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.     

Immunological detection of phytoene desaturase in algae and higher plants using an antiserum raised against a bacterial fusion-gene construct

Immunological characterization of phytoene desaturase, a key enzyme of carotenoid biosynthesis, is reported. For this purpose, a phytoene-desaturase fusion protein has been employed. For its construction 921 base pairs of the crtI gene were fused to the N-terminal region of the Escherichia coli lacZ gene. Plasmid pGABX2 resulted from insertion of a BglI - XhoI fragment from the Rhodobacter capsulatus carotenoid biosynthesis gene cluster, carrying the crtI, crtA and crtB genes, into pBR322. A 968-base-pair SalI-fragment from pGABX2 was cloned into pUR288 at the 3' end of the lacZ gene. Isopropyl-beta-D-thio-galactopyranoside-dependent activation of the lacZ fusion gene resulted in expression of a stable 150-kDa protein. After electroelution from SDS/polyacrylamide slab gels, the protein was used for antibody production. The heterogenic antiserum obtained was tested by Western blotting against proteins from Rhodobacter capsulatus and several different photoautotrophic organisms including higher plants. Apparent molecular masses of immunoreactive proteins from Rhodobacter, Aphanocapsa, rape and spinach were around 64 kDa. In Bumilleriopsis a 55-kDa protein was found instead. The antibody also inhibited in vitro desaturation of phytoene when detergent-solubilized membranes were employed.     

New functional assignment of the carotenogenic genes crtB and crtE with constructs of these genes from Erwinia species

The role of carotenoid genes crtB and crtE has been functionally assigned. These genes were cloned from Erwinia into Escherichia coli or Agrobacterium tumefaciens. Their functions were elucidated by assaying early isoprenoid enzymes involved in phytoene formation. In vitro reactions from extracts of E. coli carrying the crtE gene or a complete carotenogenic gene cluster in which crtB was deleted showed an elevated conversion of farnesyl pyrophosphate (FPP) into geranylgeranyl pyrophosphate (GGPP). These results strongly indicate that the crtE gene encodes GGPP synthase. Introduction of the crtB gene into A. tumefaciens led to the conversion of GGPP into phytoene. This activity was absent in similar transformants with the crtE gene. Thus, the crtB gene probably encodes phytoene synthase, which was further supported by demonstration that phytoene accumulated in E. coli harboring both the crtB and crtE genes.     

RegA control of bacteriochlorophyll and carotenoid synthesis in Rhodobacter capsulatus

We provide in vivo genetic and in vitro biochemical evidence that RegA directly regulates bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus. beta-Galactosidase expression assays with a RegA-disrupted strain containing reporter plasmids for Mg-protoporphyrin IX monomethyl ester oxidative cyclase (bchE), Mg-protoporphyrin IX chelatase (bchD), and phytoene dehydrogenase (crtI) demonstrate RegA is responsible for fourfold anaerobic induction of bchE, threefold induction of bchD, and twofold induction of crtI. Promoter mapping studies, coupled with DNase I protection assays, map the region of RegA binding to three sites in the bchE promoter region. Similar studies at the crtA and crtI promoters indicate that RegA binds to a single region equidistant from these divergent promoters. These results demonstrate that RegA is directly responsible for anaerobic induction of bacteriochlorophyll biosynthesis genes bchE, bchD, bchJ, bchI, bchG, and bchP and carotenoid biosynthesis genes crtI, crtB, and crtA.     

Isolation and characterization of canthaxanthin biosynthesis genes from the photosynthetic bacterium Bradyrhizobium sp. strain ORS278

A carotenoid biosynthesis gene cluster involved in canthaxanthin production was isolated from the photosynthetic Bradyrhizobium sp. strain ORS278. This cluster includes five genes identified as crtE, crtY, crtI, crtB, and crtW that are organized in at least two operons. The functional assignment of each open reading frame was confirmed by complementation studies.     

Production of lycopene by the food yeast, Candida utilis that does not naturally synthesize carotenoid

The Erwinia uredovora crtE, crtB, and crtI genes, which are responsible for the synthesis of carotenoid lycopene from farnesyl pyrophosphate, were expressed in Candida utilis under the control of the promoters and terminators derived from the C. utilis GAP, PGK, and PMA genes, respectively. The yeast transformant carrying the carotenoid biosynthesis genes produced 758 microg/g dry weight of lycopene along with 407 microg/g dry weight of phytoene in the stationary phase. It was observed in the C. utilis transformant that ergosterol content was decreased to 65% of that in the parent strain that accumulated 6.04 mg/g dry weight of ergosterol. It is therefore possible that the carbon flux for the ergosterol biosynthesis has been branched at farnesyl pyrophosphate to generate a new pathway for the lycopene production in this yeast transformant.     

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.     

Elucidation of a carotenoid biosynthesis gene cluster encoding a novel enzyme, 2,2'-beta-hydroxylase, from Brevundimonas sp. strain SD212 and combinatorial biosynthesis of new or rare xanthophylls

A carotenoid biosynthesis gene cluster mediating the production of 2-hydroxyastaxanthin was isolated from the marine bacterium Brevundimonas sp. strain SD212 by using a common crtI sequence as the probe DNA. A sequence analysis revealed this cluster to contain 12 open reading frames (ORFs), including the 7 known genes, crtW, crtY, crtI, crtB, crtE, idi, and crtZ. The individual ORFs were functionally analyzed by complementation studies using Escherichia coli that accumulated various carotenoid precursors due to the presence of other bacterial crt genes. In addition to functionally identifying the known crt genes, we found that one (ORF11, named crtG) coded for a novel enzyme, carotenoid 2,2'-beta-hydroxylase, which showed intriguingly partial homology with animal sterol-C5-desaturase. When this crtG gene was introduced into E. coli accumulating zeaxanthin and canthaxanthin, the resulting transformants produced their 2-hydroxylated and 2,2'-dihydroxylated products which were structurally novel or rare xanthophylls, as determined by their nuclear magnetic resonance and high-performance liquid chromatography/photodiode array detector/atmospheric pressure chemical ionization mass spectrometry spectral data. The new carotenoid produced was suggested to have a strong inhibitory effect on lipid peroxidation.     

