GENOA OF THE BIOSYNTHESIS OF THE MONACOLINE K

The present invention relates to the field of molecular biology and microbiology. More particularly, the present invention relates to genes of the biosynthesis of monacolin K.

Monascus The yeast is used in food production from thousands of years in China. Recently, it has been found that more bioactive Monascus product. These bioactive substances are mainly secondary metabolites Monascus, comprising substances for reducing hypertension, substances such as anti-fouling bacteria the [...], anti-cancer substances, substances for reducing the blood sugar, of 1' ergosterol, antioxidants, and inhibitors of cholesterol synthesis such that the monacolin. Therefore, Monascus has been found in recent years as a functional health food.

The monacolin K, the inhibitor of cholesterol synthesis produced by Monascus, has first been isolated from the medium by [...] Monascus rubber CO ., LTD. Merck & Co., Inc., and it has been found the same substance in the medium of Aspergillus terreus, named lovastatin and acting as an inhibitor of HMG-CoA reductase. The monacolin K is part polyketides and a structure similarities with that of HMG-CoA. Therefore, the monacolin K competitively inhibits the cholesterol synthesis with HMG-CoA, and HMG-CoA reductase cannot catalyze HMG-CoA to form the [...], leading to the reduction of the cholesterol synthesis.

Polyketide products The secondary metabolites by fungi express a variety of structural and unique characteristics which do not exist in other bacteria ('Hagan, 1995). These features are also expressed in a variety of enzymes synthesis of the polyketide. The monacolin K produced by Monascus is an element from the group of polyketides and have been observed are various polyketide products by condensation of acetyl CoA catalyzed by the polyketide synthase (PKS) (Kennedy and al, 1999; and Abe and al, 2002). From the study of the polyketide synthase associated with combinatorial biosynthesis, the development of novel polyketide represents a significant potential (Mc Daniel and al, 1999), and the new polyketides will constitute a new path for finding effective drugs.

Therefore, one embodiment of the present invention provides an isolated DNA molecule, comprising a nucleotide sequence " [...] SEQ ID: 1" or a nucleotide sequence hybridizable thereto under stringent conditions.

Another embodiment of the present invention provides an isolated DNA molecule comprising a polynucleotide selected from a group comprising: a) a polynucleotide designated [...] and comprising a nucleotide sequence "SEQ ID NO: 2", b) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 3", c) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 4", d) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 5", e) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 6", f) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 7", g) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 8", h) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 9", i) mk I and designated a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 10", and j) a polynucleotide which can be hybridized to the polynucleotides a), b), c), d), e), f), g), h), or i) under stringent conditions.

Another embodiment of the present invention provides a vector comprising the isolated DNA molecule as defined above.

Furthermore, one embodiment of the present invention provides a cell transformed with a vector comprising a polynucleotide selected from the group comprising: a) a

and comprising a nucleotide sequence "SEQ ID NO: 3", c) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 4", d) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 5", e) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 6", f) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 7", g) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 8", h) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 9", i) rnk designated I and a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 10", and j) the combination thereof.

Furthermore, one embodiment of the present invention provides a method for increasing the production of monacolin K, comprising culturing a cell transformed with a vector, and collecting the monacolin K from the cell. The vector comprising a polynucleotide is selected from a group comprising: a) a polynucleotide designated [...] and comprising a nucleotide sequence "SEQ ID NO: 2", b) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 3", c) and designated[...] a polynucleotide comprising a nucleotide sequence " [...] SEQ ID: 4", d) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 5", e) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 6", f) mk F and designated a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 7", g) and designated[...] a polynucleotide comprising a nucleotide sequence "SEQ ID NO: 9", and h) combinations thereof.

Furthermore, one embodiment of the present invention provides a method for increasing the production of the HMG-CoA reductase inhibitor, comprising culturing a cell transformed with a vector, and collecting the HMG-CoA reductase inhibitor from the cell. The vector is as defined above.

Furthermore, one embodiment of the present invention provides a method for producing the HMG-CoA reductase inhibitor. The method includes the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 2" (genomic DNA of [...] ), " [...] SEQ ID: 3" (genomic DNA of [...] ), " [...] SEQ ID: 6" (genomic DNA [...] ), and "SEQ ID NO: 7" (genomic DNA [...] ), or a nucleotide sequence sharing 95 % homology with these sequences, or a nucleotide sequence capable of hybridizing to these sequences under stringent conditions in a host cell, these sequences encode proteins having respectively the activity of the synthase [...], [...] of the synthase, of the dehydrogenase, and transesterase ; (b) culturing the transformed cell under conditions suitable for expression of the nucleotide sequences; and

(c) collecting the inhibitor of HMG-CoA.

Furthermore, one embodiment of the present invention provides a method for producing the monacolin k, comprising culturing a cell transformed with a vector and collecting the monacolin k from the cell. The vector is as defined above.

Embodiments of the present invention can be understood more completely and other advantages will appear with reference to the following description and the accompanying the drawings, on which:

the FIG. 1A~1G illustrate the sequence comparison of the polyketide synthase of Monascus or other fungi, and the unit of the fatty acid synthase rat. The FIG. IA has the comparison with the synthase [...], the FIG. IB demonstrates the comparison with the acyl transferase, FIG. 1C demonstrates the comparison with the dehydratase, FIG. 1D present the comparison with the methyl transferase, FIG. 1st demonstrates the comparison with the enoyl reductase, FIG. 1F demonstrates the comparison with the [...] and the reductase FIG. IG demonstrates the comparison with the acyl carrier protein.

FIG. 2 illustrates the expression of the gene cluster of Monascus 3~14 mk to days.

The FIG. 3A and 3B illustrate boards and [...][...] respectively.

FIG. 4 illustrates the amino acid sequence pattern assumed the binuclear MKH and Zn (II) [...] NTI analyzed by the vector. The amino acid sequence in the enclosure indicates the binuclear pattern Zn (II) [...] ; Cys 6 is marked grey.

FIG. 5 illustrates construction of the expression vector E. coli containing the gene[...] of the polyketide synthase. A group of oligonucleotide primers (p3 and p4) has been designed to amplify the gene (6.5 KB) [...] partial cDNA by RT-PCR. The primers pl p2 and have been designed so as to amplify the partial cDNA [...] (3.0 KB) gene by RT-PCR.

