The document summarizes research on the Monascus genome project and related genetic studies. Key points include: 1) The Monascus genome was sequenced to 8x coverage, revealing various genomic features. A Monacolin K biosynthesis gene cluster was identified containing genes for polyketide synthases and other enzymes. 2) An efficient method for genetic transformation of Monascus pilosus was developed using aurintricarboxylic acid. 3) Disruption of the mokA gene and overexpression of the mokH transcription factor gene resulted in loss of and increased Monacolin K production, respectively.

1. 紅麴菌基因體計畫及基因研究 Bioresource Collection and Research Center Food Industry Research and Development Institute Dec. 16, 2008 The Monascus genome project and the genes study

2. The Monascus genome project and the genes study I. Background II. The Monascus genome project III. Genetic transformation of Monascus IV. Study on the Monacolin K biosynthesis gene cluster V. Exploring the distribution of citrinin biosynthesis related genes among Monascus spp. VI. MRT, a new non-LTR retrotransposon in Monascus spp. VII. Conclusion

3. History Review Fermentation history of Monascus Previous Study Monascus strains collected Monascus mutants generated Monacolin K, GABA, Pigments, Citrinin Liquid/solid fermentation technique I. Background

4. cDNA library fosmid / BAC library EST database fosmid/BAC end sequence data whole genome 8x coverage 2x coverage shotgun library 10x coverage draft finishing candidate fosmid/BAC clone fosmid/BAC shotgun library whole genomic data functional genomic data contract out Unigene database cDNA sequence data II. The Monascus genome project

5. BCRC 38072 BCRC 38072 is a Monascus strain isolated from anka Produce monacolin K High production Mutant strain BCRC 38093 BCRC 38072 identified as Monascus pilosus * Type strain DNA hybridization II. The Monascus genome project

6. EST Analytic Platform II. The Monascus genome project cDNA Sequence EST DB Phred basecall and qualification EST EST EST EST EST Blastn Tentative Unique Genes Hit No Hit Singleton Contig Collapse the Contig Cap3 Assembly update update Functional Analysis Blastx

7.  coverage  qualified reads  average read length  genome size Sequencing Facts BAC Library mpb01-02 BCRC/FIRDI Fosmid Library mpf01 BCRC/FIRDI Plasmid Library mpg01-12, 31-34, Yang Ming University Plasmid Library mpg61-64 Vita Genomics II. The Monascus genome project 0.16x 10,451 12,048 14,506 7,253 80-100 kb mpb01-02 0.41x 22,384 24,066 27,086 13,543 30-40 kb mpf01 795,897 135,028 11,785 612,970 Qualified reads 673,853 127,250 10,663 503,105 Input reads 13.06x 860,225 451,796 Total 2.21x 175,689 79,872 8.0-10.0 kb mpg61-64 0.22x 11,959 6,624 6.0-7.0 kb mpg33-34 2.5-5.0 kb Insert Size 630,985 Reads 10.06x 344,504 mpg01-12, mpg31-32 Coverage  Clones Library Name

8. Monascus Genome Draft II. The Monascus genome project

9. Genome Features II. The Monascus genome project ** novel means no similarity to known/hypothetical proteins * by alignment of EST to genome

10. III. Genetic transformation of Monascus Heat shock protein Phosphoenolpyruvate carboxykinase Vector construction-with strong Monascus promoter

11. 帶螢光之轉形株 III. Genetic transformation of Monascus Efficiencies of Monascus Transformation 44 (colonies/ μg DNA) Electroporation 10 (colonies/μg DNA) PEG 200 (colonies/10 7 spore) Agrobacteria Frequency Method

12. Restriction-enzyme-mediated integration (REMI) & Aurintricarboxylic acid (ATA) (Cell Mol. Life Sci., 2001) ATA III. Genetic transformation of Monascus Circular vector with selectable marker Restriction enzyme linearized Enzymes digest cell wall cell membrane Nuclear membrane Genomic DNA

