Redesign,Reconstruction, and Directed Extension of the Brevibacterium linens C40 Carotenoid Pathway in Escherichia coli

In this study, the carotenoid biosynthetic pathways of Brevibacterium linens DSMZ 20426 were reconstructed, redesigned, and extended with additional carotenoid-modifying enzymes of other sources in a heterologous host Escherichia coli. The modular lycopene pathway synthesized an unexpected carotenoid structure, 3,4-didehydrolycopene, as well as lycopene. Extension of the novel 3,4-didehydrolycopene pathway with the mutant Pantoea lycopene cyclase CrtY₂ and the Rhodobacter spheroidene monooxygenase CrtA generated monocyclic torulene and acyclic oxocarotenoids, respectively. The reconstructed β-carotene pathway synthesized an unexpected 7,8-dihydro-β-carotene in addition to β-carotene. Extension of the β-carotene pathway with the B. linens β-ring desaturase CrtU and Pantoea β-carotene hydroxylase CrtZ generated asymmetric carotenoid agelaxanthin A, which had one aromatic ring at the one end of carotene backbone and one hydroxyl group at the other end, as well as aromatic carotenoid isorenieratene and dihydroxy carotenoid zeaxanthin. These results demonstrate that reconstruction of the biosynthetic pathways and extension with promiscuous enzymes in a heterologous host holds promise as a rational strategy for generating structurally diverse compounds that are hardly accessible in nature.Carotenoids, which are produced by many microorganisms and plants, belong to a class of pigment chemicals found in nature. These structurally diverse pigments have different biological functions such as coloration, photo protection, light-harvesting, and precursors for many hormones (3, 22). Carotenoids are commercially used as food colorants, animal feed supplements and, more recently, as nutraceuticals and as cosmetic and pharmaceutical compounds (19). Currently, only a few carotenoids can be produced commercially by chemical synthesis, fermentation, or isolation from a few abundant natural sources (13). The increasing industrial importance of carotenoids has led to renewed efforts to develop bioprocesses for large-scale production of a range of carotenoids, including lycopene, β-carotene, and more structurally diverse carotenoids (17, 21, 30, 31, 34). Interestingly, a recent study showed that carotenoids with more diverse structures tend to have higher biological activity than simple structures (1).Previously, in vitro evolution altered the catalytic functions of the carotenoid enzymes phytoene desaturase CrtI and lycopene cyclase CrtY (Fig. ​(Fig.1)1) and produced novel carotenoid structures of tetradehydrolycopene and torulene in Escherichia coli (27). Furthermore, these in vitro evolved pathways and redesigned C₃₀ carotenoid biosynthetic pathways were successfully extended with additional, wild-type carotenoid modifying enzymes and evolved enzymes (21), generating novel carotenoid structures (26).Open in a separate windowFIG. 1.Reconstructed and redesigned B. linens carotenoid biosynthetic pathway in the heterologous host E. coli. Carotenogenic enzymes of B. linens, P. ananatis, and R. capsulatus, which were used for the biosynthetic pathway reconstruction, are indicated by boldface letters. Idi (IPP isomerase), IspA (FPP synthase), CrtE (GGPP synthase), CrtB (phytoene synthase), CrtI (phytoene desaturase), CrtYcYd (lycopene cyclase), CrtU (β-carotene desaturase), CrtZ (β-carotene hydrolase), CrtY₂ (mutant lycopene cyclase), and CrtA (spheroidene monooxygenase). B. linens 3,3′-dihydroxyisorenieratene biosynthesis is indicated by dashed arrows.Beside in vitro evolution (23, 34), combinatorial biosynthesis with carotenoid-modifying enzymes in a heterologous host has often been used to generate structurally novel carotenoids (24, 32). This combinatorial biosynthetic approach basically relies on the functional coordination of pathway enzymes from different sources in a heterologous host (5, 19, 35). Carotenogenic enzymes tend to be promiscuous in their substrate specificity (33) and show unexpected/hidden activities (20) when expressed in heterologous host microorganisms. One example is the unusual activity of diapophytoene desaturase CrtN in E. coli, which resulted in structurally novel compounds (20). Therefore, utilizing the promiscuity of carotenogenic enzymes makes combinatorial biosynthesis one of the most powerful strategies to generate structurally novel carotenoids that cannot be accessed in nature.Yellow colored Brevibacterium linens is commonly used as a food colorant by the cheese industry (15). Interestingly, B. linens is known to synthesize aromatic ring-containing carotenoids, isorenieratene and its hydroxy derivatives (6, 7, 16). They are produced by seven carotenogenic enzymes expressed in B. linens: GGPP synthase CrtE, phytoene synthase CrtE, phytoene desaturase CrtI, lycopene cyclase CrtYcYd, β-carotene desaturase CrtU, and the cytochrome P450 (Fig. ​(Fig.1).1). Even though the carotenoid biosynthetic pathways of B. linens have been recently studied (6, 10), there have been no systematic functional study of downstream enzymes such as lycopene cyclase CrtYcYd in the biosynthetic pathway of B. linens in a heterologous environment.Therefore, in the present study, for the first time we reconstructed, redesigned, and rationally extended the B. linens carotenoids biosynthetic pathway in E. coli to investigate the flexibility of the pathway enzymes in a heterologous host. Using this approach, we obtained an unexpected structure 3,4-didehydrolycopene, 7,8-dihydro-β-carotene, torulene, and the asymmetric carotenoid, agelaxanthin A, from engineered B. linens carotenoid pathways in E. coli.     