FIG. 6 illustrates the construction of the expression vector containing the gene sfp E. coli of the 4'-phosphopantetheine transferase B. subtilis.

FIG. 7 illustrates the expression of [...] andsfp in E.coli. Proteins E. The total coli harboring the control plasmids [...] -lb and [...] (band 1) and the expression plasmids [...]sfp ( gene sfp) and[...] (gene [...] ) ( web 2) have been brought to boiling in a load buffer and subjected to SDS-PAGE analysis. The proteins have been colored with blue colloidal [...]. The arrows indicate the positions of and [...] MKA. Lysed The proteins have been purified by the purification system™ [...] ( [...] ). The strip 3 shows the centrifugation of the lysate to 3000xg during 60 min and the strip 4 corresponds to the proteins purified by Ni column.

Probes specific to Monascus have been constructed in accordance with the degenerate primers of lovastatin of Aspergillus terreus, by publication of Nicholson (2001). The genes related to the synthesis of monacolin K have been cloned from the library by hybridization to BAC of Monascus colony, block and annotated. PKS Two complete cDNAs were amplified by RT-PCR and cloned into expression vectors, respectively. The invention was then performed.

The discovery of the cholesterol inhibitors produced by Monascus in the years 1980 has received increased interest centered on the effect of Monascus on the ameliorating hypertension, hyperglycemia and cholesterol. In addition to the monacolin K, other substances for reducing cholesterol have been isolated from Monascus, for example, the monacolin J, L, M, and X (Endo and al, 1979, 1985, 1986, and and Komagata al, 1989). The [...] J and L are precursors of monacolin K. The mechanism of the inhibition of cholesterol synthesis by the monacolin K is based on structural similarities between the monacolin k and HMG-CoA. K The monacolin can bind to HMG-CoA reductase and blocking its catalyst activity for forming [...] from HMG-CoA, which will reduce dramatically the synthesis of cholesterol. Except the monacolin K, other methods adopting the biotransformation or chemical modification for the inhibition of cholesterol are also commercially available, for example, pravastatin, simvastatin, fluvastatin, or 1' atorvastatin, which all share structural similarities with HMG-CoA.

Therefore, the main object of the present invention is to provide genes related to the production of monacolin K from Monascus. The object analyzed in the present invention is Monascus sp. BCRC 38072 having the following features:

CYA, 25 °C, 7 days. Colony 25-26 mm. diameter, white mycelium initially, becoming pale red-orange and red dark orange.

MEA, 25 °C, 7 days. Colony 48 mm diameter, bright red orange, red and orange sharp.

G25N, 25 °C, 7 days. Colony 28-29 mm diameter, dark orange red, dark orange yellow to the centers.

[...] form individual or occasionally in short chain, [...] to globose, 10-13χ8-10 μτη. Globose [...], 37-72 [...] diameter. Hyaline [...], ellipsoids, 4.6-6, [...] 3, 3-4.2 μτη 3 (-6.6).

The classification system According Hawksworth & Pitt (1983), BCRC 38072 has been identified as follows:

BCRC 38072 est between m. m. pilosus and ruber.

1. BCRC 38072 est pilosus m. in similar to the color of the colonies and the growth rate.

2. Similar to m. BCRC 38072 est ruber in morphology of the ascospore.

BCRC 38072, m. ruber, andM pilosus share 100% sequence similarity in rDNA, fragment thereof and of-tubulin gene.

BCRC 38072 temporarily has been named as Monascus pilosus Sato according to K. D. [...]. & Pitt.

From analysis of BCRC 38072 of Monascus pilosus, specific probes to the conserved region of [...], a length of 226 bp ("SEQ ID [...]: 1") have been designed to serve for the hybridization in colonies, southern blot analysis and PCR. Furthermore, the probes can be used for screening of Monascus. The annotation and providing the complete DNA sequence BAC have been produced by Vector BLAST and NTI. Nine genes sharing similarities high over more than 54% with the gene cluster of lovastatin produced by Aspergillus terreus have been obtained by the bank BAC, as seen in the table 1.

The gene cluster monacolin K and the gene cluster of the compactin, synthesized from Penicillin citrinum, share similarities high more than 49%. The two new genes comprise genes of the polyketide synthase, one being responsible for the synthesis of [...] and the other, of the synthesis of [...]. Furthermore, a gene of the monooxygenase, 1 a gene ' oxidoreductase, a dehydrogenase gene, a gene of the trans-esterase, a gene HMG-CoA reductase, a transcription factor gene and a gene efflux pump are also included. A [...] of Monascus has been constructed and two clones which have been detected by the comparison of the sequences end [...] were a patent application under No. [...], comprising [...], [...], [...], [...], [...], [...] ; and [...] comprising [...], [...], [...], [...], [...], [...], [...], and[...]. [...] has been deposited at the American Type Culture Collection (ATCC) as PTA-5685, and [...], as PTA-5686. The functional regions of the gene and [...] synthase gene [...] synthase have additionally been analyzed by comparison with known polyketide genes of Aspergillus, Penicillium, Cochliobolus, andGibberella, and of the genes of the fatty acid synthesis (FAS) rat. The results indicate that the functional regions of these two genes are similar to those of the [...] produced by Aspergillus terreus and compactin produced by Penicillium citrinum, as shown in FIG 1. The Northern blot analysis of total RNA extract of Monascus highlights the expression of these genes at the transcriptional level, as shown in FIG 2.

The two genes of the polyketide synthase of the present invention are multifunctional enzymes: the gene [...] has a functionality of- [...] synthase, acetyl transferase, dehydratase, methyltransferase, ketoreductase, and carrier protein; the gene [...] 6 exerts the aforementioned functions and further, the function of 1' enoyl reductase, as shown in FIG 1. The full-length cDNA of the two genes was obtained by RT-PCR, then cloned into the Pichia pastoris expression system for expression of the polyketide. Since the gene expresses multifunctional polyketide synthase enzymes, various products of polyketides may be produced recombinantly from 1' as the DNA rearrangement from a full-length cDNA of the present invention. The method opens up a new way for drug discovery. Several patents such as U.S. patents no. 6,221 641 6,391 594 and have similar methods expression in bacteria. However, the secondary metabolites polyketides produced by fungi have a variety and a structural complexity. Therefore, [...] (FIG. 3A) and [...] (FIG. 3B) obtained in the present invention can be used as expression plasmids to express various products of polyketides by rearrangement.