13. a. The plasmid pMS-1.5hp was linearized by Fsp I restriction enzyme. b. The Fsp I-linearized pMS-1.5hp was mixed with 1 mM ATA and protoplasts. c. The Fsp I-linearized pMS-1.5hp was mixed with 10 units Fsp I restriction enzyme and protoplasts. d. The Hpa I-linearized pMS-1.5hp was mixed with 10 units Hpa I restriction enzyme and protoplasts. e. Number of transformants per 5 ug linear plasmid. The values were based on three determinations. Efficient transformation using aurintricarboxylic acid (ATA) III. Genetic transformation of Monascus 119 ± 11 48 ± 4 159 ± 24 17 ± 2 M. pilosus e Hpa I (10 units) d Fsp I (10 units) c REMI Treated by ATA (1mM) b Linear plasmid (non-REMI) a Transformation methods Experiment

14. Wild type pMS-1.5hp Transformants Southern hybridization analysis using Hin dIII in the genomes of eight transformants hybridized with hph probe Enhanced green fluorescent protein (EGFP) in transformed M. pilosus H: hyphae C: conidia Confirmation of M. pilosus transformants by Southern blot and fluorescent microscopy III. Genetic transformation of Monascus

15. Conclusions The valid and convenient gene transformation in M. pilosus was developed using aurintricarboxylic acid (ATA). The method can be applied in various fungi for efficient genetic transformation. Chen, Y. –P, Chen, I.-C., Hwang, I.-E., Yuan, G.-F., Liaw, L.-L., Tseng, C.-P. Selection of an effective red-pigment producing Monascus pilosus by efficient transformation with aurintricarboxylic acid. 2008. Biosci. Biotechnol. Biochem. (Accepted) III. Genetic transformation of Monascus

16. BAC library Screen BAC library containing putative monacolin K biosynthesis gene cluster Shotgun Sequence of complete putative monacolin K biosynthesis genes for 10 X coverage Annotation of the putative monacolin K biosynthesis gene cluster sequences by computer (Blast and Vector NTI software) Design specific probes to Monascus pilosus IV. Study on the Monacolin K biosynthesis gene cluster

17. Annotation of BAC DNA mps01 predicted by BLAST and VectorNTI ORF 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18 mokB mokI mokH mokF mokD mokA mokC mokE mokG 19 20 21 159958 bp mokA mokC mokD mokE mokF mokG mokH mokI mokB similar to Adapter-related protein complex 1 beta 1 subunit 1 NADP(H)-dependent alcohol dehydrogenase 2 late sexual development protein 3 predicted protein 4, 9 Non-LTR retrotransposon, MRT 5, 10 DNA primase small subunit 6 SUAPRGA1 7 exonuclease 8 Hypothetical protein 11, 12, 14 actin patches distal 13 ABNA-arabinanase 15 Fum12p(hydroxylase ) 16 Acetotactate synthase 17 related to GABA transport protein 18 Polyketide synthase (nonaketide) P450 monooxygenase Oxidoreductase Dehydrogenase Transesterase HMG-CoA reductase Transcription factor Efflux pump Polyketide synthase (diketide) Hypothetical protein 19 sugar transporter-like protein 20 Crh-like protein 21 IV. Study on the Monacolin K biosynthesis gene cluster

18. Monascus pilosus (Putative Monacolin K Biosynthesis Gene Cluster) Penicillium citrinum (compactin biosynthesis gene cluster) (Mol. Genet. Genomics. 2002. Abe et al.) Aspergillus terreus (lovastatin biosynthesis gene cluster) (Science. 1999. Kennedy et al.) P450 monooxygenase ( mokC ) Polyketide synthase ( mokB ) Oxidoreductase ( mokD ) Dehydrogenase ( mokE ) HMG-CoA reductase ( mokG ) Transesterase ( mokF ) Transcription factor ( mokH ) Efflux pump ( mokI ) Polyketide synthase ( mokA ) IV. Study on the Monacolin K biosynthesis gene cluster

