibited by some herbicides, leading to the bleaching of the cells due to destruction of chl. Specific point mutations in PDS confer resistance to the herbicide norflurazon, suggesting that mutated PDS could be used as a dominant selectable marker for genetic engineering of algae, for which very few selective markers are available. In this study, we report the isolation and characterization of the PDS gene from the astaxanthin‐producing green alga Chlorella zofingiensis Dönz. The open reading frame (ORF) of this PDS gene, interrupted by six introns, encoded a polypeptide of 558 amino acid residues. The deduced protein sequence showed significant homology to phytoene desaturases of algae, cyanobacteria, and higher plants. Expression of the PDS gene in Escherichia coli demonstrated that the enzyme was able to convert phytoene to ζ‐carotene. The PDS gene in Chlorella was shown to be up‐regulated by high light and glucose treatment. With a single amino acid change (L516R), the mutated PDS‐L516R was still active and exhibited ～36‐fold greater resistance to the bleaching herbicide norflurazon than the unaltered enzyme. Thus, the modified PDS gene could be a useful tool for genetic engineering of carotenoid biosynthesis in C. zofingiensis and perhaps also in other algae.     

The Effect of Salt Stress on the Production of Canthaxanthin and Astaxanthin by Chlorella zofingiensis Grown Under Limited Light Intensity

The fresh water green microalga Chlorella zofingiensisis known to accumulate ketocarotenoids – primarily astaxanthin but also canthaxanthin – when grown under stress conditions of high light irradiance and low nitrogen. We found that salt stress can replace light stress with respect to inducing carotenoid production: cells of C. zofingiensis grown under low light irradiance and subjected to salt and low nitrogen stress accumulated higher amounts of total secondary carotenoids than those growing under high light and low nitrogen stress. Furthermore, C. zofingiensis growing under conditions of salt stress and low light accumulated higher amounts of canthaxanthin than astaxanthin. It is suggested that for canthaxanthin accumulation under salt stress, light is not a limiting factor, but for astaxanthin accumulation high light irradiance is mandatory. These results may be applied in the future for the commercial production of canthaxanthin by C. zofingiensis in systems in which light availability is poor.     

Isolation and characterization of a carotenoid oxygenase gene from Chlorella zofingiensis (Chlorophyta)

The green alga Chlorella zofingiensis produces large amounts of the valuable ketocarotenoid astaxanthin under dark, heterotrophic growth conditions, making it potentially employable for commercial production of astaxanthin as feed additives, colorants, and health products. Here, we report the identification and characterization of a β-carotene oxygenase (CRTO) gene that is directly involved in the biosynthesis of ketocarotenoids in C. zofingiensis. The open reading frame of the crtO gene, which is interrupted by three introns of 243, 318, and 351 bp, respectively, encodes a polypeptide of 312 amino acid residues. Only one crtO gene was detected in the genome of C. zofingiensis. Furthermore, the expression of the crtO gene was transiently up-regulated upon glucose treatment. Functional complementation in Escherichia coli showed that the coding protein of the crtO gene not only exhibits normal CRTO activity by converting β-carotene to canthaxanthin via echinenone, but also displays a high enzymatic activity of converting zeaxanthin to astaxanthin via adonixanthin. Based on the bifunctional CRTO, a predicted pathway for astaxanthin biosynthesis in C. zofingiensis is described, and the CRTO is termed as carotenoid 4,4′-β-ionone ring oxygenase.     

The biosynthetic pathway of carotenoids in the astaxanthin-producing green alga <Emphasis Type="Italic">Chlorella zofingiensis</Emphasis>

The carotenoid composition of the astaxanthin-producing green alga Chlorella zofingiensis was investigated using high-performance liquid chromatography. Astaxanthin, adonixanthin, and zeaxanthin are the major carotenoids in this alga. The pigment pattern was characterized during the accumulation period, and in response to diphenylamine (DPA), an inhibitor of carotenoid biosynthesis. An increase in zeaxanthin followed by a decrease in xanthophyll was seen after the induction of astaxanthin biosynthesis by glucose. This biphasic kinetics of zeaxanthin was parallel to the marked increase in adonixanthin (from 0 mg g⁻¹ to 0.21 mg g⁻¹) and astaxanthin (from 0.05 mg g⁻¹ to 0.35 mg g⁻¹) and decrease of β-carotene (from 0.30 mg g⁻¹ to 0.03 mg g⁻¹). More importantly, unlike the Haematococcus alga, in which there was a high β-carotene accumulation after DPA treatment, C. zofingiensis showed an accumulation of extra zeaxanthin instead of β-carotene after treatment of the cells with DPA. All these results observed in vivo studies corroborate the observations in vitro studies at the enzyme level that zeaxanthin is a substrate for the carotenoid oxygenase in C. zofingiensis. It is suggested that zeaxanthin might be an important intermediate and not an end product of the biosynthetic pathway of astaxanthin. Therefore, a new pathway for astaxanthin formation by C. zofingiensis, which is different from that of the other astaxanthin-producing microorganisms, is proposed. An understanding of the astaxanthin biosynthetic pathway may yield important information toward the optimization of astaxanthin production, especially for the improvement of astaxanthin through genetic manipulations.     

