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Applied and Environmental Microbiology, March 2007, p. 1736-1741, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.02425-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of a Sterol {Delta}7 Reductase Gene Involved in Desmosterol Biosynthesis in Mortierella alpina 1S-4{triangledown} ,{dagger}

Shuo Zhang, Eiji Sakuradani, and Sakayu Shimizu*

Division of Applied Life of Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan

Received 16 October 2006/ Accepted 1 January 2007


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Molecular cloning of the gene encoding sterol {Delta}7 reductase from the filamentous fungus Mortierella alpina 1S-4, which accumulates cholesta-5,24-dienol (desmosterol) as the main sterol, revealed that the open reading frame of this gene, designated Mo{Delta}7SR, consists of 1,404 bp and codes for 468 amino acids with a molecular weight of 53,965. The predicted amino acid sequence of Mo{Delta}7SR showed highest homology of 51% with that of sterol {Delta}7 reductase (EC 1.3.1.21) from Xenopus laevis (African clawed frog). Heterologous expression of the Mo{Delta}7SR gene in yeast Saccharomyces cerevisiae revealed that Mo{Delta}7SR converts ergosta-5,7-dienol to ergosta-5-enol (campesterol) by the activity of {Delta}7 reductase. In addition, with gene silencing of Mo{Delta}7SR gene by RNA interference, the transformant accumulated cholesta-5,7,24-trienol up to 10% of the total sterols with a decrease in desmosterol. Cholesta-5,7,24-trienol is not detected in the control strain. This indicates that Mo{Delta}7SR is involved in desmosterol biosynthesis in M. alpina 1S-4. This study is the first report on characterization of sterol {Delta}7 reductase from a microorganism.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Sterols are major components of eukaryotic cell membranes. The end products and the sterol biosynthetic pathway differ among species. Cholesterol (cholesta-5-enol) is typical in animals, ergosterol (ergosta-5,7,22-trienol) is common in fungi, and sitosterol (24-ethyl cholesta-5-enol), campesterol (ergosta-5-enol), and stigmasterol (24-ethyl cholesta-5,22-dienol) are the main end sterols in plants (7). These end sterols are composed of a similar four-ringed structures, including an unsaturation at the {Delta}5 position on ring B and a specific branched side chain. Ergosterol, unlike other end sterols, contains an additional unsaturation at the {Delta}7 position on ring B. On the other hand, desmosterol (cholesta-5,24-dienol) was found to be an end sterol in the zygomycetes fungus Mortierella alpina (20). Recently, the sterol biosynthetic pathway in M. alpina was demonstrated to produce 13 sterols but no ergosterol (12).

The biosynthetic reactions that allow conversion of lanosterol (4,4-dimethyl cholesta-8,24-dienol) resulting from the oxidative cyclization of squalene to cholesterol have been well studied (14, 15, 18). As a representative of microorganisms, yeast, the biosynthetic steps of ergosterol and the related enzymes were characterized in Saccharomyces cerevisiae (5, 13). From zymosterol (cholesta-8,24-dienol) to cholesterol in mammals, ergosterol in fungi, and phytosterols in plants, there are multiple alternative pathways leading to terminal sterols. In the sequential steps of cholesterol biosynthesis in mammals, sterol {Delta}7 reductase ({Delta}7SR; EC 1.3.1.21) is a terminal enzyme, which is absent in yeast, catalyzes the reduction of the {Delta}7 double bond in sterol intermediates with presence of NADPH under anaerobic conditions (6, 10). A deficiency of this enzyme activity due to genetic mutation in humans has been found to cause Smith-Lemli-Opitz syndrome (SLOS) (4, 28). SLOS is an autosomal-recessive multiple congenital anomaly and/or mental retardation disorder caused by inborn error of post-squalene cholesterol biosynthesis that leads to the elevation of 7-dehydrocholesterol in serum body fluids and tissues (19, 21, 27).

