Biosynthesis of a novel C30 carotenoid in Bacillus firmus isolates.

AIMS: Pigmented Bacillus spp. with probiotic properties have been isolated. In the yellow-/orange-coloured strains, the carotenoid pigments present have been characterized. In contrast, the carotenoids present in the Bacillus isolates coloured red await identification. The present article reports progress on the elucidation of the pigment biosynthetic pathway in these red-pigmented Bacillus firmus strains. METHODS AND RESULTS: A combination of UV/Vis, chromatographic and mass spectrometry (MS) has revealed the properties of the predominant pigment and the end-point carotenoid of the pathway to be methyl 4,4'-diapolycopene-dioate after transmethylation. The diglycosyl ester of 4,4'-diapolycopene-dioate persists in vivo prior to chemical treatment. Different mutants and inhibitor treatment were employed to establish the C30 biosynthesis pathway with all precursors and intermediates to 4,4'-diapolycopene-dioate detected, which include 4,4'-diapophytene and all desaturation intermediates to 4,4'-diapolycopene and 4,4'-diapolycopene-dialdehyde. To cultures synthesizing the 4,4'-diapolycopene-dioate derivative and those in which its formation was inhibited, oxidative stress was induced by peroxide treatment. Conditions that decreased the growth rate of the pigmented cells by only 30% caused a complete growth inhibition of the culture devoid of the 4,4'-diapolycopene-dioate derivative. CONCLUSION: This finding demonstrates the diversity of C30 carotenoid biosynthesis in Bacillus species and the antioxidative function of the 4,4'-diapolycopene-dioate derivative in B. firmus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: It could be shown that the C30 4,4'-diapolycopene-dioate derivatives protect pigmented B. firmus from peroxidative reactions. Under oxidative conditions, this can be an ecological advantage over nonpigmented (=noncarotenogenic) strains that are equally abundant.

Carotenoids found in Bacillus.

AIMS: To identify the diversity of pigmented aerobic spore formers found in the environment and to characterize the chemical nature of this pigmentation. MATERIALS AND RESULTS: Sampling of heat-resistant bacterial counts from soil, sea water and the human gastrointestinal tract. Phylogenetic profiling using analysis of 16S rRNA sequences to define species. Pigment profiling using high-performance liquid chromatography-photo diode array analysis. CONCLUSIONS: The most commonly found pigments were yellow, orange and pink. Isolates were nearly always members of the Bacillus genus and in most cases were related with known species such as Bacillus marisflavi, Bacillus indicus, Bacillus firmus, Bacillus altitudinis and Bacillus safensis. Three types of carotenoids were found with absorption maxima at 455, 467 and 492 nm, corresponding to the visible colours yellow, orange and pink, respectively. Although the presence of other carotenoids cannot be ruled out, these three predominant carotenoids appear to account for the pigments obtained in most pigmented bacilli, and our analysis reveals the existence of a C30 biosynthetic pathway. Interestingly, we report the presence of a water-soluble pigment that may also be a carotenoid. The function of carotenoids is photoprotection, and carotenoid-containing spores exhibited significantly higher levels of resistance to UV radiation than non-carotenoid-containing Bacillus species. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that pigmented bacilli are ubiquitous and contain new carotenoid biosynthetic pathways that may have industrial importance.

Elevation of the provitamin A content of transgenic tomato plants.

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

Identification of metabolites associated with water stress responses in Solanum tuberosum L. clones.

Water deficiency has become a major issue for modern agriculture as its effects on crop yields and tuber quality have become more pronounced. Potato genotypes more tolerant to water shortages have been identified through assessment of yield and dry matter. In the present study, a combination of metabolite profiling and physiological/agronomical measurements has been used to explore complex system level responses to non-lethal water restriction. The metabolites identified were associated with physiological responses in three different plant tissues (leaf, root and tuber) of five different potato genotypes varying in susceptibility/tolerance to drought. This approach explored the potential of metabolite profiling as a tool to unravel sectors of metabolism that react to stress conditions and could mirror the changes in the plant physiology. The metabolite results showed different responses of the three plant tissues to the water deficit, resulting either in different levels of the metabolites detected or different metabolites expressed. The leaf material displayed the most changes to drought as reported in literature. The results highlighted genotype-specific signatures to water restriction over all three plant tissues suggesting that the genetics can predominate over the environmental conditions. This will have important implications for future breeding approaches.

