紫红曲霉PKS基因的克隆分析及MpLigD4基因缺失株的构建
摘要
本研究主要分为两部分,紫红曲霉(Monascus purpureus)中PKS基因的克隆与分析；及紫红曲霉MpLigD4基因缺失株的构建。
第一部分,本研究使用引物LC1/LC2c得到紫红曲霉中的NR-PKS的基因片段；使用引物LC3/LC5c和KAF2/KAR1得到紫红曲霉中的PR-PKS的基因片段。本研究还创建出一套扩增PKS编码基因的方法,利用此方法,根据ER的氨基酸保守序列设计简并引物,成功获得紫红曲霉中一条ER编码基因的全长核苷酸序列。在此基础上,对该ER基因进行了序列分析,发现该基因在进化上的特异性。这为进一步研究PKS提供了有效的方法,对聚酮类化合物合成的研究具有重要意义。
第二部分,使用简并引物得到目的基因片段,通过基因走读的方法得到全长及一定的上下游序列。本研究采用常规双交换的方式缺失MpLigD4基因关键区域(600 bp-1500bp),缺失掉该基因的从上游300 bp到该基因序列上的1700 bp长约2000 bp的序列片段。但是由于其同源重组效率太低,且假阳性太多,所以改进双交换的方法,在打靶质粒同源区的一侧插入oliC31基因。该基因对三乙基锡高度敏感,三乙基锡的使用浓度为10nM,通过改进后的双交换法构建了MpLigD4基因缺失的紫红曲霉菌株。本研究为进一步研究MpLigD4基因功能提供了基础,为今后的基因功能研究提供了基因操作平台。
There are two parts of work in this research. Firstly, Clone and analysis the Monascus purpureus PKS genes. Secondly, obtain MpLigD4 disruptant mutant of M. purpureus.
Part one, amplified NR-PKS gene fragmant by LC1/LC2c primer pairs and the PR-PKS by LC3/LC5c, KAF2/KAR1 primer pairs. Based on the conserved amino acid sequence codes for ER domain of HR-PKS, a partial ER nucleic acid fragment MpER1 about 300 bp was successfully cloned from M.purpureus by nested PCR, using designed degenerate primers. The analysis of the sequence showed that PKS genes have specific evolution, which provided an effective method to investigate the PKS genes.
Part two, partial MpLigD4 nucleic acid fragment was successfully cloned from Monascus purpureus by using designed degenerate primers, and the intact ORF codes for MpLigD4 domian was acquired by Genome walking. Generation of MpLigD4 deletion mutants with the key sequence of MpLigD4 which located 600 bp between 1500 bp replaced by hph gene. Construction of gene-targeting vectors of MpLigD4 which carrying 2-kbp homologous. The hph gene was used as a positive selection marker for transformants, and the oliC31 gene was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants. oliC31 codes for an triethyltin-hypersensitive form of subunit 9 of the mitochondrial ATP synthase complex. The concentration of triethyltin is 10 nM. Providing MpLigD4-deficient strains as excellent tools for gene targeting in M. purpureus.
第一部分,本研究使用引物LC1/LC2c得到紫红曲霉中的NR-PKS的基因片段；使用引物LC3/LC5c和KAF2/KAR1得到紫红曲霉中的PR-PKS的基因片段。本研究还创建出一套扩增PKS编码基因的方法,利用此方法,根据ER的氨基酸保守序列设计简并引物,成功获得紫红曲霉中一条ER编码基因的全长核苷酸序列。在此基础上,对该ER基因进行了序列分析,发现该基因在进化上的特异性。这为进一步研究PKS提供了有效的方法,对聚酮类化合物合成的研究具有重要意义。
第二部分,使用简并引物得到目的基因片段,通过基因走读的方法得到全长及一定的上下游序列。本研究采用常规双交换的方式缺失MpLigD4基因关键区域(600 bp-1500bp),缺失掉该基因的从上游300 bp到该基因序列上的1700 bp长约2000 bp的序列片段。但是由于其同源重组效率太低,且假阳性太多,所以改进双交换的方法,在打靶质粒同源区的一侧插入oliC31基因。该基因对三乙基锡高度敏感,三乙基锡的使用浓度为10nM,通过改进后的双交换法构建了MpLigD4基因缺失的紫红曲霉菌株。本研究为进一步研究MpLigD4基因功能提供了基础,为今后的基因功能研究提供了基因操作平台。
There are two parts of work in this research. Firstly, Clone and analysis the Monascus purpureus PKS genes. Secondly, obtain MpLigD4 disruptant mutant of M. purpureus.
Part one, amplified NR-PKS gene fragmant by LC1/LC2c primer pairs and the PR-PKS by LC3/LC5c, KAF2/KAR1 primer pairs. Based on the conserved amino acid sequence codes for ER domain of HR-PKS, a partial ER nucleic acid fragment MpER1 about 300 bp was successfully cloned from M.purpureus by nested PCR, using designed degenerate primers. The analysis of the sequence showed that PKS genes have specific evolution, which provided an effective method to investigate the PKS genes.
