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1.
The phytoene desaturase CrtI from Rubrivivax gelatinosus catalyzes simultaneously a three- and four-step desaturation producing both neurosporene and lycopene. These carotenes are intermediates for the synthesis of spheroidene and spirilloxanthin, respectively. Two different mutation libraries for the crtI gene from R. gelatinosus were constructed to screen for modified enzymes which synthesize almost exclusively either neurosporene or lycopene. The resulting mutants carried between one and four amino acid exchanges and at least one of them affected the secondary protein structure by shortening or extending one of the helices. A prominent amino acid which was exchanged in the neurosporene or lycopene-forming desaturase was leucine 208. Enzyme kinetic studies were carried out with the L208 modified desaturase and the specificities for phytoene and neurosporene as substrates determined. Higher and lower values correlate well with the higher or lower potential for the synthesis of lycopene from neurosporene. TopPred analysis of the mutations of L208 indicated that the location is in a highly hydrophobic membrane-integrated region which is a good candidate for the substrate-binding site of the desaturase.  相似文献   

2.
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.  相似文献   

3.
Photosynthetic organisms synthesize a diverse range of carotenoids. These pigments are important for the assembly, function and stability of photosynthetic pigment-protein complexes, and they are used to quench harmful radicals. The photosynthetic bacterium Rhodobacter sphaeroides was used as a model system to explore the origin of carotenoid diversity. Replacing the native 3-step phytoene desaturase (CrtI) with the 4-step enzyme from Erwinia herbicola results in significant flux down the spirilloxanthin pathway for the first time in Rb. sphaeroides. In Rb. sphaeroides, the completion of four desaturations to lycopene by the Erwinia CrtI appears to require the absence of CrtC and, in a crtC background, even the native 3-step enzyme can synthesize a significant amount (13%) of lycopene, in addition to the expected neurosporene. We suggest that the CrtC hydroxylase can intervene in the sequence of reactions catalyzed by phytoene desaturase. We investigated the properties of the lycopene-synthesizing strain of Rb. sphaeroides. In the LH2 light-harvesting complex, lycopene transfers absorbed light energy to the bacteriochlorophylls with an efficiency of 54%, which compares favourably with other LH2 complexes that contain carotenoids with 11 conjugated double bonds. Thus, lycopene can join the assembly pathway for photosynthetic complexes in Rb. sphaeroides, and can perform its role as an energy donor to bacteriochlorophylls.  相似文献   

4.
Phytoene desaturase Al-1 from Neurospora crassa was expressed in Escherichia coli and an active enzyme was isolated which catalyzed the stepwise introduction of up to five double bonds into the substrate phytoene. The major reaction products were 3, 4-didehydrolycopene and lycopene. Several of the desaturation intermediates, zeta-carotene, neurosporene, and lycopene, were also accepted as a substrate by Al-1. In contrast to the structurally related bacterial enzymes, the cofactor involved in the dehydrogenation reaction was NAD for Al-1. In situ competition with a neurosporene- and lycopene-converting hydratase and cyclase indicated that these enzymes can divert intermediates of the desaturation sequence. Based on the in vitro and in vivo results, the organization of the phytoene desaturase from N. crassa was proposed as an assembly of identical protein units which are responsible for the multistep reaction. However, the spatial arrangement should be loose enough to allow an exchange of individual intermediates in both directions in and out of this complex. Since gamma-carotene is not accepted as a substrate by Al-1, the formation of torulene must proceed exclusively by the cyclization of 3,4-didehydrolycopene.  相似文献   