Despite their importance, the isolation and functional analysis of the {Delta}7SR genes were just reported from the limited species. A gene encoding NADPH-dependent {Delta}7SR was first isolated from a higher plant, Arabidopsis thaliana (9). Functional analysis in a yeast expression system revealed that this plant enzyme efficiently reduced {Delta}5,7-ergosta- and cholesta-sterols in vivo, regardless of the structural variations on the side chain (9). Plant {Delta}7SR was used for steroid biosynthesis such as pregnenolone, progesterone, and hydrocortisone in yeast (3, 22). On the other hand, {Delta}7SR genes from humans (11) and rats (1) were isolated and characterized. At present, more than 100 different mutations have been identified in SLOS patients who represent a continuum of clinical severity (30).

Thus far, the microbial {Delta}7SR gene has never been isolated and characterized. In the present study, a {Delta}7SR cDNA, designated Mo{Delta}7SR, was isolated by a PCR-based cloning method and characterized by a yeast expression system and RNA interference (RNAi) in M. alpina 1S-4. This is the first report on an {Delta}7SR gene from a microorganism.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains and cultivation media.
For cloning a {Delta}7SR gene, M. alpina 1S-4, deposited in the AKU culture collection (Agriculture of Kyoto University) as AKU3998 (29), was used. The uracil auxotroph (ura5– strain) isolated previously from M. alpina 1S-4 was used for gene silencing of Mo{Delta}7SR by RNAi. Yeast S. cerevisiae SX-1065 (MAT{alpha} his3 leu2 erg5::TRP1 ura3), with its sterol {Delta}22 desaturase (ERG5) gene (8) disrupted, was kindly provided by Mitsubishi Chemical Co. (Tokyo, Japan) and used for functional analysis of the Mo{Delta}7SR gene. A complete medium (GY medium) and a selection yeast minimal medium (uracil-free SC medium) previously reported (23) were used for cultivation of M. alpina. A selection yeast minimal medium (YNBD medium) and a complete medium (YPD medium) were used for cultivation of S. cerevisiae (16).

Chemicals.
Desmosterol and campesterol were purchased from Sigma-Aldrich Co. Cholesta-5,7,24-trienol was kindly provided by Mitsubishi Chemical Co. All other chemicals were of the highest commercial grades available. The polymerase used was Takara LA Taq (Takara Bio, Inc., Shiga, Japan).

PCR-based cloning.
Degenerate oligonucleotide primers were designed based on the highly conserved amino acid sequences of {Delta}7SR from human (GenBank accession no. AF034544) and African clawed frog (GenBank accession no. BC044995). Primer {Delta}7SR-DeF1 (5'-ATGGGIATHGARTTYAAYCC-3') corresponding to amino acid sequence MGIEFNP and primer {Delta}7SR-DeR1 (5'-CATRTADATIAWRTARAARTA-3') corresponding to YFY(F/I)IYM were used. PCR amplification was carried out with the genomic DNA extracted from M. alpina 1S-4 as described previously (17). The amplified 760-bp PCR fragment was cloned into the pT7Blue T-vector (Novagen/Merck KGaA, Darmstadt, Germany) and sequenced.

The complete Mo{Delta}7SR cDNA was obtained by PCR using primers with primers designed from the sequence information mentioned above with the cDNA library as a template. The amplified 1.4-kb PCR fragment was cloned into the pT7Blue T-vector and sequenced. The resultant plasmid was designated pT7Mo7SR. Similarly, the genomic gene encoding this protein was obtained with the same primers and the genomic gene as a template. The amplified 1.9-kb PCR fragment was cloned into the pT7Blue T-vector and sequenced.