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.     

Activation and analysis of crypticcrt genes for carotenoid biosynthesis fromStreptomyces griseus

Genes encoding enzymes with sequence similarity to carotenoid biosynthetic enzymes of other organisms were cloned fromStreptomyces griseus JA3933 and transformed into the colourless (non-daunorubicin producing) mutantStreptomyces griseus IMET JA3933/956/2. Cells harbouring these genes showed an orange-red pigmentation, caused by the strongly hydrophobic, membrane-bound lycopene. The cloned fragment (9 kb) contained seven genes, four transcribed in one direction (crtEIBV) and three (crtYTU) transcribed convergently to them. Three of these genes encode polypeptides that resemble geranylgeranyl-pyrophosphate (GGPP) synthases (CrtE), phytoene synthases (PS) (CrtB) and phytoene dehydrogenases (PDH) (CrtI), respectively, of various bacteria. These enzymes are sufficient for the formation of lycopene.crtE alone was sufficient to induce zeaxanthin formation in anEscherichia coli clone containing thecrt gene cluster fromErwinia herbicola deleted forcrtE. The combination ofcrtE andcrtB led to formation of phytoene inS. griseus. The putativecrtEp promoter region was cloned and mapped by primer extension analysis. In a gel retardation experiment, this fragment was specifically shifted by an unknown protein. CrtY shows similarity to lycopene cyclases that convert lycopene intoβ-carotene, CrtT resembles various methyltransferases and CrtU a dehydrogenase. We conclude that these genes are functionally intact, but not expressed (cryptic) in the wild-typeS. griseus strain.     

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).     

Isolation and functional characterisation of a novel type of carotenoid biosynthetic gene from Xanthophyllomyces dendrorhous

The red heterobasidiomycetous yeast Xanthophyllomyces dendrorhous (perfect state of Phaffia rhodozyma) contains a novel type of carotenoid biosynthetic enzyme. Its structural gene, designated crtYB, was isolated by functional complementation in a genetically modified, carotenogenic Escherichia coli strain. Expression studies in different carotenogenic E. coli strains demonstrated that the crtYB gene encodes a bifunctional protein involved both in synthesis of phytoene from geranylgeranyl diphosphate and in cyclisation of lycopene to β-carotene. By sequence comparison with other phytoene synthases and complementation studies in E. coli with various deletion derivatives of the crtYB gene, the regions responsible for phytoene synthesis and lycopene cyclisation were localised within the protein. Received: 20 January 1999 / Accepted: 21 May 1999     

Activation and analysis of crypticcrt genes for carotenoid biosynthesis fromStreptomyces griseus