Part two, partial MpLigD4 nucleic acid fragment was successfully cloned from Monascus purpureus by using designed degenerate primers, and the intact ORF codes for MpLigD4 domian was acquired by Genome walking. Generation of MpLigD4 deletion mutants with the key sequence of MpLigD4 which located 600 bp between 1500 bp replaced by hph gene. Construction of gene-targeting vectors of MpLigD4 which carrying 2-kbp homologous. The hph gene was used as a positive selection marker for transformants, and the oliC31 gene was employed as a negative selection marker to facilitate elimination of non-homologous recombinants among the transformants. oliC31 codes for an triethyltin-hypersensitive form of subunit 9 of the mitochondrial ATP synthase complex. The concentration of triethyltin is 10 nM. Providing MpLigD4-deficient strains as excellent tools for gene targeting in M. purpureus.
引文
[1]Dufosse L, Galaup P, Yaron A, Arad SM, Blanc P, Murthy KNC, Ravishankar GA. Microorganisms and microalgae as sources of pigments for food use:a scientific oddity or an industrial reality? Trends Food Sci Technol.2005,16:389-406.
[2]Kim HS. Monascus. In:Fermentation microbiology. Seoul:Hyangmunsa.1979, p:304.
[3]Juzlova P, Martinkova L, Kern V. Secondary metabolites of the fungus Monascus:a review. J Indust Microbiol.1996,16:163-170.
[4]Endo A, Hasumi K, Yamada A, Shimoda R, Hiroshi T. The synthesis of compactin (ML-236B) and monacolin K in fungi. J Antibiot.1979,32:1609-1616.
[5]Endo A, Komagata D, Shimada H. Monacolin M, a new inhibitor of cholesterol biosynthesis. J Antibiot.1986,39:1670-1673.
[6]Sabater-Vilar M, Maas RF, Fink-Gremmels J. Mutagenicity of commercial Monascus fermentation products and the role of citrinin contamination. Mutat Res.1999,444: 7-16.
[7]O'Hagan, D. The Polyketide Metabolites. Ellis Horwood:Chichester.1991.
[8]Ma J, Li Y, Ye Q, Li J, Hua Y, Ju D, Zhang D, Cooper R, Chang M. Constituents of red yeast rice, a traditional food and medicine. J Agric Food Chem.2000,48:5220-5225.
[9]翁照南,方展瑞.液态深层发酵生产红曲色素的研究.广州食品科技,1998,29：317-320.
[10]Turner WB. In fungal metabolites. Academic Press:London and New York.1971.
[11]Wong H, Koehler PE. Production of red water-soluble Monascus pigments. J Food Sci. 1983,48:1200-1203.
[12]Lin TF, Demain AL. Leucine interference in the production of water-soluble red Monascus pigments. Arch Microbiol.1994,162:114-119.
[13]Hajjaj H, Klaebe A, Loret MO, Tzedakis T, Goma G, Blanc PJ. Production and identification of N-glucosylrubropunctamine and N-glucosylmonascorubramine from Monascus ruber and the occurrence of electron donor-acceptor complexes in these red pigments. Appl Environ Microbiol.1997,63:2671-2678.
[14]宫会会,陈惠阴.红曲与红曲色素的研究进展.武汉工业大学学报,2002,1：22-23.
[15]Lin TF, Yakushijin K, Bu"chi GH, Demain AL. Formation of water-soluble Monascus red pigments by biological and semi-synthetic processes. J Ind Microbiol.1992,9: 173-179.
[16]Pastrana L, Blanc PJ, Santerre AL, Loret MO, Goma G. Production of red pigments by Monascus ruber in synthetic media with a strictly controlled nitrogen source. Process
Biochem.1994,30:333-341.
[17]Blanc PJ, Loret MO, Santerre AL, Pareilleux A, Prome D, Prome JC, Laussac JP, Goma G. Pigments of Monascus. J Food Science.1994,59:862-865.
[18]Lin TF, Demain AL. Effect of nutrition on formation of Monascus red pigments. Appl Microbiol Biotechnol.1991,36:70-75.
[19]Martinkova L, Juzlova P, Vesely D. Biological activity of polykedite pigments produced by the funngs Monascus. J Appl Biotechnol.1995,79:609-616.
[20]Sato K, Gods Y, Sakamoto SS, Shibata H, Maitani T, Yamada T. Identification of major pigments containing D-amino acid units in commercial monascus pigments. Chem Pharm Bull.1997,45:227-229.
[21]Jung H, Kim C, Kim K, Shin CS. Color characteristics of monascus pigments derived by fermentation with various amino acids. J Agric Food Chem.2003,51:1302-1306.
[22]Kim C, Jung H, Kim JH, Shin CS. Effect of monascus pigment derivatives on the electrophoretic mobility of bacteria, and the cell adsorption and antibacterial activities of pigments. Colloids Surf B Biointerfaces.2006,47:153-159.