Functional analysis of Mo{Delta}7SR cDNA.
For the expression of Mo{Delta}7SR cDNA in yeast, an amplified product was obtained by PCR using the primer {Delta}7SR-ExF4 (5'-CTCCTCAAGCTTATGGCAGTGCAGCAGAGG-3') containing an ATG start site (underlined) and a HindIII cloning site (italics) and the primer {Delta}7SR-ExR4 (5'-CATTCTAGATTAGTAAATGTAGGGAATGAGC-3') containing a TAA stop site (underlined) and a XbaI cloning site (italics) and plasmid pT7Mo7SR as a template. The resultant PCR fragment was cloned into the pT7Blue T-vector and sequenced. The full-length gene was cloned into the HindIII/XbaI sites of yeast expression vector pYES2 (Invitrogen) to generate a Mo{Delta}7SR gene expression vector, pYEMo{Delta}7SR, followed by transformation into S. cerevisiae SX-1065 by the electroporation method (2). Transformants were selected for uracil auxotrophy on YNBD medium without uracil. To characterize the function of Mo{Delta}7SR, transformants were grown at 28°C for 24 h in YPD medium and then for 48 h after the addition of galactose (2% of final concentration) to induce gene expression, followed by whole-cell sterol analysis. The host strain transformed with pYES2 was used as a negative control in all experiments.

Mo{Delta}7SR gene silencing.
According to the previous method (26), a vector for Mo{Delta}7SR gene silencing was constructed. Four primers, {Delta}7SR-RiF5 (5'-CGGATCCTATGGCAGTGCAGCAGAGGAAAG-3'), {Delta}7SR-RiR5 (5'-GTCCATGGAACAATTCCTAGGCGACCGTTG-3'), {Delta}7SR-RiF6 (5'-GATCCCATGGCAGTGCAGCAGAGGAAAG-3'), and {Delta}7SR-RiR6 (5'-GAGCCCTAGGACTTGCGGTCGGCAGCATGC-3') containing BamHI, NcoI and BlnI, NcoI, and BlnI sites, respectively, were designed for construction of a Mo{Delta}7SR gene silencing vector. First, a PCR product (730 bp) amplified with a primer pair ({Delta}7SR-RiF5 and {Delta}7SR-RiR5) and pYEMo{Delta}7SR as a template was digested with NcoI and BamHI and then ligated to the NcoI/BamHI sites of pBlues-hpt, which was previously made for the construction of gene expression vector in M. alpina (25), resulting in the construction of pBlues{Delta}7-1. Next, the PCR product (623 bp) amplified with a primer pair ({Delta}7SR-RiF6 and {Delta}7SR-RiR6) and pYEMo{Delta}7SR as a template was digested with NcoI and BlnI and ligated to the NcoI/BlnI sites of pBlues{Delta}7-1, resulting in construction of pBlues{Delta}7-2. Finally, the DNA fragment containing histone H4 promoter and ura5 gene was prepared by digestion of pDura5 with EcoRI and BamHI, which had been previously constructed for the transformation of uracil auxotrophs of M. alpina (24) and then ligated to the EcoRV site of pBlues{Delta}7-2 with a DNA blunting kit, resulting in construction of pBlues{Delta}7U5. Plasmid pBlues{Delta}7U5, which brings about formation of double-strand RNA of the Mo{Delta}7SR gene under the control of the homologous histone H4 promoter in M. alpina, was used for transformation of the M. alpina 1S-4 ura5– strain as described previously (24). Transformants were selected on uracil-free SC agar medium, and their sterol composition was analyzed.

Sterol analysis.
The cultivated yeast and mycelia were harvested by centrifugation and vacuum filtration, respectively, and their total sterols, extracted as described previously (20), were analyzed by gas-liquid chromatography (GLC) and gas chromatography-mass spectrometry (GC-MS). The analytical conditions for the GLC were as follows: apparatus, GC-17A (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector; column, ULBON HR-52 (0.25 mm by 50 m; Shinwakakou, Kyoto, Japan); injection port temperature, 300°C; detector port temperature, 300°C; carrier gas, He; makeup gas, N2; and split ratio, 50:1. The initial column temperature of 220°C was raised at 5°C/min to 280°C and then maintained for 28 min at 280°C. A GC-MS QP5050 (Shimadzu) operating at an ionization voltage of 70 eV was used for mass spectral analyses.

