Allijn, I.E.

Arentshorst, M.

Gelber, I.B.

Patyshakuliyeva, A.

Ram, A.F.J.

Vries, R.P. de

Leiden University Scholary Publications

ORIGINAL RESEARCH PAPERImproving cellulase production by Aspergillus niger usingadaptive evolutionAleksandrina Patyshakuliyeva . Mark Arentshorst . Iris E. Allijn .Arthur F. J. Ram . Ronald P. de Vries . Isabelle Benoit GelberReceived: 12 December 2015 / Accepted: 3 February 2016 / Published online: 15 February 2016 The Author(s) 2016. This article is published with open access at Springerlink.comAbstractObjectives To evaluate the potential of adaptiveevolution as a tool in generating strains with animproved production of plant biomass degradingenzymes.Results An Aspergillus niger cellulase mutant wasobtained by adaptive evolution. Physiological proper-ties of this mutant revealed a five times highercellulose production than the parental strain. Tran-scriptomic analysis revealed that the expression ofnoxR, encoding the regulatory subunit of the NADPHoxidase complex, was reduced in the mutant comparedto the parental strain. Subsequent analysis of a noxRknockout strain showed the same phenotypic effect asobserved for the evolution mutant, confirming the roleof NoxR in cellulose degradation.Conclusions Adaptive evolution is an efficientapproach to modify a strain and activate genesinvolved in polysaccharide degradation.Keywords Aspergillus niger  Adaptive evolution Cellulose  NADPH oxidase  NoxRIntroductionCellulose is the most abundant carbohydrate in nature.It is a linear polymer of D-glucose residues linked by b-1,4-glucosidic bonds and forms major structural plantcell wall component. Due to its partially crystallinestructure, it is more recalcitrant to enzymatic degrada-tion than other plant cell wall polysaccharides. Thecellulose-degrading system in fungi includes threeclasses of hydrolytic enzymes: b-1,4-endoglucanases(EGL) and exoglucanases/cellobiohydrolases (CBH)that hydrolyze cellulose into gluco-oligosaccharideswhich are then further degraded into D-glucose by theaction of b-glucosidases (BGL) (van den Brink and deVries 2011). A fourth class of oxidative enzymes hasbeen recently described: the lytic polysaccharidemono-oxygenases (Morgenstern et al. 2014). Cellulases havewide range of applications including production ofchemicals, fuel, food, brewery and wine, animal feed,textile and laundry, and pulp and paper. However, froma commercial point of view, the cost of cellulose-degrading enzymes is a major barrier to the economicalproduction of biochemicals and second generationbiofuels. Therefore, production and optimization ofcellulases from a wide range of fungi have been studied(Lynd et al. 2002). The industrial filamentous fungus,Trichoderma reesei, has been extensively studied forA. Patyshakuliyeva  I. E. Allijn  R. P. de Vries I. B. Gelber (&)Fungal Molecular Physiology, CBS-KNAW FungalBiodiversity Centre, Utrecht University, Utrecht,The Netherlandse-mail: i.benoit@cbs.knaw.nlM. Arentshorst  A. F. J. RamMolecular Microbiology and Biotechnology, LeidenUniversity, Leiden, The Netherlands123Biotechnol Lett (2016) 38:969–974DOI 10.1007/s10529-016-2060-0the production of cellulase preparations. It produceshigh levels of endoglucanases and cellobiohydrolasesbut low levels of b-glucosidases which are essential forfull hydrolysis of cellulose (Singhania et al. 2013).Aspergillus niger is also a well-known, geneticallytractable and industrial fungus that proficientlydegrades plant cell wall polysaccharides. Its genomecontains a complete set of cellulolytic genes but A.niger grows poorly on cellulose (Coutinho et al. 2009).During growth on plant biomass A. niger secretes highBGL and low CBH and EGL levels compared to T.reesei (Hanif et al. 2004).One of the outstanding features of microorganisms istheir capability to adapt rapidly to various environmen-tal conditions. This ability has been applied to improveyeast strains for different biotechnological applicationsby conducting adaptive laboratory evolution (Bachmannet al. 2015; Kutyna et al. 2012). Aspergillus nidulanswas exposed to adaptive evolution, resulting in pheno-type changes as well as a higher fitness in the adaptedpopulation (Schoustra et al. 2009; Schoustra andPunzalan 2012). However, this evolutionary approachhas not yet been applied to generate fungal strains withenhanced enzyme production. We describe here thegeneration and characterization of a stable evolvedisolate, which showed improved cellulase production.MethodsStrains, media