Mol Genet Genomics (2002) 266: 768±777 DOI 10.1007/s004380100565
O R I GI N A L P A P E R
J. Ruiz-Albert á E. CerdaÂ-Olmedo á L.M. Corrochano
Genes for mevalonate biosynthesis in Phycomyces
Received: 18 January 2001 / Accepted: 13 July 2001 / Published online: 9 November 2001 Ó Springer-Verlag 2001
Abstract Terpenoids or isoprenoids constitute a vast family of organic compounds that includes sterols and carotenoids. The terpenoids in many organisms share early steps in their biosynthesis, including the synthesis of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) and its conversion to mevalonate. We have cloned and characterised the genes hmgS for HMG-CoA synthase and hmgR for HMG-CoA reductase from the Zygomycete Phycomyces blakesleeanus. Single copies of these genes are present in the Phycomyces genome. The predicted product of hmgS is largely hydrophilic and that of hmgR has eight putative transmembrane segments and a large hydrophilic domain. The hydrophilic domain suces for catalytic activity, as shown by expressing it in Escherichia coli. Several features in the promoter of hmgS and in HMG-CoA reductase resemble motifs known to be involved in sterol-mediated regulation and sterol sensing. Carotene-overproducing mutants contain more hmgS mRNA than the wild type, possibly in response to an increased demand for HMGCoA. Keywords HMG-CoA synthase á HMG-CoA reductase á Mevalonate á b-Carotene á Filamentous fungi
Communicated by C. A. M. J. J. van den Hondel J. Ruiz-Albert1 á E. CerdaÂ-Olmedo á L.M. Corrochano (&) Departamento de GeneÂtica, Universidad de Sevilla, Avenida Reina Mercedes 6, 41012 Sevilla, Spain E-mail:
[email protected] Tel.: +34-954-550919 Fax: +34-954-557104 Present address: 1 Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College of Science, Technology and Medicine, Armstrong Road, London SW7 2AZ, UK
Introduction The terpenoids include the carotenoids, the sterols, the dolichols, and various hormones, lipid moieties in membrane proteins, ubiquinone and chlorophyll (Conolly and Hill 1992). Many organisms synthesize the early precursors of the terpenoids by condensing acetyl coenzyme A (acetyl-CoA) with acetoacetyl-CoA, to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), and converting this intermediate to mevalonate. These steps are catalyzed by HMG-CoA synthase (EC 4.1.3.5) and HMG-CoA reductase (EC 1.1.1.34), respectively. HMG-CoA synthases are soluble enzymes with similar amino acid sequences, particularly at the N-terminal end, where the active site is located. Schizosaccharomyces pombe and Arabidopsis thaliana have a single gene for this enzyme, but mammals have separate genes for cytoplasmic and mitochondrial isoenzymes (Hegardt 1999). HMG-CoA reductase is the product of a single gene in mammals, but two genes have been described from Saccharomyces cerevisiae (Basson et al. 1986) and several ®lamentous fungi (Burmester and Czempinski 1994; Croxen et al. 1994). In plants, HMG-CoA reductase is encoded by multigene families with dierent numbers of genes, depending on the species (Stermer et al. 1994). Eukaryotic HMG-CoA reductases have an N-terminal hydrophobic domain containing several transmembrane segments and a C-terminal hydrophilic domain that contains the active site. Whereas the catalytic domain is highly conserved, the hydrophobic domain presents a variable number of transmembrane segments (Hampton et al. 1996). Transmembrane segments are required for the correct localisation of the protein in the endoplasmic reticulum (Gil et al. 1985) and are involved in the regulation of enzyme stability by mevalonate and sterol concentration in mammals (Goldstein and Brown 1990). Part of the hydrophobic domain of HMG-CoA reductase has been proposed to be involved in sensing the sterol concentration in the membrane, hence the name
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Sterol Sensing Domain for this segment (Loftus et al. 1997; Brown and Goldstein 1999; Davies and Ioannou 2000). The mode of regulation of HMG-CoA synthase and reductase is complex. In mammals, transcription of the corresponding genes depends on the concentration of sterols, which modulate the binding of speci®c transcription factors to sterol regulatory elements present in the promoters (Vallett et al. 1996; Brown and Goldstein 1997; Dooley et al. 1998). Translation, stability, and degradation of HMG-CoA reductase are also regulated by dierent metabolites of the terpenoid pathway (Goldstein and Brown 1990). The biosynthesis of the terpenoid b-carotene in the ®lamentous fungus Phycomyces blakesleeanus has been the subject of genetical, biochemical and physiological research on the wild type and many structural and regulatory mutants (CerdaÂ-Olmedo 1985; CerdaÂ-Olmedo and Lipson 1987). The Zygomycete Phycomyces is one of the organisms that may be used for the commercial production of b-carotene and other carotenoids that are in demand as dyestus and because of their favourable eects on human and animal health (Vandamme 1989). Mutants aected in the genes carS, carD, and carF are altered in the regulation of b-carotene biosynthesis and accumulate up to one hundred times more b-carotene than the wild type (Murillo and CerdaÂ-Olmedo 1976; Salgado et al. 1989; Mehta et al. 1997). Molecular characterisation has been carried out on the structural genes carB (Ruiz-Hidalgo et al. 1997) and carRA (Arrach et al. 2001), which encode the three enzymes speci®cally devoted to b-carotene biosynthesis. On the other hand, little is known about the enzymes involved in the early steps of terpenoid biosynthesis in Phycomyces or their mechanism of regulation. We have therefore cloned and characterised the Phycomyces genes for HMG-CoA synthase and HMG-CoA reductase, and analysed their expression, particularly in relation to enhanced production of b-carotene.