Therefore, the present invention provides the molecules

dNA, vectors and following methods.

The present invention has a probe for screening of genes related to the synthesis of the monacolin K, comprising a nucleotide sequence " [...] SEQ ID: 1". The probe is specific to Monascus [...] and has been designed from the degenerate primers for the gene synthesis of lovastatin d ' Aspergillus terreus designed by[...] (2001). It is easy for the skilled person or purification of isolating genes similar to those of the monacolin K by known methods such as hybridization using the probe of the present

invention.

(II) [...] synthase (DNA [...] of [...] )

One aspect of the present invention relates to a DNA sequence comprising a nucleotide sequence " [...] SEQ ID: 2" (genomic DNA [...] ), or a nucleotide sequence sharing homology with 95% " [...] SEQ ID: 2", or a nucleotide sequence hybridizable thereto under stringent conditions and encoding a protein having an activity of synthase [...]. The DNA sequence has been isolated from [...]. Furthermore, the DNA sequence of the present invention can be obtained by those skilled in the art by known methods such as PCR or hybridization according to the description of the present invention. References to the stringent conditions may be found in the document EP 1,325 959 Al P7. The [...] synthase catalyzes an acetate and eight [...] malonates to form a. Multifunctional This gene has regions, comprising the

[...] synthase, 1' acetyltransferase, the dehydratase, methyltransferase, the ketoreductase, and carrier protein. Comparison of this gene with the regions

functional [...] of Monascus [...] share a high similarity with the gene of Aspergillus terreus [...] and the gene [...] Penicilliumcitrinum.

Another aspect of the present invention provides a vector, comprising a nucleotide sequence "SEQ ID NO: 2" (genomic DNA of [...] ), or a nucleotide sequence sharing homology with 95% "SEQ ID NO: 2", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity of synthase [...]. The tools of the construction of the present invention include molecular sequences self-replicable or be integrated with the chromosome in a host cell, for example, a plasmid, a phage, or a virus; preferably, the vector is a shuttle vector. The vector of the present invention further produces various products polyketide by rearrangement. A reference to the rearrangement can be found in US patent No.: 6,221 641 and US Patent No.: 6,391 594.

Another aspect of the invention relates to a transformant, comprising a nucleotide sequence "SEQ ID NO: 2", or a nucleotide sequence sharing 95% homology with the " [...] SEQ ID: 2", or a nucleotide sequence hybridizable thereto under stringent conditions and encoding a protein having an activity of synthase [...], and a host cell to construct the transformant. The host cell comprises the prokaryotic or eukaryotic cells. Suitable host cells include but are not limited to, bacteria, yeasts, animal cells, insect cells, plant cells, or filamentous fungi. The filamentous fungi can be Monascus sp., Monascus pilosus in particular,Monascus ruber, orMonascus purpureus, more particularly, [...]. The transformation may be performed using a processing method for the filamentous fungi belonging to the genus Aspergillus using the host system-known vector. The document EP 1,325 959 See Al P5.

Another aspect of the invention relates to a method for increasing the production of [...] K, comprising the steps of: (a) transforming a nucleotide sequence of "SEQ ID NO: 2" (genomic DNA of [...] ), or a nucleotide sequence sharing homology with 95% " [...] SEQ ID: 2", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having the activity of [...] synthase in a host cell; and (b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence. Preferably, the host cell is initially a cell producing the monacolin K.

Another aspect of the invention relates to a method for increasing the production of HMG-CoA reductase inhibitor. The method includes the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 2", or a nucleotide sequence sharing homology with 95% " [...] SEQ ID: 2", or a nucleotide sequence capable of hybridizing therewith under stringent conditions and encoding a protein having the activity of [...] synthase in a host cell ; (b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence; and

(c) collecting the HMG-CoA reductase inhibitor. Preferably, the host cell is initially a cell producing the monacolin K. (III) [...] synthase (genomic DNA [...] )

The present invention relates to a DNA sequence comprising a nucleotide sequence "SEQ ID NO: 3" (genomic DNA [...] ), or a nucleotide sequence sharing homology with 95% "SEQ ID NO: 3", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity of synthase [...]. The DNA sequence has been isolated from [...]. Furthermore, the DNA sequence of the present invention can be obtained to those skilled in the art by known methods such as PCR or hybridization as described in the invention. References to the stringent conditions may be found in the document EP 1,325 959 Al P7. Synthase The [...] in the biosynthesis of a leg of the monacolin K, in other words the [...] or referred 2-methylbutyrate. Multifunctional This gene has regions comprising the- [...] synthase, acetyl transferase, the dehydratase, methyltransferase, the ketoreductase, the acyl carrier protein and 1' enoyl

functional [...] ofMonascus pilosus [...] share a high similarity with the gene of Aspergillus terreus [...] and the gene mlcB Penicilliumcitrinum.

Another aspect of the invention relates to a vector comprising a nucleotide sequence " [...] SEQ ID: 3", or a nucleotide sequence sharing homology with 95% "SEQ ID NO: 3", or a nucleotide sequence hybridizable thereto under stringent conditions and encoding a protein having an activity of synthase [...]. The tools of the construction of the present invention include molecular sequences that are self-replicable or integration with a chromosome in a host cell, for example, a plasmid, a phage, or a virus. The vector of the present invention further produces various products polyketide by rearrangement. References to rearrange may be found in the U.S. patents No.: 6,221 641 and US No.: 6,391 594.

Another aspect of the invention also provides a transformant, comprising a nucleotide sequence "SEQ ID NO: 3", or a nucleotide sequence sharing homology with 95% "SEQ ID NO: 3", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity of synthase [...], and a host cell to construct the transformant. The host cell comprises the prokaryotic or eukaryotic cells. Suitable host cells include but are not limited to, bacteria, yeasts, animal cells, insect cells, plant cells or filamentous fungi. The filamentous fungi can be Monascus sp., Monascus pilosus in particular, Monascus ruber, orMonascus purpureus, more particularly, [...]. The transformation may be performed using a process for transforming the filamentous fungi belonging to the genus Aspergillus using the host system-known vector. The document EP 1,325 959 See Al P5.