19. cDNA sequences analysis of monacolin K gene cluster 5’ GU· · ·AG 3’ (2) 113 99 2 56.9 3156 mokG (HMG-CoA reductase) 5’ GU· · ·AG 3’ (1) 5’ GC· · ·AG 3’ (1) 60.6 71 2 57.3 1575 mokC (P450 monooxygenase) 55.8 57.9 60.5 63.9 63.1 55.1 56.9 GC content(%) 5’ GU· · ·AG 3’ (7) 5’ GU· · ·AG 3’ (1) 5’ GU· · ·AG 3’ (2) 5’ GU· · ·AG 3’ (2) 0 5’ GU· · ·AG 3’ (6) 5’ GU· · ·AG 3’ (7) Splice site of introns (number) 57.5 73 7 1632 mokI (Efflux pump) 49.4 96 1 1368 mokH (Transcription factor) 46.8 91 2 1242 mokF (Transesterase) 38.9 72 2 1083 mokE (Dehydrogenase) 28.9 0 0 792 mokD (Oxidoreductase) 276 72 6 7644 mokB (Polyketide synthase) 338 67 7 9228 mokA (Polyketide synthase) molecular weight (kDa) Ave. size of intron (bp) Number of intron Full-length cDNA (bp) IV. Study on the Monacolin K biosynthesis gene cluster

20. mokA -Polyketide synthase gene (nonaketide) KS AT DH MeT KR ACP 2kb 3kb Plasmid map of pMkAko for targeted gene disruption of mokA IV. Study on the Monacolin K biosynthesis gene cluster

21. 1: wild type 2. disrupted-strain Disruption of mokA gene in M. pilosus N: Nde I, F: Fsp I Mok gene cluster Mok gene cluster pMkAko vector N N N mokA mokC HPH EGFP N N F F 7.1 kb 3.8 kb probe N N N mokC HPH EGFP N mokA IV. Study on the Monacolin K biosynthesis gene cluster

22. Monacolin K analysis of wild-type and disruptant by HPLC Monacolin K Wild type disruptant IV. Study on the Monacolin K biosynthesis gene cluster

23. Transcription factor mokH gene Zn(II)2Cys6 binuclear Cys -X 2 - Cys –X 6 - Cys -X 11 - Cys -X 2 - Cys X 6 - Cys Overexpression: lovE in A. terreus  7~10 fold lovastatin mlcR in P. citrinum  10~15% compactin 1 100 C C C C C C mlcR lovE mkH Zn Zn 26 29 36 48 51 58 N C X 2 X 6 X 11 X 2 X 6 IV. Study on the Monacolin K biosynthesis gene cluster

24. rRNA pMSmokH 8774 bp probe gpd promoter mokH EGFP HPH hsp promoter S X X X S ptrpR pgpdF pmHF pmHR PCR Northern blot mokH gene trpC terminator transformants transformants Wild-type Wild-type pMSmokH Confirmation of transformants containing extra copy mokH 4 3 2 1 5 4 3 2 1 M kb 4 3 2 1 IV. Study on the Monacolin K biosynthesis gene cluster

25. Real-time RT-PCR analysis of relative expression of transformant T-mokH1 to M. pilosus BCRC38072 IV. Study on the Monacolin K biosynthesis gene cluster

26. Time (days) Monacolin K (mg/ mL) Time (days) Dry cell mass (g/50 mL) Monacolin K analysis within transformant T-mokH1 and wild-type M. pilosus 1.7-fold monacolin K higher than wild-type 0 0.05 0.1 0.15 0.2 3 5 7 9 11 13 15 38072 T-mokH1 0 2 4 6 8 3 5 7 9 11 13 15 38072 T-mokH1 IV. Study on the Monacolin K biosynthesis gene cluster

