Molecular structure and enzymatic function of lycopene cyclase from the cyanobacterium Synechococcus sp strain PCC7942.

A gene encoding the enzyme lycopene cyclase in the cyanobacterium Synechococcus sp strain PCC7942 was mapped by genetic complementation, cloned, and sequenced. This gene, which we have named crtL, was expressed in strains of Escherichia coli that were genetically engineered to accumulate the carotenoid precursors lycopene, neurosporene, and zeta-carotene. The crtL gene product converts the acyclic hydrocarbon lycopene into the bicyclic beta-carotene, an essential component of the photosynthetic apparatus in oxygen-evolving organisms and a source of vitamin A in human and animal nutrition. The enzyme also converts neurosporene to the monocyclic beta-zeacarotene but does not cyclize zeta-carotene, indicating that desaturation of the 7-8 or 7'-8' carbon-carbon bond is required for cyclization. The bleaching herbicide 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA) effectively inhibits both cyclization reactions. A mutation that confers resistance to MPTA in Synechococcus sp PCC7942 was identified as a point mutation in the promoter region of crtL. The deduced amino acid sequence of lycopene cyclase specifies a polypeptide of 411 amino acids with a molecular weight of 46,125 and a pI of 6.0. An amino acid sequence motif indicative of FAD utilization is located at the N terminus of the polypeptide. DNA gel blot hybridization analysis indicated a single copy of crtL in Synechococcus sp PCC7942. Other than the FAD binding motif, the predicted amino acid sequence of the cyanobacterial lycopene cyclase bears little resemblance to the two known lycopene cyclase enzymes from nonphotosynthetic bacteria. Preliminary results from DNA gel blot hybridization experiments suggest that, like two earlier genes in the pathway, the Synechococcus gene encoding lycopene cyclase is homologous to plant and algal genes encoding this enzyme.     

Genes from a Dietzia sp. for synthesis of C40 and C50 beta-cyclic carotenoids

Dietzia sp. CQ4 accumulated the C(40) beta-cyclic carotenoids (canthaxanthin and echinenone) and the C(50) beta-cyclic carotenoid (C.p.450 monoglucoside). A plant-type lycopene beta-cyclase gene crtL was identified for beta-cyclization of the C(40) carotenoids. A carotenoid synthesis gene cluster was identified away from the crtL gene, which contained the crtEBI genes for the synthesis of lycopene followed by the lbtABC genes for lycopene elongation and beta-cyclization of the C(50) carotenoids. This C(50) beta-cyclic carotenoid synthesis gene cluster from Dietzia sp. CQ4 showed high homology with the gene clusters for synthesizing the C(50) epsilon-cyclic carotenoids (decaprenoxanthin and glucosides) from Corynebacterium glutamicum and Agromyces mediolanus. One unique feature of the C(50) beta-cyclic carotenoid synthesis genes in Dietzia sp. CQ4 was that the gene encoding a C(50) carotenoid beta-cyclase subunit and the gene encoding the lycopene elongase appeared to be fused as a single gene (lbtBC). Expression of the gene (lbtA) encoding another subunit of the C(50) carotenoid beta-cyclase and the lbtBC gene in lycopene-accumulating Escherichia coli produced almost exclusively the C(50) beta-cyclic carotenoid C.p.450. One gene (crtX) with high homology to glycosyl transferases was transcribed in the opposite orientation downstream of the lbtBC gene. The crtX gene was likely involved in C.p.450 glucosylation in Dietzia sp. CQ4. The pathway analogous to the synthesis of the C(50) epsilon-cyclic carotenoids was proposed for the synthesis of the C(50) beta-cyclic carotenoids.     