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.     

An ORF323 with homology to crtE, specifying prephytoene pyrophosphate dehydrogenase, is encoded by cyanelle DNA in the eukaryotic alga Cyanophora paradoxa.

Carotenoids are essential constituents of the light-harvesting and light-protective systems of photosynthetic organisms. The biochemistry of carotenoid biosynthesis in eukaryotes is known, whereas evidence for the genes specifying this biosynthetic pathway is scant. We report here the nucleotide sequence and expression of a gene likely encoding crtE (prephytoene pyrophosphate dehydrogenase). The reaction product of this enzyme is phytoene, a C40 carotenoid precursor common to all organisms. The gene is found in the cyanelle (plastid) DNA of an eukaryotic alga, Cyanophora paradoxa. The expression into protein of cyanelle crtE has been demonstrated in vitro. The identity and similarity scores of CrtE from cyanelles with the corresponding protein from the photosynthetic bacterium Rhodobacter capsulatus are 28.6 and 68.5%, respectively.     

Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level.

A carotenoid biosynthesis gene cluster for the production of astaxanthin was isolated from the marine bacterium Agrobacterium aurantiacum. This cluster contained five carotenogenic genes with the same orientation, which were designated crtW, crtZ, crtY, crtI, and crtB. The stop codons of individual crt genes except for crtB overlapped the start codons of the following crt genes. Escherichia coli transformants carrying the Erwinia uredovora carotenoid biosynthesis genes provide suitable substrates for carotenoid biosynthesis. The functions of the five crt genes of A. aurantiacum were determined through chromatographic and spectroscopic analyses of the pigments accumulated in some E. coli transformants carrying various combinations of the E. uredovora and A. aurantiacum carotenogenic genes. As a result, the astaxanthin biosynthetic pathway is proposed for the first time at the level of the biosynthesis genes. The crtW and crtZ gene products, which mediated the oxygenation reactions from beta-carotene to astaxanthin, were found to have low substrate specificity. This allowed the production of many presumed intermediates of astaxanthin, i.e., adonixanthin, phoenicoxanthin (adonirubin), canthaxanthin, 3'-hydroxyechinenone, and 3-hydroxyechinenone.     

Production of beta-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovora.

The Erwinia uredovora crtB, crtE, crtI, and crtY genes required for beta-carotene biosynthesis were introduced by conjugal transfer into an ethanol-producing bacterium, Zymomonas mobilis, and a phytopathogenic bacterium, Agrobacterium tumefaciens, in which no carotenoid is synthesized. The transconjugants of Z. mobilis and A. tumefaciens carrying these genes appeared as yellow colonies and produced 220 and 350 micrograms of beta-carotene per g of dry weight, respectively, in the stationary phase in liquid culture.     

Search for the function of the nuclease NucM of Erwinia chrysanthemi

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 ).     

Two distinct crt gene clusters for two different functional classes of carotenoid in Bradyrhizobium

Aerobic photosynthetic bacteria possess the unusual characteristic of producing different classes of carotenoids. In this study, we demonstrate the presence of two distinct crt gene clusters involved in the synthesis of spirilloxanthin and canthaxanthin in a Bradyrhizobium strain. Each cluster contains the genes crtE, crtB, and crtI leading to the common precursor lycopene. We show that spirilloxanthin is associated with the photosynthetic complexes, while canthaxanthin protects the bacteria from oxidative stress. Only the spirilloxanthin crt genes are regulated by light via the control of a bacteriophytochrome. Despite this difference in regulation, the biosyntheses of both carotenoids are strongly interconnected at the level of the common precursors. Phylogenetic analysis suggests that the canthaxanthin crt gene cluster has been acquired by a lateral gene transfer. This acquisition may constitute a major selective advantage for this class of bacteria, which photosynthesize only under conditions where harmful reactive oxygen species are generated.     

Production of beta-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovora

The Erwinia uredovora crtB, crtE, crtI, and crtY genes required for beta-carotene biosynthesis were introduced by conjugal transfer into an ethanol-producing bacterium, Zymomonas mobilis, and a phytopathogenic bacterium, Agrobacterium tumefaciens, in which no carotenoid is synthesized. The transconjugants of Z. mobilis and A. tumefaciens carrying these genes appeared as yellow colonies and produced 220 and 350 micrograms of beta-carotene per g of dry weight, respectively, in the stationary phase in liquid culture.     

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

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

Controlling the Metabolic Flux through the Carotenoid Pathway Using Directed mRNA Processing and Stabilization

A synthetic operon containing the crtI and crtY genes, encoding the phytoene desaturase and the lycopene cyclase, respectively, was placed under the control of the araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 5' and 3' ends of the genes and a putative RNase E site was placed between the genes. This construct was transformed into Escherichia coli cells harboring the genes for phytoene production. By varying the mRNA secondary structures, we were able to modulate the flux through the carotenoid pathway, resulting in a 300-fold variation in the production of beta-carotene relative to lycopene. In addition, intermediates in the pathway from phytoene to beta-carotene production that are not observed in cells expressing the recombinant operon were observed when the engineered operons were used, indicating that changes in levels of the enzymes affected the formation of intermediates. These results indicate that it is possible to coordinately regulate the genes encoding the enzymes of a metabolic pathway and balance the production of the intermediates.