Materials and methods Strains and growth conditions NRRL1555, the standard wild-type strain of Phycomyces blakesleeanus, and the mutant strains C115 [genotype carS42 mad-107(±); Murillo and CerdaÂ-Olmedo 1976] and S561 [genotype carF181(±); Mehta et al. 1997] were used in this study. Both car mutations result in overproduction of b-carotene, and the mad mutation in altered phototropism, while (±) designates one of the two mating types. Cultures were inoculated with heat-activated spores (48°C, 15 min) and grown at 22°C in the dark on either minimal or nutrient medium (CerdaÂ-Olmedo and Lipson 1987). Eschericha coli DH5a was used for cloning plasmids; strain XL1 BlueMAR (P2) (Stratagene) to amplify a Phycomyces lambda library; and strain NCM631, which carries plasmid pIZ227 (Govantes et al. 1996), to express the catalytic domain of HMGCoA reductase. Bacteria were grown at 37°C in Luria-Bertani nutrient medium supplemented with ampicillin (100 mg/l) or chloramphenicol (50 mg/l) as appropriate.
Cloning and sequencing techniques Phycomyces genomic DNA was isolated from vegetative mycelia (Weinkove et al. 1998). Phycomyces mRNA from vegetative mycelia, and cDNA, were obtained using the QuickPrep mRNA Puri®cation Kit and the TimeSaver cDNA Kit (Amersham Pharmacia Biotech) respectively. Vegetative mycelia were produced by incubating spores for 2 days in the dark on minimal agar plates (104 spores/plate). Polymerase chain reactions (PCRs) were carried out using the DNA polymerase mixture Expand High Fidelity (Roche) following the manufacturer's instructions. DNA was sequenced on both strands by the chain-termination method using synthetic oligonucleotides as primers. DNA and RNA manipulations were carried out as described in Sambrook et al. (1989). Cloning of hmgS and hmgR A fragment of hmgS was obtained by PCR using Phycomyces cDNA as template, together with a set of primers designed to anneal to conserved areas of the gene: HS-2 (5¢-GGNAAATATACNATHGG-3¢, corresponding to the amino acid sequence GKYTIG) and HS-3 (5¢-AAATCRTANGCRTGYTGCAT-3¢, aa sequence MQHAYDF). An aliquot (1 ll) of the PCR product was used in a second PCR with the nested primers HS-1 (5¢-ACCGAGACNATHATHGAYAA-3¢, aa sequence TETIID) and PIG2 (5¢-GCTCTAGAGCNGTNCCNCCRTARCANGC-3¢, aa sequence NACYGGT). This second set of nested primers should anneal to sites within the product of the ®rst PCR, thereby increasing the sensitivity and yield of the reaction. The second PCR produced a DNA of the predicted size, 130 bp, which was cloned in pGEM-T (Promega) and sequenced. The complete hmgS gene from Phycomyces was cloned by inverse PCR using the sequence of the 130-bp hmgS fragment to design the primers. As template, we used 1.8-kb PvuII DNA fragments and 4-kb DraI DNA fragments of Phycomyces genomic DNA, which were self-ligated with T4 ligase. The primers used were HS-5 (5¢-GATCGAAGGTATCGACACC3¢) and HS-6 (5¢-GTAGTCTTGACAGACTTGG-3¢) with the PvuII template, and HS-6 and HS-10 (5¢-GGAGACGCTTCTACAAGCG-3¢) with the DraI template. The products of the inverse PCR were isolated, cloned in pGEM-T (Promega) and sequenced. The Phycomyces hmgR gene was cloned by screening a genomic library made from a partial Sau3AI digest inserted in k2001 (Arrach et al. 2001), using as a probe a 315-bp fragment of the gene previously isolated by Corrochano and Avalos (1992). A clone carrying a 15.5-kb