Engineering of an endogenous phytoene desaturase gene as a dominant selectable marker for Chlamydomonas reinhardtii transformation and enhanced biosynthesis of carotenoids

A phytoene desaturase (PDS) gene was cloned and characterized from the unicellular green microalga Chlamydomonas reinhardtii. Functional complementation analysis revealed C. reinhardtii PDS (CrPDS) catalyzes the conversion of phytoene to the colored carotenoid ζ-carotene. A single amino acid substitution, L505F, enhanced its desaturation activity by 29%, as indicated by an in vitro enzymatic assay. In addition, CrPDS-L505F exhibited 27.7-fold higher resistance to the herbicide norflurazon. Glass bead-mediated delivery displayed a high transformation efficiency of C. reinhardtii with CrPDS-L505F, demonstrating clearly that the engineered endogenous CrPDS is a dominant selectable marker for C. reinhardtii and possibly for other green algae. Furthermore, the expression of PDS could enhance the intracellular carotenoid accumulation of transformants, opening up the possibility of engineering the carotenogenic pathway for improved carotenoid production in microalgae.     

The molecular basis of resistance to the herbicide norflurazon

We have cloned and sequenced a gene, pds, from the cyanobacterium Synechococcus PCC7942 that is responsible for resistance to the bleaching herbicide norflurazon. A point mutation in that gene, leading to an amino acid substitution from valine to glycine in its polypeptide product, was found to confer this resistance. Previous studies with herbicide-resistant mutants have indicated that this gene encodes phytoene desaturase (PDS), a key enzyme in the biosynthesis of carotenoids. A short amino acid sequence that is homologous to conserved motifs in the binding sites for NAD(H) and NADP(H) was identified in PDS, suggesting the involvement of these dinucleotides as cofactors in phytoene desaturation.     

Intragenic Enhancers and Suppressors of Phytoene Desaturase Mutations in Chlamydomonas reinhardtii

Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. A light-sensitive, phytoene-accumulating mutant, pds1-1, was isolated in Chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase (PDS) gene. PDS catalyzes the second step in carotenoid biosynthesis-the conversion of phytoene to ζ-carotene. Decreased accumulation of downstream colored carotenoids suggested that the pds1-1 mutant is leaky for PDS activity. A screen for enhancers of the pds1-1 mutation yielded the pds1-2 allele, which completely lacks PDS activity. A second independent null mutant (pds1-3) was identified using DNA insertional mutagenesis. Both null mutants accumulate only phytoene and no other carotenoids. All three phytoene-accumulating mutants exhibited slower growth rates and reduced plating efficiency compared to wild-type cells and white phytoene synthase mutants. Insight into amino acid residues important for PDS activity was obtained through the characterization of intragenic suppressors of pds1-2. The suppressor mutants fell into three classes: revertants of the pds1-1 point mutation, mutations that changed PDS amino acid residue Pro64 to Phe, and mutations that converted PDS residue Lys90 to Met. Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 90 and 143 are in close contact in the active PDS enzyme and have important roles in its structural stability and/or activity.     

Enhanced production of lutein in heterotrophic Chlorella protothecoides by oxidative stress

The fast growing unicellular green microalgae Chlorella protothecoides has attracted interest as a promising organism for commercial production of a high-value carotenoid, lutein, by heterotrophic fermentation. Effects of two oxidant-forming reactive oxygen species (ROS) on the biomass concentration, and yield and content of lutein in batch culture of heterotrophic Chlorella protothecoides were investigated in this study. The addition of 0.1 mmol/L H₂O₂ and 0.01 mmol/L NaClO plus 0.5 mmol/L Fe²⁺ to the culture led to the generation of ^·OH and enhanced the lutein content from 1.75 to 1.90 and 1.95 mg/g, respectively. The lutein content further increased to 1.98 mg/g when 0.01 mmol/L H₂O₂ and 0.5 mmol/L NaClO were added to generate ¹O₂. The maximum yield of lutein (28.5, 29.8 and 31.4 mg/L) and a high biomass concentration (15.0, 15.3 and 15.9 g/L) were also achieved through the above treatments. The results indicated that 1O2 could promote lutein formation and enhance lutein production in heterotrophic Chlorella protothecoides. Moreover, ¹O₂ produced from the reaction of H₂O₂ and NaClO was more effective in enhancing lutein production and reducing biomass loss than ^·OH from the reaction of H₂O₂ or NaClO plus Fe²⁺. Supported by the National Key Project of Sci & Tech Supporting Programs Funded by Ministry of Science & Technology of China (Grant No. 2006BAD27B03), Sci & Tech Project of Guangzhou (Grant No. 2005Z3-E0331) and Sci & Tech Project of Guangdong (Grant No. 20052050166)     