27. Conclusions: The monacolin K biosynthesis gene cluster was found within a 42-kb region in the mps01 clone, designated as mokA - mokI . The mokA -disrupted strain did not produce monacolin K, indicating that mokA was responsible for monacolin K biosynthesis. The transformant containing two copies of the mokH gene-encoded transcription factor shows higher production of monacolin K than wild type. Chen, Y. –P, Tseng, C.-P., Liaw, L.-L., Wang, C.-L., Chen, I.-C., Wu, W.-J., Wu, M.-D., Yuan, G.-F. Cloning and characterization of monacolin K biosynthetic gene cluster from Monascus pilosus . J. Agric. Food Chem. 2008. 56, 5639-5646. IV. Study on the Monacolin K biosynthesis gene cluster

28. 1kb orf1 ctnA orf3 orf4 orf5 pksCT H H H H H H H H H H P P P F1 R1 F2 R2 F3 R3 F4 R4 R5 F5 R6 F6 F7 R7 F8 R8 F9 R9 F10 R10 F11 R11 Citrinin gene cluster in M. purpureus (Appl. Environ. Microbiol. 2005, 2007) H: Hin dIII V. Exploring the distribution of citrinin biosynthesis related genes among Monascus spp. orf5 : transporter pksCT : polyketide synthase orf4 : oxidoreductase orf3 : oxygenase ctnA : transcription factor orf1 : dehydrogenase

29. 3 kb 2 kb 6 kb 6 kb (A) M. pilosus M. ruber M. purpureus M. kaoliang M. sanguineus pksCT : polyketide synthase ctnA : transcription factor orf3 : oxygenase (C) (B) V. Exploring the distribution of citrinin biosynthesis related genes among Monascus spp. 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

30. Phylogeny of PKS from Monascus and various organisms pksCT based on AT domain V. Exploring the distribution of citrinin biosynthesis related genes among Monascus spp.

31. a.a. DNA a.a. DNA M. purpureus BCRC33325 M. sanguineus BCRC33446 Sequence analyses of the pksCT KS domain V. Exploring the distribution of citrinin biosynthesis related genes among Monascus spp. S S S C A T D L T L S S S C P T D L G C A C A G T T C A C A G T C T A C C T A C T G C T C G T A C G G C A C A G T T C A A G T C T A C C T A C T A C T C G T A C C L

32. V. Exploring the distribution of citrinin biosynthesis related genes among Monascus spp.

33. Phylogeny of Monascus spp. based on the β-tubulin gene V. Exploring the distribution of citrinin biosynthesis related genes among Monascus spp. M. pilosus BCRC38072 M. ruber BCRC33323 M. ruber BCRC31534 M. barkeri BCRC33309 M. ruber BCRC31535 M. ruber BCRC33314 M. ruber BCRC31523 M. pilosus BCRC31502 M. ruber BCRC31533 M. sanguineus BCRC33446 M. purpureus BCRC31541 M. purpureus BCRC31542 M. kaoliang BCRC31506 M. purpureus BCRC31615 M. purpureus BCRC33325 M. pallens BCRC33641 M. floridanus BCRC33310 M. lunisporas BCRC33640 A. fumigatus A. parasiticus A. flavus 100 99 99 95 99 67 100 100 0.01 + ctnA + orf3 + pksCT + mokA + mokE

34. Conclusions PCR and Southern blot clearly indicate that the citrinin gene cluster in M. purpureus and M. kaoliang carry out citrinin biosynthesis. A citrinin producing phenotype was detected only in M. purpureus and M. kaoliang . According to the phylogenetic subgroups established with the β-tubulin gene, the citrinin genes can group the species of Monascus . Chen Y.-P., Tseng C.-P., Chien I.-L., Wang W.-Y., Liaw L.-L., Yuan G.-F. Exploring the distribution of citrinin biosynthesis related genes among Monascus species. 2008. J. Agric. Food Chem. (Accepted) V. Exploring the distribution of citrinin biosynthesis related genes among Monascus spp.