5.
The biosynthesis of the major carotenoid spirilloxanthin by the purple nonsulfur bacterium Rhodospirillum rubrum is thought to occur via a linear pathway proceeding through phytoene and, later, lycopene as intermediates. This assumption is based solely on early chemical evidence (B. H. Davies, Biochem. J. 116:93–99, 1970). In most purple bacteria, the desaturation of phytoene, catalyzed by the enzyme phytoene desaturase (CrtI), leads to neurosporene, involving only three dehydrogenation steps and not four as in the case of lycopene. We show here that the chromosomal insertion of a kanamycin resistance cassette into the crtC-crtD region of the partial carotenoid gene cluster, whose gene products are responsible for the downstream processing of lycopene, leads to the accumulation of the latter as the major carotenoid. We provide spectroscopic and biochemical evidence that in vivo, lycopene is incorporated into the light-harvesting complex 1 as efficiently as the methoxylated carotenoids spirilloxanthin (in the wild type) and 3,4,3′,4′-tetrahydrospirilloxanthin (in a crtD mutant), both under semiaerobic, chemoheterotrophic, and photosynthetic, anaerobic conditions. Quantitative growth experiments conducted in dark, semiaerobic conditions, using a growth medium for high cell density and high intracellular membrane levels, which are suitable for the conventional industrial production in the absence of light, yielded lycopene at up to 2 mg/g (dry weight) of cells or up to 15 mg/liter of culture. These values are comparable to those of many previously described Escherichia coli strains engineered for lycopene production. This study provides the first genetic proof that the R. rubrum CrtI produces lycopene exclusively as an end product.  相似文献   

6.
八氢番茄红素脱氢酶的研究进展北大核心CSCD   总被引:1,自引:0,他引:1  
李春季  李炳学  韩晓日 《微生物学报》2016,56(11):1680-1690
类胡萝卜素是一类超过700种的萜烯基团类不饱和化合物的总称,根据结构可分为胡萝卜素族和叶黄素族,具有较高的营养价值。八氢番茄红素脱氢酶是类胡萝卜素生物合成途径中的首要限速酶,它参与催化无色的八氢番茄红素转变成有色类胡萝卜素,发挥着中心调控作用。不同生物源的八氢番茄红素脱氢酶在功能上呈现多样性,在大多数蓝细菌,藻类和高等植物的类胡萝卜素生物合成途径中,由Crt P,Crt Q和异构酶Crt H或PDS,ZDS和异构酶Z-ISO、Crt ISO共同参与番茄红素的形成,而在大多数微生物中只有Crt I-type一种酶来完成八氢番茄红素的脱氢反应,且根据脱氢步骤的不同分别可生成链孢红素、番茄红素或脱氢番茄红素。本文阐述了不同生物源八氢番茄红素脱氢酶的基因分离与鉴定,功能多样性及表达调控机制等最新研究进展,并进行了进化分析,为八氢番茄红素脱氢酶的深入研究及利用基因工程策略生产类胡萝卜素的应用提供重要信息。  相似文献   

7.
Evolution of carotene desaturation: the complication of a simple pathway   总被引:2,自引:0,他引:2  
In a series of desaturation reactions, the trienoic structures of phytoene and diapophytoene are extended to a maximum of 15 or 11 conjugated double bonds, respectively. After the cloning of several genes from bacteria and eukaryotes, the desaturation reactions were first analyzed in a heterologous host by functional genetic complementation. In addition, different desaturases were heterologously expressed and the reactions studied in vitro. This revealed that in archaea, non-photosynthetic prokaryotes and fungi the desaturases differ significantly from convergently evolved desaturases in cyanobacteria, Chlorobaculum (old name Chlorobium) species and eukaryotic photosynthetic organisms including plants. Detailed analysis of the desaturation reactions including the determination of the substrates converted by the enzymes, the intermediates and the products formed in the reactions revealed the bacterial all-trans desaturation pathway catalyzed by a single enzyme and the cyanobacterial/plant type poly-cis desaturation pathway which involves two closely related desaturases. This indicates that in the course of evolution of carotenogenesis from bacteria via cyanobacteria to plants, the simple situation of one enzyme for the entire reaction sequence from phytoene to all-trans lycopene changed to a more complex process. Three individual enzymes, newly acquired phytoene and ζ-carotene desaturases, as well as a carotene isomerase which is phylogenetically related to CrtI are involved. Only the CrtI-type enzymes seem to have the property to catalyze cis to trans conversion of carotenes.  相似文献   

