20081216_04陳福生_紅麴菌分子生物學研究進展

1. College of Food Science and Technology 紅曲菌分子生物學研究進展 Review of Molecular Biology of Monascus spp. Professor Fusheng Chen 陳福生 博士 教授 HAZHONG AGRICULTURAL UNIVERSITY Wuhan, Hubei Province, R. P. China 華中農業大學食品科技學院 武漢 湖北

2. 3. Our Researches on Monascus spp. 2. Review of Molecular Biology of Monascus spp. 1. Brief Introduction of Huazhong Agricultural University MAIN CONTENTS

3. 1 Brief Introduction of HZAU and CFST 1.1 Brief Introduction of HZAU Huazhong Agricultural University (HZAU) has a long history of more than one hundred years. Its predecessor, Huazhong Agricultural College before 1985, was derived from Hubei Farming School established in 1898.

4. HZAU covering an area of 4.95 square kilometers, is located in Wuhan city, the capital of Hubei Province, in the center of China, sitting at the confluence of the Han River and the Yangtze River, the world's third longest river.

5. By its beautiful surroundings e nclosed by fascinating waters on three sides with emerald tree-covered Lion Hill, the campus serves as an ideal place for education and scientific researches.

13. HZAU boasts a highly qualified teaching contingent of 1,400 teachers, of which 260 are full professors, 360 full associate professors. Moreover, embodied in the teachers' rank are academicians, one is the Chinese Academy of Sciences, four are the Chinese Academy of Engineering. In general, HZAU ranks at the 2nd or 3rd position among about 100 agricultural and forest universities in China.

14. College of Food Science & Technology (CFST) of HZAU came into existence in 1984. In 1985, CFST began to recruit the undergraduate students of the food science specialty. Now, CFST has 3 departments.They are The Department of Food Chemistry and Molecular Biology, The Department of Food Safety and Microbiology , and The Department of Food Science and Engineering. CFST also has 2 centers. One is The Central Lab and another is The Center of Experiment and Practice for Undergraduate Students . 1.2 Brief Introduction of CFST

15. CFST holds one key lab of The Ministry of Agriculture, two engineering technology centers of Hubei Province, 2 research laboratories approved by The Ministry of Agriculture. There are also 1 first-rank subject doctoral authorization station including 6 doctor specialties and 7 master specialties, 1 post-doctor circulating station, 2 undergraduate specialties. There are 70 faculties, 1400 full-time students including about 450 graduate students. In general, CFST ranks at the 5th or 6th position in China.

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17. College of Food Science and Technology 2 Review of Molecular Biology of Monascus spp. 2.1 Molecular markers of Monascus spp. 2.2 Genetic transformation of Monascus spp. 2.3 Clone of some genes from Monascus spp.

18. 2.1 Molecular markers of Monascus spp. Random amplification polymorphism DNA(RAPD ) D1/D2 region of the large subunit (LSU) rRNA Internal transcription spacer (ITS ) Intergenic spacers (IGS) Inter-simple sequence repeat (ISSR ) Sequence related amplification polymorphism (SRAP ) All above molecular markers were applied as alternative tools to classify Monascus spp.. MRT elements with the partialβ-tubulin gene

19. 2.1.1 Random amplification polymorphism DNA(RAPD ) A collection of 25 isolates of Monascus from red yeast rice and sofu was assessed with RAPD markers, the result showed that five distinct clusters were revealed based on genetic distances between and among clusters （ Lakrod etal ， 2000 ） . Lakrod et al , RAPD analysis of genetic variation within a collection of Monascus spp. isolated from red yeast rice (ang-kak) and sofu. Mycol Res, 2000,104:403-408 Fig.1 Graph of the first three dimensions from a multiple correspondence analysis of RAPD fragment occurrence in Monascus isolates from red rice and sofu. Clusters were assigned with Ward’s minimum variance method. Mean genetic distances within each cluster are indicated. The co-occurrence index (COI) is the mean proportion of 1000 resampling cycles in which the isolates in the cluster were grouped together. MCG21 is an outgroup isolate.