Genes encoding enzymes with sequence similarity to carotenoid biosynthetic enzymes of other organisms were cloned fromStreptomyces griseus JA3933 and transformed into the colourless (non-daunorubicin producing) mutantStreptomyces griseus IMET JA3933/956/2. Cells harbouring these genes showed an orange-red pigmentation, caused by the strongly hydrophobic, membrane-bound lycopene. The cloned fragment (9 kb) contained seven genes, four transcribed in one direction (crtEIBV) and three (crtYTU) transcribed convergently to them. Three of these genes encode polypeptides that resemble geranylgeranyl-pyrophosphate (GGPP) synthases (CrtE), phytoene synthases (PS) (CrtB) and phytoene dehydrogenases (PDH) (CrtI), respectively, of various bacteria. These enzymes are sufficient for the formation of lycopene.crtE alone was sufficient to induce zeaxanthin formation in anEscherichia coli clone containing thecrt gene cluster fromErwinia herbicola deleted forcrtE. The combination ofcrtE andcrtB led to formation of phytoene inS. griseus. The putativecrtEp promoter region was cloned and mapped by primer extension analysis. In a gel retardation experiment, this fragment was specifically shifted by an unknown protein. CrtY shows similarity to lycopene cyclases that convert lycopene into-carotene, CrtT resembles various methyltransferases and CrtU a dehydrogenase. We conclude that these genes are functionally intact, but not expressed (cryptic) in the wild-typeS. griseus strain.     

Investigation of cellular targeting of carotenoid pathway enzymes in Pichia pastoris

Cellular targeting of lycopene biosynthetic enzymes was investigated in Pichia pastoris X-33. Three lycopene pathway enzymes, CrtE, CrtB, and CrtI, were fused to fluorescent EGFPs with or without a peroxisomal targeting sequence (PTS1) and then expressed in P. pastoris. When P. pastoris was grown in YPD, the PTS1 fusion enzymes were found to be localized in peroxisomes, whereas the enzymes not fused with PTS1 were equally distributed throughout the entire cell. A similar targeting pattern was also observed in P. pastoris strains that were grown in peroxisome-proliferating medium, YPOT. Analysis of the fluorescent images of isolated peroxisomes showed that the PTS1 fused enzymes were dominantly present in peroxisomes whereas small amount of the enzymes not fused with PTS1 were non-specifically sent to peroxisomes. These results indicate that PTS1 specifically target lycopene pathway enzymes into peroxisomes and this targeting pathway was strong enough to overcome their inherent targeting program. In conclusion, we first showed that carotenogenic enzymes can be targeted into the specific cellular location of recombinant hosts and this targeting strategy can serve as the basis for the subsequent development of sophisticated pathway engineering in microorganisms.     

Engineering the lycopene synthetic pathway in <Emphasis Type="Italic">E. coli</Emphasis> by comparison of the carotenoid genes of <Emphasis Type="Italic">Pantoea agglomerans</Emphasis> and <Emphasis Type="Italic">Pantoea ananatis</Emphasis>

The lycopene synthetic pathway was engineered in Escherichia coli using the carotenoid genes (crtE, crtB, and crtI) of Pantoea agglomerans and Pantoea ananatis. E. coli harboring the P. agglomerans crt genes produced 27 mg/l of lycopene in 2YT medium without isopropyl-beta-d-thiogalactopyranoside (IPTG) induction, which was twofold higher than that produced by E. coli harboring the P. ananatis crt genes (12 mg/l lycopene) with 0.1 mM IPTG induction. The crt genes of P. agglomerans proved better for lycopene production in E. coli than those of P. ananatis. The crt genes of the two bacteria were also compared in E. coli harboring the mevalonate bottom pathway, which was capable of providing sufficient carotenoid building blocks, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), with exogenous mevalonate supplementation. Lycopene production significantly increased using the mevalonate bottom pathway and 60 mg/l of lycopene was obtained with the P. agglomerans crt genes, which was higher than that obtained with the P. ananatis crt genes (35 mg/l lycopene). When crtE among the P. ananatis crt genes was replaced with P. agglomerans crtE or Archaeoglobus fulgidus gps, both lycopene production and cell growth were similar to that obtained with P. agglomerans crt genes. The crtE gene was responsible for the observed difference in lycopene production and cell growth between E. coli harboring the crt genes of P. agglomerans and P. ananatis. As there was no significant difference in lycopene production between E. coli harboring P. agglomerans crtE and A. fulgidus gps, farnesyl diphosphate (FPP) synthesis was not rate-limiting in E. coli. Sang-Hwal Yoon and Ju-Eun Kim: These authors contributed equally to this work.     