Construction of the astaxanthin biosynthetic pathway in a methanotrophic bacterium Methylomonas sp. strain 16a

Methylomonas sp. strain 16a is an obligate methanotrophic bacterium that uses methane or methanol as the sole carbon source. An effort was made to engineer this organism for astaxanthin production. Upon expressing the canthaxanthin gene cluster under the control of the native hps promoter in the chromosome, canthaxanthin was produced as the main carotenoid. Further conversion to astaxanthin was carried out by expressing different combinations of crtW and crtZ genes encoding the β-carotenoid ketolase and hydroxylase. The carotenoid intermediate profile was influenced by the copy number of these two genes under the control of the hps promoter. Expression of two copies of crtZ and one copy of crtW led to the accumulation of a large amount of the mono-ketolated product adonixanthin. On the other hand, expression of two copies of crtW and one copy of crtZ resulted in the presence of non-hydroxylated carotenoid canthaxanthin and the mono-hydroxylated adonirubin. Production of astaxanthin as the predominant carotenoid was obtained in a strain containing two complete sets of carotenoid biosynthetic genes. This strain had an astaxanthin titer ranging from 1 to 2.4 mg g⁻¹ of dry cell biomass depending on the growth conditions. More than 90% of the total carotenoid was astaxanthin, of which the majority was in the form of E-isomer. This result indicates that it is possible to produce astaxanthin with desirable properties in methanotrophs through genetic engineering.     

Astaxanthin Accumulation in the Green Alga Haematococcus pluvialis： Effects of Cultivation Parameters

The green alga, Haematococcus pluvlalis Flotow is used as a source of the ketocarotenoid astaxanthin for application in fish aquaculture, pharmaceutical and cosmetic industries. Ceils of the green alga were induced by the application of different light and starvation conditions to evaluate the effect in astaxanthin accumulate. The conditions used for the Induction were high light intensity （170 μmol·m^-2·s^-1）, iron starvation, sulfur starvation and phosphate starvation. The results show that stresses applied in culture, which interfere with cell division, trigger the accumulation of astaxanthin. Notably, sulfur starvation results in a massive accumulation of this commercially important carotenoid.     

Expression of novel carotenoid biosynthesis genes from Enterococcus gilvus improves the multistress tolerance of Lactococcus lactis

Aims

To determine whether the carotenoid production improves stress tolerance of lactic acid bacteria, the cloned enterococcal carotenoid biosynthesis genes were expressed in Lactococcus lactis ssp. cremoris MG1363, and the survival rate of carotenoid‐producing engineered MG1363 strain under stress condition was investigated.

Methods and Results

We cloned carotenoid biosynthesis genes from yellow‐pigmented Enterococcus gilvus. The cloned genes consisted of crtN and crtM and its promoter region were inserted into the shuttle vector pRH100, and the resulting plasmid was named pRC. The cloned crtNM was expressed using pRC in noncarotenoid‐producing L. lactis ssp. cremoris MG1363. The expression of crtNM led to the production of C₃₀ carotenoid 4,4′‐diaponeurosporene. After exposure to 32 mmol l^?1 H₂O₂, low pH (1.5, acidified with HCl), 20% bile acid and 12 mg ml^?1 lysozyme, the survival rates of the MG1363 strain harbouring pRC were 18.7‐, 6.8‐, 8.8‐ and 4.4‐fold higher, respectively, than those of MG1363 strain harbouring the empty vector pRH100.

Conclusions

The expression of carotenoid biosynthesis genes from Ent. gilvus improves the multistress tolerance of L. lactis.

Significance and Impact of the study

First report of the improvement of multistress tolerance of lactic acid bacteria by the introduction of genes for carotenoid production.     