20. Fig. 2 Dendrogram showing five clusters and four proposed lineages of red rice and sofu M isolates. The numbers at the nodes indicate the mean proportion of times the isolates in the corresponding clusters were grouped together throughout 1000 cycles of resampling and cluster analysis.

21. Based on the sequence information of D1/D2 regions of the large subunit (LSU) rRNA genes, 65 strains of Monascus and Xeromyces ( 耐干霉菌 ) were clustered into five groups. The sequences of Aspergillus anthodesmis , Penicillium inflatum , Cephalotheca sulfurea , Albertiniella polyporicola , and Aporothielavia leptoderma were used as outgroup because they are closely related taxonomically with the genus Monascus . （ H.G. Park · S.-C. Jong ， 2003 ， ATCC) 2.1.2 D1/D2 region of the large subunit (LSU) rRNA Houng G. Park and Shung-Chang Jong, Molecular characterization of Monascus strains based on the D1/D2 regions of LSU rRNA genes, Mycoscience ,2003, 44:25–32.

22. Fig. 3 A strict consensus phylogenetic tree obtained from five parsimonious trees of all 65 strains studied. The sequences (GenBank accession no. in parentheses) of Aspergillus anthodesmis NRRL 22884 (U17916), Penicillium inflatum NRRL 5179 (AF033393), Cephalotheca sulfurea (AF096188), Albertiniella polyporicola (AF096185), and Aporothielavia leptoderma (AF096186) were used as outgroup because they are closely related taxonomically with the genus Monascus . The number at the left side of each tree node is a bootstrap value based on a heuristic search of the data with 1000 bootstrap replications; it was 95% for the tree node of M. sanguineus, M. purpureus, M. ruber, M. eremophilus , and M. pilosus . Bootstrap values less than 50% are not shown. The consistency index, retention index, rescaled consistency index, and homoplasy index for this tree were 0.811, 0.854, 0.693, and 0.189, respectively

23. 2.1.3 Internal transcription spacer (ITS) and ß-tubulin gene ITS and partial β-tubulin genes of 17 ATCC strains of Monascus species were PCR amplified and sequenced. The result showed analyses with the ITS sequences were incongruent with the analyses using the partial β-tubulin genes (Park et al , 2004, ATCC). Park et al , Phylogenetic relationships of Monascus species inferred from the ITS and the partial β-tubulin gene, Bot. Bull. Acad. Sin. 2004,45: 325-330

24. Fig.4 Most parsimonious tree postulated with the ITS sequences . Total numbers of ingroup taxa and outgroup were 24 and one (Penicillium digitatum AY373851), respectively. Out of 672 characters, 399 were constant, 131 variable characters were parsimony-uninformative, and 142 variable characters were parsimony-informative. At each node, a bootstrap value larger than 50 percent from 1000 replicates is shown. Three sequences, Aspergillus fumigatus (AY373851), A. flavus (AY521473), and A. parasiticus (AY373859), and four sequences of Monascus (AF451856, AF451855, AF451859, and AF458473) from GenBank were incorporated into the database. ATCC strain numbers or GenBank accession numbers for the sequences obtained from GenBank are specified after the species name.

25. Fig.5 Most parsimonious tree inferred from analysis with partial β-tubulin genes . Total number of ingroup taxa was 20, and total number of outgroup taxa was one ( Penicillium digitatum D78154). Out of 852 characters, 549 were constant, 119 variable characters were parsimony-uninformative, and 184 variable characters were parsimony-informative. At each node, a bootstrap value larger than 50 percent from 1000 replicates is shown. Bootstrap values less than 50 percent are not shown. Three sequences, Aspergillus flavus (M38265), A. parasiticus (L49386), and P. fumigatus (AY048754) from GenBank were incorporated into the database. ATCC strain numbers or GenBank accession numbers for the sequences obtained from GenBank are specified after the species name.