8.
At least 700 natural carotenoids have been characterized; they can be classified into C(30), C(40) and C(50) subfamilies. The first step of C(40) pathway is the combination of two molecules of geranylgeranyl pyrophosphate to synthesize phytoene by phytoene synthase (CrtB or PSY). Most natural carotenoids originate from different types and levels of desaturation by phytoene desaturase (CrtI or PDS+ZDS), cyclization by lycopene cyclase (CrtY or LYC) and other modifications by different modifying enzyme (CrtA, CrtU, CrtZ or BCH, CrtX, CrtO, etc.) of this C(40) backbone. The first step of C(30) pathway is the combination of two molecules of FDP to synthesize diapophytoene by diapophytoene synthase (CrtM). But natural C(30) pathway only goes through a few steps of desaturation to form diaponeurosporene by diapophytoene desaturase (CrtN). Natural C(50) carotenoid decaprenoxanthin is synthesized starting from the C(40) carotenoid lycopene by the addition of 2 C(5) units. Concerned the importance of carotenoids, more and more attention has been concentrated on achieving novel carotenoids. The method being used successfully is to construct carotenoids biosynthesis pathways by metabolic engineering. The strategy of metabolic engineering is to engineer a small number of stringent upstream enzymes (CrtB, CrtI, CrtY, CrtM, or CrtN), then use a lot of promiscuous downstream enzymes to obtain large number of novel carotenoids. Two key enzymes phytoene desaturase (CrtI(m)) and lycopene cyclase (CrtY(m)) have been modified and used with a series of downstream modifying enzymes with broad substrate specificity, such as monooxygenase (CrtA), carotene desaturase (CrtU), carotene hydroxylase (CrtZ), zeaxanthin glycosylase (CrtX) and carotene ketolase (CrtO) to extend successfully natural C(30) and C(40) pathways in E. coli. Existing C(30) synthase CrtM to synthesize carotenoids with different chain length have been engineered and a series of novel carotenoids have been achieved using downstream modifying enzymes. C(35) carotenoid biosynthesis pathway has been constructed in E. coli as described. C(45) and C(50) carotenoid biosynthesis pathways have also been constructed in E. coli, but it is still necessary to extend these two pathways. Those novel acyclic or cyclic carotenoids have a potential ability to protect against photooxidation and radical-mediated peroxidation reactions which makes them interesting pharmaceutical candidates.  相似文献   

9.
Abstract A screening procedure for carotenoid genes involving heterologous complementation with two different plasmid constructs was developed. The plasmids contained the crtE and crtB genes from Erwinia unredovora together with the phytoene desaturase gene from either Rhodobacter capsulatus or Synechococcus PCC 7942. Transformation in E. coli led to the accumulation of neurosporene and ζ-carotene, respectively. Co-transformation with an Anabaena plasmid library resulted in the isolation of the two plasmids, pZDS1 and pZDS1. Their gene products showed the ability to convert neurosporene and ζ-carotene into lycopene. In contrast, accumulated phytoene could not be converted. We conclude that the cloned gene codes for the carotenoid biosynthesis gene ζ-carotene desaturase ( zds ).  相似文献   

10.
11.
Diseases caused by rust fungi pose a significant threat to global plant production. Although carotenoid pigments are produced in spores of nearly all rust species, the corresponding biosynthesis pathway(s) have not been investigated. Here, candidate genes for carotenoid biosynthesis in Puccinia graminis f. sp. tritici (Pgt) were identified, cloned and functionally complemented using specifically engineered strains of Escherichia coli. A part of the carotenoid biosynthesis pathway in rust fungi was elucidated, with only two genes, CrtYB and CrtI, catalysing the reactions from geranyl–geranyl diphosphate (GGPP) to γ-carotene. The CrtYB gene encodes a bi-functional lycopene cyclase/phytoene synthase, which catalyses the condensation of two GGPP into phytoene, as well as the cyclisation of the ψ-end of lycopene to form γ-carotene. The CrtI gene encodes a phytoene desaturase that carries out four successive desaturations of phytoene, through the intermediates phytofluene and neurosporene to lycopene. The evolution of carotenoid pigmentation in rust fungi, including Pgt, P. graminis avenae, P. graminis secalis (Pgs), P. graminis lolli, P. striiformis f. sp. tritici, P. striiformis f. sp. pseudohordei, P. striiformis f. sp. hordei, the “scabrum” rust (putative hybrids between Pgt and Pgs), P. triticina, and P. hordei, was investigated by phylogenetic analysis. Both CrtYB and CrtI were found to be closely related among rust fungi, other pathogenic fungi, and some aphids. Our results provide a springboard to increase the understanding of the physiological role(s) of carotenoid pigments in rust fungi, to better understand evolution within the Pucciniales, and to develop robust molecular diagnostics for rust fungi.  相似文献   