Another aspect of the invention relates to a method for increasing the production of monacolin K, comprising the steps of: (a) transforming a nucleotide sequence " [...] SEQ ID: 3", or a nucleotide sequence sharing homology with 95% " [...] SEQ ID: 3", or a nucleotide sequence hybridizable thereto under stringent conditions and encoding a protein having the activity of [...] synthase in a host cell; and (b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence. Preferably, the host cell is initially a cell producing the monacolin K.

Another aspect of the present invention relates to a method for increasing the production of HMG-CoA reductase. The method includes the steps of: (a) transforming a nucleotide sequence "SEQ ID NO:3 ", or a nucleotide sequence sharing homology with 95% "SEQ ID NO: 3", or a nucleotide sequence hybridizable thereto under stringent conditions and encoding a protein having the activity of [...] synthase in a host cell ; (b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence; and (c) collecting the HMG-CoA reductase inhibitor. Preferably, the cell

host is initially a cell producing the monacolin K.

The present invention relates to a DNA sequence comprising a nucleotide sequence "SEQ ID NO: 9" (genomic DNA [...] DNA), or a nucleotide sequence sharing homology with 95% " [...] SEQ ID: 9", or a nucleotide sequence hybridizable thereto under stringent conditions and encoding a protein having the activity of transcription factor. The DNA sequence has been isolated from [...]. Furthermore, the DNA sequence of the present invention can be obtained to those skilled in the art by known methods such as PCR or hybridization as described in the invention. References to the stringent conditions may be found in the document EP 1,325 959 Al P7. The gene [...] binuclear pattern is identified as Zn (II) [...] from the comparison with the vector NTI. The conserved sequence of this gene is Cys-X 2-Cys-X 6-Cys-balance-Cys-X 2-Cys-X 6-Cys, as shown in FIG 4.

Another aspect of the present invention relates to a vector comprising a nucleotide sequence "SEQ ID NO: 9", or a nucleotide sequence sharing homology with 95% "SEQ ID NO: 9", or a nucleotide sequence hybridizable thereto under stringent conditions and encoding a protein having the activity of transcription factor. The tools of the construction of the present invention include molecular sequences self-replicable or be integrated with the chromosome in a host cell, for example, a plasmid, a phage, or a virus. Preferably, the vector of the present invention is an expression vector.

Another aspect of the invention also provides a transformant, comprising a nucleotide sequence "SEQ ID NO: 9", or a nucleotide sequence sharing homology with 95% "SEQ ID NO: 9", or a nucleotide sequence hybridizable thereto under stringent conditions and encoding a protein having the activity of transcription factor, and a host cell for the construction the transformant. The host cell comprises the prokaryotic or eukaryotic cells. Suitable host cells include but are not limited to, bacteria, yeasts, animal cells, insect cells, plant cells or filamentous fungi. The filamentous fungi can be Monascus sp., Monascus pilosus in particular, Monascus ruber, orMonascus purpureus, more particularly[...]. The transformation may be performed using a process for transforming the filamentous fungi belonging to the genus Aspergillus using the host system-known vector. The document EP 1,325 959 See Al P5.

Another aspect of the invention relates to an expression system. The expression system comprises a nucleotide sequence "SEQ ID NO:9 ", or a nucleotide sequence sharing homology with 95% "SEQ ID NO: 9", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having the activity of transcription factor, and a host cell for the expression of the nucleotide sequence. The sequence is transformed in the host cell by transformation. Suitable host cells include bacteria, yeast, animal cells, insect cells, plant cells or filamentous fungi. The filamentous fungi can be Monascus sp., Monascus pilosus in particular,Monascus ruber, or Monascus purpureus, more particularly[...].

Another aspect of the invention relates to a method for increasing the production of monacolin K, comprising the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 9", or a nucleotide sequence sharing homology with 95% "SEQ ID NO: 9", or a nucleotide sequence hybridizable thereto under stringent conditions and encoding a protein having the activity of transcription factor in a host cell; and (b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence. Preferably, the host cell is initially a cell producing the monacolin K.

Another aspect of the invention relates to a method for increasing the production of the inhibitor HMG-CoA reductase. The method includes the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 9", or a nucleotide sequence sharing homology with 95% "SEQ ID NO: 9", or a nucleotide sequence hybridizable thereto under stringent conditions and encoding a protein having the activity of transcription factor in a host cell;

(b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence; and

(c) collecting the HMG-CoA reductase inhibitor. The host cell comprises the prokaryotic or eukaryotic cells. Suitable host cells include but are not limited to, bacteria, yeasts, animal cells, insect cells, plant cells or filamentous fungi. The filamentous fungi can be Monascus sp., Monascus pilosus in particular, Monascus

ruber, orMonascus purpureus, more particularly [...].

The present invention relates to a method for producing the HMG-CoA reductase inhibitor, preferably used for the production of K. [...] The method includes the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 2" (genomic DNA [...] ), "[...] SEQ ID: 3" (DNA [...][...] ), "SEQ ID NO: 6" (genomic DNA [...] ), and "SEQ ID NO: 7" ( [...][...] DNA), or a nucleotide sequence that can be hybridized to these sequences under stringent conditions in a host cell, these sequences encode proteins having the activity of [...] respectively synthase, synthase of [...], dehydrogenase, and transesterase ; (b) culturing the transformed cell under conditions suitable for expression of the nucleotide sequences; and (c) collecting the monacolin K. The host cell comprises prokaryotic or eukaryotic cells. Suitable host cells include but are not limited to, bacteria, yeasts, animal cells, insect cells, plant cells or filamentous fungi. The filamentous fungi can be Monascus sp., Monascus pilosus in particular, Monascus ruber, orMonascus purpureus, more particularly[...].

Hutchinson and coll. (2000) have suggested that four genes required for the synthesis of lovastatin when heterologously expressed, in other words, those of the [...] synthase, synthase the [...], the dehydrogenase, and the transesterase. Amongst them, the synthase the dehydrogenase [...] and contribute to the production of the precursor of the lovastatin, the synthase [...] aids the production of 2-methylbutyrate, and the product 2-methylbutyrate binds to the [...] by transesterase and forms the complete lovastatin.

The above method further comprises a nucleotide sequence " [...] SEQ ID: 4" (genomic DNA [...] ), " [...] SEQ ID: 5" (genomic DNA [...] ), "SEQ ID NO: 8" (DNA [...][...] ), " [...] SEQ ID: 9" (genomic DNA [...] ), and " [...] SEQ ID: 10" (genomic DNA [...] ), or a nucleotide sequence sharing 95 % homology with these sequences, or a nucleotide sequence capable of hybridizing to these sequences under stringent conditions, these sequences encode proteins having respectively the activities of P450 monooxygenase, oxidoreductase, HMG-CoA reductase, transcription factor and efflux pump activity.