26. 2.1.4 Monascus species were grouped by MRT The species of Monascus can be grouped by the presence or absence of MRT elements in the hybridization pattern according to phylogenetic subgroups established with the partialβ-tubulin gene (Chen, et al , 2007). Chen et al , Characterization of MRT, a new non-LTR retrotransposon in Monascus spp. Botanical Studies, 2007, 48: 377-385

27. 2 .1.5 Inter-simple sequence repeat (ISSR) Strain No. Species AS 3.2666 、 AS 3.554 、 M-4 、 M-5 、 M-17 、 M-18 、 M-19 、 M-20 、 M-22 、 M-23 、 M-24 、 M-25 、 M-27 、 M-28 、 M-29 、 M-30 、 M-31 、 M-32 、 M-33 、 M-34 、 M-35 、 M-36 、 M-37 、 M-38 、 M-39 、 M-40 、 M-41 、 M-42 、 M-43 、 M-44 、 M-45 、 M-46 、 M-47 、 M-48 、 M-49 、 M-50 、 M-51 、 M-52 、 M-53 安卡红曲菌（ M. anka ） 39 OUT2011 、 OUT2012 、 CICC5016 、 M-6 、 M-8 、 M-9 、 M-10 、 M-11 、 M-21 紫色红曲菌（ M. purpureus ） 9 OUT2015 、 OUT2137 、 M-1 、 M-2 、 M-7 、 M-13 、 M-15 丛毛红曲菌 （ M. pilosus ） 7 OUT2012 、 M-12 、 M-14 、 M-16 巴克红曲菌（ M. barkeri ） 4 CICC5015 橙色红曲菌（ M. aurantiacus ） 1 CICC5017 发白红曲菌（ M. albidus ） 1 CICC5020 烟灰色红曲菌（ M. fuliginosus ） 1 AS 3.549 红色红曲菌（ M. ruber ） 1 CBS113675 新月红曲菌（ M. lunisporas ） 1 CBS109402 阿根廷红曲菌（ M. argentinensis ） 1 M-3 、 M-26 暂不确定 2

28. Lane 1 ～ 51 ： OUT2011, OUT2012, OUT2014, OUT2015, OUT2137, CICC5015, CICC5016, CICC5017, CICC5020, AS 3.2666, AS 3.549, AS 3.554, CBS113675, CBS109402, M-1~M-37 ISSR amplification results Fig.6 Amplification products generated from tested strains with primer 808 #

29. Table 1 Total bands and polymorphic bands resulting from ISSR Analysis of results of polymorphic bands of ISSR Primer Total bands Polymorphic bands Polymorphism （ % ） 808 ＃ 14 13 92.9 810 ＃ 13 12 92.3 811 ＃ 11 11 100.0 834 ＃ 21 21 100.0 835 ＃ 12 12 100.0 841 ＃ 18 17 94.4 842 ＃ 19 19 100.0 Total 108 105 97.2

30. ISSR c luster analysis Fig. 7 Dendrogram based on ISSR fingerprints at the similarity level of 55%, 2 groups genetic similarity coefficient: 0.48~1.00 at the similarity level of 70%, 4 groups Ⅰ Ⅱ Ⅲ Ⅳ

31. 2.1.6 SRAP amplification results Fig. 8 Amplification products generated from tested strains with primer me1-em2 Lane 1 ～ 51 ： OUT2011, OUT2012, OUT2014, OUT2015, OUT2137, CICC5015, CICC5016, CICC5017, CICC5020, AS 3.2666, AS 3.549, AS 3.554, CBS113675, CBS109402, M-1~M-37

32. Table2 Total bands and polymorphic bands resulting from SRAP Analysis of result of polymorphic bands of SRAP 红曲菌的 SRAP 分析 Primer Total bands Polymorphic bands Polymorphism （ % ） me1-em2 19 17 89.5 me1-em4 23 20 87.0 me1-em5 24 22 91.7 me2-em4 30 30 100.0 me3-em2 18 17 94.4 me3-em6 17 17 100.0 me4-em4 23 23 100.0 me5-em6 29 27 93.1 Total 183 173 94.5

33. SRAP c luster analysis Fig. 9 Dendrogram based on SRAP fingerprints genetic similarity coefficient: 0.46~1.00 at the similarity level of 55%, 2 groups at the similarity level of 70%, 4 groups Ⅰ Ⅱ Ⅲ Ⅳ

34. Conclusion: From the results of above ISSR and SRAP molecular markers, they shared the same results for the tested Monascus strains. Compared with the results of morphologic identification, ISSR and SRAP are good alternative methods to assist Monascus species classification.