[23]Hiroi S, Shima T, Suzuki T. Hyperpigment productive mutant of Monascus anka for solid culture. Agric Biol Chem.1979,43:1975-1976.
[24]Wong HC, Koehler PE. Mutant for Monascus pigment production. J Food Sci.1981,46: 956-957.
[25]Blanc PJ, Laussac JP, Le Bars J, Le Bars P, Loret MO. Pareilleux A. Characterization of monascidin A from Monascus as citrinin. J Food Microbiol.1995,27:201-213.
[26]Endo A. Monacolin K, a new hypocholesterolemic agent produced by Monascus species. J Antibiot.1979,32:852-854.
[27]Endo A, Hasumi K, Nakamura T, Kunishima M, Masuda M. Dihydromonacolin L and monacolin X, new metabolites that inhibit cholesterol biosynthesis. J Antibiot.1985,38: 321-327.
[28]Hajjaj H, Klaebe A, Loret MO, Goma G, Blanc PJ, Francois J. Biosynthetic pathway of citrinin in the filamentous fungus Monascus ruber as revealed by 13C nuclear magnetic resonance. Appl Environ Microbiol.1999,65:311-314.
[29]Rasheva TV, Nedeva TS, Hallet JN, Kujumdzieva AV. Characterization of a non-pigment producing Monascus purpureus mutant strain. Antonie van Leeuwenhoek.2003,83: 333-340.
[30]章克第.红曲霉及红曲色素.中国调味品.1989,2：1-5.
[31]Beale MH. Biosythesis of C5-C20 terpenoid compounds. Nat Prod Rep.1991,8:409.
[32]Endo A. Compactin(ML-236) and related compounds as potential cholesterol-lowering agent that inhibite HMG-CoA reductase. J Med Chem.1985,28:401.
[33]Endo A, Hasumi K. HMG-CoA reductase inhibiters. Nat Prod Rep.1993,10:541.
[34]高蓝,李浩明.洛伐他汀生物合成及其相关基因研究进展.药物生物技术.2005,12：201-206.
[35]Manzoni M, Rollini M. Biosythesis and biotechnological production of statins by filamentous fungi and application of these cholesterol-lowing drugs. Appl Microbiol Biotechnol.2002,58:555.
[36]Alberts AW,Chen J, Springer J. M evinolin:A highly poten t competitive inhibitor of cholesterol-lowering agent. Proc Natl Acad Sci USA.1980,77:3957.
[37]Kenndy J, Auclair K, Kendrew SG. Modulation of polyketides synthase activity by accessory proteins during lovastatin biosynthesis. Science.1999,284:1368.
[38]Sutherland A, Auclair K, Vederas JC. Recent advances in the biosynthetic studies of Lovastatin. Curr Opin Drug Discov Devel.2001,4:229.
[39]Chan JK, Moore RN, Nakashima TT. Biosynthesis of mevinol in Spectral Assignment by double-quantum coherence NMR after high carbon-13 incorporation. J Am Chem Soc. 1983,105:3334.
[40]Moore RN, Bigam G, Chan JK. Biosynthesis of the hypocholecsterolemic agent Mevinolin by Aspergillum terreus. Determination of the origin of carbon and oxygen atoms by 13C NMR and mass spectrometry. J Am Chem Soc.1985,107:3694.
[41]Hetherington AC, Raistrick H. Studies in biochemistry of microorganism XI. On the production and chemical constitution of a new yellow colouring matter, citrinin, produced from glucose by Penicillium citrinum. Thom Phil Trans R Soc London, Ser B.1931,220:269-297.
[42]Ei-Banna AA, Pitt JI, Leistner L. Production of mycotoxins by Penicillium species. Syst Appl Microbiol.1987,10:42-46.
[43]Kurata H. Mycotoxins and mycotoxicoses. In:Microbial toxins in foods and Feeds. New York, USA:Plenum Press.1990, p:249-259.
[44]Xu B, Jia X, Gu L, Sung C. Review on the qualitative and quantitative analysis of the mycotoxin citrinin. Food Control.2006,17:271-285.
[45]Chagas GM, Oliveira MBM, Campello AP, Kluppel M LW. (1995). Mechanism of citrinin-induced dysfunction of mitochondria.Ⅲ. Effects on renal cortical and liver Mitochondria swelling. J Appl Toxicol.1995,15:91-95.
[46]Deshpande SS. Handbook of Food Toxicology. New York, USA:Marcel Dekker.2002, p:424.
[47]Poupko R, Luz Z, Destro R. Carbon-13 NMR of citirnin in the solid state and in solutions. J Phys Chem A.1997,101:5097-5102.
[48]Barber J, Staunton J. New insights into polyketide metabolism; the use of protium as a
tracer in the biosynthesis of citrinin by Penicillium citrinum. J Chem Soc Perkin Trans.1980,1980:2244-2248.
[49]Sankawa U, Ebizuka Y, Noguchi H, Isikawa Y, Kitaghawa S, Yamamoto Y, Kobayashi T, Iitak Y. Biosynthesis of citrinin in Aspergillus terreus. Tetrahedron.1983,39: 3583-3591.