甜橙辣椒红／辣椒玉红素合成酶同源基因的克隆（简报）

The complete sequence of orange homologous capsanthin/capsorubin synthase gene is 3788 bp long with a coding sequence of 1512 bp, which encodes a polypeptide of 503 amino acids. The 5' upstream sequence is 1721 bp long and the 3' downstream sequence is 555 bp long. The amino acid sequence of this gene is 78% and 69% identical to the genes from carrot and pepper, respectively. It is also partially homologous to plant neoxanthin synthase, lycopene beta-cyclase and lycopene epsilon cyclase genes. Isolation of the gene provides a framework for elucidation of the mechanisms involved in inability of citrus to produce capsanthin and capsorubin.     

Coordinate expression of multiple bacterial carotenoid genes in canola leading to altered carotenoid production

Carotenoids have drawn much attention recently because of their potentially positive benefits to human health as well as their utility in both food and animal feed. Previous work in canola (Brassica napus) seed over-expressing the bacterial phytoene synthase gene (crtB) demonstrated a change in carotenoid content, such that the total levels of carotenoids, including phytoene and downstream metabolites like beta-carotene, were elevated 50-fold, with the ratio of beta- to alpha-carotene being 2:1. This result raised the possibility that the composition of metabolites in this pathway could be modified further in conjunction with the increased flux obtained with crtB. Here we report on the expression of additional bacterial genes for the enzymes geranylgeranyl diphosphate synthase (crtE), phytoene desaturase (crtI) and lycopene cyclase (crtY and the plant B. napus lycopene beta-cyclase) engineered in conjunction with phytoene synthase (crtB) in transgenic canola seed. Analysis of the carotenoid levels by HPLC revealed a 90% decrease in phytoene levels for the double construct expressing crtB in conjunction with crtI. The transgenic seed from all the double constructs, including the one expressing the bacterial crtB and the plant lycopene beta-cyclase showed an increase in the levels of total carotenoid similar to that previously observed by expressing crtB alone but minimal effects were observed with respect to the ratio of beta- to alpha-carotene compared to the original construct. However, the beta- to alpha-carotene ratio was increased from 2:1 to 3:1 when a triple construct consisting of the bacterial phytoene synthase, phytoene desaturase and lycopene cyclase genes were expressed together. This result suggests that the bacterial genes may form an aggregate complex that allows in vivo activity of all three proteins through substrate channeling. This finding should allow further manipulation of the carotenoid biosynthetic pathway for downstream products with enhanced agronomic, animal feed and human nutritional values.     

A novel type of lycopene ε-cyclase in the marine cyanobacterium<Emphasis Type="Italic"> Prochlorococcus marinus</Emphasis> MED4

Chlorophyll- b-possessing cyanobacteria of the genus Prochlorococcus share the presence of high amounts of alpha- and beta-carotenoids with green algae and higher plants. The branch point in carotenoid biosynthesis is the cyclization of lycopene, for which in higher plants two distinct enzymes are required, epsilon- and beta-lycopene cyclase. All cyanobacteria studied so far possess a single beta-cyclase. Here, two different Prochlorococcus sp. MED4 genes were functionally identified by heterologous gene complementation in Escherichia coli to encode lycopene cyclases. Whereas one is both functionally and in sequence highly similar to the beta-cyclase of Synechococcus sp. strain PCC 7942 and other cyanobacteria, the other showed several intriguing features. It acts as a bifunctional enzyme catalyzing the formation of epsilon- as well as of beta-ionone end groups. Expression of this cyclase in E. coli resulted in the simultaneous accumulation of alpha- beta-, delta-, and epsilon-carotene. Such an activity is in contrast to all lycopene epsilon-cyclases known so far, including those of the higher plants. Thus, for the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either beta- or epsilon-end groups. These two genes are suggested to be designated as crtL-b and crtL-e. The results indicate that both enzymes might have originated from duplication of a single gene. Consequently, we suggest that multiple gene duplications followed by functional diversification resulted several times, and in independent lineages, in the appearance of enzymes for the biosynthesis of cyclic carotenoids.     