12.
Phytoene desaturases occurring in nature convert phytoene to either neurosporene or lycopene in most eubacteria. Approximately 10% of known phytoene desaturases, as in Rhodobacter, produce neurosporene, whereas the rest produce lycopene. These two types of enzymes, although similar in function, have relatively low similarity (below 60%) in terms of nucleotide or amino acid sequence. The mechanism controlling the product specificity of these enzymes is unclear. Here we used directed evolution to change the product of Rhodobacter sphaeroides phytoene desaturase (crtI gene product), a neurosporene-producing enzyme, to lycopene. Two generations of random mutagenesis were performed, from which three positive mutants were isolated and sequenced. We then used site-directed mutagenesis to determine the effect of each amino acid change. Gathering information from random mutagenesis, we further recombined the beneficial mutations by site-directed mutagenesis and increased the percent of lycopene production to 90%.  相似文献   

13.
Carotenoids are essential photoprotective and antioxidant pigments synthesized by all photosynthetic organisms. Most carotenoid biosynthetic enzymes were thought to have evolved independently in bacteria and plants. For example, in bacteria, a single enzyme (CrtI) catalyzes the four desaturations leading from the colorless compound phytoene to the red compound lycopene, whereas plants require two desaturases (phytoene and zeta-carotene desaturases) that are unrelated to the bacterial enzyme. We have demonstrated that carotenoid desaturation in plants requires a third distinct enzyme activity, the carotenoid isomerase (CRTISO), which, unlike phytoene and zeta-carotene desaturases, apparently arose from a progenitor bacterial desaturase. The Arabidopsis CRTISO locus was identified by the partial inhibition of lutein synthesis in light-grown tissue and the accumulation of poly-cis-carotene precursors in dark-grown tissue of crtISO mutants. After positional cloning, enzymatic analysis of CRTISO expressed in Escherichia coli confirmed that the enzyme catalyzes the isomerization of poly-cis-carotenoids to all-trans-carotenoids. Etioplasts of dark-grown crtISO mutants accumulate acyclic poly-cis-carotenoids in place of cyclic all-trans-xanthophylls and also lack prolamellar bodies (PLBs), the lattice of tubular membranes that defines an etioplast. This demonstrates a requirement for carotenoid biosynthesis to form the PLB. The absence of PLBs in crtISO mutants demonstrates a function for this unique structure and carotenoids in facilitating chloroplast development during the first critical days of seedling germination and photomorphogenesis.  相似文献   

14.
The zeta-carotene desaturase from Capsicum annuum (EC 1.14.99.-) was expressed in Escherichia coli, purified and characterized biochemically. The enzyme acts as a monomer with lipophilic quinones as cofactors. Km values for the substrate zeta-carotene or the intermediate neurosporene in the two-step desaturation reaction are almost identical. Product analysis showed that different lycopene isomers are formed, including substantial amounts of the all-trans form, together with 7,7',9,9'-tetracis prolycopene via the corresponding neurosporene isomers. The application of different geometric isomers as substrates revealed that the zeta-carotene desaturase has no preference for certain isomers and that the nature of the isomers formed during catalysis depends strictly on the isomeric composition of the substrate.  相似文献   

