The gene crtI mediates the conversion of phytoene into colored carotenoids in Rhodopseudomonas capsulata

Carotenoids are membrane pigments present in all photosynthetic organisms, providing essential photoprotective functions. The first carotenoid formed in the pathway is phytoene, a colorless compound which is then converted into colored carotenoids by a series of dehydrogenation reactions. In the photosynthetic bacterium Rhodopseudomonas capsulata mutations that affect carotenoid biosynthesis before colored carotenoids are formed have a "blue-green" phenotype as opposed to the "red" of wild type cells. We have extracted carotenoids from several blue-green mutants and found that two strains (BPY69 and BPY102) accumulate phytoene and no colored carotenoids. These mutants failed to dehydrogenate phytoene in an in vitro assay. However, dehydrogenation of this compound can be achieved in vitro by adding a cell-free extract from another blue-green mutant blocked earlier in the pathway. Genetic complementation and deletion mapping indicate that the gene crtI is responsible for the conversion of phytoene into colored carotenoids in these mutants.     

Carotenoid biosynthesis and overproduction in Corynebacterium glutamicum

ABSTRACT: BACKGROUND: Corynebacterium glutamicum contains the glycosylated C50 carotenoid decaprenoxanthin as yellow pigment. Starting from isopentenyl pyrophosphate, which is generated in the non-mevalonate pathway, decaprenoxanthin is synthesized via the intermediates farnesyl pyrophosphate, geranylgeranyl pyrophosphate, lycopene and flavuxanthin. RESULTS: Here, we showed that the genes of the carotenoid gene cluster crtE-cg0722-crtBIYeYfEb are co-transcribed and characterized defined gene deletion mutants. Gene deletion analysis revealed that crtI, crtEb, and crtYeYf, respectively, code for the only phytoene desaturase, lycopene elongase, and carotenoid C45/C50 epsilon-cyclase, respectively. However, the genome of C. glutamicum also encodes a second carotenoid gene cluster comprising crtB2I2-1/2 shown to be co-transcribed, as well. Ectopic expression of crtB2 could compensate for the lack of phytoene synthase CrtB in C. glutamicum DeltacrtB, thus, C. glutamicum possesses two functional phytoene synthases, namely CrtB and CrtB2. Genetic evidence for a crtI2-1/2 encoded phytoene desaturase could not be obtained since plasmid-borne expression of crtI2-1/2 did not compensate for the lack of phytoene desaturase CrtI in C. glutamicum DeltacrtI. The potential of C. glutamicum to overproduce carotenoids was estimated with lycopene as example. Deletion of the gene crtEb prevented conversion of lycopene to decaprenoxanthin and entailed accumulation of lycopene to 0.03 +/- 0.01 mg/g cell dry weight (CDW). When the genes crtE, crtB and crtI for conversion of geranylgeranyl pyrophosphate to lycopene were overexpressed in C. glutamicum DeltacrtEb intensely red-pigmented cells and an 80 fold increased lycopene content of 2.4 +/- 0.3 mg/g CDW were obtained. CONCLUSION: C. glutamicum possesses a certain degree of redundancy in the biosynthesis of the C50 carotenoid decaprenoxanthin as it possesses two functional phytoene synthase genes. Already metabolic engineering of only the terminal reactions leading to lycopene resulted in considerable lycopene production indicating that C. glutamicum may serve as a potential host for carotenoid production.     

Mutants of the carotene cyclase domain of al-2 from Neurospora crassa

Phytoene synthase and carotene cyclase, two key enzymes in carotenoid biosynthesis, are encoded by two separate genes in bacteria and plants, but by a single bifunctional gene in fungi. The cyclase function has been demonstrated for the products of the genes crtYB from the basidiomycete Xanthophyllomyces dendrorhous, and for carRA and carRP from the zygomycetes Phycomyces blakesleeanus and Mucor circinelloides, respectively. These three genes are highly similar to al-2 from Neurospora crassa. Taking advantage of the high proportion of the final product of the carotenoid pathway that accumulates Neurospora when mycelium is illuminated at low temperature, we have isolated two mutants with a pale reddish pigmentation. Both mutants are complemented by the wild-type al-2 gene, and carry mutations in the al-2 domain to which cyclase activity has been attributed in other fungi. The mutants lack neurosporaxanthin and accumulate an unidentified reddish carotenoid, as shown by column chromatography and HPLC. The chemical and spectrophotometrical properties of this carotenoid are consistent with the absence of carotenoid cyclization, and indicate that the product of al-2 is bifunctional. The existence of a single gene responsible for phytoene synthase and carotene cyclase thus seems to be a widespread trait among filamentous fungi, as shown by the examples now known in a basidiomycete, two zygomycetes and one ascomycete.     