Functional analysis of combinations in astaxanthin biosynthesis genes from<Emphasis Type="Italic">Paracoccus haeundaensis</Emphasis>

Carotenoids are important natural pigments produced by many microorganisms and plants. We have previously reported the isolation of a new marine bacterium,Paracoccus haeundaensis, which produces carotenoids, mainly in the form of astaxanthin. The astaxanthin biosynthesis gene cluster, consisting of six carotenogenic genes, was cloned and characterized from this organism. Individual genes of the carotenoid biosynthesis gene cluster were functionally expressed inEscherichia coli and each gene product was purified to homogeneity. Their molecular characteristics, including enzymatic activities, were previously reported. Here, we report cloning the genes for crtE, crtEB, crtEBI, crtEBIY, crtEBIYZ, and crtEBI-YZW of theP. haeundaensis carotenoid biosynthesis genes inE. coli and verifying the production of the corresponding pathway intermediates. The carotenoids that accumulated in the transformed cells carrying these gene combinations were analyzed by chromatographic and spectroscopic methods.     

Turning a green alga red: engineering astaxanthin biosynthesis by intragenic pseudogene revival in Chlamydomonas reinhardtii

The green alga Chlamydomonas reinhardtii does not synthesize high‐value ketocarotenoids like canthaxanthin and astaxanthin; however, a β‐carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C‐terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic redesign of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to reddish‐brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.3 mg/L/day. Astaxanthin productivity in engineered C. reinhardtii shown here might be competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio‐accessibility of these pigments were much higher in cell wall‐deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor‐made pigments from this host.     

Effect of the carbon source on the carotenoid profiles of Phaffia rhodozyma strains

Phaffia rhodozyma strains ATCC 24202, ATCC 24203, ATCC 24228, ATCC 24229, ATCC 24261, NRRL Y-10921, NRRL Y-10922 and NRRL Y-17268 were grown on culture media containing glucose, sucrose or xylose as carbon sources. Carotenoids were extracted from biomass and analyzed by HPLC with diode-array detection. The carotenoid profiles depended on both the strain considered and the carbon source employed. Astaxanthin, the main pigment found in P. rhodozyma, accounted for 42–91% of total carotenoids. Other carotenoids such as canthaxanthin, echinenone, 3-hydroxyechinenone, lycopene, 4-hydroxy-3′, 4′-didehydro-β-ψ-carotene and phoenicoxanthin were detected. The highest volumetric carotenoid concentration (3.60 mg L⁻¹) was obtained with strain NRRL Y-17268 growing on xylose. In this case, astaxanthin accounted for 82% of total carotenoids. Received 29 May 1997/ Accepted in revised form 08 August 1997     

Growth-associated biosynthesis of astaxanthin in heterotrophic Chlorella zofingiensis (Chlorophyta)

The green microalga Chlorella zofingiensis can produce the ketocarotenoid astaxanthin under heterotrophic culture conditions. Here we report the growth-associated biosynthesis of astaxanthin in this biotechnologically important alga. With glucose as sole carbon and energy source, C. zofinginesis grew fast in the dark with rapid exhaustion of nitrogen and carbon sources from media, leading to a high specific growth rate (0.034 h⁻¹). Cultures started at a cell concentration of about 3.4 × 10⁹ cells l⁻¹ reached, after 6 days, standing biomass values of 1.6 × 10¹¹ cells or 8.5 g dry weight l⁻¹. Surprisingly, the biosynthesis of astaxanthin was found to start at early exponential phase, independent of cessation of cell division. A general trend was observed that the culture conditions benefiting cell growth also benefited astaxanthin accumulation, indicating that astaxanthin was a growth-associated product in this alga. The maximum cell dry biomass and astaxanthin yield were 11.75 g l⁻¹ and 11.14 mg l⁻¹ (about 1 mg g⁻¹), simultaneously obtained in the fed-batch culture with a combined glucose–nitrate mixture addition, which were the highest ever reported in dark-heterotrophic algal cultures. The possible reasons why dark-heterotrophic C. zofingiensis could produce astaxanthin during the course of cell growth were discussed.     

Flower color alteration in <Emphasis Type="Italic">Lotus japonicus</Emphasis> by modification of the carotenoid biosynthetic pathway

To establish a model system for alteration of flower color by carotenoid pigments, we modified the carotenoid biosynthesis pathway of Lotus japonicus using overexpression of the crtW gene isolated from marine bacteria Agrobacterium aurantiacum and encoding β-carotene ketolase (4,4′-β-oxygenase) for the production of pink to red color ketocarotenoids. The crtW gene with the transit peptide sequence of the pea Rubisco small subunit under the regulation of the CaMV35S promoter was introduced to L. japonicus. In most of the resulting transgenic plants, the color of flower petals changed from original light yellow to deep yellow or orange while otherwise exhibiting normal phenotype. HPLC and TLC analyses revealed that leaves and flower petals of these plants accumulated novel carotenoids, believed to be ketocarotenoids consisting of including astaxanthin, adonixanthin, canthaxanthin and echinenone. Results indicated that modification of the carotenoid biosynthesis pathway is a means of altering flower color in ornamental crops.