Identification and characterization of starch and inulin modifying network of Aspergillus niger by functional genomics

Identification and characterization of starch and inulin modifying network of Aspergillus niger by functional genomics

Yuan, X.L.

Citation

Yuan, X. L. (2008, January 23). Identification and characterization of starch and inulin modifying network of Aspergillus niger by functional genomics.

Institute of Biology Leiden (IBL), Group of Molecular Microbiology, Faculty of Science, Leiden University. Retrieved from

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Summary









Aspergillusniger is an ubiquitous filamentous fungus and commonly present on decaying

plant debris. As a saprophytic fungus, A.nigerproduces a variety of hydrolytic enzymes

thatareabletobreakdowntheplantpolysaccharidesintosmallermoleculestoserveasits

nutrient and energy sources.  Many enzymes secreted by A. niger have potential

applicationsinthebaking,starch,textile,foodandfeedindustries.Thishasattractedalot

researchattentiontoexploreA.nigerforbetterunderstandingthecarbohydratemodifying

networkinA.niger,inordertoimprovetheenzymeproductionandsubstrateutilization.In

addition, the discovery of the novel enzyme activities produced by A.niger is a subject of

greatinterest.BoththediscoveryofnewenzymesintheA.niger,aswellasagenomewide

analysis of transcriptional networks related to degradation of carbohydrates has become

possible through recent  sequencing of A.niger genome by DSM and DOE Joint Genome

Institute (Pel et al., 2007, Baker et al., 2007)and the availability of Affymetrix microarrays

(Peletal.,2007).

Inthisthesis,weareinterestedinthemolecularmechanismsbywhichtheA.niger

can sense thepresence ofdifferent (often complex) carbon sources inits vicinity andhow

the presence of a certain carbohydrate activates the expression of a network of genes

encodingenzymestodegradethecarbonsource.Specialfocusisontheidentificationofso

called pathway inducing molecules and transcription factors that are required for

expressionofenzymenetworksinvolvedinthebreakdownanduptakeofstarchandinulin,

and their derivatives. In addition, attention is also paid to how the expressed enzymes

encodedbystructuralgenesreacttothecarbonsourcesandtodeterminetheirphysiological

rolesinA.niger.

Chapter 1 outlines the importance of A. niger as an enzyme producer and the current

knowledge on enzymes involved in the starch and inulin degradation or modification in

Aspergilli. The expression and production of enzymes involved in carbohydrate

degradation is highly regulated and controlled by various widedomain transcription

factors (CreA, AreA, PacC), and pathway specific transcriptional activators (AmyR and

InuR).ThisChaptergivesthereaderanoverviewandmorebackgroundinformationabout

thevarioustranscriptionfactorsthatareinvolvedintheregulationofgeneexpression.The

regulation mechanisms of genes encoding the enzymes involved in polysaccharides

modification or degradation and the important features of the different transcriptional

factorsinvolvedintheregulationaredescribed.

Summary

Toidentifynovelenzymesactingonstarchandinulin,twoapproacheshavebeenfollowed.

ThefirstapproachisdescribedinChapter2andisbasedontheconstructionofstarchand

inulin specific cDNA expression libraries using Gateway cloning technology. Expression

librariesforscreeningbothinbacteria(E.coli)andyeast(S.cerevisiae)wereconstructedand

characterized in detail. The libraries have been transformed to E.coli and S.cerevisae, and

transformants have been screened. Due to problems in both the expression level of the

proteins encoded by the cDNAs and the viability of the transformants only a limited

numberoftransformantshavebeenscreened.Wepresentseveralsuggestionstoimprove

themethodsforconstructionandscreeningofthecDNAexpressionlibraries.

The second approach to identify new enzymatic activities is by database mining

using functionally characterized enzymes related to starch or inulin metabolism. As

described inChapter 3, starch can be degraded by the concerted action of amylase,

glucoamylase and glucosidase enzymes, members of the glycoside hydrolase (GH)

families 13, 15 and 31, respectively. Thus, a full set of GH13, 15 and 31 family members

were recognized in A.nigergenome and 17 new members were identified.  Combination

analysis of the genome sequence of A. niger CBS 513.88 with transcriptome expression

profiles reveals a large number of these genes with unexpected transcriptional regulation:

theywereneitherinducedbystarchdegradationpathwayinducer,maltose,nordependent

on the presence of starch utilization activator, AmyR. Only two of the newly identified

enzymes, a putative glucosidase (AgdB) and an amylase (AmyC), were predicted to

play a role in starch degradation. The possible physiological functions of other predicted

familyGH13,GH15andGH31enzymes,includingpredictedintracellularenzymesandcell

wallassociatedproteins,inalternativeglucanmodifyingprocessesarediscussed.

