Exploring the potential of the bacterial carotene desaturase CrtI to increase the beta-carotene content in Golden Rice

To increase the beta-carotene (provitamin A) content and thus the nutritional value of Golden Rice, the optimization of the enzymes employed, phytoene synthase (PSY) and the Erwinia uredovora carotene desaturase (CrtI), must be considered. CrtI was chosen for this study because this bacterial enzyme, unlike phytoene synthase, was expressed at barely detectable levels in the endosperm of the Golden Rice events investigated. The low protein amounts observed may be caused by either weak cauliflower mosaic virus 35S promoter activity in the endosperm or by inappropriate codon usage. The protein level of CrtI was increased to explore its potential for enhancing the flux of metabolites through the pathway. For this purpose, a synthetic CrtI gene with a codon usage matching that of rice storage proteins was generated. Rice plants were transformed to express the synthetic gene under the control of the endosperm-specific glutelin B1 promoter. In addition, transgenic plants expressing the original bacterial gene were generated, but the endosperm-specific glutelin B1 promoter was employed instead of the cauliflower mosaic virus 35S promoter. Independent of codon optimization, the use of the endosperm-specific promoter resulted in a large increase in bacterial desaturase production in the T(1) rice grains. However, this did not lead to a significant increase in the carotenoid content, suggesting that the bacterial enzyme is sufficiently active in rice endosperm even at very low levels and is not rate-limiting. The endosperm-specific expression of CrtI did not affect the carotenoid pattern in the leaves, which was observed upon its constitutive expression. Therefore, tissue-specific expression of CrtI represents the better option.     

High-Level Production of the Industrial Product Lycopene by the Photosynthetic Bacterium Rhodospirillum rubrum

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.     

The cyanobacterium Gloeobacter violaceus PCC 7421 uses bacterial-type phytoene desaturase in carotenoid biosynthesis

Carotenoid composition and its biosynthetic pathway in the cyanobacterium Gloeobacter violaceus PCC 7421 were investigated. beta-Carotene and (2S,2'S)-oscillol 2,2'-di(alpha-L-fucoside), and echinenone were major and minor carotenoids, respectively. We identified two unique genes for carotenoid biosynthesis using in vivo functional complementation experiments. In Gloeobacter, a bacterial-type phytoene desaturase (CrtI), rather than plant-type desaturases (CrtP and CrtQ), produced lycopene. This is the first demonstration of an oxygenic photosynthetic organism utilizing bacterial-type phytoene desaturase. We also revealed that echinenone synthesis is catalyzed by CrtW rather than CrtO. These findings indicated that Gloeobacter retains ancestral properties of carotenoid biosynthesis.     

Biosynthesis of novel carotenoid families based on unnatural carbon backbones: a model for diversification of natural product pathways

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.     

Functional assembly of the foreign carotenoid lycopene into the photosynthetic apparatus of Rhodobacter sphaeroides, achieved by replacement of the native 3-step phytoene desaturase with its 4-step counterpart from Erwinia herbicola

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.     

Effect of hydroxylated carotenoid deficiency on ABA accumulation in Arabidopsis

Abscisic acid (ABA) is a sesquiterpene compound (C₁₅) derived from C₄₀ carotenoids. The immediate carotenoid precursors for ABA biosynthesis, 9- cis -violaxanthin and 9'- cis -neoxanthin, are produced from β -carotene by a series of hydroxylation, epoxidation, and isomerization reactions. Carotenoid hydroxylase deficient mutants contain severely reduced levels of violaxanthin and neoxanthin ( < 20% of wild type level) and provide a unique system to correlate carotenoid substrate availability and ABA production in photosynthetic tissues under non-stressed conditions. Quantitative measurements indicated that ABA levels in the carotenoid hydroxylase mutants are reduced nearly 50% compared to the wild type plants under non-stressed conditions. When drought-stressed, wild type plants showed up to a 17-fold increase in ABA levels, while ABA levels in the carotenoid hydroxylase mutants were only increased 6- to 7-fold (25% of wild type drought-stressed ABA levels). Expression of AtNCED3 ( Arabidopsis thaliana nine- cis -epoxycarotenoid dioxygenase 3, the rate-limiting activity for ABA biosynthesis) was induced in the carotenoid hydroxylase mutants, but to a lesser extent than the 40-fold increase in wild type plants. Therefore, the reduced ABA accumulation in response to drought-stress is at least partially due to the attenuated increase in AtNCED3 gene expression in the carotenoid hydroxylase mutants. The remaining violaxanthin and neoxanthin in the carotenoid hydroxylase mutants can not be converted into ABA, indicating that there is probably a separate pool of violaxanthin and neoxanthin that is not accessible to the cleavage enzymes, because it is sequestered in the light-harvesting complexes.     

Identification of the carotenoid isomerase provides insight into carotenoid biosynthesis, prolamellar body formation, and photomorphogenesis

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.     

