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DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
Genome sequencing and analysis of theversatile cell factory
Aspergillus niger CBS 513.88
Noël van Peij
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
TheThe plupluGbGbugugTMTM conceptconcept
•Beta-lactams Penicillium chrysogenum•Biomass Saccharomyces cerevisiae•Enzymes Aspergillus niger
Kluyveromyces lactisBacillus sp.
DSM Life Sciences produces a variety offermentation products with a limited set of
microorganisms: the pluGbugs
Objective:
Production with a limited number of optimized strainsin standardized processes
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
Strategic choice forStrategic choice for GenomicsGenomics
Processimprovement
Classical strainimprovement
rDNA strainmodification
ORFs
Functionalgenomics
•Strengthen portfolio new products•Rationalize strain & process optimization
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
MoreMore thenthen ½½ decade ofdecade of DSMDSM AspergillusAspergillus ““omicsomics””
1999 : start A. niger sequencing project (CBS 513.88)
2002 : sequencing of EST libraries - 20,000 EST clones
2003 : Affymetrix GeneChips for A. niger
2002-2007 : Functional Genomics
- Transcriptomics
- Proteomics
- Bio-Informatics
2005 : Decision to publish proprietary A. niger genome
2007 : Release of genome sequence @ publication
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
•Secretion•Development•Proteases
A.A. nigerniger research at DSMresearch at DSM
pluG
bug
DNA
mRNA
metabolites
regulation
prot
ein
•A. niger a versatile cell factory•Safety
•Genome-scalemetabolic model
•Enzymes•Proteomics
•Transcriptomics
•Annotation•Sequence analysis
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
A. niger genomeA. niger genome characteristicscharacteristics
• Sequenced genome (Mb): 33.9• Coding (%) 55• Predicted genes: 14,165• Gene density (kb): 2.4• < Gene length > : 1,572• < CDS length > : 1,320 (440 aa)• Genes with introns (%): 87.3• < Exons / gene > : 3.6• < Exon length > : 370• < Intron length > : 97
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
A glimpse: Genome annotationA glimpse: Genome annotation
• Annotation of 14.165 predicted ORF’s• 50% (7092) functionally classified (FunCat)• Metabolism, Cellular transport, Protein fate are large…
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
GenomeGenome…… phylogeneticphylogenetic analysisanalysis…… ..
• Phylogenetic relationshipbased on 20 panorthologousgenes(housekeeping functions)
• Max likelihood tree
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
A.A. nigerniger scaffoldscaffold organisationorganisation && synthenysyntheny
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
DNA coding is biasedDNA coding is biased
• The 4-letter DNA code codes for the 20natural amino acids by 61 codons
• Coding is biased for organisms
• Also within organism, bias exists(homologous genes); namely highlyexpressed genes (Affymetrix data, 2-DPX data) show extreme codon preference