35. 2.2 Genetic transformation of Monascus spp. Protoplast transformation Electroporation transformation Bombardment transformation Restricted-enzyme mediaed integration (REMI) Agrobacterium -mediated T-DNA transformation (ATMT)

36. 2.2.1 Protoplast transformation PEG and CaCl 2 -mediated protoplast transformation of M. aurantiacus AS3.4384 with hygromycin B as selective marker is established (Li etal , 2007, Nanchang University). Li et al , Transformation of Protoplast from Monascus aurantiacus AS3.4384, Food Science (Chinese), 2007, 28, ( 10): 317-321. Protoplast transformation of Monascus purpureus was performed, and 60% of transformants were found to be mitotically stable and retained the plasmid inserted in the chromosome after repeated sporulation cycles (Campoy etal ,2003, Spain). Campoy et al , Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium -mediated DNA transfer, Curr Genet 2003,43: 447–452.

37. A UV-induced albino strain of Monascus purpureus was subjected to electroporation in the presence of genomic DNA from a wild-type red strain of the fungus. Eight colonies expressed color after several weeks of growth (Lakrod etal , 2003 ). Lakrod et al , Expression of pigmentation genes following electroporation of albino Monascus purpureus, J Ind Microbiol Biotechnol ,2003, 30: 369–374. 2.2.2 Electroporation transformation Electroporation was used as a genetic transformation tool with hph as selection marker to Monascus purpureus (Kim etal ,2003, Korea). Kim et al , Genetic transformation of Monascus purpureus DSM1379 , Biotechnology Letters 2003, 25: 1509–1514.

38. 2.2.3 Bombardment transformation Lakrod et al , Transformation of Monascus purpureus to hygromycin B resistance with cosmid pMOcosX reduces fertility. Electronic Journal of Biotechnology, 2003, 6(2):143-147 Albino strain KB20M1 of M. purpureus was genetically transformed to hygromycin B resistance with cosmid pMOcosX, using biolistic bombardment. The result showed two of the independent transformants formed cleistothecia but ascospore formation was greatly reduced or absent (Lakrod etal , 2003 ).

39. 2.2.4 Restriction enzyme-mediated integration ( REMI ) In order to facilitate the producer of polyketide pathway , four different transformation methods (conventional transformation, electroporation based on protoplast , electroporation based on germinated conidia , and restriction enzyme-mediated integration) were tested and compared in an attempt to develop the genetic transformation system of M. spp. The result showed that REMI technique would be very beneficial to the establishment of the genetic transformation system of M. spp. (Zhou etal ,2006, Southern Yangtze University) . Zhou et al , Comparison of Different Transformation Methods for Monascus spp. HEREDITAS, 2006, 28(4) :479 ～ 485.

40. The transformants obtained by Agrobacterium -mediated DNA transfer remained fully stable (98%) after four sporulation rounds and showed bands of hybridization corresponding to integration of the plasmid in different sites of the genome (Campoy etal, 2003). Campoy et al , Stable transformants of the azaphilone pigment-producing Monascus purpureus obtained by protoplast transformation and Agrobacterium -mediated DNA transfer, Curr Genet 2003,43: 447–452. 2.2.5 Agrobacterium -mediated T-DNA transformation (ATMT) Agrobacterium tumefaciens -mediated transformation (ATMT) was successfully applied to Monascus ruber. The stability of transformants was over 98% after five generations (Yang, Yun-Jung and Inhyung Lee, 2008, Korea). Yang, Yun-Jung and Inhyung Lee Agrobactrium tumefaciens -Mediated Transformation of Monascus ruber , J. Microbiol. Biotechnol. (2008), 18(4), 754–758

41. T-DNA insertional library of Monascus ruber mediated by Agrobacterium tumefaciens was constructed, which consisted of about 10 000 transformants. (Shao etal, 2006) Shao et al, Construction of T-DNA insertional library of Monascus mediated by A. tumefaciens and characteristic analysis of the color mutants, Mycosystema, 2006 , 25, 2: 247~255 (Chinese)

