Genome sequencing and analysis of the versatile cell factory

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


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


Strategic choice forStrategic choice for GenomicsGenomics

Processimprovement

Classical strainimprovement

rDNA strainmodification

ORFs

Functionalgenomics

•Strengthen portfolio new products•Rationalize strain & process optimization


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


•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


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


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…


GenomeGenome…… phylogeneticphylogenetic analysisanalysis…… ..

• Phylogenetic relationshipbased on 20 panorthologousgenes(housekeeping functions)

• Max likelihood tree


A.A. nigerniger scaffoldscaffold organisationorganisation && synthenysyntheny


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


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


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


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™


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


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


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--

:*** * *:*: : .


Protein secretion in A.Protein secretion in A. nigerniger

• Secretion pathways compared

Yeast-like26S proteasome

Mammalian-likeGlycoprotein glucosyltransferaseP58 (No PERK)


Genome scale metabolic networkGenome scale metabolic network

C, H, N, O, S...

BiomassEnzymes

Citric Acid

Nucleus

Endoplasmic reticulum

Golgicomplex

Transportvesicles

Cytosol

Mitochondrion


•High degree ofredundancy in centralcarbon metabolism


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


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



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Genome sequencing and analysis of the versatile cell factory