G-protein/cAMP signaling pathway-mediated repression of genes
encoding polysaccharide-degrading enzymes in filamentous fungi
Emi KUNITAKE
Graduate School of Bioresources, Mie University
Report: Carbon catabolite repression (CCR) of cellulase and hemicellulase genes is regulated
by cAMP signaling factors independent of the transcription factor CreA in Aspergillus
nidulans. The degree to which each factor contributes to CCR varies depending on culture
conditions (plate or submerged culture, and the type of inducer and/or repressing carbon
source added to growth media).
Research aims
Fungal cellulases and hemicellulases are important industrial enzymes for effective
biomass utilization, and there is strong demand for improved productivity. Since expressionof cellulase and hemicellulase genes in filamentous fungi is induced by sugars derived from
cellulose and hemicellulose, cellulosic biomass-derived substrates are used as carbon sources
for production of these enzymes. However, monosaccharides, the degradation products ofinducer substrates, can cause carbon catabolite repression (CCR). Since increased enzyme
productivity can induce CCR, CCR-defective strains become essential to further improve
enzyme productivity. In the model fungus Aspergillus nidulans, it has long been known thatthe transcriptional repressor CreA mediates CCR, whereas we found that CCR of cellulase
genes is mainly regulated via cAMP-dependent protein kinase (PkaA), independently ofCreA. We also identified GanB as the main G protein α subunit involved in CCR. The aim of
this study is to clarify the CCR mechanisms mediated by CreA and PkaA/G proteins.
Methods
In addition to creA, pkaA, and Gα (ganA, ganB, fadA) gene deletion strains (ΔcreA,ΔpkaA, ΔganA, ΔganB, and ΔfadA, respectively), double deletion strains ΔcreA/ΔpkaA andΔcreA/ΔganB have been constructed. Cellulase production by each deletion strain wasexamined by plate assay for plate culture and by zymography of culture supernatants forsubmerged culture. Carboxymethyl cellulose (CMC) and ball-milled cellulose (BMC) wereused as inducing substrates, respectively. Cellulase genes were subjected to
RT-qPCR-mediated transcriptional analysis using RNA extracted from mycelia grown on theinducers, cellobiose or BMC. Glucose, fructose, mannose, xylose, and 2-Deoxyglucose(2DG) were used as repressing carbon sources. Xylanase and mannanase production wereanalyzed by plate assay using AZCL-xylan and locust bean gum (LBG) as enzyme substrates,
respectively. Zymography using Azo-xylan and Azo-carob galactomannan was performed forsupernatants of cultures with xylose and LBG. For transcriptional analysis, xylose andβ-mannobiose were used to induce xylanase and mannanase gene expressions, respectively.
Results
De-repression of cellulase production by CCR was observed by plate assay inΔpkaA and ΔganB but not ΔcreA strains, suggesting that cAMP signaling is the main
mediator of CCR of cellulase genes in plate culture. To
clarify the relationship between CreA and PkaA/GanB,ΔcreA/ΔpkaA and ΔcreA/ΔganB strains were also
examined (Fig. 1). Cellulase production in the presence ofglucose was higher in ΔcreA/ΔpkaA and ΔcreA/ΔganB thanin ΔpkaA and ΔganB, indicating that CreA is partiallyinvolved in CCR independently of PkaA/GanB. In contrastwith results obtained on agar plates, in submerged cultureusing BMC as inducer and glucose as the repressing carbon

Fig. 1 Cellulase production by thedeletion strains on agar plates
containing CMC with glucose.

source, cellulase gene expression and enzymatic activity were detected in ΔcreA but not inΔpkaA and ΔganB strains. In the double deletants, both gene expression and enzyme activity
were detected earlier than in the ΔcreA strain. Transcriptional analysis using cellobiose as aninducer and 2DG as the repressing carbon source revealed nearly equal involvement of CreAand PkaA in CCR. Double deletion of creA and pkaA caused stronger de-repression,confirming the presence of cAMP-signaling
dependent and CreA-independent CCR ofcellulase genes (Fig. 2). Because othercarbon sources also repress expression of thecellulase genes, we further examined CCR
by fructose, mannose, and xylose in addition
to glucose. CCR by hexoses was partiallyabolished in ΔcreA and ΔpkaA strains, asobserved when glucose was the repressing

Fig. 2 Transcriptional analysis of eglA encoding anendoglucanase under cellobiose-induction (black)
supplemented with 2DG (dark gray), glucose (lightgray), and xylose (white). Relative expression levelsof the genes were normalized with actA

carbon source. By contrast, CCR by xylose
was completely abolished by the creA
deletion, whereas the pkaA deletion yielded very minor de-repression if any (Fig. 2). Theseobservations imply that CCR caused by hexoses is mediated by the additive actions of CreA
and PkaA, while xylose-induced CCR is mainly CreA mediated. Interestingly, higher
de-repression was obtained with ΔganB than ΔpkaA, implying that GanB, independent of

PkaA, has a role in CCR of cellulase gene expression.
Expression analyses of xylanase and mannanase genes were also performed.
Xylanase expression displayed CreA, PkaA, GanB, and FadA-dependent CCR, and thecontribution of each factor varied among xylanase genes. Involvement of CreA and cAMPsignaling factors in CCR of mannanase gene expression depended on culture conditions as
was the case for cellulase gene expression, in which both CreA and PkaA/GanB regulated
glucose-mediated CCR, while CreA was the main mediator of xylose-induced CCR.
As described above, GanB appears to regulate both PkaA-dependent and-independent CCR. To clarify the PkaA-independent CCR mechanism, we examined FbxA,
which is an F-box protein with cNMP-binding motifs. However total cellulase, xylanase, andmannanase production in the presence of glucose were unaffected by fbxA deletion (ΔfbxA)and by mutation in its cNMP binding domain (fbxAΔCNBD) on agar plates.
Conclusions
In A. nidulans, CCR of cellulase and hemicellulase is mediated by the CreA system
as well as cAMP signaling factors, such as PkaA and GanB. Each factor plays distinct roles
in CCR that are dependent on the repressing carbon source and culture conditions1). FbxA,
which has been reported to be involved in xylanase expression2), appears not to be involvedin cellulase gene expression. From a biotechnological point of view, the double knockout of
creA and pkaA exhibited the maximal cellulase gene expression, however, the significantly
impaired strain growth would limit industrial application. Further research to identify PkaA
and Gα targets is underway for deeper understanding of CCR mechanisms and forconstruction of a CCR-free strain with more robust growth for industrial use.
References
1) Kunitake, E. et. al. (2019) CreA-independent carbon catabolite repression of cellulase
genes by trimeric G-protein and protein kinase A in Aspergillus nidulans. Curr. Genet. 65:
941-952
2) Colabardini, A. C. et al. (2012) Molecular characterization of the Aspergillus nidulans
fbxA encoding an F-box protein involved in xylanase induction. Fungal Genet. Biol. 49:
130-140.