[50]Shimizu T, Kinoshita H, Ishihara S, Sakai K, Nagai S, Nihira T. (2005) Polyketide synthase gene responsible for citrinin biosynthesis in Monascus purpureus. Appl Environ Microbiol.2005,71:3453-3457.
[51]Kroken S, Glass NL, Taylor JW, Yoder OC, Turgeon BG. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycete. Proc Natl Acad Sci USA.2003,100:15670-15675.
[52]Shen B. Biosynthesis of aromatic polyketides. Top Curr Chem.2000,209:1-51.
[53]Shen B. Polyketide biosynthesis beyond the type Ⅰ, Ⅱ and Ⅲ polyketide synthase paradigms.Curr Opin Chem Biol.2003,7:285-295.
[54]Staunton J, Weissman KJ. Polyketide biosynthesis:a millenium review. Nat Prod Rep. 2001,18:380-416.
[55]Schumann J, Hertweck C. Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes. J Biotechnol.2006,124:690-703.
[56]Hutchinson CR, Kennedy J. Aspects of the biosynthesis of non-aromatic fungal polyketides by iterative polyketide synthases. Antonie van Leeuwenhoek.2000,78: 287-295.
[57]Fujii I, Watanabe A. Identification of a Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans. Chem Biol.2001,8: 189-197.
[58]Cox RJ. Polyketides, proteins and genes in fungi:programmed nano-machines begin to reveal their secrets. Org Biomol Chem.2007,5:2010-2026.
[59]Nicholson TP, Rudd PAM. Design and utlity of oligonucleotide gene probes for fungal polyketide synthases. Chem. Biol.2001,8:157-178.
[60]Fujii I, Watanabe A. Structures and functional analyses of fungal polyketide synthase genes. Actinomycetologica.1998,12:1-14.
[61]Rawlings BJ. Biosynthesis of polyketides (other than actinomycete macrolides). Nat Prod Rep.1999,16:425-484.
[62]Muller R. Don't classify polyketide synthases. Chem Biol.2004,11:4-6.
[63]Beck J, Ripka S. The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases. J Biochem. 1990,192:487-498.
[64]Hendrickson L, Davis CR. Lovastatin biosynthesis in Aspergillus terreus: characterization of blocked mutants, enzyme activities and a multifunctional polyketide synthase gene. Chem Biol.1999,6:429-439.
[65]Graziani S, Vasnier C. Novel polyketide synthase from Nectria haemotococca. Appl Environ Microbiol.2004,70:2984-2988.
[66]Yang G, Rose MS. A polyketide synthase is required for fungal virulence and production of the polyketide T-toxins. Plant Cell.1996,8:2139-2150.
[67]Keller NP, Brown D. A conserved polyketide mycotoxin gene cluster in Aspergillus nidulans. In:Molecular Approaches to Food Safety Issues Involving Toxic Microorganisms. Alaken Inc:Fort Collins.1995:p263-277.
[68]Bingle LEH, Simpson TJ. Ketosynthase domain probes identify two subclasses of fungal polyketide synthase genes. Fungal Genet Biol.1999,26:209-223.
[69]Partida-Martinez LP, Hertweck C. Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature.2005,437:884-888.
[70]Case ME, Schweizer M. Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci USA.1979,76:2563-5259.
[71]Ballance DJ, Buxton FP. Transformation of Aspergillus nidulans by the orotidine-5-phosphate decarboxylase gene of Neurospora crassa. Biochem Biophys Res Commun.1983,112:284-289.
[72]Brakhage AA, Langfelder K. Menacing mold:the molecular biology of Aspergillus fumigatus. Ann Rev Microbiol.2002,56:433-455.
[73]Weidner G, d'Enfert C. Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5'-monophosphate decarboxylase. Curr Genet.1998,33:378-385.
[74]de Groot MJ, Bundock P. Agrobacterium tumefaciens mediated transformation of filamentous fungi. Nat Biotechnol.1998,16:839-842.
[75]Talbot N. Molecular and Cellular Biology of Filamentous Fungi. Oxford University Press.2001.
[76]Brown JS, Aufauvre-Brown A. Insertional mutagenesis of Aspergillus fumigatus. Mol Gen Genet.1998,259:327-335.
[77]Nakayashiki H, Hanada S. RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet Biol.2005,42:275-283.
[78]Schumann J, Hertweck C. Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes. J Biotechnol.2006,124:690-703.
[79]Cox RJ, Glod F. Rapid cloning and expression of a fungal polyketide synthase gene involved in squalestatin biosynthesis. Chem Commun.2004,20:2260-2261.
[80]Krappmann S, Sasse C, Braus GH. Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous End-Joining-Deficient genetic background. Eukaryot cell.2006,5:212-215.
[81]Kanaar R, Hoeijmakers JH, van Gent DC. Molecular mechanisms of DNA double strand break repair. Trends Cell Biol.1998,8:483-489.
[82]Hua S, Qiu M, Chan E. Minimum length of sequence homology required for in vivo cloning by homologous recombination in Yeast. Plasmid.1997,38:91-96.