DNA sequence.
The nucleotide sequences were determined with a CEQ dye terminator cycle sequencing kit (Beckman Coulter, Inc., Fullerton, CA) and an automated sequencer DNA analysis system CEQ 2000XL (Beckman Coulter, Inc.).

Nucleotide sequence accession number.
The genomic gene sequence coding for Mo{Delta}7SR has been registered in the DDBJ database under nucleotide sequence accession number AB270696.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Isolation of a {Delta}7SR gene from M. alpina 1S-4.
A PCR-based cloning strategy was adopted to obtain a gene encoding {Delta}7SR from the genomic DNA of M. alpina 1S-4. The amino acid sequences of two {Delta}7SR derived from human and African clawed frog were aligned, and degenerate oligonucleotides were designed based on the conserved regions. The single band obtained on PCR amplification contained a putative {Delta}7SR gene fragment. This fragment was used to isolate the full-length genes from the genomic gene and the cDNA library. The open reading frame in the genomic gene was 1,886 bp in length, contained three introns (227, 142, and 113 bp) with 5'-GT and 3'-AG ends, and encoded a protein of 467 amino acids with a molecular weight of 53,965.

The deduced amino acid sequence of the cloned Mo{Delta}7SR exhibited 52, 50, and 40% identity with those of {Delta}7SR from African clawed frog, human, and higher plant Arabidopsis thaliana (GenBank accession no. ATU49398), respectively. Mo{Delta}7SR possesses an identical sequence from 431 to 454 amino acid residues with a sterol reductase family signature 2 (Prosite accession no. PS01018) and a similar sterol reductase family signature 1 from 205 to 220 residues with a sterol reductase family signature 1 (Prosite accession no. PS01017). The amino acid residue at position 212 in a similar sequence of the sterol reductase family signature 1 of Mo{Delta}7SR is Leu, whereas the corresponding residue in signature 1 is Phe, Tyr, Trp, or Met. A hydropathy plot indicates that there is a large proportion (>55%) of hydrophobic amino acids throughout the polypeptide backbone (except for the region of amino acid residues 4 to 35 and 434 to 467) of the deduced amino acid sequence (data not shown). The existence of at least nine transmembrane {alpha}-helical domains is predicted by Kyte and Doolittle hydrophobicity analysis, which is identical with the profiles of animal and plant {Delta}7SR (data not shown).

Functional analysis of Mo{Delta}7SR cDNA in yeast ERG5 disruptant.
To identify the function of Mo{Delta}7SR cDNA, an expression vector, pYEMo{Delta}7SR, constructed by ligation of Mo{Delta}7SR cDNA under GAL1 promoter of yeast expression vector pYES2, was transformed into ERG5-disrupted yeast S. cerevisiae SX-1065. As shown in Fig. 1A, control strain transformed with pYES2 accumulated ergosta-5,7-dienol as the main sterol at 19.1 min of the GLC chromatogram, instead of ergosterol, because of its ERG5 disruption. GC-MS analysis of ergosta-5,7-dienol revealed a mass peak of m/z 398 and specific m/z 253 and 271 peaks. Ergosta-5,7-dienol is assumed to be derived from {Delta}24 reduction of ergosta-5,7,24(28)-trienol. On the other hand, GLC analysis of total sterols from the Mo{Delta}7SR transformant revealed accumulation of another sterol (the main sterol of the transformant) at the retention time of 18.5 min as shown in Fig. 1B. Through further GC-MS analysis, this sterol showed specific ion peaks of m/z 255, 273, and 400, which were identical to the corresponding mass peaks of authentic campesterol. Moreover, it also showed identical retention time with the authentic campesterol in high-pressure liquid chromatography and GLC analysis (see the supplemental material). These results proved that ergosta-5,7-dienol was converted to campesterol as the reduction of a {Delta}7 double band by Mo{Delta}7SR in the transgenic yeast.