The PufX protein of Rhodobacter capsulatus affects the properties of bacteriochlorophyll a and carotenoid pigments of light-harvesting complex 1

A pufX gene deletion in the purple bacterium Rhodobacter capsulatus causes a severe photosynthetic defect and increases core light-harvesting complex (LH1) protein and bacteriochlorophyll a (BChl) levels. It was suggested that PufX interrupts the LH1 alpha/beta ring around the reaction centre, allowing quinone/quinol exchange. However, naturally PufX(-) purple bacteria grow photosynthetically with an uninterrupted LH1. We discovered that substitutions of the Rhodobacter-specific LH1 alpha seryl-2 decrease carotenoid levels in PufX(-)R. capsulatus. An LH1 alphaS2F mutation improved the photosynthetic growth of a PufX(-) strain lacking the peripheral LH2 antenna, although LH1 BChl absorption remained above wild-type, suggesting that Rhodobacter-specific carotenoid binding is involved in the PufX(-) photosynthetic defect and LH1 expansion is not. Furthermore, PufX overexpression increased LH1-like BChl absorption without inhibiting photosynthetic growth. PufX(+) LH1 alphaS2-substituted mutant strains had wild-type carotenoid levels, indicating that PufX modulates LH1 carotenoid binding, inducing a conformational change that favours quinone/quinol exchange.     

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.     

Comparison of carotenoid content,gene expression and enzyme levels in tomato (Lycopersicon esculentum) leaves

Physiological conditions which lead to changes in total carotenoid content in tomato plantlets were identified. Carotenoid levels were found to increase after the onset of a dark period during a normal 24 h cycle. This rapid initial increase is followed by a steady decrease in carotenoid content throughout the night. A decrease in the expression of several carotenogenic genes, namely pds, zds (carotenoid desaturases) and ptox (plastid terminal oxidase), was observed following the removal of the light (when carotenoid content is at its highest). An increase in gene expression was observed before the return to light for pds and zds (when carotenoid levels were at their lowest), or following the return to light for ptox. The phytoene desaturation inhibitor norflurazon leads to a decrease coloured carotenoid content and, in the light, this correlated with pds and zds gene induction. In the dark, norflurazon treatment led to only a weak decrease in carotenoid content and only a small increase in pds and zds gene expression. The striking absence of phytoene accumulation under norflurazon treatment in the dark suggests a down-regulation of carotenoid formation in darkness However, prolonged dark conditions, or treatment with photosynthetic inhibitors, surprisingly led to higher carotenoid levels, which correlated with decreased expression of most examined genes. In addition to light, which acts in a complex way on carotenoid accumulation and gene expression, our results are best explained by a regulatory effect of carotenoid levels on the expression of several biosynthetic genes. In addition, monitoring of protein amounts for phytoene desaturase and plastid terminal oxidase (which sometimes do not correlate with gene expression) indicate an even more complex regulatory pattern.     

Progress on molecular breeding and metabolic engineering of biosynthesis pathways of C(30), C(35), C(40), C(45), C(50) carotenoids

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.     

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.     

Nucleotide sequence of the methoxyneurosporene dehydrogenase gene from Rhodobacter sphaeroides: comparison with other bacterial carotenoid dehydrogenases.

The nucleotide sequence of the 1794-bp fragment containing the crtD gene from Rhodobacter sphaeroides 2.4.1 encoding for methoxyneurosporene dehydrogenase has been determined. A 63% sequence identity was found when compared with the nucleotide sequence of the crtD gene from Rhodobacter capsulatus. A putative regulatory palindromic motif present in the crtD gene from R. capsulatus also exists in this gene from R. sphaeroides. The translated open reading frame of the crtD gene of R. sphaeroides has identified a polypeptide of 495 amino acids which shares a 56% sequence identity with the same CrtD protein of R. capsulatus. The N- and C-termini of these CrtD proteins present a high degree of similarity with the N- and C-termini of other carotenoid dehydrogenases including those encoded by crtI genes. This is in good agreement with the previously hypothesized homology between CrtI and CrtD proteins.     

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.     

Kinetic variations determine the product pattern of phytoene desaturase from Rubrivivax gelatinosus

In bacteria and fungi, the degree of carotenoid desaturation is determined by a single enzyme, the CrtI-type phytoene desaturase. In different organisms, this enzyme can carry out either three, four or even five desaturation steps. The purple bacterium Rubrivivax gelatinosus is the only known species in which reaction products of a 3-step and a 4-step desaturation (i.e. neurosporene and lycopene derivatives) accumulate simultaneously. The properties of this phytoene desaturation to catalyze neurosporene or lycopene were analyzed by heterologous complementations in Escherichia coli and by in vitro studies. They demonstrated that high enzyme concentrations or low phytoene supply favor the formation of lycopene. Under these conditions, CrtI from Rhodobacter spheroides can be forced in vitro to lycopene formation although this carotene is not synthesized in this species. All results can be explained by a model based on the competition between phytoene and neurosporene for the substrate binding site of phytoene desaturase. Mutations in CrtI from Rvi. gelatinosus have been generated resulting in increased lycopene formation in Escherichia coli. This modification in catalysis is due to increased amounts of CrtI protein.     

Heterologous Carotenoid-Biosynthetic Enzymes: Functional Complementation and Effects on Carotenoid Profiles in Escherichia coli

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.