• Info can be used to make genescompatible with highly expressedgroup in host organism
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
Versatility:Versatility: ExtracellularExtracellular proteomeproteome
Total lane digestion
LC-MS/MS analysis
Cell wall-degrading enzyme expression by A. niger
substrate A
strain 1
strain 2
substrate B
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
CarbohydrateCarbohydrate--active enzymes (CAZY)active enzymes (CAZY)
Glycosyl Hydrolases A.nig A.nid A.fum A.oryTotal 171 186 186 217Extracell 131 134 124 159Intracell 40 52 62 58GH28 21 9 12 18GH13 18 13 15 17
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
ProteasesProteases inin A.nigerA.niger
Proteolysis (total) about 198 genes 100%
Cytosolic, nuclear degradation 84 42%
Proteasome 36 18%
Lysosomal degradation 14 7%
Vacuolar degradation 20 10%
Other proteolytic proteins 44 22%
An example: Brewers Clarex™
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
TransportersTransporters
Secondary transporters
A.nig A.nid A.fum A.ory N.cra S.cerATPases 94 73 71 100Ion channels 44 24 36 272nd Transport 718 572 490 793
MFS 296 216 188 176 89 53Total 865 673 601 924
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
Zn(II)Zn(II)22CysCys66 TFTF’’ss inin A.A. nigerniger
Fungal Zn(2)-Cys(6) binuclear cluster domain
A.nig A.nid A.fum A.ory N.cra S.cer296 216 188 176 89 53
An example: prtT protease regulatorProtease regulator
prtT
N-sourcepH
C-source
Protease regulator
prtT
Genes encoding proteases
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
Fungal PrtTFungal PrtT’’ss
Zn(II) 2Cys6 DNA binding domain:CX2CX6CX8CX2CX6C
•PrtT present in:
A. terreus, A. fumigatus, A. flavus,
A. oryzae, A. niger, P. chrysogenum
•PrtT not present in other fungi:
A. nidulans, M. grisea, T. reesei
C. neoformans, F. graminearum,
A.oryzae MTRT-TVEPIKYEAP-SWEHKSVHVSDDHRRIIPNVGDDATRPKGRIRRSMTACNTCRKLA.niger MTR--TVDEIKYETPSSWEHKSLDVAEDGRRLAPHS--DTARPKGRIRRSMTACHTCRKLA.fumigatus MTRTTSVEDVKFEIP-AWDNSNVDVADGSGRPESSTSGDTIRPKGRIRRSMTACNTCRKL *** :*: :*:* * :*::..:.*::. * . *: *************:*****
A.oryzae KTRCDLDPRGHACRRCLSLRIDCQLPETSERFQDSTPMWSDATTAIPSIEERLTSLERSMA.niger KTRCDLDPRGHACRRCLSLRIDCKLPETTDRFQDSAAMWPDATSAIPSIEERLTSLERCMA.fumigatus KTRCDLDPRGHACRRCLSLRIECKLPETAERFQDNASMWSDATAAIPSIEERLISLERSM *********************:*:****::****.:.**.***:********* ****.*
A.oryzae REMTGMLRQILNQSPSVSNISVPPLARSVHTEETASIEGNSFGPFLPKPVRLIQDLQSEFA.niger REMTGMMRQMLDHSPGFANASVPHLTKSIITDENASMEGSPSSPFLPKPVRLIQDLQSDFA.fumigatus TEMTSMMRRMMDRSPSISGSSVSMLTRSGITDETASIEGSQSSSFAPRPIRLFQDLQSDF ***.*:*:::::**..:. **. *::* *:*.**:**. ..* *:*:**:*****:*
A.oryzae FGETNRIPVESPFLGNSFEKGILDSKLSLKLVQLFVDNFGPLVSINNQSDFHNEMRNTDSA.niger FGEAETSPVDSPLSSDGNAKGAIDSKLSLKLLQTFVDHFGACVSIYNLSDIHNDMKAPDSA.fumigatus TGEANVLPADSRSLGDLFTKGIIDPKLSQKLIQLFVDHFGIWISVDNPSDIHNELRATDP **:: *.:* .: ** :*.*** **:* ***:** :*: * **:**::: .*.