[83]Adachi N, So S, Koyama H. Loss of nonhomologous end joining confers camptothecin resistance in DT40 cells. Implications for the repair of topoisomerase I-mediated DNA damage. J Biol Chem.2004,279:37343-37348.
[84]Van Dyck E, Stasiak AZ, Stasiak A, West SC. Binding of double-strand breaks in DNA by human Rad52 protein. Nature.1999,398:728.
[85]Petrini JH, Bressan DA, Yao MS. Semin Immunol.1997,9:181-188.
[86]Meyer V. Genetic engineering of filamentous fungi-progress, obstacles and future trends. Biotechnol Adv.2008,26:177-185.
[87]Mizutani O, Kudo Y, Saito A, Matsuura T, Inoue H, Abe K, Gomi K. A defect of LigD (human Lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae. Fungal Genet Biol.2008,45:878-889.
[88]Ishibashi K, Suzuki K, Ando Y. Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora. Proc Natl Acad Sci.2006,103:14871.
[89]Martinkova L, Patakova-Juzlova P, Krent V, Kucerova Z, Havlicek V, Olsovsky P, Hovorka O, Rihova B, Vesely D, Ulrichova J, Prikrylova V. Biological activities of oligoketide pigments of Monascus purpureus. Food Addit Contam.1999,16:15-24.
[90]Brown M P, Brown-Jenco C S, Payne G A. Genetic and molecular analysis of aflatoxin biosynthesis. Fungal Genet Biol.1999,26:81-98.
[91]Charles L H. Metabolic engineering of polyketlde biosynthesis. Curr Opin Biotech.1996, 7:560-562.
[92]Sims J W, Fillmore J P, Warner D D, Schmidt E W. Equisetin biosynthesis in Fusarium heterosporum. Chem Commun.2005,2:186-188.
[93]Abe Y, Suzuki T, Ono C, Iwamoto K, Hosobuchi M, Yoshikawa H. Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum. Mol Gen Genomics.2002,267:636-646.
[94]Amnuaykanjanasin A, Punya J, Paungmoung P, Rungrod A, Tachaleat A, Pongpattanakitshote, S, Cheevadhanarak S, Tanticharoen M. Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC 1067. FEMS Microbiol Lett.2005,251:125-36.
[95]Sambrook J, Russell DW. Molecular Cloning:a Laboratory Manual. Cold Spring Harbor, NY, USA, Cold Spring Harbor Laboratory Press.2001.
[96]Kumar S, Tamura K, Nei M. MEGA3:Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform.2004,5:150-163.
[97]Ward M, Wilkinson B, Turner G Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene. Mol Gen Genet.1986,202:265-270.
[2]Kim HS. Monascus. In:Fermentation microbiology. Seoul:Hyangmunsa.1979, p:304.
[3]Juzlova P, Martinkova L, Kern V. Secondary metabolites of the fungus Monascus:a review. J Indust Microbiol.1996,16:163-170.
[4]Endo A, Hasumi K, Yamada A, Shimoda R, Hiroshi T. The synthesis of compactin (ML-236B) and monacolin K in fungi. J Antibiot.1979,32:1609-1616.
[5]Endo A, Komagata D, Shimada H. Monacolin M, a new inhibitor of cholesterol biosynthesis. J Antibiot.1986,39:1670-1673.
[6]Sabater-Vilar M, Maas RF, Fink-Gremmels J. Mutagenicity of commercial Monascus fermentation products and the role of citrinin contamination. Mutat Res.1999,444: 7-16.
[7]O'Hagan, D. The Polyketide Metabolites. Ellis Horwood:Chichester.1991.
[8]Ma J, Li Y, Ye Q, Li J, Hua Y, Ju D, Zhang D, Cooper R, Chang M. Constituents of red yeast rice, a traditional food and medicine. J Agric Food Chem.2000,48:5220-5225.
[9]翁照南,方展瑞.液态深层发酵生产红曲色素的研究.广州食品科技,1998,29：317-320.
[10]Turner WB. In fungal metabolites. Academic Press:London and New York.1971.
[11]Wong H, Koehler PE. Production of red water-soluble Monascus pigments. J Food Sci. 1983,48:1200-1203.
[12]Lin TF, Demain AL. Leucine interference in the production of water-soluble red Monascus pigments. Arch Microbiol.1994,162:114-119.
[13]Hajjaj H, Klaebe A, Loret MO, Tzedakis T, Goma G, Blanc PJ. Production and identification of N-glucosylrubropunctamine and N-glucosylmonascorubramine from Monascus ruber and the occurrence of electron donor-acceptor complexes in these red pigments. Appl Environ Microbiol.1997,63:2671-2678.
[14]宫会会,陈惠阴.红曲与红曲色素的研究进展.武汉工业大学学报,2002,1：22-23.
[15]Lin TF, Yakushijin K, Bu"chi GH, Demain AL. Formation of water-soluble Monascus red pigments by biological and semi-synthetic processes. J Ind Microbiol.1992,9: 173-179.