42. Monascus ruber Co-IM （ 200mol/L AS ） 25℃ ， 4days A. tumefaciens ATMT Procedure in our lab

43. 22℃ ， 4-10d Co-IM (200 mol/L AS ) PDA (HygB- 100 μ L /mL, Cefotaxim-5 μ L /mL )

44. Stored at 4℃ Fig.10 Transformation process PDA(HygB-100 μ L /mL , Cefotaxim-5 μ L /mL ) PDA(HygB- 100 μ L /mL)

45. Incubation time of conidia (d) Concentration of A.tumefaciens (OD 600nm ) Concentration of AS(mmol/L) Co-Incubation temperature( ℃ ) 35 Concentration of conidia ( / mL ） 14, 17, 20 , 23 10 5 ， 10 6 ， 10 7 0.1, 0.3, 0.5 , 0.7 100, 200 , 400 30 , Co-Incubation time (d) 3 ， 4 ， 5 ， 6 Study on the transformation conditions 20, 25,

46. At the optimization conditions, a T-DNA insertion library of Monascus. ruber containing more than 10 000 transformants was established in our lab.

47. Fig.11 Transformants identification with T-DNA by PCR amplification Identification of transformants by PCR T-BORDER(R ) T-BORDER(L ) pBR322ori SacI XhoI KpnI Gus Kan(R) 35SPolyA TtrpC HYG PtrpC pTFCM 95℃, 5 min 94℃, 60 sec 55℃, 40 sec 34cycles 72℃, 70 sec 72℃, 10 min 。 M-7

48. Fig.12 Identification of the copy numbers of transformants by T-DNA insertion Southern blot analysis of transformants to show T-DNA copies

49. Colony I Some typical transformants M-7 MZ734 MZ128 MZ130 MZ791

50. Colony II MZ146 MZ515 MZ619 MZ805 MZ1263 MZ2454 M-7

51. Colony III M-7 MZ 192 MZ 189 MZ1149 MZ283 MZ1132 MZ2431 MZ1358 MZ1295

52. Colony IV MZ959 MZ733 MZ 2066 M-7

53. Cleistothecia

54. Hypha and granules on hypha surface

55. 2.3 Study on the important genes of M. spp. Recently, a polyketide synthase gene of M. purpureus, a monacolin K synthesase gene of Monascus spp. and genes of regulating genes of G-protein signal transduction were cloned.

56. In order to screening genes involved in citrinin biosynthetic pathway , two cDNA subtractive libraries of Monascus were constructed by suppression subtractive hybridization (SSH) (Lai etal , 2005, Nanchang University) Lai et al , Screening correlative genes from citrinin biosynthetic pathway in Monascus Species of by SSH, Food Science, 2005, 26(3),63-65. (Chinese) SAGE library were constructed of Monasccus ruber (Xiong etal , 2004, Nanchang University ). Xiong et al , Preparation of ditags in constructing SAGE library of Monasccus ruber, Chinese Journal of Eco-Agriculture, 2004, 12(1): 19-22. 2.3.1 cDNA subtractive and SAGE libraries

57. ( A polyketidesynthase (PKS) gene from M. purpureus were cloned , with degenerate primers designed to amplify the conserved region of a ketosynthase domain of a fungal PKS. A 13-kb genomic DNA fragment was identified that contained a full-length PKS gene ( pksCT ) of 7,838 bp with a single 56-bp intron. pksCT encodes a 2,593-amino-acid protein that contains putative domains for ketosynthase, acyltransferase, acyl carrier protein (ACP), and a rare methyltransferase (Shimizu etal , 2005). Shimizu et al , Polyketide Synthase Gene Responsible for Citrinin Biosynthesis in Monascus purpureus, Applied and Environmental Microbiology, 2005, 7: 3453–3457 2.3.2 Cloning a polyketidesynthase (PKS) gene