(VI) [...] synthase ( [...] cDNA)

The invention relates to a DNA sequence comprising a nucleotide sequence "SEQ ID NO: 19" ([...] cDNA), or a nucleotide sequence having 95% homology with the "SEQ ID NO: 19", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity [...] synthase. The DNA sequence has been isolated from [...]. Furthermore, the DNA sequence of the invention may be obtained by the person skilled in the art from known methods such as PCR amplification or hybridization, as described in 1' invention.

Another aspect of the invention provides a vector, comprising a nucleotide sequence "SEQ ID NO: 19" ([...] cDNA), or a nucleotide sequence having 95% homology with the "SEQ ID NO: 19", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity [...] synthase. The tools for constructing the vector of the invention include self-replicable molecular sequences or integration with a chromosome in a host cell, for example, a plasmid, a phage, or a virus. Preferably, the vector of the invention is an expression vector. More preferably, the vector of the invention is the vector [...] A ( [...] ). The construct of the invention is [...] comprising the nucleotide sequence "SEQ ID NO: 19". The construct [...] has been obtained by cloning the full length cDNA of the gene in [...][...] A according to the methods described in the document Molecular Cloning (Cloning molecular). The use of the vector of the invention provides, by random recombination ( [...] ), different polyketide products. There are references to the random recombination searchable in U.S. Patent No.: 6,221 641 and in U.S. Patent No.: 6,391 594.

Another aspect of the invention also provides a transformant, comprising a nucleotide sequence "SEQ ID NO: 19", or a nucleotide sequence having 95% homology with the "SEQ ID NO: 19", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity [...] synthase, and a host cell to construct the transformant. The host cell is a prokaryotic cell or a eukaryotic cell. Suitable host cells include, but not limited to, bacteria, yeasts, animal cells, insect cells, plant cells, or filamentous fungi. The filamentous fungi may be derived from Monascus species, in particular theMonascus pilosus,Monascus ruber or the the Monascus purpureus, and more particularly the [...]. The transformation may be performed by applying a transformation method for filamentous fungi belonging to the genus Aspergillus utilizing the host-vector system long known date. EP 1,325 959 See Document Al P5.

Another aspect of the present invention relates to a method for increasing the production of monacolin K, comprising the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 19", or a nucleotide sequence having 95% homology with the "SEQ ID NO: 19", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity [...] synthase in a host cell; and

(b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence. Preferably, the host cell is, originally, a cell producing monacolin K.

Another aspect of the invention relates to a method for increasing the production of an inhibitor of HMG-CoA reductase. The method includes the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 19", or a nucleotide sequence having 95% homology with the "SEQ ID NO: 19", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity [...] synthase in a host cell ; (b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence (c) and collecting the HMG-CoA reductase inhibitor. Preferably, the host cell is, originally, a cell producing monacolin K.

(VII) [...] synthase ( [...] cDNA)

The invention relates to a DNA sequence comprising a nucleotide sequence "SEQ ID NO: 20" ([...] cDNA), or a nucleotide sequence having 95% homology with the "SEQ ID NO: 20", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity [...] synthase. The DNA sequence has been isolated from [...]. Furthermore, the DNA sequence of the invention may be obtained by the person skilled in the art from known methods such as PCR amplification or hybridization according to the description of the invention.

Another aspect of the invention provides a vector, comprising a nucleotide sequence "SEQ ID NO: 20" ([...] cDNA), or a nucleotide sequence having 95% homology with the "SEQ ID NO: 20", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity [...] synthase. The tools for constructing the vector of the invention include self-replicable molecular sequences or integration with a chromosome in a host cell, for example, a plasmid, a phage, or a virus. Preferably, the vector of the invention is an expression vector. More preferably, the vector of the invention is the vector [...] C ( [...] ). The construct of the invention is [...] comprising the nucleotide sequence of "SEQ ID NO: 20". The construct [...] has been obtained by cloning the full length cDNA of the gene in the vector [...][...] C according to the methods described in the document Molecular Cloning (Cloning molecular). The use of the invention vector gives through random recombination ( [...] ), different polyketide products. There are references to the random recombination searchable in U.S. Patent No.: 6,221 641 and in U.S. Patent No.: 6,391 594.

Another aspect of the invention also provides a transformant, comprising a nucleotide sequence "SEQ ID NO: 20", or a nucleotide sequence having 95% homology with the "SEQ ID NO: 20", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity [...] synthase, and a host cell to construct the transformant. The host cell is a prokaryotic cell or a eukaryotic cell. Suitable host cells include, but not limited to, bacteria, yeasts, of

filamentous may be from the species [...] s, in particular the Monascus pilosus, Monascus ruber or the the Monascus purpureus, and more particularly the [...]. The transformation may be performed by applying a transformation method for filamentous fungi belonging to the genus Aspergillus utilizing the host-vector system long known date. EP 1,325 959 See Document Al P5.

Another aspect of the present invention relates to a method for increasing the production of monacolin K, comprising the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 20", or a nucleotide sequence having homology with the 95 I "SEQ ID NO: 20", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity [...] synthase in a host cell; and

(b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence. Preferably, the host cell is, originally, a cell producing monacolin K.

Another aspect of the invention relates to a method for increasing the production of an inhibitor of HMG-CoA reductase. The method includes the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 20", or a nucleotide sequence having 95% homology with the "SEQ ID NO: 20", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having an activity [...] synthase in a host cell ; (b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence (c) and collecting the HMG-CoA reductase inhibitor. Preferably, the host cell is, originally, a cell producing monacolin K.

(VIII) Transcription factor ( [...] cDNA)

The invention relates to a DNA sequence comprising a nucleotide sequence of "SEQ ID NO: 25" ([...] cDNA), or a nucleotide sequence having 95% homology with the "SEQ ID NO: 25", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having the activity of a transcription factor. The DNA sequence has been isolated from [...]. Furthermore, the DNA sequence of the invention may be obtained by the person skilled in the art from known methods such as PCR amplification or hybridization according to the description of the invention. There are references to the searchable stringent conditions in the document EP 1,325 959 Al P7.