Figure 1
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  		FIG. 1. GLC chromatogram of total sterols and GC-MS analysis of the main sterol from the S. cerevisiae ERG5 mutant. (A) Control strain (with the vector only); (B) Mo{Delta}7SR transformant.

 
Effect of gene silencing of Mo{Delta}7SR on sterol composition.
M. alpina 1S-4 ura5– strain was transformed with plasmid pBlues{Delta}7U5 constructed for Mo{Delta}7SR gene silencing by biolistic transformation. The sterol composition of the obtained Mo{Delta}7SR-RNAi transformant and the control strain with an empty vector, pDura5, were analyzed by GLC (Fig. 2A). When these transformants were cultivated in GY medium at 28°C for 5 days, desmosterol was the main sterol (75.7%) in the total sterols of the control strain, whereas the proportion of desmosterol decreased to 21.0% in the total sterols of the Mo{Delta}7SR-RNAi transformant and some unidentified sterols were detected. One of the unidentified sterols, designated peak V in Fig. 2A, consisted of 9.9% of the total sterols. On GC-MS analysis, the mass spectra of peak V exhibited a mass peak of m/z 382, and the fragmentation pattern was identical with that of the authentic of cholesta-5,7,24-trienol (data not shown). Furthermore, the retention time of peak V on both GLC and HPLC analyses was identical to that of the authentic cholesta-5,7,24-trienol. These results indicate that peak V was identified as cholesta-5,7,24-trienol, which is converted to desmosterol by reduction at the {Delta}7 position of the sterol skeleton as shown in Fig. 2B. RNAi analysis revealed that Mo{Delta}7SR was involved in desmosterol biosynthesis in M. alpina 1S-4.


Figure 2
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  		FIG. 2. (A) GLC chromatograms of Mortierella transformants. (B) Putative biosynthesis of desmosterol from cholesta-5,7,24-trienol by Mo{Delta}7SR.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
{Delta}7SR genes have been isolated from mammals (1, 11), frogs, amoebas, and higher plants (9). It catalyzes reduction of a double bond at the {Delta}7 position on the sterol skeleton and, in particular, exhibits a significant function in the biosynthesis from 7-dehydrocholesterol to cholesterol. SLOS in humans was characterized as an abnormal accumulation of 7-dehydrocholesterol, which was caused by congenital mutation of the {Delta}7SR gene (30). On the other hand, plant {Delta}7SR gene has been used for steroid production in recombinant yeast (3, 22). With respect to microorganisms, M. alpina and Thraustochytrium sp. were found to produce desmosterol and cholesterol, respectively, whereas the microbial {Delta}7SR gene has never been reported.

Mo{Delta}7SR exhibits 39 to 51% homology with other {Delta}7SR proteins at the amino acid level and shows two typical sterol family signatures. However, the Leu residue at position 212 in Mo{Delta}7SR is not consistent with the amino acid residue of the sterol family signature 1. This indicates that the sterol family signature 1 may be revised based on the Leu residue in Mo{Delta}7SR. Mo{Delta}7SR is inferred to be present in the membrane of endoplasmic reticulum because of the nine putative transmembrane domains. We propose a phylogenetic tree of representative {Delta}7SR, sterol {Delta}14 reductases ({Delta}14SR), and sterol {Delta}24 reductases ({Delta}24SR) as shown in Fig. 3. Mo{Delta}7SR was found to be closer to mammal and frog {Delta}7SR than to A. thaliana and Oryza sativa {Delta}7SR. {Delta}7SR exhibits a relatively high similarity with {Delta}14SR and {Delta}24SR at the polypeptide level. In particular, Mo{Delta}7SR exhibited high identities with {Delta}7SR (40 to 52%) from other organisms and some identities with {Delta}14SR (34 to 38%) and {Delta}24SR (28%). There is no information about fungal {Delta}7SR other than the present study, although some fungal {Delta}14SR and {Delta}24SR were reported. To elucidate sterol biosynthetic pathways in M. alpina 1S-4, isolation and characterization of {Delta}14SR and {Delta}24SR genes from M. alpina 1S-4 is also considered very important.