58. ctnA flanking pksCT was an activator gene involved in citrinin biosynthesis in Monascus purpureus (Shimizu etal , 2007). Shimizu et al , Identification and In Vivo Functional Analysis by Gene Disruption of ctnA , an Activator Gene Involved in Citrinin Biosynthesis in M. purpureus, Applied and Environmental Microbiology, 2007,7: 5097–5103 Exploring the Distribution of Citrinin Biosynthesis Related Genes among Monascus Species (Chen etal , 2008). Chen et al , Exploring the Distribution of Citrinin Biosynthesis Related Genes among M. Species, J Agric Food Chem. 2008,11, 14 Construction of a replacement vector to disrupt pksCT gene for the mycotoxin citrinin biosynthesis in M. aurantiacus and maintain food red pigment production (Fu etal ,2007, Nanchang University). Fu etal, Construction of a replacement vector to disrupt pksCT gene for the mycotoxin citrinin biosynthesis in Monascus aurantiacus and maintain food red pigment production, Asia Pac J Clin Nutr. 2007;16 Suppl 1:137-42

59. TAIL-PCR principle Some arbitrary degenerate (AD) primers 2.3.3 Clones of regulating genes of G-protein signal transduction T-DNA 左臂 T-DNA 右臂 HYG Gus PtrpC TtrpC 35sPolyA Lsp1 Lsp3 Lsp2 Rsp1 Rsp2 Rsp3 138bp 100bp

61. Similarity analysis of 13 DNA sequences amplified by TAIL-PCR No similar sequence 无 None 340 AD6 4963 # No similar sequence 无 None 806 AD6 No similar sequence 无 None 749 AD5 3257 # 548 AD2 No similar sequence 无 None 604 AD3 2606 # No similar sequence 无 None 548 AD6 无 None 495 AD2 1164 # 127 84 1205 AD4 flbA of A. fumigatus A f293 127 84 665 AD3 805 # No similar sequence 无 None 697 AD5 635 # No similar sequence 无 None 1295 AD4 533 # No similar sequence 无 None 549 AD7 No similar sequence 无 None 612 AD2 189 # Predicted function Overlapping (bp) Similarity of the functional domain (%) Analysis by bioinformatics Amplified Length (bp) Arbitrary Primers No. Strain No.

62. The full length cDNA of 805# was achieved by RACE. Analysis for conserved domain of translated amino acid

63. Distance tree of results of amino acid sequence deduced according to cDNA sequence from 805 ＃ by PSTI and PSI Blast

64. The colony morphologies of Mgkf40 and Mgkf57 Mgkf40 Mgkf57 M-7 805 FlbA-like gene of M-7 was knocked out. Mgkf40 Mgkf57

66. 3. Our Researches on Monascus spp. 3.1 Breeding and classification Studies on the Screening, Induction and Fermentation Conditions of Monascus spp. with High-Monacolin K, Hu and Chen, 2003. Application of Molecular Markers for the Identification Strains in Monascus spp., Xu and Chen,2007 .

67. 3.2 T-DNA insertial library and genes cloned by TAIL-PCR Constructon of Transformation Library of Monascus ruber mediated by Agobacterium tumefaciens and Study on Transformants’ Characters , Wang and Chen,2005 . Study on Citrinin Related Genes in Monascus spp., Ding and Chen,2006 . Preliminary Study on GABA Related Genes in Monascus spp., Zhao and Chen, 2007. Study on Characters of Some Mutants from Transformation Library of Monascus M-7 Mediated by Agrobacterium tumefaciens , Xu and Chen,2008 . Cloning of the Pigment-Producing Gene (s) with Their Functions in M onacus spp ., Shao and Chen, 2007.

68. 3.3 Study on the related genes of G-Protein signaltransduction of Monascus ruber Isolation and analysis of mrf A involved in aerial hyphae development of Monascus ruber, Shao et al, 2008 . Signal Transduction by Mga1, a Group I G Protein Alpha Subunit of Monascus ruber, Li and Chen, 2008 . 3.4 Cloning relative genes to pigments synthesis of M. ruber Study on the relative genes to pigments synthesis of Monascus ruber , Xie and Chen, 2008.

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