Genetic basis of microbial carotenogenesis

The synthesis of carotenoids begins with the formation of a phytoene from geranylgeranyl pyrophosphate, a well conserved step in all carotenogenic organisms and catalyzed by a phytoene synthase, an enzyme encoded by the crtB (spy) genes. The next step is the dehydrogenation of the phytoene, which is carried out by phytoene dehydrogenase. In organisms with oxygenic photosynthesis, this enzyme, which accomplishes two dehydrogenations, is encoded by the crtP genes. In organisms that lack oxygenic photosynthesis, dehydrogenation is carried out by an enzyme completely unrelated to the former one, which carries out four dehydrogenations and is encoded by the crtI genes. In organisms with oxygenic photosynthesis, dehydrogenation of the phytoene is accomplished by a ζ-carotene dehydrogenase encoded by the crtQ (zds) genes. In many carotenogenic organisms, the process is completed with the cyclization of lycopene. In organisms exhibiting oxygenic photosynthesis, this step is performed by a lycopene cyclase encoded by the crtL genes. In contrast, anoxygenic photosynthetic and non-photosynthetic organisms use a different lycopene cyclase, encoded by the crtY (lyc) genes. A third and unrelated type of lycopene β-cyclase has been described in certain bacteria and archaea. Fungi differ from the rest of non-photosynthetic organisms in that they have a bifunctional enzyme that displays both phytoene synthase and lycopene cyclase activity. Carotenoids can be modified by oxygen-containing functional groups, thus originating xanthophylls. Only two enzymes are necessary for the conversion of β-carotene into astaxanthin, using several ketocarotenoids as intermediates, in both prokaryotes and eukaryotes. These enzymes are a β-carotene hydroxylase (crtZ genes) and a β-carotene ketolase, encoded by the crtW (bacteria) or bkt (algae) genes. Electronic Publication     

Construction of new <Emphasis Type="Italic">Pichia pastoris</Emphasis> X-33 strains for production of lycopene and β-carotene

In this study, we used the non-carotenogenic yeast Pichia pastoris X33 as a receptor for β-carotene-encoding genes, in order to obtain new recombinant strains capable of producing different carotenoidic compounds. We designed and constructed two plasmids, pGAPZA-EBI* and pGAPZA-EBI*L*, containing the genes encoding lycopene and β-carotene, respectively. Plasmid pGAPZA-EBI*, expresses three genes, crtE, crtB, and crtI*, that encode three carotenogenic enzymes, geranylgeranyl diphosphate synthase, phytoene synthase, and phytoene desaturase, respectively. The other plasmid, pGAPZA-EBI*L*, carried not only the three genes above mentioned, but also the crtL* gene, that encodes lycopene β-cyclase. The genes crtE, crtB, and crtI were obtained from Erwinia uredovora, whereas crtL* was cloned from Ficus carica (JF279547). The plasmids were integrated into P. pastoris genomic DNA, and the resulting clones Pp-EBI and Pp-EBIL were selected for either lycopene or β-carotene production and purification, respectively. Cells of these strains were investigated for their carotenoid contents in YPD media. These carotenoids produced by the recombinant P. pastoris clones were qualitatively and quantitatively analyzed by high-resolution liquid chromatography, coupled to photodiode array detector. These analyses confirmed that the recombinant P. pastoris clones indeed produced either lycopene or β-carotene, according to the integrated vector, and productions of 1.141 μg of lycopene and 339 μg of β-carotene per gram of cells (dry weight) were achieved. To the best of our knowledge, this is the first time that P. pastoris has been genetically manipulated to produce β-carotene, thus providing an alternative source for large-scale biosynthesis of carotenoids.     