Isolation and physiological and biochemical characterization of the Chlamydomonas reinhardtii C-41 mutant,a superproducer of ζ-carotene

The composition of the carotenes and xanthophylls of Chlamydomonas reinhardtii Dang. C-41, a mutant of a unicellular green alga and a superproducer of ζ-carotene, was studied. The light-harvesting complexes and a complex of the PS-II reaction center were established to be disrupted in the C-41 mutant. However, the mutant retained a high (up to 46%) photosynthetic activity and the capacity to accumulate chlorophylls and carotenoids (up to 50%). The composition of carotenes was studied, and it was shown that, in contrast to wild-type K(+) cells, which accumulate up to 95% of β-carotene and 5% α-carotene, cells of the C-41 mutant contained 43% β-carotene, 19% β-zeacarotene, and 38% ζ-carotene. The high level of C-41 mutant biomass accumulation made it possible to recommend the mutant as a superproducer of ζ-carotene in phytobiotechnology.     

ζ-Carotene and other carotenes in a Phycomyces mutant

Strain S442, a new mutant of the fungus Phycomyces blakesleeanus, has a greenish colour and a distinct green fluorescence under long-UV light. Carotene analyses reveal the presence of phytoene, ζ-carotene, phytofluene, an unidentified compound, and neurosporene (in descending order of abundance). Genetic analysis shows that the new mutation occurs at gene carB, whose protein product catalyses the four dehydrogenations of phytoene to lycopene via phytofluene, ζ-carotene, and neurosporene. S442 offers no indication of a specific ζ-carotene dehydrogenase. The residual dehydrogenase activity in S442 is inhibited by diphenylamine. The high ζ-carotene content makes S442 a good source of this compound.     

Contribution of two ζ-carotene desaturases to the poly-cis desaturation pathway in the cyanobacterium Nostoc PCC 7120

The presence of two completely unrelated ζ-carotene desaturases CrtQa and CrtQb in some Nostoc strains is unique. CrtQb is the ζ-carotene desaturase, which was acquired by almost all cyanobacteria. The additional CrtQa can be regarded as an evolutionary relict of the CrtI desaturase present in non-photosynthetic bacteria. By reconstruction of the carotene desaturation pathway, we showed that both enzymes from Nostoc PCC 7120 were active. However, they differed in their preferred utilization of ζ-carotene Z isomers. CrtQa converted ζ-carotene isomers that were poorly metabolized by CrtQb. In this respect, CrtQa complemented the reactions of CrtQb, which is an advantage avoiding dead ends in the poly-cis desaturation pathway. In addition to ζ-carotene desaturation, CrtQa still possesses the Z to E isomerase function of the ancestral desaturase CrtI. Biochemical characterization showed that CrtQb is an enzyme with one molecule of tightly bound FAD and acts as a dehydrogenase transferring hydrogen to oxidized plastoquinone.     

Characterization of an ammonium transporter in the oleaginous alga Chlorella protothecoides

A suppression subtractive hybridization cDNA library was used to screen the differently expressed (up-regulated) genes in the photosynthesis–fermentation approach (PFA) of Chlorella protothecoides cultivation. A total of 87 clones were obtained and sequenced, in which 78 clones were homologous to known genes in databases. Among them, the ammonium transporter gene (CpAMT1) was characterized in detail. Quantitative real-time PCR showed that the expression of CpAMT1 was significantly induced by PFA and correlated with lipid accumulation. The up-regulation of CpAMT1 was suppressed by glutamine, while the lipid biosynthesis was also inhibited. Further analysis showed that the expression of CpAMT1 was correlated with glutamine synthetase activity, suggesting that CpAMT1, along with glutamine synthetase/glutamate synthase, may be responsible for nitrogen sensing in C. protothecoides. Together, these results imply that the ammonium transporter CpAMT1 could be the initial sensor of nitrogen deficiency and channels the carbon excess toward lipid biosynthesis.     