A.oryzae LLYSTACLLASRYVPGIPPPIVHTMNLQVRHKAVNLLWEEPPLKYESLQALALLCLWPAAA.niger LLYNTACLLASRYVPGIPTSTVHAIYLQVRHAVVNILWEKPPLKYETLQALALLCLWPATA.fumigatus LLYSTACLLASRYVPGIPLSVIHAMYLQIRHATVNVLWNKTPLKHETLQALALLALWPTA ***.************** . :*:: **:** .**:**::.***:*:*******.***::
A.oryzae GQKEFPIDGWLLSGTAINHALVSFDFLNHVPSELLIDNDIAAQLRLWNAFCLTQLHFAVGA.niger AQKEPPMDSWLLSGISINHAIIALDFLNYAPSEVMVDNETAAQLRLWNTYCLTQLHFAVGA.fumigatus VQKETPMDSWLLSGISINHAIISFDFLNHAPSDLIVDNDMVAKLRVWNALCLTQLQSAIG *** *:*.***** :****::::****:.**::::**: .*:**:**: *****: *:*
A.oryzae NARPFHLPQRYLDYCPRLLEHPAATVEDGKVVAEIQLYLITLRLQANEQRMRFAEVEYEEA.niger NARPFHIQQRYLDHCPRILEHPAATLEDARVVAEIQLYLMTLRLQSNSSRMRLADLDYEEA.fumigatus NARPFHIQQRYLEHCPRLLEHPAATFEDGKIVAEIQLYLIALKLQNFSHRMRLGDFEYEE ******: ****::***:*******.**.::********::*:** . ***:.:.:***
A.oryzae IERWKVEWAHLLAGDENSTFELSLWFCQILLHRTAMRFQAESERLTSEILQGSRLIISKFA.niger IERWKREWAHLFSG-ESSTLELSLWFCQTLLHRTAMRLQPRSDRLASEVLQTSRLIISRFA.fumigatus IERWKMEWAHLLTGEQHSTLELSLWYCQLLLYRTAMRFHWESEHLISEILRNSRLILSKF ***** *****::* : **:*****:** **:*****:: .*::* **:*: ****:*:*
A.oryzae LQLRFVTALRVVDQAYFIVGYAALNLCDFNFLDPLIDQIQMFLLHLSPNEDHIAYRFSCMA.niger LQIRYSTALSLVDQVYFIVGYAALNLCDFNLMDPLIEQVQMFLLHLSPNEDHIAYRFSCMA.fumigatus LLVRFPNALAFPDQIYYIVGYAALNLCDFSPMDPLIDQVQTFLLHLSPNEDHIAYRFSYT * :*: .** . ** *:************. :****:*:* *****************
A.oryzae IAEFKRRC--AECNDPCSAVDGSQCS-FGDARKMSME-QVQFVPPLVDSMIGGYSALEQLA.niger VAEFKRRCGSAECNDPSSTVKGSPLSSYGDSRKMSMG-QAPFMPPLMDGMIEGYG-FEQLA.fumigatus ITELKRRC--ATGPNPHNVVKGA----FGDTRKLSMGQQIPFMNPLMDTMMGEYGGLEHL ::*:**** * :* ..*.*: :**:**:** * *: **:* *: *. :*:*
A.oryzae IPEVMPHSFPESVISGMAVTEAIPVGSAPYA.niger MPEVMPSSFPDGILNGMPVTGLAAYRSATLA.fumigatus IPEVPPNSLPDMLTSVAGELQAFRTAIL--
:*** * *:*: : .
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
Protein secretion in A.Protein secretion in A. nigerniger
• Secretion pathways compared
Yeast-like26S proteasome
Mammalian-likeGlycoprotein glucosyltransferaseP58 (No PERK)
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
Genome scale metabolic networkGenome scale metabolic network
C, H, N, O, S...
BiomassEnzymes
Citric Acid
Nucleus
Endoplasmic reticulum
Golgicomplex
Transportvesicles
Cytosol
Mitochondrion
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
•High degree ofredundancy in centralcarbon metabolism
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
Conclusions A. niger genome… ..ConclusionsConclusions A.A. nigerniger genomegenome…… ....
• Releasing A. niger sequence will speed up researchand genome annotations
• Genomic diversity reflects saprophytic lifestyle andpotential new products
• Genome enrichment confirming the (metabolic &nutritional) versatile A. niger cell factory?
• Efficient combination of available “Omics”tools leads tonew and improved strains and processes for protein andmetabolite production
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
People involved from DSMPeople involved from DSM
Proteomics Food SpecialtiesMarijke MissetSindy GetrouwMarga HerweijerDenise JacobsMaurien OlsthoornMichiel AkeroydSophie PorretOlaf SchoutenRob van der HoevenMarcel van Tilborg
Proteomics Nutritional ProductsIsabelle MailletThomas Lapointe
Omics AspergillusLucie Pa enicováCees SagtNoël van PeijRogier MeulenbergStefaan BreestraatHilly MenkeSerge DonkersHans RoubosAb van OoijenHerman PelHein Stam
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
DSM Life Science ProductsAspergillus systematics in the genomic era –An international workshop– April 12-14, 2007
Thank you !!
Thank you!Thank you!