[16]Pastrana L, Blanc PJ, Santerre AL, Loret MO, Goma G. Production of red pigments by Monascus ruber in synthetic media with a strictly controlled nitrogen source. Process
Biochem.1994,30:333-341.
[17]Blanc PJ, Loret MO, Santerre AL, Pareilleux A, Prome D, Prome JC, Laussac JP, Goma G. Pigments of Monascus. J Food Science.1994,59:862-865.
[18]Lin TF, Demain AL. Effect of nutrition on formation of Monascus red pigments. Appl Microbiol Biotechnol.1991,36:70-75.
[19]Martinkova L, Juzlova P, Vesely D. Biological activity of polykedite pigments produced by the funngs Monascus. J Appl Biotechnol.1995,79:609-616.
[20]Sato K, Gods Y, Sakamoto SS, Shibata H, Maitani T, Yamada T. Identification of major pigments containing D-amino acid units in commercial monascus pigments. Chem Pharm Bull.1997,45:227-229.
[21]Jung H, Kim C, Kim K, Shin CS. Color characteristics of monascus pigments derived by fermentation with various amino acids. J Agric Food Chem.2003,51:1302-1306.
[22]Kim C, Jung H, Kim JH, Shin CS. Effect of monascus pigment derivatives on the electrophoretic mobility of bacteria, and the cell adsorption and antibacterial activities of pigments. Colloids Surf B Biointerfaces.2006,47:153-159.
[23]Hiroi S, Shima T, Suzuki T. Hyperpigment productive mutant of Monascus anka for solid culture. Agric Biol Chem.1979,43:1975-1976.
[24]Wong HC, Koehler PE. Mutant for Monascus pigment production. J Food Sci.1981,46: 956-957.
[25]Blanc PJ, Laussac JP, Le Bars J, Le Bars P, Loret MO. Pareilleux A. Characterization of monascidin A from Monascus as citrinin. J Food Microbiol.1995,27:201-213.
[26]Endo A. Monacolin K, a new hypocholesterolemic agent produced by Monascus species. J Antibiot.1979,32:852-854.
[27]Endo A, Hasumi K, Nakamura T, Kunishima M, Masuda M. Dihydromonacolin L and monacolin X, new metabolites that inhibit cholesterol biosynthesis. J Antibiot.1985,38: 321-327.
[28]Hajjaj H, Klaebe A, Loret MO, Goma G, Blanc PJ, Francois J. Biosynthetic pathway of citrinin in the filamentous fungus Monascus ruber as revealed by 13C nuclear magnetic resonance. Appl Environ Microbiol.1999,65:311-314.
[29]Rasheva TV, Nedeva TS, Hallet JN, Kujumdzieva AV. Characterization of a non-pigment producing Monascus purpureus mutant strain. Antonie van Leeuwenhoek.2003,83: 333-340.
[30]章克第.红曲霉及红曲色素.中国调味品.1989,2：1-5.
[31]Beale MH. Biosythesis of C5-C20 terpenoid compounds. Nat Prod Rep.1991,8:409.
[32]Endo A. Compactin(ML-236) and related compounds as potential cholesterol-lowering agent that inhibite HMG-CoA reductase. J Med Chem.1985,28:401.
[33]Endo A, Hasumi K. HMG-CoA reductase inhibiters. Nat Prod Rep.1993,10:541.
[34]高蓝,李浩明.洛伐他汀生物合成及其相关基因研究进展.药物生物技术.2005,12：201-206.
[35]Manzoni M, Rollini M. Biosythesis and biotechnological production of statins by filamentous fungi and application of these cholesterol-lowing drugs. Appl Microbiol Biotechnol.2002,58:555.
[36]Alberts AW,Chen J, Springer J. M evinolin:A highly poten t competitive inhibitor of cholesterol-lowering agent. Proc Natl Acad Sci USA.1980,77:3957.
[37]Kenndy J, Auclair K, Kendrew SG. Modulation of polyketides synthase activity by accessory proteins during lovastatin biosynthesis. Science.1999,284:1368.
[38]Sutherland A, Auclair K, Vederas JC. Recent advances in the biosynthetic studies of Lovastatin. Curr Opin Drug Discov Devel.2001,4:229.
[39]Chan JK, Moore RN, Nakashima TT. Biosynthesis of mevinol in Spectral Assignment by double-quantum coherence NMR after high carbon-13 incorporation. J Am Chem Soc. 1983,105:3334.
[40]Moore RN, Bigam G, Chan JK. Biosynthesis of the hypocholecsterolemic agent Mevinolin by Aspergillum terreus. Determination of the origin of carbon and oxygen atoms by 13C NMR and mass spectrometry. J Am Chem Soc.1985,107:3694.
[41]Hetherington AC, Raistrick H. Studies in biochemistry of microorganism XI. On the production and chemical constitution of a new yellow colouring matter, citrinin, produced from glucose by Penicillium citrinum. Thom Phil Trans R Soc London, Ser B.1931,220:269-297.