35. Annotation of BAC DNA mps01 predicted by BLAST and VectorNTI 159958 bp mokA mokC mokD mokE mokF mokG mokH mokI mokB ORF 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17 18 mokB mokI mokH mokF mokD mokA mokC mokE mokG 19 20 21 similar to Adapter-related protein complex 1 beta 1 subunit 1 NADP(H)-dependent alcohol dehydrogenase 2 late sexual development protein 3 predicted protein 4, 9 Non-LTR retrotransposon, MRT 5, 10 DNA primase small subunit 6 SUAPRGA1 7 exonuclease 8 Hypothetical protein 11, 12, 14 actin patches distal 13 ABNA-arabinanase 15 Fum12p(hydroxylase ) 16 Acetotactate synthase 17 related to GABA transport protein 18 Polyketide synthase (nonaketide) P450 monooxygenase Oxidoreductase Dehydrogenase Transesterase HMG-CoA reductase Transcription factor Efflux pump Polyketide synthase (diketide) Hypothetical protein 19 sugar transporter-like protein 20 Crh-like protein 21 VI. MRT, a new non-LTR retrotransposon in Monascus spp.

36. Structure of the non-LTR retrotransposon, MRT APE: apurinic/apyrimidinic endonuclease domain CYS: cysteine –rich region CCHC: Cys-His region RT: reverse transcriptase RNH: RNaseH VI. MRT, a new non-LTR retrotransposon in Monascus spp.

37. Phylogeny of non-LTR retrotransposons based on RT domain VI. MRT, a new non-LTR retrotransposon in Monascus spp.

38. Monascus pilosus Monascus ruber Monascus sanguineus Monascus barkeri Monascus pilosus Monascus ruber Monascus sanguineus Monascus barkeri Southern analysis of MRT within Monascus spp. Eco RI Bam HI Monascus purpureus Monascus purpureus BCRC31503 BCRC31503 VI. MRT, a new non-LTR retrotransposon in Monascus spp.

39. + MRT — MRT M. pilosus BCRC38072 M. ruber BCRC31523 M. ruber BCRC33314 M. ruber BCRC33323 M. barkeri BCRC33309 M. pilosus BCRC31502 M. ruber BCRC31533 M. ruber BCRC31534 M. ruber BCRC31535 M. sanguineus BCRC33446 M. purpureus BCRC31615 M. purpureus BCRC33325 M. pilosus BCRC31503 M. purpureus BCRC31542 M. kaoliang BCRC31506 M. purpureus BCRC31541 M. pallens BCRC33641 M. lunisporas BCRC33640 M. floridanus BCRC33310 P. digitatum A. fumigatus A. flavus A. parasiticus 100 99 95 97 94 100 67 100 100 0.01 Phylogeny of Monascus spp. based on the β -tubulin gene VI. MRT, a new non-LTR retrotransposon in Monascus spp.

40. Conclusions A new non-LTR retrotransposon, named MRT, was discovered. 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 partial β-tubulin gene Chen, Y.-P., Tseng, C.-P., Liaw, L.-L., Wang, C.-L., Yuan, G.-F. Characterization of MRT, a new non-LTR retrotransposon in Monascus spp. Bot. Bull. Acad. Sinica. 2007. 48, 377-385. VI. MRT, a new non-LTR retrotransposon in Monascus spp.

41. Acknowledgement 基因體醫學國家型計畫 National Research Program for Genomic Medicine 基因庫及遺傳資源的收集保存研發與應用 紅麴菌基因體計畫 Collection, Preservation, Researches and application on Gene Bank and Genetic Resources The Monascus Genome project 行政院科發基金 National commission for science and technology development fund 經濟部技術處科專計畫 The Department of Industrial Technology of the Ministry of Economic Affairs

42. 第 頁 BEC / ITRI Bioinformatic Proteomic Libraries and Sequencing Functional Genomics Mycology and Fermentation TEAMS

43. 六、紅麴菌之 MRT, non-LTR retrotransposon