and growth conditionsAll cultures were performed at 30 C. The A. nigerstrains, listed in Table 1, were grown on malt extract/agar (MEA) to produce spores, which were harvestedusing ACES buffer [10 mM N-(2-acetamido)-2-aminoethanesulfonic acid (Sigma-Aldrich), 0.02 %Tween 80, pH 6.1–7.5]. For liquid cultures, 50 ml ofaspergillus minimal medium (MM) (de Vries et al.2004) containing 1 % (w/v) a-cellulose (Sigma-Aldrich) in a 250 ml flask was inoculated with 106spores ml-1 and incubated at 30 C, 250 rpm.For agar plates, 1.5 % agar was added to MMcontaining 1 % (w/v) a-cellulose or 1 % (w/v) a-cellulose and 1 % (w/v) glucose. Agar plates wereinoculated in the center with 2 ll spore suspension(106 ml-1) and incubated at 30 C.Adaptive evolution experimentA. niger N402 was inoculated with 5 ll sporesuspension at (106 ml-1) in a Petri dish containingMMwith 1 % (w/v) a-cellulose and 1.5 % (w/v) agar.We propagated this lineage by serial transfers every5 days for 24 weeks. Each generation, the completespore plate was harvested, the spore suspension mixedextensively and 5 ll was plated on fresh medium. Twoevolving lineages were conducted in parallel. Thefitness of the colonies gradually increased until a goodgrowth on a-cellulose was observed. Several colonieswere purified and the colony showing the best growthon a-cellulose was selected for further studies. Theresulting improved strain was deposited at the CBScollection, referred as CBS 140717.RNA isolation and microarray analysisLiquid cultures of A. niger N402 and CBS 140717were pre-grown for 16 h in MM with 2 % (w/v)fructose after which mycelial samples were trans-ferred to MM with 25 mM xylose and incubated for2 h. The mycelium was harvested and used for RNAisolation. The concentration of RNA was measured atA260, while the quality was analyzed with an Agilent2100 bioanalyzer, using an RNA6000 LabChip kit(Agilent Technology, Palo Alto, CA, USA). Microar-ray analysis was performed as previously described(Levin et al. 2007).Table 1 List of A. niger strainsStrain Genotype Description ReferenceN402 cspA1 Parental strain (Bos et al. 1988)CBS 140717 cspA1 Cellulose adaptive evolved strain This studyMA78.6 DkusA::amdS Reference DnoxR (ref_ DnoxR) (Carvalho et al. 2010)MA75.2 DkusA::amdS; DnoxR::AopyrG DnoxR (Kwon et al. 2011)MA 82.2 DkusA::amdS; DnoxA::AopyrG DnoxA (Kwon et al. 2011)970 Biotechnol Lett (2016) 38:969–974123Enzyme assays and protein profilesCulture samples (1.5 ml) from 1 % a-cellulose shake-cultures were taken each day for 10 days andcentrifuged for 10 min, at *10,0009g, 4 C toseparate the solid fraction from the supernatant. Thefiltrates from duplicate flask cultures were assayedusing p-nitrophenol-linked substrates (4-nitrophenylb-D-xylopyranoside, 4-nitrophenyl b-D-cellobioside,4-nitrophenyl b-D-glucopyranoside; Sigma-Aldrich).The assays contained in 100 ll, 5-40 ll sample, 10 ll0.01 % p-nitrophenol-linked substrates, 0-35 ll Mili-Q water and 50 mM sodium acetate buffer (pH 5.0).Samples were incubated in microtiter plates for 2 h at30 C. Reactions were stopped by addition of 100 ll0.25 M Na2CO3. Absorbance was measured at405 nm in a microtiter plate reader. The activitieswere calculated using a standard curve ranging from 0to 20 nM p-nitrophenol per assay volume.Culture filtrates, 20 ll, taken at day 10 of cultiva-tion were loaded onto 12 % (w/v) SDS-PAGE gels.The gel was silver stained. Total protein concentrationwas measured by BCA method (Pierce BCA ProteinAssay Kit, ThermoFisher).Results and discussionAdaptive evolution of the strainIn order to evaluate the potential of adaptive evolutionin filamentous fungi, we chose to apply this approachon A. niger N402 and select for strains with improvedcellulase activity (see Materials and methods). Thepoor growth of A. niger N402 on cellulose suggestedthis substrate as a good substrate for selective muta-tions caused by adaptive evolution. Several colonieswere purified and the colony showing the best growthon a-cellulose was selected for further studies. Theselected evolved cellulose mutant, CBS 140717,showed similar growth to N402 on cellulose in thepresence of glucose but a clear improved growth wasvisible on cellulose as sole carbon source (Fig. 1).Gene expression profile of the parentaland evolved strainsCellulolytic genes in A. niger are under control of the(hemi-)cellulolytic regulator XlnR (van Peij et al.1998) that responds to the presence of xylose.Fig. 1 A. niger celluloseevolved strain CBS 140717and parental strain N402grown on a-cellulose andglucose ? a-celluloseBiotechnol Lett (2016) 38:969–974 971123Therefore, to obtain insight in the factor causing theimproved cellulase production, a single samplemicroarray was performed on this mutant grown inxylose and compared to the parental strain grown inthe same conditions. Overall, no significant increase incellulolytic gene expression was observed. InductionFig. 