Fusion-type lycopene beta-cyclase from a thermoacidophilic archaeon Sulfolobus solfataricus

Examination of the sequence of a hypothetical gene with an unknown function included in the carotenogenic gene cluster in the genome of a thermoacidophilic archaeon Sulfolobus solfataricus led to the prediction that the gene encodes a novel-type lycopene beta-cyclase, whose N- and C-terminal halves are homologous to the subunits of the bacterial heterodimeric enzymes. The recombinant expression of the gene in lycopene-producing Escherichia coli resulted in the accumulation of beta-carotene in the cells, which verifies the function of the gene. Homologues of the archaeal lycopene beta-cyclase from various organisms such as bacteria, archaea, and fungi have been reported. Although their primary structures are clearly specific to the biological taxa, a phylogenetic analysis revealed the unexpected complicity of the evolutional route of these enzymes.     

Expression and functional analysis of a gene cluster involved in the synthesis of decaprenoxanthin reveals the mechanisms for C50 carotenoid formation.

Corynebacterium glutamicum accumulates the C50 carotenoid decaprenoxanthin. Rescued DNA from transposon color mutants of this Gram-positive bacterium was used to clone the carotenoid biosynthetic gene cluster. By sequence comparison and functional complementation, the genes involved in the synthesis of carotenoids with 50 carbon atoms were identified. The genes crtE, encoding a geranylgeranyl pyrophosphate synthase, crtB, encoding a phytoene synthase, and crtI, encoding a phytoene desaturase, are responsible for the formation of lycopene. The products of three novel genes, crtYe and crtYf, with sequence similarities to heterodimeric lycopene cyclase crtYc and crtYd, together with crtEb which exhibits a prenyl transferase motif, were involved in the conversion of C40 acyclic lycopene to cyclic C50 carotenoids. Using functional complementation in Escherichia coli, it could be shown that the elongation of lycopene to the acyclic C50 carotenoid flavuxanthin by the addition of C5 isoprenoid units at positions C-2 and C-2' is catalyzed by the crtEb gene product. Subsequently, the gene products of crtYe and crtYf in a concerted action convert the acyclic flavuxanthin into the cyclic C50 carotene, decaprenoxanthin, forming two epsilon-ionone groups. The mechanisms, involving two individual steps for the formation of cyclic C50 carotenoids from lycopene, are proposed on the basis of these results.     

Identification of a lycopene beta-cyclase required for bacteriorhodopsin biogenesis in the archaeon Halobacterium salinarum

Biogenesis of the light-driven proton pump bacteriorhodopsin in the archaeon Halobacterium salinarum requires coordinate synthesis of the bacterioopsin apoprotein and carotenoid precursors of retinal, which serves as a covalently bound cofactor. As a step towards elucidating the mechanism and regulation of carotenoid metabolism during bacteriorhodopsin biogenesis, we have identified an H. salinarum gene required for conversion of lycopene to beta-carotene, a retinal precursor. The gene, designated crtY, is predicted to encode an integral membrane protein homologous to lycopene beta-cyclases identified in bacteria and fungi. To test crtY function, we constructed H. salinarum strains with in-frame deletions in the gene. In the deletion strains, bacteriorhodopsin, retinal, and beta-carotene were undetectable, whereas lycopene accumulated to high levels ( approximately 1.3 nmol/mg of total cell protein). Heterologous expression of H. salinarum crtY in a lycopene-producing Escherichia coli strain resulted in beta-carotene production. These results indicate that H. salinarum crtY encodes a functional lycopene beta-cyclase required for bacteriorhodopsin biogenesis. Comparative sequence analysis yields a topological model of the protein and provides a plausible evolutionary connection between heterodimeric lycopene cyclases in bacteria and bifunctional lycopene cyclase-phytoene synthases in fungi.     