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.     

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.     

Elucidation of the Erwinia uredovora carotenoid biosynthetic pathway by functional analysis of gene products expressed in Escherichia coli.

The most important function of carotenoid pigments, especially beta-carotene in higher plants, is to protect organisms against photooxidative damage (G. Britton, in T. W. Goodwin, ed., Plant Pigments--1988, 1988; N. I. Krinsky, in O. Isler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). beta-Carotene also functions as a precursor of vitamin A in mammals (G. A. J. Pitt, in I. Osler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). The enzymes and genes which mediate the biosynthesis of cyclic carotenoids such as beta-carotene are virtually unknown. We have elucidated for the first time the pathway for biosynthesis of these carotenoids at the level of enzyme-catalyzed reactions, using bacterial carotenoid biosynthesis genes. These genes were cloned from a phytopathogenic bacterium, Erwinia uredovora 20D3 (ATCC 19321), in Escherichia coli and located on a 6,918-bp fragment whose nucleotide sequence was determined. Six open reading frames were found and designated the crtE, crtX, crtY, crtI, crtB, and crtZ genes in reference to the carotenoid biosynthesis genes of a photosynthetic bacterium, Rhodobacter capsulatus; only crtZ had the opposite orientation from the others. The carotenoid biosynthetic pathway in Erwinia uredovora was clarified by analyzing carotenoids accumulated in E. coli transformants in which some of these six genes were expressed, as follows: geranylgeranyl PPiCrtB----prephytoene PPiCrtE----phytoeneCrtI---- lycopeneCrtY----beta-caroteneCrtZ----zeaxanthinCrtX--- -zeaxanthin-beta- diglucoside. The carotenoids in this pathway appear to be close to those in higher plants rather than to those in bacteria. Also significant is that only one gene product (CrtI) for the conversion of phytoene to lycopene is required, a conversion in which four sequential desaturations should occur via the intermediates phytofluene, zeta-carotene, and neurosporene.     

A relationship between carotenoid accumulation and the distribution of species of the fungus Neurospora in Spain

The ascomycete fungus Neurospora is present in many parts of the world, in particular in tropical and subtropical areas, where it is found growing on recently burned vegetation. We have sampled the Neurospora population across Spain. The sampling sites were located in the region of Galicia (northwestern corner of the Iberian peninsula), the province of Cáceres, the city of Seville, and the two major islands of the Canary Islands archipelago (Tenerife and Gran Canaria, west coast of Africa). The sites covered a latitude interval between 27.88° and 42.74°. We have identified wild-type strains of N. discreta, N. tetrasperma, N. crassa, and N. sitophila and the frequency of each species varied from site to site. It has been shown that after exposure to light Neurospora accumulates the orange carotenoid neurosporaxanthin, presumably for protection from UV radiation. We have found that each Neurospora species accumulates a different amount of carotenoids after exposure to light, but these differences did not correlate with the expression of the carotenogenic genes al-1 or al-2. The accumulation of carotenoids in Neurospora shows a correlation with latitude, as Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains isolated from higher latitudes. Since regions of low latitude receive high UV irradiation we propose that the increased carotenoid accumulation may protect Neurospora from high UV exposure. In support of this hypothesis, we have found that N. crassa, the species that accumulates more carotenoids, is more resistant to UV radiation than N. discreta or N. tetrasperma. The photoprotection provided by carotenoids and the capability to accumulate different amounts of carotenoids may be responsible, at least in part, for the distribution of Neurospora species that we have observed across a range of latitudes.     

Phytoene desaturase, CrtI, of the purple photosynthetic bacterium, Rubrivivax gelatinosus, produces both neurosporene and lycopene.