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.     

Why Is Golden Rice Golden (Yellow) Instead of Red?

The endosperm of Golden Rice (Oryza sativa) is yellow due to the accumulation of beta-carotene (provitamin A) and xanthophylls. The product of the two carotenoid biosynthesis transgenes used in Golden Rice, phytoene synthase (PSY) and the bacterial carotene desaturase (CRTI), is lycopene, which has a red color. The absence of lycopene in Golden Rice shows that the pathway proceeds beyond the transgenic end point and thus that the endogenous pathway must also be acting. By using TaqMan real-time PCR, we show in wild-type rice endosperm the mRNA expression of the relevant carotenoid biosynthetic enzymes encoding phytoene desaturase, zeta-carotene desaturase, carotene cis-trans-isomerase, beta-lycopene cyclase, and beta-carotene hydroxylase; only PSY mRNA was virtually absent. We show that the transgenic phenotype is not due to up-regulation of expression of the endogenous rice pathway in response to the transgenes, as was suggested to be the case in tomato (Lycopersicon esculentum) fruit, where CRTI expression resulted in a similar carotenoid phenomenon. This means that beta-carotene and xanthophyll formation in Golden Rice relies on the activity of constitutively expressed intrinsic rice genes (carotene cis-trans-isomerase, alpha/beta-lycopene cyclase, beta-carotene hydroxylase). PSY needs to be supplemented and the need for the CrtI transgene in Golden Rice is presumably due to insufficient activity of the phytoene desaturase and/or zeta-carotene desaturase enzyme in endosperm. The effect of CRTI expression was also investigated in leaves of transgenic rice and Arabidopsis (Arabidopsis thaliana). Here, again, the mRNA levels of intrinsic carotenogenic enzymes remained unaffected; nevertheless, the carotenoid pattern changed, showing a decrease in lutein, while the beta-carotene-derived xanthophylls increased. This shift correlated with CRTI-expression and is most likely governed at the enzyme level by lycopene-cis-trans-isomerism. Possible implications are discussed.     

Carbohydrate metabolism of the phase variants of purple photosynthetic bacteria

The R and M phase variants of Rhodobacter sphaeroides and Rhodobacter capsulatus were isolated. The growth rates in the dark and in the light in glucose-containing media were much higher for the Rba. sphaeroides R variant than for the M variant. For the Rba. capsulatus R and M variants, growth rates in the dark and in the light in fructose- or glucose-containing media differed insignificantly. The cells of Rba. sphaeroides and Rba. capsulatus phase variants growing in media with glucose and fructose exhibited differences in activity of the key enzymes of the Embden–Meyerhof–Parnas (EMP) and Entner–Doudoroff (ED) pathways. The oxidative pentose phosphate pathway (PPP) does not participate in glucose and fructose metabolism in the studied bacteria. Specific activity of the ED pathway enzymes was higher in dark-grown R and M variants of both Rba. sphaeroides and Rba. capsulatus than in the cells grown under light. Specific activity of the EMP enzymes was higher for the R and M variants of both cultures grown in the light than for those grown in the dark. Activities of the 2-keto-3-deoxy-6-phosphogluconate and fructose bisphosphate aldolases, the key enzymes of the ED and EMP pathways in Rba. sphaeroides M variant grown in the medium with glucose in the light or in the dark, were approximately twice those of the R variant. In the medium with fructose activities of these enzymes in both R and M variants did not change significantly depending on growth conditions. Activities of the enzymes of the EMP and ED pathways in the extracts of the Rba. capsulatus R and M cells grown with glucose or fructose did not change significantly. Cultivation of Rba. sphaeroides and Rba. capsulatus phase variants in the medium with fructose resulted in a considerably increased synthesis of 1-phosphofructokinase. Induction of 1-phosphofructokinase synthesis in Rba. sphaeroides occurred only in the light, while in Rba. capsulatus induction of this enzyme in the medium with fructose was observed both in the dark and in the light. Thus, under aerobic conditions in the dark the phase variants of both bacteria probably assimilated glucose and fructose via the ED pathway, while in the light the EMP pathway was active.     