The purification of phytoene dehydrogenase from Phycomyces blakesleeanus.

The carotenogenic enzyme phytoene dehydrogenase has been purified from the C9carR21(-) (lycopene-accumulating) mutant of the filamentous fungus Phycomyces blakesleeanus. Solubilization of the membrane-bound enzyme with 1% Tween-60 was followed by a 250-fold purification to homogeneity using polyethylene glycol precipitation, CM-Sepharose, gel filtration and isoelectric focusing. Multiple peaks of enzymic activity were found in eluates from ion-exchange and gel filtration chromatography, with the lowest molecular weight fraction having an apparent molecular mass of approx. 14 kDa. All active fractions catalyzed the dehydrogenation of 15-cis phytoene into all-trans lycopene, with a cis-trans isomerization occurring at phytofluene. Both NADP+ and FAD were required for the dehydrogenation reaction. The presence of > 0.5% Tween-60 was necessary to maintain enzymic activity, although in its absence lipids restored some activity. The enzyme could be stored for at least 6 weeks at -70 degrees C in the presence of 20% (v/v) glycerol.

Expression of an active phytoene synthase from Erwinia uredovora and biochemical properties of the enzyme.

The crtB gene encoding phytoene synthase from the carotenogenic enterobacterium Erwinia uredovora was overexpressed to about 20% of the total cellular protein in Escherichia coli. Formation of the active phytoene synthase had the effect of suppressing the growth of the expressing strain. Presumably inhibition of growth arose from the depletion of the substrate geranylgeranyl pyrophosphate (GGPP) which, in E. coli, is necessary for the synthesis of essential prenylpyrophosphate derivatives. In order to overcome the poor growth characteristics of the phytoene synthase expressing strain, GGPP levels were increased by co-expressing the isoprenoid biosynthetic genes crtE and idi, encoding the Erwinia GGPP synthase and Rhodobacter isopentenyl pyrophosphate isomerase, respectively. The crude enzyme preparation was partially purified 15-fold by chromatography on a DEAE column. A non-radioactive assay was developed that enabled the conversion of GGPP to phytoene. The reaction product was identified by co-chromatography with authentic standards on HPLC systems and comparison of spectral characteristics. The phytoene formed in vitro was present in both a 15-cis and all-trans isomeric configuration. The essential cofactors required were ATP in combinations with either Mn2+ or Mg2+. The Km value for GGPP was determined as 41 microM. Phytoene synthesis was inhibited by phosphate ions and squalestatin. The I50 value for the latter inhibitor was 15 microM. Lineweaver-Burk plots showed constant Km values in the presence or absence of squalestatin.

Carotenoid biosynthesis in wild type and mutant strains of Mucor circinelloides.

Carotenoid biosynthesis in wild type Mucor circinelloides has been investigated and the biochemical characterisation of the MS1 and MS9 mutant strains, impaired in carotenoid formation, carried out. In liquid cultures, all strains produced carotenoids (mainly beta-carotene, but also xi-carotene, lycopene and gamma-carotene) at the onset of stationary phase of growth. Carotenogenesis was light dependent. In liquid cultures carotenoid formation in wild type was affected by diphenylamine, which prevented desaturation, nicotine, resulting in reduced carotenoid levels, but CPTA caused an increase in the total carotenoid content but a reduced beta-carotene level, with the accumulation of lycopene and gamma-carotene. The mutant strains MS1 and MS9 contained only 5.0 and 11.5% of wild type carotenoid levels, respectively. Cell extracts of light-grown mycelia, incubated with 3(R)-[2-14C] mevalonic acid, produced beta-carotene, but incorporations into carotenoids were substantially reduced in the cell extracts of MS1 and MS9. Analysis of prenyl diphosphate intermediates indicated that, compared to wild type, geranylgeranyl diphosphate accumulated in MS1. MS9 extracts produced a larger amount of prenyl phosphates and a more even distribution of radioactivity from mevalonic acid into farnesyl and geranylgeranyl diphosphates. Squalene and long chain prenyl phosphates were formed by the cell extracts of all strains. It is proposed that the MS1 strain possesses a mutation in a gene responsible for phytoene formation, whilst a regulatory mutation, affecting prenyl transferase activities has occurred in MS9.

Carotenoid Biosynthesis during Tomato Fruit Development (Evidence for Tissue-Specific Gene Expression).

Tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) fruit, at five stages of development, have been analyzed for their carotenoid and chlorophyll (Chl) contents, in vitro activities of phytoene synthase, phytoene desaturase, and lycopene cyclase, as well as expression of the phytoene synthase (Psy) and phytoene desaturase (Pds) genes. During ripening, the total carotenoids increased with a concomitant decrease in Chl. Although the highest carotenoid content (consisting mainly of lycopene and [beta]-carotene) was found in ripe fruit, the greatest carotenogenic enzymic activities were found in green fruit. Phytoene synthase was located in the plastid stroma, whereas the metabolism of phytoene was associated with plastid membranes during all stages of fruit development. The in vitro products of phytoene desaturation altered from being predominantly phytofluence and [zeta]-carotene in chloroplasts to becoming mainly lycopene in chromoplasts. The expression of Psy was detected in breaker and ripe fruit, as well as flowers, but was not detectable by northern blot analysis in leaves or green fruits. The Pds gene transcript was barely detectable in green fruit and leaves but was expressed in flowers and breaker fruit. These results suggest that transcription of Psy and Pds is regulated developmentally, with expression being considerably elevated in chromoplast-containing tissues. Antiserum to the Synechococcus phytoene synthase cross-reacted with phytoene synthase of green fruit only on western blots and not with the enzyme from ripe fruit. In contrast, a monoclonal antibody to the Psy gene product only cross-reacted with phytoene synthase from ripe fruit. The enzymes from green and ripe fruit had different molecular masses of 42 and 38 kD, respectively. The absence of detectable Psy and Pds mRNA in green tissues using northern blot analyses, despite high levels of phytoene synthase and desaturase activity, lends support to the hypothesis of divergent genes encoding these enzymes.

Expression of a tomato cDNA coding for phytoene synthase in Escherichia coli, phytoene formation in vivo and in vitro, and functional analysis of the various truncated gene products.

Full length and truncated cDNA expression constructs of the phytoene synthase (psy) gene from tomato have been ligated into a pUC8 cloning vector. One of the truncated constructs was introduced into Escherichia coli carrying the Erwinia uredovora GGPP synthase gene. This transformant produced 15,15'-cis-phytoene, which was identified on the basis of its UV and IR spectral data, from geranylgeranyl diphosphate. The function of this gene product was further confirmed by in vitro assay using cell-free extract of E. coli harboring the construct. On transformation with the above constructs together with a plasmid containing the carotenoid gene cluster from E. uredovora devoid of the phytoene synthase (crtB) gene, yellow, carotenoid-containing, E. coli colonies were produced. The amounts of carotenoids synthesized by the transformed cells, related to the steady-state levels of psy mRNA, varied depending upon the psy constructs. The full-length psy clone produced 16-fold less carotenoids per unit amount of RNA than cells containing phytoene synthase without the first 114 N-terminal amino acids. Removal of further amino acids from the N-terminus caused a large decrease in carotenogenesis. A Western blot of ripe fruit stroma with a monoclonal antibody raised against phytoene synthase revealed a single protein band of apparent molecular mass 38 kDa. Based upon this immunological evidence, we conclude that the size of the transit peptide of phytoene synthase from ripe tomato fruit is approximately 9 kDa, corresponding to about 80 amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the Cnr mutation on carotenoid formation during tomato fruit ripening.

The characteristic pigmentation of ripe tomato fruit is due to the deposition of carotenoid pigments. In tomato, numerous colour mutants exist. The Cnr tomato mutant has a colourless, non-ripening phenotype. In this work, carotenoid formation in the Cnr mutant has been studied at the biochemical level. The carotenoid composition of Ailsa Craig (AC) and Cnr leaves was qualitatively and quantitatively similar. However, Cnr fruits had low levels of total carotenoids and lacked detectable levels of phytoene and lycopene. The presence of normal tocopherols and ubiquinone-9 levels in the ripe Cnr fruits suggested that other biosynthetically related isoprenoids were unaffected by the alterations to carotenoid biosynthesis. In vitro assays confirmed the virtual absence of phytoene synthesis in the ripe Cnr fruit. Extracts from ripe fruit of the Cnr mutant also revealed a reduced ability to synthesise the carotenoid precursor geranylgeranyl diphosphate (GGPP). These results suggest that besides affecting the first committed step in carotenoid biosynthesis (phytoene synthase) the Cnr mutation also affects the formation of the isoprenoid precursor (GGPP).