Biosynthetic pathways for carotenoids in the purple photosynthetic bacterium, Rubrivivax gelatinosus, which synthesizes spirilloxanthin in addition to spheroidene and OH-spheroidene, were investigated by means of genetic manipulation. A phytoene desaturase gene (crtI) found in the photosynthesis gene cluster of this bacterium was expressed in an Escherichia coli strain that can produce phytoene. Both neurosporene and lycopene were synthesized in the recombinant, probably by three- and four-step desaturation reactions of CrtI. A mutant of RVI: gelatinosus lacking the crtI gene produced only phytoene, indicating that this organism had no other phytoene desaturases. When the crtI deletion mutant was complemented by the three-step phytoene desaturase of Rhodobacter capsulatus, spirilloxanthin and its precursors were not synthesized, although spheroidene and OH-spheroidene were accumulated. It was concluded that neurosporene and lycopene are produced by a single phytoene desaturase in RVI: gelatinosus resulting in the synthesis of spheroidene and spirilloxanthin, and that there are no pathways for spirilloxanthin synthesis via spheroidene.     

甲基营养菌 Methylobacterium sp MB200 中crtI基因的克隆及表达

八氢番茄红素脱氢酶( CrtI)催化八氢番茄红素经过4次脱氢合成番茄红素,或者经过3次脱氢合成链孢红素,在类胡萝卜素的生物合成中发挥重要的作用.以甲基营养菌Methylobacterium sp MB200为原始菌株,首先采用转座子突变技术构建部分突变体库共11552株,筛选得到33株颜色发生变化的目的突变体,随后利用分子克隆技术从目的突变体中获得crtI基因的完整ORF,长为1539 bp,编码512个氨基酸.与来自M.populi BJ001、M.chloromethanicum CM4和M.extorquens AM1的crtI一致性均为93％.将crtI与载体pCM80连接得到重组质粒pCM80-crtI,导入原始菌株中得到重组菌MB200/pCM80-crtI.测定原始菌株与重组菌株的CrtI酶活,结果发现,重组菌株CrtI的酶活与原始菌株相比约提高了40％.实验结果为完善甲基营养菌中类胡萝卜素的生物合成代谢途径提供了理论参考.     

The biotechnological potential of the al-2 gene from Neurospora [correction of Neurospra] crassa for the production of monocyclic keto hydroxy carotenoids

The al-2 cDNA from Neurospora crassa was cloned, expressed and functionally characterized. The enzyme comprised the two catalytic activities of a phytoene synthase and a lycopene cyclase. In contrast to most other lycopene cyclases, single cyclizations were preferentially catalyzed. This N. crassa enzyme is the first CrtYB-type monocyclic-acting lycopene cyclase. Therefore, this cDNA has been evaluated for the heterologous synthesis of monocyclic hydroxy-keto carotenoids by combination with other carotenogenic genes in Escherichia coli. Depending on the degree of desaturation, 4-keto derivatives of gamma-carotene and torulene with additional 2-hydroxy, 3-hydroxy and/or 1'-HO groups were generated and the following asymmetrical carotenoids identified and quantitated: 3-HO-4-keto-gamma-carotene, 2-HO-4-keto-gamma-carotene, 4-keto-1'-HO-gamma-carotene, 3,1'-(HO)(2)-4-keto-gamma-carotene, 3-HO-4-keto-torulene and 2-HO-4-keto-torulene. Among them all the monocyclic gamma-carotene derivatives with 9 conjugated double bonds were not found naturally before. Furthermore, 2-HO-4-keto-torulene with 10 conjugated double bonds is another novel carotenoid.     

Construction and characterization of genetically modified synechocystis sp. PCC 6803 photosystem II core complexes containing carotenoids with shorter pi-conjugation than beta-carotene

Beta-carotene has been identified as an intermediate in a secondary electron transfer pathway that oxidizes Chl(Z) and cytochrome b(559) in Photosystem II (PS II) when normal tyrosine oxidation is blocked. To test the redox function of carotenoids in this pathway, we replaced the zeta-carotene desaturase gene (zds) or both the zds and phytoene desaturase (pds) genes of Synechocystis sp. PCC 6803 with the phytoene desaturase gene (crtI) of Rhodobacter capsulatus, producing carotenoids with shorter conjugated pi-electron systems and higher reduction potentials than beta-carotene. The PS II core complexes of both mutant strains contain approximately the same number of chlorophylls and carotenoids as the wild type but have replaced beta-carotene (11 double bonds), with neurosporene (9 conjugated double bonds) and beta-zeacarotene (9 conjugated double bonds and 1 beta-ionylidene ring). The presence of the ring appears necessary for PS II assembly. Visible and near-infrared spectroscopy were used to examine the light-induced formation of chlorophyll and carotenoid radical cations in the mutant PS II core complexes at temperatures from 20 to 160 K. At 20 K, a carotenoid cation radical is formed having an absorption maximum at 898 nm, an 85 nm blue shift relative to the beta-carotene radical cation peak in the WT, and consistent with the formation of the cation radical of a carotenoid with 9 conjugated double bonds. The ratio of Chl(+)/Car(+) is higher in the mutant core complexes, consistent with the higher reduction potential for Car(+). As the temperature increases, other carotenoids become accessible to oxidation by P(680)(+).     