Another aspect of the invention provides a vector, comprising a nucleotide sequence "SEQ ID NO: 25", or a nucleotide sequence having 95% homology with the "SEQ ID NO: 25", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having the activity of a transcription factor. The tools for constructing the vector of the invention include self-replicable molecular sequences or integration with a chromosome in a cell

expression. More preferably, the vector of the invention is the vector pMS. The construct of the invention is [...] comprising the nucleotide sequence "SEQ ID NO: 25". The construct [...] has been obtained by cloning the full length cDNA of the gene in the vector [...] pMS according to the methods described in the document Molecular Cloning (Cloning molecular).

Another aspect of the invention also provides a transformant, comprising a nucleotide sequence "SEQ ID NO: 25", or a nucleotide sequence having 95% homology with the "SEQ ID NO: 25", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having the activity of a transcription factor, and a host cell to construct the transformant. The host cell is a prokaryotic cell or a eukaryotic cell. Suitable host cells include, but not limited to, bacteria, yeasts, animal cells, insect cells, plant cells, or filamentous fungi. The filamentous fungi may be derived from Monascus species, in particular theMonascus pilosus, Monascus ruber or the the Monascus purpureus, and more particularly the [...]. The transformation may be performed by applying a transformation method for filamentous fungi belonging to the genus Aspergillus utilizing the host-vector system long known date. EP 1,325 959 See Document Al P5.

thereof under stringent conditions, and a suitable host cell for expression of a nucleotide sequence, wherein the sequence is transformed in the host cell by a transformation. Suitable host cells include bacteria, yeasts, animal cells, insect cells, plant cells, or filamentous fungi. The filamentous fungi may be derived from Monascus species, in particular theMonascus pilosus,Monascus ruber or the the Monascus purpureus, and more particularly the [...].

Another aspect of the present invention relates to a method for increasing the production of monacolin K, comprising the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 25", or a nucleotide sequence having 95% homology with the "SEQ ID NO: 25", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having the activity of a transcription factor in a host cell; and

(b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence. Preferably, the host cell is, originally, a cell producing monacolin K.

Another aspect of the invention relates to a method for increasing the production of an inhibitor of HMG-CoA reductase. The method includes the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 25", or a nucleotide sequence having 95% homology with the "SEQ ID NO: 25", or a nucleotide sequence capable of hybridizing thereto under stringent conditions and encoding a protein having the activity of a transcription factor in a host cell ; (b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence (c) and collecting the HMG-CoA reductase inhibitor. Preferably, the host cell is, originally, a cell producing monacolin K.

The present invention relates to a method for producing an inhibitor of HMG-CoA reductase, preferably the production of monacolin K. The method includes the steps of: (a) transforming a nucleotide sequence "SEQ ID NO: 19" ([...] cDNA), "SEQ ID NO: 20" ([...] cDNA), "SEQ ID NO: 23" ([...] cDNA) and "SEQ ID NO: 24" ([...] cDNA), or a nucleotide sequence capable of hybridizing to these sequences under stringent conditions in a host cell, wherein these sequences encode proteins having activity [...] respectively synthase, synthase [...], and transesterase dehydrogenase ; (b) culturing the transformed cell under conditions suitable for the expression of the nucleotide sequence (c) and collecting the monacolin K. The host cell is a prokaryotic cell or a eukaryotic cell. Suitable host cells include, but not limited to, bacteria, yeasts, of

filamentous can be those Monascus species, in particular theMonascus pilosus, Monascus ruber or the the Monascus purpureus, and more particularly the [...]. Hutchinson and According Al. (2000), four genes are required for the synthesis of lovastatin in case of heterologous expression, which are the [...] synthase, synthase the [...], and the transesterase dehydrogenase. Of these, the synthase dehydrogenase [...] and contribute to the production of the precursor of the lovastatin, the [...] synthase participates in the production of the 2-methylbutyrate, and the product 2-methylbutyrate and binds to the [...] by the transesterase, and form the complete lovastatin.

The aforesaid method includes, further, a nucleotide sequence "SEQ ID NO: 21" ([...] cDNA), " SEQ ID NO: 22" ([...] cDNA), "SEQ ID NO: 25" ([...] cDNA) and "SEQ ID NO: 26" ([...] cDNA), or a nucleotide sequence having 95% homology with these sequences, or a nucleotide sequence capable of hybridizing to these sequences under stringent conditions, wherein these sequences encode proteins having respectively mono-oxygenase activity (cytochrome Ρ450), oxidoreductase, a transcription factor activity and efflux pump.

Monascus The has been inoculated into agar PDA (Dextrose) inclined and cultured at 30 °C. The hyphae and spores have been collected by scraping, inoculated in medium 50°ml (7% glycerol, 3% glucose, 3% MSG, 1.2% of polypeptone, 0.2% of [...]_3, 0.1% of [...] 7:00_2*0), cultured under vibration and to 25 °C.

A Production of the nuclei of Monascus

Monascus cells have been collected, washed with [...] and dried by extracting air. Is added [...] volumes of washing buffer pre-cooled (H3+1.5% of buffer

mercaptoethanol) to the dried cells and the cells have been homogenized in a mixer. Monascus The cores have been obtained by filtering [...].

B. Production of the semi-solid matrices and chromosomal DNA

Prepared a solution of agarose to 1.8% low melting point with a buffer HB and the water bath to 50 °C equal volumes of a solution of agarose and cores have been completely mixed and added in a die mold (Bio-Rad). Impurities have been treated with a proteinase K (l ° mg/ml). The semi-solid matrices have been spliced to small pieces and partially digested by [...]. A the end of the reaction of restriction, the semi-solid matrices have been analyzed in to 1% agarose gel for electrophoresis in a pulsed field. DNA fragments having a size of 200°kb have been recovered by electro-elution.

C. construction of a library of Monascus

The vector was used pIndigoBAC -5 digested by [...] ready for use (Epicentre) as a vector for ligation with 1' DNA collected. Ligature The reagent was then electroporated into competent cells (E. coli [...][...] ™ EC 100™, the Company Epicentre.). The colonies have been stored on 384 well microplate.

The PCR amplification and sequencing have been performed by degenerate primers and the DNA sequence obtained was used as a basis for the design of primers from a probe base pairs 226, as shown in the annex 1. [...] the software was used to design primers, and designed primers are presented in the list below.