Production and characterisation of monoclonal antibodies to phytoene synthase of Lycopersicon esculentum.

Monoclonal antibodies have been prepared against the tomato (Lycopersicon esculentum Mill.) fruit ripening-enhanced phytoene synthase (PSY1). The antigen was prepared as a beta-galactosidase fusion protein by cloning a 1.13 kb fragment of Psy1 cDNA into pUR291, followed by transformation of E. coli. The fusion protein, induced by IPTG, was purified by preparative SDS-PAGE and used to elicit an immune response. The cell lines were screened for cross-reactivity against beta-galactosidase-phytoene synthase fusion protein in E. coli extracts using western blotting and ELISA detection procedures. Positive clones were further screened for their ability to cross-react with the mature phytoene synthase protein on western blots as well as their ability to inhibit enzyme activity. Eleven monoclonal lines were obtained. Nine of these, all of the IgM isotype, exhibited strong responses to phytoene synthase of ripe tomato fruit on western blots, but did not inhibit enzyme activity effectively. The other two lines (IgG/la 2 isotypes) inhibited phytoene synthase activity in ripe tomato stroma, but produced a poor response to the protein on western blots. The monoclonals identified a ripe fruit phytoene synthase of 38 kDa, exclusively located in the chromoplast. In contrast, antibodies were unable to detect microbial phytoene synthases, nor phytoene synthase of maize leaf, tomato chloroplast or mango fruit extracts, either on western blots or from inhibition of phytoene synthase activity. However, they did cross-react with a 44 kDa protein from carrot leaf stroma and with three different proteins (44, 41, and 37 kDa) in carrot root. Cross-reactivity was also found with a 37 kDa protein from pumpkin fruit stroma.

Proteomic approach for the detection of chicken mechanically recovered meat.

Mechanically recovered meat is cheaper than raw meat and thus has been incorporated into many meat-derived products. EU regulations exclude mechanically recovered meat from the definition of meat; as a consequence analytical procedures are needed to differentiate it from hand-deboned meat. The present pilot study has utilized a proteomic approach to find potential markers for the detection of chicken mechanically recovered meat. Intact proteins were extracted from raw meat and then analyzed with OFF-GEL electrophoresis followed by SDS-PAGE and identification of potential markers by nano-LC-MS/MS. It was shown that it is possible to extract, separate and identify key proteins from processed meat material. Potential chicken mechanically recovered meat markers--hemoglobin subunits and those similar to myosin-binding protein C were also identified.

In vitro assays of three carotenogenic membrane-bound enzymes from Escherichia coli transformed with different crt genes.

In vitro assays have been developed for three membrane-bound carotenogenic enzymes, phytoene desaturase, lycopene cyclase and beta-carotene hydroxylase, expressed in Escherichia coli. Transformants of E. coli containing different deletion constructs of the Erwinia herbicola carotenogenic gene cluster were employed, allowing the estimation of enzyme activities without interference from subsequent reactions. New HPLC systems were developed to separate substrates and reaction products enabling the determination of radioactivity on-line. The newly developed assays facilitate the purification of these enzymes which have never been isolated before.

Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli.

Escherichia coli expressing the Erwinia carotenoid biosynthesis genes, crtE, crtB, crtI and crtY, form yellow-coloured colonies due to the presence of beta-carotene. This host was used as a visible marker for evaluating regulatory systems operating in isoprenoid biosynthesis of E. coli. cDNAs enhancing carotenoid levels were isolated from the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis. Nucleotide sequence analysis indicated that they coded for proteins similar to isopentenyl diphosphate (IPP) isomerase of the yeast Saccharomyces cerevisiae. Determination of enzymic activity confirmed the identity of the gene products as IPP isomerases. The corresponding gene was isolated from the genomic library of S. cerevisiae based on its nucleotide sequence, and was confirmed to have the same effect as the above two IPP isomerase genes when introduced into the E. coli transformant accumulating beta-carotene. In the three E. coli strains carrying the individual exogenous IPP isomerase genes, the increases in carotenoid levels are comparable to the increases in IPP isomerase enzyme activity with reference to control strains possessing the endogenous gene alone. These results imply that IPP isomerase forms an influential step in isoprenoid biosynthesis of the prokaryote E. coli, with potential for the efficient production of industrially useful isoprenoids by metabolic engineering.