[42]Ei-Banna AA, Pitt JI, Leistner L. Production of mycotoxins by Penicillium species. Syst Appl Microbiol.1987,10:42-46.
[43]Kurata H. Mycotoxins and mycotoxicoses. In:Microbial toxins in foods and Feeds. New York, USA:Plenum Press.1990, p:249-259.
[44]Xu B, Jia X, Gu L, Sung C. Review on the qualitative and quantitative analysis of the mycotoxin citrinin. Food Control.2006,17:271-285.
[45]Chagas GM, Oliveira MBM, Campello AP, Kluppel M LW. (1995). Mechanism of citrinin-induced dysfunction of mitochondria.Ⅲ. Effects on renal cortical and liver Mitochondria swelling. J Appl Toxicol.1995,15:91-95.
[46]Deshpande SS. Handbook of Food Toxicology. New York, USA:Marcel Dekker.2002, p:424.
[47]Poupko R, Luz Z, Destro R. Carbon-13 NMR of citirnin in the solid state and in solutions. J Phys Chem A.1997,101:5097-5102.
[48]Barber J, Staunton J. New insights into polyketide metabolism; the use of protium as a
tracer in the biosynthesis of citrinin by Penicillium citrinum. J Chem Soc Perkin Trans.1980,1980:2244-2248.
[49]Sankawa U, Ebizuka Y, Noguchi H, Isikawa Y, Kitaghawa S, Yamamoto Y, Kobayashi T, Iitak Y. Biosynthesis of citrinin in Aspergillus terreus. Tetrahedron.1983,39: 3583-3591.
[50]Shimizu T, Kinoshita H, Ishihara S, Sakai K, Nagai S, Nihira T. (2005) Polyketide synthase gene responsible for citrinin biosynthesis in Monascus purpureus. Appl Environ Microbiol.2005,71:3453-3457.
[51]Kroken S, Glass NL, Taylor JW, Yoder OC, Turgeon BG. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycete. Proc Natl Acad Sci USA.2003,100:15670-15675.
[52]Shen B. Biosynthesis of aromatic polyketides. Top Curr Chem.2000,209:1-51.
[53]Shen B. Polyketide biosynthesis beyond the type Ⅰ, Ⅱ and Ⅲ polyketide synthase paradigms.Curr Opin Chem Biol.2003,7:285-295.
[54]Staunton J, Weissman KJ. Polyketide biosynthesis:a millenium review. Nat Prod Rep. 2001,18:380-416.
[55]Schumann J, Hertweck C. Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes. J Biotechnol.2006,124:690-703.
[56]Hutchinson CR, Kennedy J. Aspects of the biosynthesis of non-aromatic fungal polyketides by iterative polyketide synthases. Antonie van Leeuwenhoek.2000,78: 287-295.
[57]Fujii I, Watanabe A. Identification of a Claisen cyclase domain in fungal polyketide synthase WA, a naphthopyrone synthase of Aspergillus nidulans. Chem Biol.2001,8: 189-197.
[58]Cox RJ. Polyketides, proteins and genes in fungi:programmed nano-machines begin to reveal their secrets. Org Biomol Chem.2007,5:2010-2026.
[59]Nicholson TP, Rudd PAM. Design and utlity of oligonucleotide gene probes for fungal polyketide synthases. Chem. Biol.2001,8:157-178.
[60]Fujii I, Watanabe A. Structures and functional analyses of fungal polyketide synthase genes. Actinomycetologica.1998,12:1-14.
[61]Rawlings BJ. Biosynthesis of polyketides (other than actinomycete macrolides). Nat Prod Rep.1999,16:425-484.
[62]Muller R. Don't classify polyketide synthases. Chem Biol.2004,11:4-6.
[63]Beck J, Ripka S. The multifunctional 6-methylsalicylic acid synthase gene of Penicillium patulum. Its gene structure relative to that of other polyketide synthases. J Biochem. 1990,192:487-498.
[64]Hendrickson L, Davis CR. Lovastatin biosynthesis in Aspergillus terreus: characterization of blocked mutants, enzyme activities and a multifunctional polyketide synthase gene. Chem Biol.1999,6:429-439.
[65]Graziani S, Vasnier C. Novel polyketide synthase from Nectria haemotococca. Appl Environ Microbiol.2004,70:2984-2988.
[66]Yang G, Rose MS. A polyketide synthase is required for fungal virulence and production of the polyketide T-toxins. Plant Cell.1996,8:2139-2150.
[67]Keller NP, Brown D. A conserved polyketide mycotoxin gene cluster in Aspergillus nidulans. In:Molecular Approaches to Food Safety Issues Involving Toxic Microorganisms. Alaken Inc:Fort Collins.1995:p263-277.
[68]Bingle LEH, Simpson TJ. Ketosynthase domain probes identify two subclasses of fungal polyketide synthase genes. Fungal Genet Biol.1999,26:209-223.
[69]Partida-Martinez LP, Hertweck C. Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature.2005,437:884-888.