类胡萝卜素生物合成途径及其控制与遗传操作

类胡萝卜素在真菌和植物细胞胞液／内质网上是由乙酰CoA经甲羟戊酸途径合成的,在细菌与植物质体中由磷酸甘油醛与丙酮酸经1-脱氧木酮糖-5-磷酸途径合成。形成的异戊烯基焦磷酸经多次缩合生成第一个类胡萝卜素八氢番茄红素,再经脱氢、环化、羟基化、环氧化等转变为其它类胡萝卜素。类胡萝卜素生物合成中涉及的酶都是膜结合的或整合入膜中的。类胡萝卜素合成是通过底物可利用性与环化分支方式进行控制的。白色体到叶绿体的转变以及花与果实成熟时类胡萝卜素合成增加是在基因转录水平调节的。进行类胡萝卜素合成酶基因的转化,可增加转化体类胡萝卜素的积累。     

Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene.

We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase. Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria. Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes. Southern analysis indicated that the C. nicotianae phytoene dehydrogenase gene is present in a single copy. Extraction of beta-carotene, the sole carotenoid accumulated by C. nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light. Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures.     

Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.     

Elevation of the provitamin A content of transgenic tomato plants

Tomato products are the principal dietary sources of lycopene and major source of beta-carotene, both of which have been shown to benefit human health. To enhance the carotenoid content and profile of tomato fruit, we have produced transgenic lines containing a bacterial carotenoid gene (crtI) encoding the enzyme phytoene desaturase, which converts phytoene into lycopene. Expression of this gene in transgenic tomatoes did not elevate total carotenoid levels. However, the beta-carotene content increased about threefold, up to 45% of the total carotenoid content. Endogenous carotenoid genes were concurrently upregulated, except for phytoene synthase, which was repressed. The alteration in carotenoid content of these plants did not affect growth and development. Levels of noncarotenoid isoprenoids were unchanged in the transformants. The phenotype has been found to be stable and reproducible over at least four generations.     

High-level production of beta-carotene in Saccharomyces cerevisiae by successive transformation with carotenogenic genes from Xanthophyllomyces dendrorhous

To determine whether Saccharomyces cerevisiae can serve as a host for efficient carotenoid and especially beta-carotene production, carotenogenic genes from the carotenoid-producing yeast Xanthophyllomyces dendrorhous were introduced and overexpressed in S. cerevisiae. Because overexpression of these genes from an episomal expression vector resulted in unstable strains, the genes were integrated into genomic DNA to yield stable, carotenoid-producing S. cerevisiae cells. Furthermore, carotenoid production levels were higher in strains containing integrated carotenogenic genes. Overexpression of crtYB (which encodes a bifunctional phytoene synthase and lycopene cyclase) and crtI (phytoene desaturase) from X. dendrorhous was sufficient to enable carotenoid production. Carotenoid production levels were increased by additional overexpression of a homologous geranylgeranyl diphosphate (GGPP) synthase from S. cerevisiae that is encoded by BTS1. Combined overexpression of crtE (heterologous GGPP synthase) from X. dendrorhous with crtYB and crtI and introduction of an additional copy of a truncated 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene (tHMG1) into carotenoid-producing cells resulted in a successive increase in carotenoid production levels. The strains mentioned produced high levels of intermediates of the carotenogenic pathway and comparable low levels of the preferred end product beta-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of beta-carotene, up to 5.9 mg/g (dry weight), which was accomplished by the introduction of an additional copy of crtI and tHMG1 into carotenoid-producing yeast cells. This transformant is promising for further development toward the biotechnological production of beta-carotene by S. cerevisiae.