Figure 3
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  		FIG. 3. Phylogenetic analysis of {Delta}7SR, sterol {Delta}24 reductase ({Delta}24SR), and sterol {Delta}14 reductase. The phylogenetic tree was prepared by using the GENETYX version 7.0.3 (Genetyx Co.). For each numbered sequence, the EMBL (em) or TrEMBL (tr) accession no. is as follows: sequence 1, em, AF048704; sequence 2, em, AF480070; sequence 3, em, AF256535; sequence 4, tr, Q59LS4; sequence 5, em, M99419; sequence 6, tr, Q41852; sequence 7, tr, Q4WW43; sequence 8, em, S58126; sequence 10, em, AB016800; sequence 13, em, AY653733; and sequence 15, em, AP005514. The gene accession numbers of sequences 9, 11, 12, and 14 are noted in the text.

 
The ERG5 disruptant SX-1065 from S. cerevisiae accumulates ergosta-5,7-dienol instead of ergosterol. Campesterol, which is the reduction product of ergosta-5,7-dienol, is produced in the presence of Mo{Delta}7SR in transformed SX-1065. Both ergosta-5,7-dienol and campesterol are not detected in M. alpina 1S-4. Furthermore, almost all ergosta-5,7-dienol was converted to campesterol in the transformed SX-1065. These points suggest that Mo{Delta}7SR has wide substrate specificity and high activity for {Delta}7 reductase. M. alpina 1S-4 accumulates more than 70% of desmosterol in the total sterols. RNAi analysis revealed that Mo{Delta}7SR plays a significant function in vivo, converting cholesta-5,7,24-trienol to desmosterol. The GLC chromatogram of the Mo{Delta}7SR-RNAi transformant was very different from that of the control strain. Some unidentified peaks other than cholesta-5,7,24-trienol were detected in the Mo{Delta}7SR-RNAi transformant. We hypothesize that these compounds have a {Delta}5,7 structure and are converted to ergosta-5,24(25)-dienol, ergosta-5,25-dienol, 24,25-methylenecholest-5-enol, and so on by {Delta}7 double bond reduction. Mo{Delta}7SR can tolerate various sterol side chains while being specific for the {Delta}7 double bond reduction in a {Delta}5,7 structure, as shown in Fig. 4.


Figure 4
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  		FIG. 4. Partial biosynthetic pathways of the sterols in Mo{Delta}7SR transgenic yeast and M. alpina 1S-4.

 
In conclusion, our results on the molecular characterization of Mo{Delta}7SR gene may provide some useful opportunities with regard to (i) the study of steroid production by microbial fermentation, (ii) investigation of structure-function relationships in {Delta}7SR, and (iii) the elucidation of evolution of sterol reductases in various organisms.


   ACKNOWLEDGMENTS
 
We thank Makoto Ueda, Masahiro Yamagishi, and Takeshi Sakamoto, Mitsubishi Chemical Co., for GC-MS analysis of the sterols and kind suggestions regarding identification of the sterols.

This study was supported in part by a grant-in-aid for scientific research (grant 15658024) to S.S. from the Ministry of Education, Science, Sports, and Culture of Japan; a COE award for Microbial-Process Development Pioneering Future Production Systems (COE Program of the Ministry of Education, Science, Sports, and Culture of Japan) to S.S.; and the Project of the New Energy and Industrial Technology Development Organization of Japan (grant 05A07003d to E.S.).


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Applied Life of Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan. Phone: 81757536114. Fax: 81757536128. E-mail: sim{at}kais.kyoto-u.ac.jp. Back

{triangledown} Published ahead of print on 12 January 2007. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back


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 Abstract
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 Materials and Methods
 Results
 Discussion
 References
 

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Applied and Environmental Microbiology, March 2007, p. 1736-1741, Vol. 73, No. 6
0099-2240/07/$08.00+0     doi:10.1128/AEM.02425-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.




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