In vitro characterization of astaxanthin biosynthetic enzymes.

Escherichia coli strains expressing the marine bacteria (Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) astaxanthin biosynthetic genes (crtZ and W), Haematococcus pluvialis bkt, and Erwinia uredovora crtZ genes were used for in vitro characterization of the respective enzymes. Specific enzyme assays indicated that all of the enzymes are bifunctional, in that the CrtZ enzymes formed zeaxanthin from beta-carotene via beta-cryptoxanthin, as well as astaxanthin from canthaxanthin via phoenicoxanthin (adonirubin). The BKT/CrtW enzymes synthesized canthaxanthin via echinenone from beta-carotene and 4-ketozeaxanthin (adonixanthin) with trace amounts of astaxanthin from zeaxanthin. Comparison of maximum catalytic activities as well as selectivity experiments carried out in the presence of both utilizable substrates indicated that the CrtZ enzymes from marine bacteria converted canthaxanthin to astaxanthin preferentially, whereas the Erwinia CrtZ possessed a favorability to the formation of zeaxanthin from beta-carotene. The CrtW/BKT enzymes were not so defined in their substrate preference, responding readily to fluctuations in substrate levels. Other properties obtained indicated that the enzymes were strictly oxygen-requiring; and a cofactor mixture of 2-oxoglutarate, ascorbic acid, and Fe2+ was beneficial to activity. Based on enzymological data, a predicted pathway for astaxanthin biosynthesis is described, and it is proposed that CrtZ-like enzymes be termed carotenoid 3, (3')-beta-ionone ring hydroxylase and CrtW/BKT carotenoid 4, (4')-beta-ionone ring oxygenase.

Phytoene synthase from tomato (Lycopersicon esculentum) chloroplasts--partial purification and biochemical properties.

Phytoene synthase activity in tomato chloroplasts is membrane-associated, requiring treatment with high ionic strength buffer or mild non-ionic detergent for solubilisation. Using a combination of ammonium sulphate precipitation, cation and anion exchange, dye-ligand and hydrophobic interaction chromatography, phytoene synthase has been purified 600-fold from tomato (Lycopersion esculentum Mill.) chloroplasts. The native molecular mass of the enzyme was 43 kDa. with an isoelectric point of 4.6. Although phytoene synthase was functional in a monomeric state, under optimal native conditions it was associated with a large (at least 200 kDa) protein complex which contained other terpenoid enzymes such as isopentenyl diphosphate isomerase and geranylgeranyl diphosphate (GGPP) synthase. Both Mn2+ and ATP, in combination, were essential for catalytic activity; their effect was stochiometric from 0.5 to 2 mM, with Km values for Mn2+, ATP and the substrate GGPP of 0.4 mM, 2.0 mM and 5 microM, respectively. The detergents Tween 60 and Triton X-100 (0.1 w/v) stimulated (5-fold) enzyme activity, but lipids (crude chloroplast lipids and phospholipids) had no such effect and could not compensate for the absence of detergent. A number of metabolites with possible regulatory effects were investigated, including beta-carotene, which reduced enzyme activity in vitro some 2-fold. A comparison of phytoene synthase activity from partially purified chloroplast and chromoplast preparations indicated biochemical differences.

Purification and reactivation of recombinant Synechococcus phytoene desaturase from an overexpressing strain of Escherichia coli.

The Synechococcus phytoene desaturase has been isolated from an overexpressing strain of Escherichia coli. The plasma pPDSde135 mediated the overexpression of the full-length polypeptide directly. The recombinant protein comprised 5% of the total cellular protein and was found predominantly in the inclusion body fraction. Urea was used to solubilize the recombinant protein from the inclusion fraction and the protein was subsequently purified to homogeneity on a DEAE-cellulose column. The purification scheme yielded 4.0 mg of homogeneous desaturase protein after a 20-fold purification, recovering 40% of the original protein from a 100 ml suspension culture of E. coli. The recombinant desaturase had an apparent molecular mass of 53 kDa on SDS/PAGE and crossreacted with an antiserum raised against the expressed protein. Desaturase activity was restored upon the removal of urea. The enzyme catalysed the conversion of phytoene to zeta-carotene via phytofluene. These products of the desaturase reaction existed predominantly in a cis configuration. Lipid replenishment enhanced activity. NAD+ and NADP+ were observed to be involved, whilst FAD was an ineffective electron acceptor.