[...] 5 ' [...] 3' (SEQ ID NO:29)

[...] 5 ' [...] 3' (seq id no:30)

A DNA sequence containing the DIG-ll-dUTP (Roche, PCR Probe DIG Synthesis Kit) has been amplified by PCR and used as

probe.

Having been cut a corner of the nylon membrane (Roche) in order to find the direction, is then placed the nylon membrane on a plate containing the colonies. The nylon membrane on top of which the colony has been deposited, has been contacted sequentially with a lysis solution (NaOH 2N, 0.1% SDS) during 5 minutes, a solution of NaOH/NaCl 0.5 1.5 M M for 5 minutes, a buffer/NaCl 1.5 Μ Tris-HCl (pH 7.4) 0.5 Μ during 5 minutes, and a buffer [...] SSC for 5 minutes. 1/DNA was then immobilized on the nylon membrane by exposure to UV light. The nylon membrane obtained was used for hybridization and 1' immunoassay (Roche). After this, the resulting colonies from the reaction were cultured and the DNA of the BAC has been extracted by using the kit Broadband construct A kit (Qiagen).

A. Preparation of the shotgun sequencing library

3 to 5°pg BAC DNA were ultrasonic treatment to have sizes to 2°kb 1 under conditions of suitable ultra-sonication and the results were confirmed by electrophoresis. DNA was then repaired by the nuclease Bal 31 and the T4 DNA polymerase to be ends francs and the DNA fragments of 1 to 2°kb have been collected by electrophoresis. Ligation is made using the vector pUC18/SmaI/ [...] (50 pg/pL) (Pharmacia) as a carrier and ligature the reagent has been electroporated in bacteria strains E. Coli DH5.

B. and sequencing DNA sequence analysis

The plasmid DNA has been extracted with high yield by using a microplate well to 96 and plasmid DNA obtained was sequenced using the kit [...] v3.0. Sequence analysis have been carried out by a sequencer * ABI3700 with a cover 10.

The assembly DNA sequences has been provided by software Phred., [...], developed by Phil Green [...] the lab.

The BAC full-length then been annotated using the software and [...] BLAST.

0,2 hyphae ° g Monascus have been placed in a mortar, frozen liquid nitrogen and then ground into a powder. Total RNA was extracted using the reagent [...] ( [...] ) and chloroform, and then dissolved in DEPC-H 2O. RNA was used as a model for reverse transcription (system [...] - [...] Reverse Transcription, unique) to obtain a full length cDNA. The full length cDNA has been ligated to a vector TA (system pGEM-T vector, unique).

A. RNA Electrophoresis

We prepared a to 1.2% agarose gel containing a buffer gel migration formaldehyde and formaldehyde. RNA has been thoroughly mixed with the gel migration buffer formaldehyde, formaldehyde (37%) and formamide. Electrophoresis the RNA has been made and the gel has been dyed with [...].

B. Transfer RNA

The nylon membrane has been cut to the size of the gel and a corner of the nylon membrane has been cut in order to find the direction. RNA has been transferred from the gel on the nylon membrane and the RNA has been immobilized on the nylon membrane by exposure to UV light. The nylon membrane obtained was used for hybridization and 1' immunoassay (Roche).

The probes have been designed according to the DNA sequence of the BAC for preparing probes used for the Northern blot analysis. The probes are presented in the list below.

[...] gene

Direct 5 'ATA AGA ATG GCT CCG GOCT CC 3' (SEQ ID NO:31) Reverse 5 'CCA TCA AGG ATG CTC TGT CG 3' (SEQ ID NO:32) A length of the probe: 229 base pairs

[...] gene

Direct 5 'CTA GAC TTT GCT TCC CAC GCC A 3' (SEQ ID NO:33) Reverse 5 'CAT TGT CGA GCG TTG GAG TC 3' (SEQ ID NO:34) of the probe^{0 Length:} 167 base pairs

[...] gene

A length of the probe^{0 :} 175 base pairs

A length of the probe^{0 :} 246 base pairs

[...] gene

Direct 5 'GCC CCG AAT CCT ACA TGA AG 3' (SEQ ID NO:41)

Reverse 5 'GGC CCA CCG ATG TAG TTG GT 3' (SEQ ID NO:42)

A length of the probe^{0 :} 166 base pairs

[...] gene

A length of the probe^{0 :} 179 base pairs

A length of the probe^{0 :} 127 base pairs

The full length cDNA PKS { [...] and[...] ) have been ligated to [...] and C ( [...] ), respectively. The transformation has been performed by using the transformation kit Pichia [...]. A kit ( [...] ).

The depurination to the acid was accomplished by immersing the gel in HCl 0.25 M, under stirring for 10°min in a dish, and the gel has been washed with [...]. Denaturation was then performed by soaking the gel in a solution of NaCl/NaOH (NaCl 1.5 M > NaOH 0.5 N) by stirring during 15°minutes twice and the gel has been washed with [...]_{2 0}. Neutralization has finally been performed by soaking the gel in a solution of NaCl/Tris-HCl (NaCl 1.5 M ' Tris-HCl > pH 7.4 IM) by stirring during 15°minutes twice. A nylon membrane the same size as the gel has been prepared and a corner of the nylon membrane has been cut to locate the direction. The DNA has been transferred from the gel to the nylon membrane and immobilized on the nylon membrane by exposure to UV light. The nylon membrane obtained can be used for hybridization and 1' immunoassay (Roche). The primers presented in the list below have been designed for the preparation of probes used for Northern blotting,

[...] gene

[...] 5 'TGA ACA GCA AGC CAT AGG GG 3' (SEQ ID NO:49)

[...] 5 'GCA GAA GAC GGC GCC ATT AT 3' (SEQ ID NO:50)

A length of the probe^{0 :} 293 base pairs

[...] gene

Furthermore, the polyketide synthases DHADs were successfully expressed in Streptomyces and E. coli and a low level of yield of polyketides has been [...]. Generally, the lack of post-translational modifications in E. coli prevents the heterologous expression of the functional polyketide synthase. Therefore, it is reasonable to the co-expression of PKS with a 4'-phosphopantetheine transferase ( [...] ) to produce a domain holo-ACP (acyl carrier protein) PKS for the production of [...] (and [...] al., 2001). For further investigating the effect of expression of the expression vector of the invention, the gene sfp ([...] ) of Bacillus subtilis has been successfully cloned into the expression vector [...] -lb (Figure 6) and co-express with PKS, the gene [...] (Figure 5), in E. coli. The result of the SDS-PAGE analysis showed a low level of efficiency of the soluble protein PKS (342°kDa) (Figure 7). However, the majority of the soluble proteins were [...] resulting in protein expression 29°kDa.