2 b-1,4-D-Glucosidase (a), cellobiohydrolase (b) and b-xylosidase (c) activities of culture filtrates produced by A. nigerstrains grown in cellulose shaken cultures. Extracellular proteinprofile on SDS-PAGE and total protein concentration (d) ofCBS 140717, N402, DnoxR and reference strain (ref_ DnoxR) ofA. niger972 Biotechnol Lett (2016) 38:969–974123for 2 h with xylose may have been too short to see animmediate effect on cellulolytic gene expression.Interestingly, the expression level of the A. nigernoxR homolog has strongly decreased in the CBS140717 strain, from a signal value of around 50–1.5. InPodospora anserina deletion of this gene (PaNoxR)resulted in reduced cellulase production (Brun et al.2009). Thus, we hypothesized a similar role in theregulation of cellulase production in A. niger.Characterization of the A. niger noxR knockoutstrainTo confirm the role of noxR in cellulase production inA. niger, we compared CBS 140717 and its parentalstrain to the knockout mutant noxR (Kwon et al. 2011)and its parental strain.BXL, CBH and BGL activities were measuredduring 10 days of cultivation, demonstrating increaseof these activities over time (Fig. 2a–c). All threeenzyme activities showed higher expression for CBS140717 and DnoxR strains compared to their referencestrains.The SDS-PAGE patterns of CBS 140717and N402show distinct differences in terms of absence andaltered intensity of protein bands (Fig. 2d). A similarpattern was observed when comparing DnoxR andref_DnoxR strains with the absence of some proteinsor reduced intensity of protein bands in ref_DnoxR(Fig. 2d). Higher secreted protein concentrations (6-7-fold) were measured for both CBS 140717 and DnoxRcompared to N402 and ref_DnoxR strains, respec-tively. As NoxR interacts with NoxA in A.niger(Kwon et al. 2011), we also tested the DnoxA strain(data not shown), but this strain was comparable to thereference strain.Our adaptive evolution approach was efficient tomodify A. niger N402 in a milder way than theclassical random mutagenesis (e.g. UV), which wasfor instance used to obtain the T. reesei RUT-C30cellulose producer (Montenecourt and Eveleigh1977). Advantageously, this method is likely to induceless randommutations because mutations are linked tothe selection for the improved fitness of the strain.These results demonstrate the value of adaptiveevolution as a tool to generate strains with improvedenzyme production. In addition, these strains can beused to identify novel factors that affect enzymeproduction, such as NoxR. In future studies, we aim toaddress the molecular mechanism of NoxR in thisprocess.In conclusion, the evolved strain, CBS 140717,showed an increase of about five-fold in cellulaseproduction compared to A. niger N402 and analysis ofthis strain revealed a role for NoxR (RiaA) in cellulaseproduction in A. niger.Acknowledgments AP was supported by a grant of the DutchTechnology Foundation STW, Applied Science division ofNWO and the Technology Program of theMinistry of EconomicAffairs UGC11108. IB was supported by a grant of the DutchTechnology Foundation STW, Applied Science division ofNWO and the Technology Program of theMinistry of EconomicAffairs UGC 07938 to RPdV.Compliance with ethical standardsConflict of interest The authors declare no financial orcommercial conflict of interest.Open Access This article is distributed under the terms of theCreative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrest-ricted use, distribution, and reproduction in any medium, pro-vided you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons license, andindicate if changes were made.ReferencesBachmann H, Pronk JT, Kleerebezem M, Teusink B (2015)Evolutionary engineering to enhance starter culture per-formance in food fermentations. 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Improving cellulase production by Aspergillus niger using adaptive evolution

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Improving cellulase production by Aspergillus niger using adaptive evolution

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