Enzymic confirmation of reactions involved in routes to astaxanthin formation, elucidated using a direct substrate in vitro assay.

An in vitro assay procedure for the carotenoid (beta-ionone ring) 3,3'-hydroxylase and 4,4'-oxygenase has been developed that enables efficient conversion of non-radiolabeled carotenoid substrates added directly into aqueous solution. The following enzymic conversions were demonstrated and apparent kinetic constants (Vmax, Km, and specificity constants) obtained: (a) 3,3'-hydroxylase (from Agrobacterium aurantiacum and Alcaligenes sp. strain PC-1) converted phoenicoxanthin (adonirubin) to astaxanthin, 3-hydroxyechinenone to 4-ketozeaxanthin (adonixanthin), 3'-hydroxyechinenone to 4-ketozeaxanthin, as well as echinenone to 4-ketozeaxanthin via 3- and 3'-hydroxyechinenone; (b) 4,4'-Oxygenase (from A. aurantiacum, Alcaligenes sp. strain PC-1 and Haematococcus pluvialis) converted 4-ketozeaxanthin to astaxanthin, 3-hydroxyechinenone to phoenicoxanthin, 3'-hydroxyechinenone to phoenicoxanthin, and echinenone to canthaxanthin. Determination of substrate specifities allowed assessment of biosynthetic routes to astaxanthin formation and demonstrated that pathways via mono-hydroxylated and ketolated products are enzymically feasible.

Increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway.

The yeast Candida utilis does not possess an endogenous biochemical pathway for the synthesis of carotenoids. The central isoprenoid pathway concerned with the synthesis of prenyl lipids is present in C. utilis and active in the biosynthesis of ergosterol. In our previous study, we showed that the introduction of exogenous carotenoid genes, crtE, crtB, and crtI, responsible for the formation of lycopene from the precursor farnesyl pyrophosphate, results in the C. utilis strain that yields lycopene at 1.1 mg per g (dry weight) of cells (Y. Miura, K. Kondo, T. Saito, H. Shimada, P. D. Fraser, and N. Misawa, Appl. Environ. Microbiol. 64:1226-1229, 1998). Through metabolic engineering of the isoprenoid pathway, a sevenfold increase in the yield of lycopene has been achieved. The influential steps in the pathway that were manipulated were 3-hydroxy methylglutaryl coenzyme A (HMG-CoA) reductase, encoded by the HMG gene, and squalene synthase, encoded by the ERG9 gene. Strains overexpressing the C. utilis HMG-CoA reductase yielded lycopene at 2.1 mg/g (dry weight) of cells. Expression of the HMG-CoA catalytic domain alone gave 4.3 mg/g (dry weight) of cells; disruption of the ERG9 gene had no significant effect, but a combination of ERG9 gene disruption and the overexpression of the HMG catalytic domain yielded lycopene at 7.8 mg/g (dry weight) of cells. The findings of this study illustrate how modifications in related biochemical pathways can be utilized to enhance the production of commercially desirable compounds such as carotenoids.

Expression in Escherichia coli, purification, and reactivation of the recombinant Erwinia uredovora phytoene desaturase.

A plasmid has been constructed by cloning the complete crtI gene encoding phytoene desaturase from Erwinia uredovora behind the lac Z promoter of pUC18 resulting in a reading frame for the full polypeptide with additional 9 amino acids at the N terminus. This plasmid mediated the overexpression of phytoene desaturase in transformed Escherichia coli. The overexpressed enzyme was sequestrated into inclusion bodies requiring urea treatment for solubilization. Purification to homogeneity was subsequently performed on a DEAE-cellulose column and by SDS-polyacrylamide gel electrophoresis. The purification scheme allowed the isolation of 5.3 mg of homogeneous desaturase protein from 100 ml of E. coli cell suspension. On SDS-polyacrylamide gel electrophoresis an apparent molecular mass of 56.2 kDa was determined. An antiserum raised against phytoene desaturase cross-reacted with the expressed protein and was employed to monitor the isolation steps. Upon removal of urea, desaturase activity was restored. The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene. FAD was involved in desaturation, whereas NAD and NADP were inhibitory. This is the first time that a membrane-integrated carotenogenic enzyme has been purified and finally obtained in an active state.