A kinetic model for lutein production by the green microalga Chlorella protothecoides in heterotrophic culture

Production of lutein by the green microalga Chlorella protothecoides grown heterotrophically in a fermentor using glucose as the carbon source and urea as the nitrogen source was investigated. An unstructured kinetic model was proposed to describe the microalgal culture system including cell growth, lutein formation, as well as glucose and nitrogen consumption. The inhibition potentials of biomass, product and substrates on growth and lutein formation were examined and incorporated into the kinetic model. Values of the kinetic model parameters were estimated. The resulting model predictions were in good agreement with the experimental results. The model can be helpful in scale-up, optimization and control of the C. protothecoides culture process, and can also be used as a guideline for similar microalgal cultivation systems. Received 28 January 1999/ Accepted in revised form 27 August 1999     

Production of biomass and lutein by Chlorella protothecoides at various glucose concentrations in heterotrophic cultures

The influence of initial glucose concentrations on the production of biomass and lutein by Chlorella protothecoides CS-41 was investigated in batch cultures using both shake flasks and fermentors. The maximum biomass concentration increased from 4·9 to 31·2 g litre⁻¹ dry cells with an increase in initial glucose concentration from 10 to 80 g litre⁻¹. An even higher initial glucose concentration (100 g litre⁻¹) resulted in a lower biomass concentration, a lower specific growth rate, a lower growth yield coefficient and a considerably longer lag phase, which might be due to substrate inhibition. The initial glucose level also had a significant effect on the production of lutein. In a 3·7-litre fermentor an increase in lutein production from 19·39 to 76·56 mg litre⁻¹ was obtained with an increase in initial glucose concentration from 10 to 40 g litre⁻¹, within which range, lutein yield coefficient was constant (Y_Itn=1·90±0·02 mg g⁻¹). A simple substrate inhibition model was developed, which fitted the experimental data better than the classical Haldane model. A group of time-dependent kinetic models for algal cultivation in fermentors were also constructed, which were in good agreement with the experimental results and could be employed to predict the production of biomass and lutein, and the consumption of glucose in fermentor cultures.     

Heterotrophic production of lutein by selected Chlorella strains

Seven Chlorella strains representing three species obtained from culture collections and research laboratories were screened for their potential of heterotrophic production of lutein on two different media (Basal and Kuhl) containing glucose. While both media supported good growth and lutein formation of the seven strains in darkness, higher biomass concentrations and lutein content were achieved on Basal medium. Chlorella protothecoides CS-41 was chosen from the seven strains for further investigation due to its higher productivities of both biomass and lutein. The maximal biomass concentration and lutein content of C. protothecoides cultivated heterotrophically with 9 g L^-1 glucose in a 3.7-L fermentor were respectively 4.6 g dry cells L^-1 and 4.60 mg lutein g^-1 dry cells on Basal medium, and 4.0 g dry cells L^-1 and 4.36 mg lutein g^-1 dry cells on Kuhl medium. The heterotrophic cultivation process was scaled up successfully to 30 L using a fermentor, in which the Basal medium containing 36 g L^-1 glucose was used; the maximal biomass concentration of 16.4 g dry cells L^-1, specific growth rate of 0.92 d^-1,lutein content of 4.85 mg lutein g^-1 dry cells,growth yield of 0.47 g dry cells g^-1 glucose and lutein yield of 1.93 mg lutein g^-1 glucose were respectively achieved. This revised version was published online in August 2006 with corrections to the Cover Date.     