insert was isolated, and the restriction fragments that hybridised with the hmgR probe were subcloned and sequenced. cDNAs for hmgS and hmgR were isolated by PCR using cDNA as template, together with the appropriate primers. Construction of pJR21 A DNA fragment encoding the catalytic domain of Phycomyces HMG-CoA reductase was obtained by PCR from cDNA using HR-15 (5¢-CGTACACCGCAT ATGTTGAACG-3¢) and HR-1 (5¢-TGTAGGTGTGTAAGTGAATG-3¢) as primers. HR-15 corresponds to the amino acid sequence 738-RTPEMLN-744 with a GAA to CAT substitution (underlined) that introduces a NdeI site. The resulting 1430-bp fragment, encoding the last 435 residues of HMG-CoA reductase, was digested with SmaI, partially digested with NdeI to avoid restriction at an internal NdeI site and cloned into SmaI/NdeI sites of pT7-7 (Tabor 1994), generating pJR21. This plasmid carries the sequence encoding the catalytic domain of HMG-CoA reductase under the control of the phage T7 /10 promoter. DNA sequence analysis and phylogenetic analysis The DNA sequences obtained have been deposited in the EMBL Nucleotide Sequence Database under the Accession Nos. AJ297414 (hmgS) and X58371 (hmgR).
770 Coding sequences were predicted by similarity with those of other HMG-CoA synthases or reductases in the database using the program Gapped-Blast (Altschul et al. 1997). PEST®nd was used to search for PEST sequences in the proteins (Rechsteiner and Rogers 1996). TMPred (Hofmann and Stoel 1993) was used to calculate hydrophobicity pro®les and to predict transmembrane segments. All the programs used default parameters, and were accessed through the ExPASy server (Swiss Institute of Bioinformatics, Geneva, Switzerland, http://www.expasy.ch). For sequence alignment and phylogenetic analysis we used the programs Clustal W and Clustal X (Thompson et al. 1994, 1997). Gaps in the alignments were disregarded. The leader peptide of the mitochondrial HMG-CoA synthase and the N-terminal extension of the animal cytosolic HMG-CoA synthase were not considered. For HMG-CoA reductase alignments only the catalytic domain was used: amino acids 836±1152 of the Phycomyces protein for general alignment, residues 836±940 for fungal alignment, and the corresponding sequences of the homologous proteins in each case. Distance matrices obtained from the alignments were corrected for multiple substitutions. Cladograms were built by neighbourjoining using the corrected matrices, and the reliability of each node was established by bootstrap methods. Nucleic acid hybridisations For Southern hybridisations, Phycomyces genomic DNA (5 lg per sample) was treated with the appropriate restriction enzyme, fractionated by electrophoresis (0.8% agarose), and transferred onto nylon membranes. Membranes were prehybridised at 42°C (high stringency) or 37°C (low stringency) in 50% formamide (v/ v), 5´SSC, 0.02% SDS (w/v) and 5% (w/v) Blocking Reagent (Roche). Hybridisations were carried out overnight under the same conditions using as probes the DNA segments of 130 bp (hmgS) and 315 bp (hmgR). Probes were labelled with digoxigenin-11-dUTP (Roche). Membranes were washed twice in 2´SSC, 0.1% SDS (w/v) at room temperature for 5 min, and twice in 0.1´SSC, 0.1% SDS (w/v) at 65°C (high stringency) or 55°C (low stringency) for 20 min. The digoxigenin-dUTP detection system was used, following procedures recommended by the manufacturer (Roche). Northern hybridisations were performed following procedures recommended by Sambrook et al. (1989). Aliquots (2 lg) of each mRNA sample were fractionated by electrophoresis (in 1.2% agarose), transferred to a nylon membrane, and probed with the DNA segments of 130 bp (hmgS) and 315 bp (hmgR) labelled with [a-32P]dCTP by random priming. RNA size was estimated by comparison with ribosomal RNAs in total RNA samples. As a control for loading, membranes were stripped and rehybridised with a fragment of pyrG, the Phycomyces gene encoding orotidine-5¢-phosphate decarboxylase (DõÂ az-MõÂ nguez et al. 1990). Relative amounts of mRNA in each hybridisation were calculated by densitometric analysis performed on a Macintosh computer using the NIH Image 1.6 program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). The Northern hybridization experiments were performed three times with independent mRNA samples. HMG-CoA reductase labelling, assay, and puri®cation Plasmid pJR21 was introduced into E. coli NCM631 carrying pIZ227, and the expression of the catalytic domain of HMG-CoA reductase was induced with isopropylthio-b-D-galactoside (IPTG) (Govantes et al. 1996) for 90 min. After a further 30 min in the presence of rifampicin (200 mg/l), the culture (1 ml) was mixed with 1 ml (3.7´105 Bq) of a solution of [35S]methionine and [35S]cysteine (in vivo cell labelling grade, Amersham Pharmacia Biotech) and incubated for 5 min. Cells were collected by centrifugation for 5 min at 7,000´g and 4°C, and lysed in 4% SDS. Cell extracts were fractionated in polyacrylamide-SDS electrophoresis following standard procedures (Sambrook et al. 1989).
To measure HMG-CoA reductase activity, E. coli NCM631 cells carrying pIZ227 and either pJR21 or pT7-7 were grown in inducing conditions for 90 min, and cells were collected by centrifugation (7,000´g, 5 min, 4°C). Cells were then resuspended (0.25 g of wet mass per ml) in ice-cold buer [0.25 M saccharose, 10 mM N-(2-hydroxyethyl)piperazine-N¢-(2-ethanesulfonic acid), 50 mM NaCl, 20 mM EDTA, 2 mM EGTA, 5 mM dithiothreitol, 50 mg/ml aprotinin, 20 mg/ml leupeptin, 10 mM phenanthroline, 1 mM benzamidine, 50 mg/ml trypsin inhibitor (Sigma) and 50 mM phenylmethylsulfonyl ¯uoride], and sonicated at 60 W for 30 s per ml of buer, at 4°C. Homogenates were cleared by centrifugation (1,000´g, 1 min, 4°C) and the protein concentration was determined (Bio-Rad Protein assay). HMG-CoA reductase activity was measured as the rate of conversion of 3-hydroxy-3-methyl-[3-14C]glutaryl-CoA to [14C]mevalonate as described by PenÄa-DõÂ az et al. (1997). For protein puri®cation, E. coli NCM631 carrying pIZ227 and pJR21 was grown under inducing conditions for 4 h in a volume of 1 l, collected by centrifugation (7,000´g, 5 min, 4°C) and resuspended (0.25 g wet mass per ml) in homogenisation buer A (Dale et al. 1995). The cell suspension was homogenised in a French press (American Instrument Company), pelleted by centrifugation (15,000´g, 20 min, 4°C), resuspended in 50 ml of ice-cold lysozyme (0.2 mg/ml), 1 mM EDTA, and incubated at 4°C for 1 h with gentle shaking. This ®nal step was repeated twice, resuspending the pellet successively in 10 ml of 4 M and 6 M urea. The extract was cleared by centrifugation (25,000´g, 15 min, 4°C), dialysed against buer C (Dale et al. 1995) and concentrated by ultra®ltration (Centripep-10, Amicon).