15.
In situ location of phytoene desaturase, a key enzyme in the carotenoid biosynthesis pathway, has been investigated in chloroplasts from higher plants. For this purpose, an antiserum has been raised against the phytoene desaturase from the cyanobacterium Synechococcus PCC 7942 overexpressed in E. coli . The specifity of this antiserum was demonstrated by inhibition of the enzymatic desaturation reaction in vitro. The antiserum was further purified and immunoabsorbed with E. coli proteins. The resulting IgG-fraction was tested by western blotting against membrane proteins from chloroplasts of tobacco ( Nicotiana tabacum L. cv. Samsun) and spinach ( Spinacia oleracea L. cv. Atlanta). Apparent molecular masses of immunoreactive proteins were 62 and 64 kDa. A western blot of different membrane fractions of spinach chloroplasts (inner and outer envelopes, and thylakoids) indicated a localization of the phytoene desaturase in thylakoids. A post embedding immunogold microscopy procedure was employed. In these experiments the main labelling (79%) was associated with thylakoid membranes of tobacco chloroplasts. Of the counted colloidal gold particles, 16% were found in the stroma. Only 5% were detected in the envelope membranes. These results give clear evidence that at least the majority of phytoene desaturase molecules is localized within thylakoid membranes of higher plant chloroplasts and that the presence of the enzyme in the envelope is of minor significance.  相似文献   

16.
A limited number of carotenoid pathway genes from microbial sources have been studied for analyzing the pathway complementation in the heterologous host Escherichia coli. In order to systematically investigate the functionality of carotenoid pathway enzymes in E. coli, the pathway genes of carotenogenic microorganisms (Brevibacterium linens, Corynebacterium glutamicum, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodopirellula baltica, and Pantoea ananatis) were modified to form synthetic expression modules and then were complemented with Pantoea agglomerans pathway enzymes (CrtE, CrtB, CrtI, CrtY, and CrtZ). The carotenogenic pathway enzymes in the synthetic modules showed unusual activities when complemented with E. coli. For example, the expression of heterologous CrtEs of B. linens, C. glutamicum, and R. baltica influenced P. agglomerans CrtI to convert its substrate phytoene into a rare product—3,4,3′,4′-tetradehydrolycopene—along with lycopene, which was an expected product, indicating that CrtE, the first enzyme in the carotenoid biosynthesis pathway, can influence carotenoid profiles. In addition, CrtIs of R. sphaeroides and R. capsulatus converted phytoene into an unusual lycopene as well as into neurosporene. Thus, this study shows that the functional complementation of pathway enzymes from different sources is a useful methodology for diversifying biosynthesis as nature does.  相似文献   

17.
The desaturation reactions of C30 carotenoids from diapophytoene to diaponeurosporene was investigated in vitro and by complementation in Escherichia coli. The expressed diapophytoene desaturase from Staphylococcus aureus inserts three double bonds in an FAD-dependent reaction. The enzyme is inhibited by diphenylamine. In the complementation experiment diapophytoene desaturase was able to convert C40 phytoene to some extend but exhibited a high affinity to ζ-carotene. Comparison to the reaction of a phytoene desaturase from Rhodobacter capsulatus catalyzing a parallel three-step desaturation sequence with the corresponding C40 carotenes revealed that this desaturase can also convert C30 diapophytoene. Other homologous bacterial C40 carotene desaturases could also utilize C30 substrates, including one type of ζ-carotene desaturase which converted diaponeurosporene to diapolycopene. Further complementation experiments including the diapophytoene synthase gene from S. aureus revealed that the C30 carotenogenic pathway is determined by this initial enzyme which is highly homologous to C40 phytoene synthases.  相似文献   

18.
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.  相似文献   

19.
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.  相似文献   

20.
We show that the C40 carotenoid desaturase CrtI from Pantoea ananatis (Erwinia uredovora) is capable of desaturating unnaturally long C45 and C50 carotenoid backbones in recombinant E. coli. Desaturation step number in these pathways is not very specific, and at least ten new C45 and C50 carotenoids were synthesized. We also present evidence for a novel asymmetric C40 backbone formed by the condensation of farnesyl diphosphate (C15PP) with farnesylgeranyl diphosphate (C25PP), and the subsequent desaturation of this backbone by CrtI in an atypical manner. Under some conditions, the C40, C45, and C50 carotenoid backbones synthesized in E. coli were monohydroxylated; their desaturation by CrtI in vitro led to yet more novel carotenoids. Challenging CrtI with larger-than-natural substrates in vivo has allowed us to show that this enzyme regulates desaturation step number by sensing the end groups of its substrate. Analysis of the mechanisms by which chemical diversity is generated and propagated through the nascent pathways provides insight into how natural product diversification occurs in nature.  相似文献   

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