[70]Case ME, Schweizer M. Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci USA.1979,76:2563-5259.
[71]Ballance DJ, Buxton FP. Transformation of Aspergillus nidulans by the orotidine-5-phosphate decarboxylase gene of Neurospora crassa. Biochem Biophys Res Commun.1983,112:284-289.
[72]Brakhage AA, Langfelder K. Menacing mold:the molecular biology of Aspergillus fumigatus. Ann Rev Microbiol.2002,56:433-455.
[73]Weidner G, d'Enfert C. Development of a homologous transformation system for the human pathogenic fungus Aspergillus fumigatus based on the pyrG gene encoding orotidine 5'-monophosphate decarboxylase. Curr Genet.1998,33:378-385.
[74]de Groot MJ, Bundock P. Agrobacterium tumefaciens mediated transformation of filamentous fungi. Nat Biotechnol.1998,16:839-842.
[75]Talbot N. Molecular and Cellular Biology of Filamentous Fungi. Oxford University Press.2001.
[76]Brown JS, Aufauvre-Brown A. Insertional mutagenesis of Aspergillus fumigatus. Mol Gen Genet.1998,259:327-335.
[77]Nakayashiki H, Hanada S. RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet Biol.2005,42:275-283.
[78]Schumann J, Hertweck C. Advances in cloning, functional analysis and heterologous expression of fungal polyketide synthase genes. J Biotechnol.2006,124:690-703.
[79]Cox RJ, Glod F. Rapid cloning and expression of a fungal polyketide synthase gene involved in squalestatin biosynthesis. Chem Commun.2004,20:2260-2261.
[80]Krappmann S, Sasse C, Braus GH. Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous End-Joining-Deficient genetic background. Eukaryot cell.2006,5:212-215.
[81]Kanaar R, Hoeijmakers JH, van Gent DC. Molecular mechanisms of DNA double strand break repair. Trends Cell Biol.1998,8:483-489.
[82]Hua S, Qiu M, Chan E. Minimum length of sequence homology required for in vivo cloning by homologous recombination in Yeast. Plasmid.1997,38:91-96.
[83]Adachi N, So S, Koyama H. Loss of nonhomologous end joining confers camptothecin resistance in DT40 cells. Implications for the repair of topoisomerase I-mediated DNA damage. J Biol Chem.2004,279:37343-37348.
[84]Van Dyck E, Stasiak AZ, Stasiak A, West SC. Binding of double-strand breaks in DNA by human Rad52 protein. Nature.1999,398:728.
[85]Petrini JH, Bressan DA, Yao MS. Semin Immunol.1997,9:181-188.
[86]Meyer V. Genetic engineering of filamentous fungi-progress, obstacles and future trends. Biotechnol Adv.2008,26:177-185.
[87]Mizutani O, Kudo Y, Saito A, Matsuura T, Inoue H, Abe K, Gomi K. A defect of LigD (human Lig4 homolog) for nonhomologous end joining significantly improves efficiency of gene-targeting in Aspergillus oryzae. Fungal Genet Biol.2008,45:878-889.
[88]Ishibashi K, Suzuki K, Ando Y. Nonhomologous chromosomal integration of foreign DNA is completely dependent on MUS-53 (human Lig4 homolog) in Neurospora. Proc Natl Acad Sci.2006,103:14871.
[89]Martinkova L, Patakova-Juzlova P, Krent V, Kucerova Z, Havlicek V, Olsovsky P, Hovorka O, Rihova B, Vesely D, Ulrichova J, Prikrylova V. Biological activities of oligoketide pigments of Monascus purpureus. Food Addit Contam.1999,16:15-24.
[90]Brown M P, Brown-Jenco C S, Payne G A. Genetic and molecular analysis of aflatoxin biosynthesis. Fungal Genet Biol.1999,26:81-98.
[91]Charles L H. Metabolic engineering of polyketlde biosynthesis. Curr Opin Biotech.1996, 7:560-562.
[92]Sims J W, Fillmore J P, Warner D D, Schmidt E W. Equisetin biosynthesis in Fusarium heterosporum. Chem Commun.2005,2:186-188.
[93]Abe Y, Suzuki T, Ono C, Iwamoto K, Hosobuchi M, Yoshikawa H. Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum. Mol Gen Genomics.2002,267:636-646.
[94]Amnuaykanjanasin A, Punya J, Paungmoung P, Rungrod A, Tachaleat A, Pongpattanakitshote, S, Cheevadhanarak S, Tanticharoen M. Diversity of type I polyketide synthase genes in the wood-decay fungus Xylaria sp. BCC 1067. FEMS Microbiol Lett.2005,251:125-36.
[95]Sambrook J, Russell DW. Molecular Cloning:a Laboratory Manual. Cold Spring Harbor, NY, USA, Cold Spring Harbor Laboratory Press.2001.
[96]Kumar S, Tamura K, Nei M. MEGA3:Integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief Bioinform.2004,5:150-163.
[97]Ward M, Wilkinson B, Turner G Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene. Mol Gen Genet.1986,202:265-270.