Xylanbackbone

β4β4β4β4

α3 α3

Lignin

FerFer

Substrate FaeA FaeB FaeCChlorogenic acid - 24% -Ethyl coumarate - 36% 42%Ethyl ferulate 13% 1% 88%Methyl-3,4-dimethoxycinnamate 6% 0.1% 100%Methyl caffeate - 14% 34%Methyl ferulate 55% 1% 74%Methyl hydroxybenzoate - - -Methyl p-coumarate - 100% 66%Methyl sinapate 100% - 41%The highest activity for each enzyme was set to 100% and used to comparerelative activities.

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SubstrateInsoluble wheat arabinoxylan

Wheat bran Sugar beetpectin 1Solubilized Not solubilized

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Figure 4 Relative expres-sion patterns for faeA, faeB, faeC on feruloylated polysac-charides. The cultures were grown in minimal medium containing 1% wheat bran or 1% sugar beet pulp with or without calcium lignin sup-plementation in a course of three days, and the gene ex-pression was quantitated us-ing RT-qPCR. ▲, wheat bran; ■, sugar beet pulp; ∆, wheat bran with lignin; □, sugar beet pulp with lignin. Standard de-viations from two biological and three technical replicates are shown as error bars.

Compound Struc-ture R1 R2 R3 R4

1 Benzoic acid Ι H H H COOH

2 Cinnamic acid ΙΙ H H H COOH

3 p-Hydroxybenzoic acid Ι H OH H COOH

4 p-Coumaric acid ΙΙ H OH H COOH

5 Protocatechuic acid Ι OH OH H COOH

6 Caffeic acid ΙΙ OH OH H COOH

7 Vanillic acid Ι OCH3 OH H COOH

8 Ferulic acid ΙΙ OCH3 OH H COOH

9 Veratric acid Ι OCH3 OCH3 H COOH

10 3,4-Dimethoxycinnamic acid ΙΙ OCH3 OCH3 H COOH

11 Syringic acid Ι OCH3 OH OCH3 COOH

12 Sinapic acid ΙΙ OCH3 OH OCH3 COOH

13 3,4-Dimethylbenzyl alcohol Ι CH3 CH3 H CH2COOH

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Figure 5 Relative expression patterns for faeA, faeB, faeC on monomeric phenolic compounds. The cultures were grown on monomeric phenolic compounds for two hours and gene expression was quantified using RT-qPCR. Standard deviations from two biological and three technical repli-cates are shown as error bars.

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Compound 1 2 3 4 5 6 7 8 9 10 11 12 13Structure Ι ΙΙ Ι ΙΙ Ι ΙΙ Ι ΙΙ Ι ΙΙ Ι ΙΙ Ι

Figure 1 Phylogenetic relation-ships among the (putative) fun-gal FAEs from subfamily 5.

The phylogenetic analysis was performed by Neighbor-joining implemented in MEGA6 with pairwise deletion of gaps and the Poisson correction distance of substitution rates. Statistical support for phylogenetic group-ing was estimated by 1000 boot-strap resamplings.

Characterized FAEs are shown in bold. ‘Uncharacterized FAEs’ indicate multiple FAEs in these branches. Only the bootstrap above 40% were shown on the branches.

Introduction: Ferulic acid (FA) is a major phenolic acid component of plant cell walls. FA and to a lesser extent other hydroxycinnamic acids (e.g. p-coumaric acid) are ester-linked to plant cell wall polymers, mainly to (hetero)xylans and pectins, forming polysaccharide-polysaccharide and polysaccharide-lignin cross-links. Phenolic cross-links increase the physical strength and integrity of plant cell walls and reduce their biodegradability by microorganisms. Feruloyl esterases (FAEs) [E.C. 3.1.1.73] are able to release FA and other phenolic acids from natural plant sources and agro-industrial by-products and are therefore widely used in food, feed, pulp-paper, bioethanol and pharmaceutical industries. The broad fields require various types of FAEs to fit specific pH, temperature and other conditions.

Conclusion: - Heterologously produced A. niger FaeC possesses a broad substrate specificity towards synthetic FAE substrate compounds. - FaeC acts synergistically with xylanase to release ferulic acid from xylan. - FaeC showed promising potential particularly because of its broad substrate profile and the maximal activity at neutral pH.- The three fae genes from A. niger respond differently towards the feruloylated polysaccharides and monomeric phenolic compounds indicating that the FAE isoenzymes may target different substrates in a complementarily manner, contributing to the efficient degradation of diverse plant biomass.