Functional properties of diapophytoene and related desaturases of C30 and C40 carotenoid biosynthetic pathways

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.     

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.     

Phytoene synthase and phytoene dehydrogenase associated with envelope membranes from spinach chloroplasts

Envelope membranes of spinach chloroplasts contain appreciable activities of the carotenogenic enzymes phytoene synthase (formation of phytoene by condensation of two molecules geranylgeranyl pyrophosphate) and phytoene dehydrogenase (formation of lycopene from phytoene), plus a phosphatase activity. These results were obtained by coincubation experiments using isolated envelope membranes and either a phytoene-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate) or [¹⁴C]geranylgeranyl pyrophosphate or a geranylgeranyl-pyrophosphate-forming in vitro system (from [1-¹⁴C]isopentenyl pyrophosphate). Within thylakoids carotenogenic enzymes could not be detected. It is concluded that the chloroplast envelope is at least a principal site of the membrane-bound steps of carotenoid biosynthesis in chloroplasts.Abbreviastions Chlorophyll a_GC Chlorophyll a, esterified with geranylgeraniol - GGPP geranylgeranyl pyrophosphate - HPLC high pressure liquid chromatography - IPP isopentenyl pyrophosphate     

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.     

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 β-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 β-carotene, as determined by high-performance liquid chromatography. We finally succeeded in constructing an S. cerevisiae strain capable of producing high levels of β-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 β-carotene by S. cerevisiae.     

Catalytic properties of the expressed acyclic carotenoid 2-ketolases from Rhodobacter capsulatus and Rubrivivax gelatinosus

Purple photosynthetic bacteria synthesize the acyclic carotenoids spheroidene and spirilloxanthin which are ketolated to spheroidenone and 2,2′-diketospirilloxanthin under aerobic growth. For the studies of the catalytic reaction of the ketolating enzyme, the crtA genes from Rubrivivax gelatinosus and Rhodobacter capsulatus encoding acyclic carotenoid 2-ketolases were expressed in Escherichia coli to functional enzymes. With the purified enzyme from the latter, the requirement of molecular oxygen and reduced ferredoxin for the catalytic activity was determined. Furthermore, the putative intermediate 2-HO-spheroidene was in vitro converted to the corresponding 2-keto product. Therefore, a monooxygenase mechanism involving two consecutive hydroxylation steps at C-2 were proposed for this enzyme. By functional pathway complementation studies in E. coli and enzyme kinetic studies, the product specificity of both enzymes were investigated. It appears that the ketolases could catalyze most intermediates and products of the spheroidene and spirilloxanthin pathway. This was also the case for the enzyme from Rba. capsulatus from which spirilloxanthin synthesis is absent. In general, the ketolase of Rvi. gelatinosus had a better specificity for spheroidene, HO-spheroidene and spirilloxanthin as substrates than the ketolase from Rba. capsulatus.     

Carotenoid biosynthesis in photosynthetic bacteria. Genetic characterization of the Rhodobacter capsulatus CrtI protein

Carotenoids are photoprotective pigments present in many photosynthetic and nonphotosynthetic organisms. The desaturation of phytoene into phytofluene is an early step in the biosynthetic pathway that in the photosynthetic bacterium Rhodobacter capsulatus is mediated by the product of the crtI gene. Here we report the sequence of this gene and the identification of CrtI as a membrane protein of approximate Mr 60,000. Mutant strains with 5-fold lower or 10-fold higher levels of CrtI with respect to wild type have only small differences in their carotenoid content, indicating that the cellular concentration of CrtI is not a limiting factor in carotenoid biosynthesis. However, a correlation was found between the levels of CrtI and the formation of a photosynthetic antenna system.