Results Genes hmgS and hmgR HMG-CoA synthases are proteins with conserved amino acid sequences, particularly around the active site. Using a set of primers designed to anneal to conserved segments of the gene, we cloned a 130-bp PCR fragment of the hmgS gene encoding HMG-CoA synthase from Phycomyces, as con®rmed by sequence similarity. The rest of the gene was obtained by inverse PCR and sequenced to assemble a 2528-bp DNA segment containing the Phycomyces hmgS gene and its ¯anking regions (EMBL Accession No. AJ297414). The gene hmgR for HMG-CoA reductase was cloned by screening a Phycomyces genomic library with a fragment of that gene previously cloned by Corrochano and Avalos (1992). A DNA segment of 5456 bp was sequenced and found to contain the entire hmgR gene and its ¯anking regions (EMBL Accession No. X58371). Coding sequences were predicted by similarities with other HMG-CoA synthases and reductases. The corresponding cDNAs were sequenced to determine the presence of introns and their boundaries and to identify putative start and stop codons for translation. The hmgS gene comprises a total of 1635 bp including the coding sequence for 450 amino acids and three introns (56, 97, and 129 bp). The putative protein product has a predicted molecular mass of 50.1 kDa. The hmgR gene is 3978 bp long, including the coding sequence for 1176 amino acids and four introns (82, 74, 155, and 136 bp). Its protein product has a predicted molecular mass of 127.7 kDa (Fig. 1). The Phycomyces
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The 5¢-untranslated region of hmgS contains a putative sterol regulatory element (5¢-CTCACCCTAG-3¢) starting 89 bp before the initiating ATG codon, and a putative binding site (5¢-ATTGGC-3¢) for nuclear factor Y (NF-Y), starting 126 bp before the initiating ATG codon. The Phycomyces HMG-CoA synthase and HMG-CoA reductase Fig. 1 Structure of the DNA fragments that contain hmgS and hmgR. Non-coding sequences are depicted as lines. Coding sequences are presented as boxes, with the arrowhead indicating the direction of translation. The scheme shows the coding sequences for the three domains of HMG-CoA reductase: the hydrophobic domain in grey, the linker in white, and the catalytic domain in black. The positions of the probes used for hybridizations are indicated by the short lines above the genes. Some of the restriction sites used during the cloning of the genes are also indicated
genome contains single copies of hmgS and hmgR, as revealed by Southern hybridisations at high and low stringencies, which gave similar results (Fig. 2). The exons of hmgS and hmgR contain 50% and 51% G+C, respectively, much more than the non-coding sequences of the genes (35% for hmgS and 27% for hmgR). A marked preference for pyrimidines in the third position is seen in both genes, as in other Phycomyces genes.
Fig. 2 The Phycomyces genome contains single copies of hmgS and hmgR. The panels show hybridisations of Phycomyces genomic DNA with hmgS and hmgR probes at high stringency. Each lane contains genomic DNA digested with the indicated restriction enzyme. The lanes marked - contained undigested genomic DNA; lanes labelled pl were loaded with 0.2 ng of linearized plasmids carrying the fragments used as probes; M, molecular size markers. The enzymes used (B, BamHI; D, DraI; E, EcoRV; H, HindIII; N, NdeI; P, PvuII; S, ScaI; Ss, SspI; X, XbaI; Xh, XhoI) do not cut the probes, with one exception: the hmgR probe contains a single recognition site for HindIII
The coding sequences of the genes hmgS and hmgR are similar to those of other genes for HMG-CoA synthases and reductases, respectively. The predicted hmgS protein product is identical in 52% and 47% of its amino acids with the human cytosolic and mitochondrial gene products, respectively; the corresponding value for the hmgR gene product is 40%. The C-terminal region (catalytic domain) of this latter protein is more highly conserved than the rest. The predicted hmgS gene product contains the amino acids that are believed to be involved in catalysis by HMG-CoA synthases: cysteine 123 for acetyl-CoA binding (Misra et al. 1993), histidine 254 for acetoacetyl-CoA binding (Misra and Miziorko 1996), and glutamate 89 for C-C bond formation (Chun et al. 2000). The Phycomyces enzyme is largely hydrophilic and lacks the leader peptide characteristic of the animal enzymes which are located in the mitochondria (Hegardt 1999). The predicted hmgR gene product (Fig. 3A) contains features observed in HMG-CoA reductases, such as binding sites for HMG-CoA and NAD(P)H, and the sequence ENVIGXXXIP, which has been proposed to be a key dimerization element (Istvan et al. 2000). Residues involved in catalytic activity are conserved: glutamate 841 (Frimpong and Rodwell 1994), lysine 1016 (Lawrence et al. 1995, Istvan et al. 2000), aspartate 1048 (Frimpong and Rodwell 1994) and histidine 1146 (Darnay and Rodwell 1993). The hydrophobicity pro®le of the hmgR gene product distinguishes the three domains found in all eukaryotic HMG-CoA reductases: an
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N-terminal hydrophobic domain (644 aa), a linker (100 aa), and a C-terminal catalytic domain (432 aa) (Fig. 3B). The N-terminal region of the Phycomyces protein is predicted to include eight transmembrane segments separated by hydrophilic loops which may be used to anchor the enzyme to the plasma membrane, as in other HMG-CoA reductases (Gil et al. 1985; Olender and Simoni 1992). The hydrophobic domain contains a sequence that is very similar to the Sterol Sensing Domain described in other HMG-CoA reductases and sterol-interacting proteins (Loftus et al. 1997; Brown and Goldstein 1999; Davies and Ioannou 2000) (Fig. 3C). The 26-residue C-terminal sequence of the hmgR gene product resembles PEST sequences, which act as signals for rapid protein degradation (Rechsteiner and Rogers 1996).