Phytoene,phytofluene and ζ-carotene isomers from a Scenedesmus obliquus mutant

A number of mutant strains of the green alga, Scenedesmus obliquus, when grown in the dark, accumulated ζ-carotene as their major carotenoid together with appreciable concentrations of phytoene and phytofluene. The phytoene was almost entirely the 15-cis isomer, and phytofluene was also present mainly as the 15-cis form, whereas the ζ-carotene could be separated into three isomers, predominantly all-trans ζ-carotene accompanied by the 15-cis and an unidentified cis isomer. All the ζ-carotene isomers, when illuminated in the presence of iodine, gave the same equilibrium mixture of stereo-isomers, including a product with unusual spectroscopic and chromatographic properties, which may be a cyclic compound. The pathway of carotenoid biosynthesis in S. obliquus is discussed. On illumination, most of the ζ-carotenic strains were killed, but PGI strain survived, due to the production of cyclic carotenoids with chromophores long enough to protect chlorophyll from photosensitized oxidation.     

CD36 homolog divergence is responsible for the selectivity of carotenoid species migration to the silk gland of the silkworm Bombyx mori

Dietary carotenoids are absorbed in the intestine and delivered to various tissues by circulating lipoproteins; however, the mechanism underlying selective delivery of different carotenoid species to individual tissues remains elusive. The products of the Yellow cocoon (C) gene and the Flesh (F) gene of the silkworm Bombyx mori determine the selectivity for transport of lutein and β-carotene, respectively, to the silk gland. We previously showed that the C gene encodes Cameo2, a CD36 family member, which is thought to function as a transmembrane lipoprotein receptor. Here, we elucidated the molecular identity of the F gene product by positional cloning, as SCRB15, a paralog of Cameo2 with 26% amino acid identity. In the F mutant, SCRB15 mRNA structure was severely disrupted, due to a 1.4 kb genomic insertion in a coding exon. Transgenic expression of SCRB15 in the middle silk gland using the binary GAL4-UAS expression system enhanced selective β-carotene uptake by the middle silk gland, while transgenic expression of Cameo2 enhanced selective lutein uptake under the same GAL4 driver. Our findings indicate that divergence of genes in the CD36 family determines the selectivity of carotenoid species uptake by silk gland tissue and that CD36-homologous proteins can discriminate among carotenoid species.     

A simple purification method for the preparation of solubilized chlorophyllase from Chlorella protothecoides

A simple and rapid purification procedure is described for the routine preparation of large quantities of purified chlorophyllase (chlorophyll chlorophyllido-hydrolase, EC 3.1.1.14) from Chlorella protothecoides. The enzyme with specific activity of 960 nmol chlorophyll a hydrolyzed (mg protein)^?1 min^?1 was prepared by treating the homogenate with n-butanol, ammonium sulfate fractionations and gel filtration through Sephadex G-200 and Sepharose CL-6B, with a yield of 53% of activity based on the butanol extract. The enzyme preparation showed apparent homogeneity as judged by polyacrylamide gel electrophoresis. The procedures take only 4 days and can be operated routinely without column repacking.     

Kinetic flux profiling dissects nitrogen utilization pathways in the oleaginous green alga Chlorella protothecoides

As a promising candidate for biodiesel production, the green alga Chlorella protothecoides can efficiently produce oleaginous biomass and the lipid biosynthesis is greatly influenced by the availability of nitrogen source and corresponding nitrogen assimilation pathways. Based on isotope‐assisted kinetic flux profiling (KFP), the fluxes through the nitrogen utilization pathway were quantitatively analyzed. We found that autotrophic C. protothecoides cells absorbed ammonium mainly through glutamate dehydrogenase (GDH), and partially through glutamine synthetase (GS), which was the rate‐limiting enzyme of nitrogen assimilation process with rare metabolic activity of glutamine oxoglutarate aminotransferase (GOGAT, also known as glutamate synthase); whereas under heterotrophic conditions, the cells adapted to GS‐GOGAT cycle for nitrogen assimilation in which GS reaction rate was associated with GOGAT activity. The fact that C. protothecoides chooses the adenosine triphosphate‐free and less ammonium‐affinity GDH pathway, or alternatively the energy‐consuming GS‐GOGAT cycle with high ammonium affinity for nitrogen assimilation, highlights the metabolic adaptability of C. protothecoides exposed to altered nitrogen conditions.     

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.