1. Construction of expression plasmids

The partial cDNA fragment (6.5 ° kb cDNA) with the forward primer p3 °: 5 '- [...] -3' (SEQ ID NO:49) and the reverse primer p4 °: 5 '- [...] -3' (SEQ ID NO °:

50) [...] gene has been amplified from the first strand of Monascus cDNA by RT-PCR amplification. The cDNA fragment 6.5 ° kb has been introduced into the vector pGEM-T to obtain the fragment EcoRI -Not I by a reaction of restriction. Then the fragment was ligated with [...] to give [...]. A set of oligonucleotide primers with the forward primer pl °: 5 ' [...] -3' (SEQ ID NO:51) contains 23 bases complementary to the gene [...] 5 'and introduces a restriction site EcoRI and the reverse primer p2 °: 5' [...] -3' (SEQ ID NO:52) was designed to amplify the partial cDNA of 3.0 ° kb by RT-PCR amplification. The cDNA fragment EcoRI-EcoRI of 3.0 ° kb has been ligated with [...] to give [...] (Figure 6).

A set of oligonucleotide primers with the forward primer 5 '- [...] -3' (SEQ ID NO:53) contained 20 bases complementary to the gene sfp 5 'and introduces a BamHI restriction site and the reverse primer 5' [...] -3 '(SEQ ID NO:54) contained 20 bases complementary to the gene sfp 5' and introduces a restriction site [...] was designed to amplify the sfp gene from the genomic DNA of said Bacillus subtilis 168. Sfp The gene was ligated with BamHI- [...][...] -lb

to give [...] (Figure 7).

2. Co-expression of [...] and sfp in E. coli

[...][...] The and both have been co-transformed into E. coli BL21 (DE3) lb and a medium has been inoculated with a single colony to be cultured to the morning after. Lb Then the medium has been transferred in a medium ATCC 765 enriched with glycerol and 10% of the culture was continued until an optical density at 600 nm (D0600) of 0.6 to 0.8. The expression of the gene has been induced by the addition of [...] -thiogalactoside (IPTG) to 1.0 mm (final concentration) and the culture was incubated for 48 hours at 20 °C. The cell pellet was collected and frozen in liquid nitrogen then thawed at 42 °C to lyse cells. To facilitate lysis, lysozyme is added to the cell pellet. Expression of proteins was monitored by SDS-PAGE (7.5% gels) of total cellular proteins and soluble proteins, followed by a colloidal by coomassie blue staining.

1. Y. Abe, Suzuki t., Ono C., [...] K, [...] M, Yoshikawa H. Molecular cloning and characterization of an ml-236B

(compactin) gene cluster [...] in Penicillium citrinum.

2002, Mol. Genet. Genomics. 267: 636-646.

2. Bedford D. J., Schweizer [...]., D. A. Hopwood, Khosla C.:

Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3 (2), 1995, J. Microbiol.

177, 4544-4548.

3. Endo A.: [...] Κ, new [...] agent has produced by a Monascus species. J [...] (Tokyo) 1979, August;

32 (8): 852-4.

4. Endo A., D. Komagata, [...] Η.: [...] Μ ., a new inhibitor of cholesterol biosynthesis. J [...] (Tokyo), Dec 1986 ; 39 (12): 1670-3.

5. Endo A, [...] K, Negishi, S.: [...] J and L, new inhibitors of cholesterol biosynthesis by Monascus ruber produced. J [...] (Tokyo), March 1985 ; 38 (3): 420-2.

6. Endo A, [...] Κ, Nakamura Τ, [...] Μ, Masuda Μ.

[...][...] L and X, new metabolites which inhibit cholesterol biosynthesis. J [...] (Tokyo), March 1985 ; 38 (3): 321-7.

7. Gokhale, R. S., Tsuji, S. Y., Cane, D. E., Khosla, C. [...] communication and exploiting [...] in polyketide synthases. 1999. Science. 284, 482-485.

8. Hendrickson L, Davis CR, Roach C, Nguyen DK, Aldrich Τ, & Reeves [...] PC CD.: Lovastatin biosynthesis in Aspergillus terreus: [...] characterization of mutants, enzyme activities and a [...] polyketide synthase gene. 1999, Chem.

Biol. 6: 429-439.

9. Hutchinson C. R., Kennedy J., Park C., [...] S., Auclair Κ.

vederas and J.: Aspects of the biosynthesis of non-aromatic by iterative fungal polyketide synthases. Antonie van Leeuwenhoek 2000,78: 287-295.

10. Kennedy J., Auclair Κ ., S. g. [...], Park C., J. C. Vederas, Hutchinson C. R.: Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis. Science 1999, May 21 ; 284 (5418): 1368-72.

11. D. Komagata, [...] Η ., [...] S., A. Endo: Biosynthesis of [...]: conversion of [...] J L to [...] by a monooxygenase of Monascus ruber. J. [...] (Tokyo), March 1989;

42 (3): 407-12.

12. McDaniel R., A. [...], Gustafsson C., Fu Η ., [...] Μ ., Ashley g.: Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel ' [...]' natural products. Proc Natl Acad Sci USA 1999,96:

1846-1851.

13. [...] H. D., R. [...], [...] Μ. Α.: 4' [...] transfer in primary and secondary metabolism of [...] subtilis. 2001. J. Biol. Chem. 40,37289-37298.

14. Nicholson TP, Rudd BA, Dawson Μ, Lazarus Cm, Simpson TJ, Cox RJ. Design and utility of oligonucleotide probes gene for fungal polyketide synthases. Chem Biol 2001, Feb; 8 (2): 157-78.

15. D. 'Hagan Biosynthesis of fatty acid and polyketide metabolites. Nat Prod Rep. 2001,12:1-32.

16. Pfeifer B. A., S. J. [...], Gramajo Η ., D. E. Cane, C. Khosla Biosynthesis of complex polyketides in a [...][...] of [...]. coli strain. 2001. Science. 291, 1790-1792.