Expression of the hmgS and hmgR genes Northern hybridisation experiments were performed to analyse the expression of hmgS and hmgR in the wild type and in b-carotene overproducing mutants. Strain C115 (carS) and strain S561 (carF) accumulate similar amounts of b-carotene ± about 2 mg/g of dry mass compared to 40 lg/g dry mass in the wild type (Murillo and CerdaÂ-Olmedo 1976; Mehta et al. 1997). mRNA was isolated from 2-day-old mycelia, and hybridised with either the hmgS or hmgR probe (Fig. 4). The sizes of the hmgS (1.5 kb) and hmgR (3.6 kb) mRNAs were determined by comparison with ribosomal RNAs. Increased amounts of hmgS mRNA were found in b-carotene overproducing strains, especially in the carF mutant (4.6 times the wild-type value), but also in the
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Fig. 3A±C Phycomyces HMG-CoA reductase. A Sequence comparison of the substrate-binding sequences from Phycomyces with equivalent sequences from other HMG-CoA reductases. Identical residues are marked in grey. The glutamate residue marked in dark grey is believed to be essential for catalysis. The sequence ENVIGXXXIP is also involved in dimerization. B Hydrophobicity plot of Phycomyces HMG-CoA reductase obtained using TMPred. The ordinate represents the TMPred score divided by 1000. Positive scores suggest hydrophobic regions. The putative membrane-spanning regions are marked by arrows. The scheme shows the predicted structure of Phycomyces HMG-CoA reductase, with three domains: the hydrophobic domain (in grey with transmembrane segments depicted as black lines), the linker (in white) and the hydrophilic catalytic domain (in dark grey). The Sterol Sensing Domain is marked by the double-headed arrow. C Sequence comparison of the putative Sterol Sensing Domain from Phycomyces HMG-CoA reductase with the equivalent domain from the human proteins HMGR (HMG-CoA reductase), NPC±1 (Niemann-Pick C1), and SCAP (SREBP cleavage activating protein). Identical amino acids are marked in grey. Similar amino acids conserved in at least 2/3 of the sequences are boxed. The proteins used for the alignments are from Phycomyces blakesleeanus, Fusarium (Gibberella) fujikuroi, Saccharomyces cerevisiae, Arabidopsis thaliana, and Homo sapiens, and were obtained from the EMBL Database, where the corresponding references can be found
segment of the hmgR gene encoding the last 435 amino acids of the protein was placed under the control of a T7 promoter in the pT7-7 expression vector. The resulting plasmid, pJR21, was introduced into a strain of E. coli that contains the T7 RNA polymerase gene under the control of an IPTG-inducible promoter. Under appropriate inducing conditions, pJR21 directed the synthesis of a major polypeptide of about 45.5 kDa, the size expected for the truncated Phycomyces protein (Fig. 5A). Protein extracts from similar cultures showed detectable HMG-CoA reductase activity (56 pmol/s per g protein). The enzyme activity and the polypeptide were not detected in control cultures transformed with the expression vector only. The protein responsible for the enzyme activity could not be puri®ed using published methods for HMG-CoA reductase (Ness et al. 1979; Dale et al. 1995). Gross overexpression of the protein resulted in the formation of inclusion bodies in which the truncated Phycomyces protein constituted the majority of the proteins. Solubilisation of the inclusion bodies using increasing concentrations of urea resulted in the partial puri®cation of the catalytic domain of Phycomyces HMG-CoA reduc-
carS mutant (1.8 times the wild-type value). Expression of hmgR was not aected in the carS mutant (0.98 times the wild-type value) but a slight increase was detected in the carF mutant (1.8 times the wild-type value). Expression of the catalytic domain of HMG-CoA reductase in E. coli The identity of the polypeptide encoded by the hmgR gene was con®rmed by expressing it in bacteria. The large hydrophobic domain of HMG-CoA reductase may cause the enzyme to aggregate when expressed in large quantities in E. coli (Ferrer et al. 1990). However, the hydrophilic C-terminal domain of HMG-CoA reductase from other organisms has been shown to be sucient for catalytic activity (Gil et al. 1985; Dale et al. 1995). A