重叠延伸PCR克隆三孢布拉霉carRA基因及其功能活性检测系统的构建

三孢布拉氏霉菌CarRA蛋白,既有番茄红素环化酶功能活性又有八氢番茄红素合成酶功能活性,为了对CarRA蛋白进行双功能活性分析,及探测CarRA蛋白的番茄红素环化酶功能活性位点,构建了在大肠杆菌体内通过颜色互补检测两种酶活性的系统。通过重叠延伸PCR的方法克隆得到了carRA基因,并构建原核表达载体pET28a-carRA,与携带crtI/crtB/crtE基因簇的质粒pAC-LYC共转化BL21(DE3),验证番茄红素环化酶功能活性;与以pAC-LYC为基础构建的携带crtI/crtE基因簇的质粒pAC     

Cloning,sequencing and expressing the carotenoid biosynthesis genes,lycopene cyclase and phytoene desaturase,from the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 in Escherichia coli

Two genes which encode the enzymes lycopene cyclase and phytoene desaturase in the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 have been cloned and sequenced. The gene for lycopene cyclase, designated crtY, was expressed in a strain of Escherichia coli which contained the crtE, B, I and Z genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and β-carotene hydroxylase, respectively. As a result, zeaxanthin production was observed in E. coli transformants. In addition, expression of the E. longus gene crtI for phytoene desaturase in E. coli containing crtE and B resulted in the accumulation of lycopene in transformants. Zeaxanthin and lycopene were also determined by mass spectrum. Nucleotide sequence similarities between E. longus crtY gene and other microbial lycopene cyclase genes are 40.2% (Erwinia herbicola), 37.4% (Erwinia uredovora) and 22.9% (Synechococcus sp.), and those between phytoene desaturase genes are 50.3% (E. herbicola), 54.7% (E. uredovora) and 39.6% (Rhodobacter capsulatus).     

A lycopene β‐cyclase/lycopene ε‐cyclase/light‐harvesting complex‐fusion protein from the green alga Ostreococcus lucimarinus can be modified to produce α‐carotene and β‐carotene at different ratios

Biosynthesis of asymmetric carotenoids such as α‐carotene and lutein in plants and green algae involves the two enzymes lycopene β‐cyclase (LCYB) and lycopene ε‐cyclase (LCYE). The two cyclases are closely related and probably resulted from an ancient gene duplication. While in most plants investigated so far the two cyclases are encoded by separate genes, prasinophyte algae of the order Mamiellales contain a single gene encoding a fusion protein comprised of LCYB, LCYE and a C‐terminal light‐harvesting complex (LHC) domain. Here we show that the lycopene cyclase fusion protein from Ostreococcus lucimarinus catalyzed the simultaneous formation of α‐carotene and β‐carotene when heterologously expressed in Escherichia coli. The stoichiometry of the two products in E. coli could be altered by gradual truncation of the C‐terminus, suggesting that the LHC domain may be involved in modulating the relative activities of the two cyclase domains in the algae. Partial deletions of the linker region between the cyclase domains or replacement of one or both cyclase domains with the corresponding cyclases from the green alga Chlamydomonas reinhardtii resulted in pronounced shifts of the α‐carotene‐to‐β‐carotene ratio, indicating that both the relative activities of the cyclase domains and the overall structure of the fusion protein have a strong impact on the product stoichiometry. The possibility to tune the product ratio of the lycopene cyclase fusion protein from Mamiellales renders it useful for the biotechnological production of the asymmetric carotenoids α‐carotene or lutein in bacteria or fungi.     

Metabolism of cyclic carotenoids: a model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts

The biosynthetic pathway of cyclic carotenoid is known to be quantitatively and qualitatively different in the non-green plastids of Capsicum annuum fruits compared with chloroplasts. Here, the cloning is described of a novel cDNA from this organism, which encodes an enzyme catalyzing the cyclization of lycopene to β-carotene when expressed in Escherichia coli . The corresponding gene is constitutively expressed during fruit development. Significant amino acid sequence identity was observed between this enzyme and capsanthin/capsorubin synthase which is involved in the synthesis of the species-specific red carotenoids of C. annuum fruits. The latter enzyme was found also to possess a lycopene β-cyclase activity when expressed in E. coli . A model is proposed for the origin of the capsanthin/capsorubin synthase gene and the role of this enzyme, together with the newly cloned lycopene cyclase, in the specific re-channeling of linear carotenoids into β-cyclic carotenoids in C. annuum ripening fruits.