Discovery of FaeC:Aspergillus niger is one of the most well-known and important industrial fungi, which produces a wide range of plant cell wall-degrading enzymes. Two FAEs from A. niger (FaeA and FaeB) have been char-acterized. Phylogenetic analysis of fungal FAEs performed by Benoit et al. (2008) and Dilokpimol et al. (2016) has led us to numerous novel FAE candidates and several putative FAEs from A. niger including FaeC.

Table 1 Substrate specificity of A. niger FAEs towards different synthetic substrates

tFaeC

Activity of FaeC:FaeC was produced recombinantly by Pichia pastoris X-33 and the culture supernatant was used for further characterization.FaeC showed broad substrate specificity and catalyzed the hydrolysis of almost all tested substrates, except for chlorogenic acid and methyl hydroxybenzoate. FaeB also showed broad substrate specific-ity but with preference for methyl p-coumarate, ethyl coumarate and chlorogenic acid; whereas FaeA showed quite narrow substrate specificity and could hydrolyze only methyl sinapate, methyl ferulate, ethyl ferulate and methyl 3,4-dimethoxycinnamate.

FaeC was most active at pH 7.0 and retained >85% of its activity from pH 5.0 to 7.0. FaeA and FaeB were most active at pH 5.0 and 6.0, respectively, and retained >85% of their activity from pH 5.0 to 6.0. All three FAEs showed maximal activity at 50°C and retained >85% of their activity for 2 h up to 45°C.

Figure 2 The effect of pH and tem-perature on activity and stability towards methyl ferulate.

A) pH-dependence for activity.B) Temperature-dependence for activity.C) pH-stability after 2 h incubation at 37°C.D) Thermal stability after 2 h incu-bation at pH 6.

Each experiment was made in trip-licate. Standard deviations are shown as error bars

A) B)

C) D)

tFaeB

tFaeA

Release of ferulic acid by FaeC:Highest ferulic acid amount was released from wheat arabinoxylan (up to 3 mg/g substrate) which al-most all ferulic acid was released when treated with xylanase.FaeC released similar amounts of ferulic acid from all sugar beet pectin samples (prepared with endo-polygalacturonase or rhamnogalacturonan hydrolase and rhamnogalacturonan acetyl esterase). No diferulic acids (i.e. 8-O-4-, 5,5- and 8,5- diferulic acids) were detected, although these dimers were demonstrated to occur in wheat and sugar beet.

Figure 3 Release of ferulic acid from wheat arabinoxylan, wheat bran and sugar beet pectin using FaeC. FaeC was used with pretreatment or co-incubation with xylanase (X) for wheat arabinoxylan and wheat bran, with endopolygalactu-ronase (P) for sugar beet pectin 1, and with rhamnogalacturonan hydrolase and rhamnogalacturonan acetylesterase (RR) for pectin 2. Each experiment was made in triplicate. Standard deviations are shown as error bars. The values were subtracted from the negative control.

Expression of faeC on polysaccharides:On both wheat bran (WB) and sugarbeet pulp (SBP), faeC was expressed lowly, but showed increase in expression level over a three day period. In contrast, both faeA and faeB were highly expressed on the first day and declined gradually in the course of time. Supplement the medium with lignin helped increasing the expression of all fae genes.

Expression of faeC on phenolic monomeric compounds:faeC was expressed very lowly in general on aromatic compounds. However, faeC was expressed high-er when A. niger grew on e.g. cinnamic acid and syringic acid. The three fae genes were differently ex-pressed on the tested aromatic compounds, which could represent the specific response of each FAE.

Acknowledgement: This work was supported by the European Union, Grant agreement no: 613868 (OPTIBIOCAT).

Fungal PhysiologyWesterdijk Fungal Biodiversity Institute

Uppsalalaan 8, 3584 CT Utrecht, the Netherlands www.westerdijkinstitute.nl, info@westerdijkinstitute.nl

Utrecht University

Expanding feruloyl esterase gene family of Aspergillus niger: characterization of a new feruloyl esterase, FaeC Adiphol Dilokpimol1, Miia R. Mäkelä1,2, Sadegh Mansouri2, Olga Belova1, Martin Waterstraat3, Mirko Bunzel3, Ronald P. de Vries1,2 and Kristiina Hilden2

1 Fungal Physiology, Westerdijk Fungal Biodiversity Institute & Fungal Molecular Physiology, Utrecht University, the Netherlands 2 Division of Microbiology and Biotechnology, Department of Food and Environmental Sciences, University of Helsinki, Finland 3 Department of Food Chemistry and Phytochemistry, Institute of Applied Biosciences, Karlsruhe Institute of Technology (KIT), Germany

E-mail: Kristiina.S.Hilden@helsinki.fi