Fig. 4 Expression of the hmgS and hmgR genes in Phycomyces. mRNA from wild-type and the b-carotene overproducing carF and carS strains was fractionated according to size in an agaroseformaldehyde gel, transferred to a nylon membrane, and hybridized with 32P-labelled probes for the hmgS and hmgR genes. A 32 P-labelled probe for the pyrG gene was used as a loading control
Fig. 5A, B Expression and puri®cation of the catalytic domain of Phycomyces HMG-CoA reductase in E. coli. A Synthesis of the truncated Phycomyces protein. Cultures of bacteria that carried pJR21, and were thus able to express a fragment of hmgR, or pT77, as a negative control, were induced, proteins were labelled with [35S]methionine and [35S]cysteine, and fractionated according to size by SDS-polyacrylamide electrophoresis. B Puri®cation of the Phycomyces protein expressed in E. coli. Lane 1: protein sample obtained from bacterial cultures expressing the hmgR fragment after cell lysis and centrifugation. Lane 2: protein sample obtained from inclusion bodies puri®cation by two cycles of precipitation with urea. Lane 3: protein sample after dialysis and concentration. Unlabelled proteins were detected by staining with Coomassie blue
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Fig. 6A±C Phylogeny of HMG-CoA synthases and HMG-CoA reductases. A Cladogram for HMG-CoA synthases. B Cladogram for the catalytic domain of HMG-CoA reductases. C Cladogram for a portion of the catalytic domain of fungal HMG-CoA reductases. The proteins used for the alignments are from Halobacterium volcanii, Sulfolobus solfataricus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida utilis, Fusarium (Gibberella) fujikuroi, Sphaceloma manihoticola, Phycomyces blakesleeanus, Blakeslea trispora, Mucor mucedo, Parasitella parasitica, Absidia glauca, Ustilago maydis, Pinus sylvestris, Solanum tuberosum, Hevea brasiliensis, Arabidopsis thaliana, Drosophila melanogaster, Blattella germanica, Caenorhabditis elegans, Xenopus laevis, Gallus gallus, Sus scrofa, Cricetulus griseus, Rattus norvegicus, and Homo sapiens, and were obtained from the EMBL Database, where the corresponding references can be found
tase (Fig. 5B). This puri®cation method may not be suitable for precise biochemical characterization of the enzyme but will allow the production of speci®c antibodies for Western analysis and for the localisation of the HMG-CoA reductase in the Phycomyces mycelium.
Evolution of HMG-CoA synthase and HMG-CoA reductase Many organisms have several copies of genes for HMGCoA synthase and HMG-CoA reductase, and they use the isoenzymes in specialized and regulated ways. To investigate the origin and extent of hmgS and hmgR gene duplication and their evolution we have constructed evolutionary cladograms using the available protein sequences. The comparison of the amino acid sequences of several HMG-CoA synthases was used to infer a phylogeny for these proteins (Fig. 6A). As expected, Phycomyces HMG-CoA synthase is grouped with other fungal enzymes. The cladogram shows the monophyletic origin of the mitochondrial HMG-CoA synthases and the duplication of the cytosolic HMG-CoA synthase gene in the cockroach Blattella germanica (Buesa et al. 1994). The phylogeny of HMG-CoA reductases was inferred using the same method, but only the amino acid
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sequences of the catalytic domain were compared, because these are best conserved among species. The major evolutionary groups can be seen in this cladogram, with the fungal HMG-CoA reductases showing a large degree of divergence (Fig. 6B). We also constructed a phylogeny of fungal HMG-CoA reductases using partial sequences of the catalytic domains from various fungi, including several Zygomycetes. This fungal phylogeny (Fig. 6C) clearly separates all three major fungal groups represented in the comparison: Ascomycetes, Zygomycetes and Basidiomycetes. This is at variance with the phylogeny obtained using the orotidine-5¢-phosphate decarboxylase where Basidiomycetes and Zygomycetes are more closely related to each other than to Ascomycetes (Radford 1993). The cladogram shows a recent duplication in the lineage leading to S. cerevisiae, and another duplication occurring before Zygomycetes diverged.