Among the newly identified GH13 family enzymes, three members (AgtA, AgtB

and AgtC), sharing high similarity to fungal amylases were predicted to be

glycosylphosphatidylinositolanchored and lacked some highly conserved amino acids of

the amylase family.Chapter 4 describes the functional characterization of the purified

proteins (AgtA and AgtB) from A. niger overexpression strains, and the phenotytic

characterization of gene deletion (agtA) and gene overexpression (agtA and agtB) strains.

Both AgtA and AgtB displayed a unique type of (1, 4) glucanotransferase (EC 2.4.1.25)

activity.DeletionofagtAinA.nigerresultedinanincreasedsusceptibilitytowardsthecell

wall disrupting compound Calcofluor White, indicating a cell wall integrity defect in this

strain.Interestingly,homologuesofAgtAandAgtBarealsopresentinotherfungalspecies

with glucans in their cell walls, but not in yeast species lacking cell wall glucan.

Therefore the possible role for these enzymes in the synthesis and/or maintenance of the

fungalcellwallissuggestedanddiscussedinthischapter.

InChapter5,wesurveyA.nigergenomeforthepresenceofGH32familyenzymes,which

arethoughttobeinvolvedinutilizationofinulin,anditssyntheticsubstrate,sucrose.Two

new intracellular proteins SucB and SucC were identified, in addition to three known

extracellularinulinolyticenzymes,SucA(invertase),InuE(exoinulinase)andInuA(endo

inulinase).Transcriptionalanalysisrevealedthattheextracellularenzymesarecoordinately

regulatedandinducedbysucroseandinulin.Theirtranscriptionwasalsoundercontrolof

thecarboncataboliterepressorCreA.Furtheranalysisindicatedthatsucrose,orasucrose

derivedintermediate,butnotfructose,actsasaninducerfortheexpressionofinulinolytic

genesinA.niger.

InChapter 6, we examine if SucB, one of the two novel intracellular invertases,

plays an essential role in generating pathway inducing molecule involved in inulin or

sucrose catabolism. Therefore the phylogenetic, molecular and biochemical characteristics

ofSucBwerestudied.TheoverexpressedandpurifiedSucBproteinwasshowntoactasan

invertase with transfructosylating activity. Disruption of sucB in A.nigerdid not result in

alterationofgrowthininulinorsucroseindicatingSucBdoesnotplayanessentialrolein

inulin or sucrose catabolism in A. niger. However, SucB may be needed for intracellular

conversionofsucrosetofructose,glucose,andsmalloligosaccharides.

AsshowninChapter5,theextracellularinulinolyticgeneswerecoregulatedand

inducedbysucroseandinulin,suggestingacommontranscriptionalactivatormightexistto

activate the expression of the structural genes in the presence of inducing molecules. In

Chapter 7 we describe the identification and characterization of the Zn(II)2Cys6 type,

inulinolytictranscriptionactivatorInuR.TheidentificationofinuRwasbasedoninspection

of inulinolytic gene clusters in A. niger genome. inuR gene is clustered with sucB, which

encodes an intracellular invertase with transfructosylation activity (Chapter 6) and a

putative sugar transporter encoding gene (An15g00310). Deletion of the inuR gene

displayedaseveredefectivegrowthoninulinandsucroseassolecarbonsource.Induction

of the extracellular inulinolytic genes and the sugar transporter gene An15g00310 in the

presenceofsucroseandinulinwasdependentonInuR.Genomewideexpressionanalysis

revealed three additional putative sugar transporter encoding genes (An15g04060,

An15g03940 and An17g01710), which were strongly induced by sucrose in an InuR

dependent way. The specificity of these putative sugar transporters, especially the

An15g00310 encoded sugar transporter is an interesting topic and is now under the

investigationbybiochemicalanalysis.InsilicoanalysisofthestronglyInuRregulatedgenes

revealed a putative binding site for InuR consisting of two CGG triplets separated by 8

nucleotides, which is very similar to the AmyR binding site CGGN8(C/A)GG. Analysis of

thedoublemutant(amyRinuR)revealedtheirindependentregulatingfunction.Howthe

InuR and AmyR recognize their target genes and what determines the specificity of the

bindingisaninterestingtopicforfurtherresearch.

AmongtheinulinolyticgenespresentinA.niger,weidentifiedtheinuEgeneasthe

most strongly induced gene in the presence of the pathway inducing poly and

disaccharides inulin and sucrose, respectively. InChapter 8, using inuE promoter region

combinedwithracAG12VorGFPreportergenes,wepresentanovelscreeningmethodfor

the isolation of mutants involved in inulin or sucrose signalling. Such a genetic screen

followed by identification of the mutants would allow the identification of proteins that

generate inducing molecules, transport or sense the inducing molecules as well as the

proteins that activate the pathway transcription factor InuR involved in the inulin

catabolism. This method can be generally used for identification of transcription factor

Summary

mutants. In addition, during the setup of screening, we found that raffinose is able to

inducetheexpressionofinuEgene.

Finally, based on last four chapters, Chapter 5, 6, 7 and 8, a speculative working

modelofinulinsignallingpathwaywaspresentedattheendofChapter8.