Discussion The genetic machinery for mevalonate biosynthesis in Phycomyces is among the simplest known, consisting of a single gene for each of the enzymes HMG-CoA synthase and HMG-CoA reductase. The existence of two or more genes for these enzymes is common, even among the Zygomycetes (Burmester and Czempinski 1994). The cladogram in Fig. 6 suggests an explanation: a duplication of the hmgR gene occurred in an ancestor of the Zygomycetes, and one of the copies was later lost in some of the lineages. These enzymes and the corresponding genes have retained their general structure, down to such details as intron position, over a long period of evolutionary time. The four introns of Phycomyces hmgR coincide in their positions with four of the eight introns in the homologous gene from hamster, Cricetulus griseus (Reynolds et al. 1984). The isolation and characterization of the hmgS and hmgR genes from Phycomyces has allowed the identi®cation of sequences in the promoter of the Phycomyces hmgS gene, and in the HMG-CoA reductase, that may be relevant for their regulation. The promoter of the Phycomyces hmgS gene contains a putative sterol regulatory element and a putative binding site for the transcriptional co-regulator NF-Y. Both sequences are located at relative positions found in other genes to be appropriate for their interaction in vivo (Ericsson et al. 1996; Dooley et al. 1998). The Phycomyces hmgS promoter is thus likely to be regulated by sterol concentration. In contrast, no obvious regulatory elements were detected in the hmgR promoter. The dierences in the promoters correlate with differences in transcription. The two b-carotene overproducing strains that were tested contained more hmgS mRNA than the wild type, and one of them showed an increase in hmgR mRNA level as well. We propose that the increased synthesis of b-carotene in these mutants
may deplete the HMG-CoA in the cell. The subsequent reduction in sterol biosynthesis would activate the promoters containing sterol regulatory elements, including that of hmgS. This would explain how the carS mutant could accumulate 50 times more b-carotene while maintaining its ergosterol concentration at a level similar to that of the wild type (Bejarano and CerdaÂ-Olmedo 1992). This hypothesis is also compatible with a putative role for carS and carF gene products as repressors of hmgS transcription, as proposed for the carS gene product in the transcriptional regulation of the genes carB and carRA (Salgado et al. 1991). The Phycomyces HMG-CoA reductase may be regulated at a post-transcriptional level. It contains the characteristic set of putative transmembrane segments that allow eukaryotic HMG-CoA reductases to anchor to membranes. Some of these segments are similar to the Sterol Sensing Domain of mammalian HMG-CoA reductases, which regulate the activity of the protein in response to the sterol concentration in the membrane (Loftus et al. 1997; Brown and Goldstein 1999). Furthermore, the C-terminal end of Phycomyces HMGCoA reductase contains a putative PEST sequence like those involved in determining the stability of various proteins (Rechsteiner and Rogers 1996) and found in plant HMG-CoA reductases (Caelles et al. 1989; Chye et al. 1992). The isolation of antibodies obtained with the puri®ed HMG-CoA reductase catalytic domain will help to elucidate whether the protein has a particularly short half-life. The isolation of the Phycomyces genes for HMGCoA synthase and HMG-CoA reductase represents a ®rst step toward the investigation of the regulation of mevalonate biosynthesis in this fungus. Our results suggest that sterol concentrations in Phycomyces may regulate directly the activity and stability of HMG-CoA reductase and the transcription of the mRNA for HMGCoA synthase. If this hypothesis is veri®ed experimentally, it will be relevant for the genetic engineering of the b-carotene pathways in fungi, since the transcription of hmgS, and perhaps HMG-CoA reductase activity, may be bottlenecks that limit carotene yields. Genetic manipulations designed to increase carotene production should include hmgS and hmgR among the genes to be modi®ed and optimised. Acknowledgements We are grateful to Walter Giordano for his help with the enzymatic assays, Rainer Hertel for his advice and hospitality during enzyme puri®cation, Eduardo Santero, Fausto G. Hegardt, Javier Avalos, Carmen BeuzoÂn, and Steve Garvis for providing materials and for helpful discussions. J.R.A. was supported by a Fellowship from the Spanish Ministerio de EducacioÂn y Cultura. This work was supported by the European Union (FAIR CT96-1633), the Spanish Ministerio de EducacioÂn y Cultura (PB96± 1336, APC1999-0151), the Acciones Integradas between Spain and Germany, and the Junta de AndalucõÂ a (Group CVI 0119).
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