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Deciphering domain structures of Aspergillus and Streptomyces GH3-β-Glucosidases: a screening system for enzyme engineering and biotechnological applications

BMC Research Notes volume 17, Article number: 257 (2024) Cite this article

Abstract

The glycoside hydrolase family 3 (GH3) β-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 β-glucosidases. Two distinct configurations were identified in fungal GH3 β-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in β-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger β-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric β-glucosidases enzymes for industrial application.

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Introduction

β-glucosidases play an important role for the complete breakdown of cellulose by hydrolysing cellobiose into glucose [1]. Cellobiose is an intermediate product resulting from the action of endoglucanases and exoglucanases on cellulose. Through the presence of β-glucosidases, the inhibited complete breakdown caused by cellobiose accumulation can be alleviated [2, 3], making these enzymes targets for improving cellulolytic enzyme cocktails.

Filamentous fungi such as those from the Aspergillus genera, are recognized for their production of β-glucosidase [4, 5]. According to the carbohydrate-active enzyme (CAZyme) database, the majority of fungal β-glucosidases are listed as GH3 [6]. The GH3 β-glucosidases from filamentous fungi commonly are associated with an accessory Fn3 domain at the C-terminus [7,8,9,10,11]. Notably, β-glucosidase BGLA from Aspergillus niger is the major β-glucosidase protein used industrially and has the GH3-Fn3 domain structure (2,4). However, the precise function of the Fn3 domain remains uncertain, as different effects on the activity of the associated CAZymes were observed [12,13,14,15,16,17,18,19].

In filamentous Actinomycete bacteria, GH3 modular domain organization was found not only with a C-terminal Fn3 domain but also with a C-terminal CBM2 domain [20]. It has been reported that accessory domains can significantly impact enzymes properties [12, 21]. As a result, various studies have focused on developing novel enzymes by modifying enzyme domains for enhanced functionality [22,23,24]. This research aimed to explore the variation in domain organization within GH3 β-glucosidase from fungi and actinomycete, focusing on representatives from Aspergillus and Streptomyces genera.

Furthermore, a screening system was developed based on the enzyme domain of the well-known fungal GH3-Fn3 β-glucosidase gene (bglA) from A. niger. In this approach, the native bglA in A. niger was knocked out to create an A. niger mutant strain devoid most of its extracellular β-glucosidase activity which was then used as a recipient strain for reintroducing the GH3-Fn3 gene. This approach established the foundation for further investigating the role of different accessory domains on β-glucosidase activity towards different substrates.

Materials and methods

Gene, microbial strains, and substrates

The A. niger β-glucosidase gene (An18g03570; bglA) served as the basis for designing the screening system. Substrates used included esculin (Sigma, E8250), p-nitrophenyl β-D-glucopyranoside (pNPGlc) (Sigma, N7006), and 4-methylumbeliferyl-β-d-glucopyranoside (MUGlc) (Carl Roth), while 4 nitrophenol (Sigma, 241326) was used as a standard for some of the activity assays.

Deletion of wild type (WT) bglA gene reintroducing GH3-Fn3 gene transformation screening and cultivation

To generate A. niger MGG029 ΔbglA, the native bglA gene was deleted using the split marker method [25] (Additional File 1, Fig. S1). The split marker fragments containing 5’ and 3’ flanks of the bglA and hygB selection (Additional File 1, Fig. S1a) were transformed into A. niger MGG029 to create A. niger MGG029 ΔbglA strain, as described by [25, 26]. A colony PCR was conducted to confirm the deletion bglA in A. niger strain using primers listed in Additional File 1: Table S1. For reintroducing the GH3-Fn3 gene, the gene was generated through PCR amplification of bglA gene (An18g03570), using genomic DNA of A. niger N402 as a template. Subsequently, the GH3-Fn3 gene fragment was cloned into pMA351 plasmid derived from pAN52-1Not [27] to create the GH3-Fn3 expression construct (Additional File 1, Fig. S2). This plasmid carries the gpdA promoter and trpC terminator from Aspergillus nidulans. The A. niger pyrE gene (An04g08330) including promoter and terminator region was amplified using primers pyrEP13f and pyrEP14r listed in Additional File 1: Table S1 and genomic DNA of A. niger N402 as a template. The pyrE PCR product (2764 bp) was ligated into pJET1.2/blunt (Thermofisher) and was subsequently used as selection marker.

Fungal transformation was conducted according to the procedure essentially described [26, 28]. The GH3-Fn3 expression construct as well as the vector containing pyrE gene was introduced into A. niger MGG029 Δbgl pyrE−, as recipient host in transformation [24]. This pyrE− strain, derived from A. niger MGG029 [29], was prepared following the method outlined in [26]. Twenty randomly selected transformants were purified by single colony streaks on minimal medium plates and streaked on esculin-containing plates for activity screening, in which positive transformants will produce a dark halo around the colony. The GH3-Fn3 gene was sequenced for verification using primers listed in Additional File 1: Table S1. Cultivation was conducted in 300 mL Erlenmeyer flasks with a 100 mL working volume of complete medium, inoculated with 1 × 108A. niger spores, and incubated at 180 rpm, 30 °C for 3 days. The spent medium was collected by filtration.

Enzyme activity assay and protein gel detection

Qualitative enzyme activity assay was carried out on minimal medium agar containing 0.5% esculin and 2% ferric sulphate (FeSO4) as described by [30]. The quantitative β-glucosidase assay was performed as essentially described by [31] with pNPGLc as substrate. Moreover, SDS-PAGE was performed in 10% precast polyacrylamide gels (Bio-Rad, #4561033) as described in [24] using spent medium and mycelium extract of different transformants. Zymogram analysis was carried out for the detection of β-glucosidase activity following the running of native PAGE as described by [24]. After electrophoresis, the gel was overlaid with either 4 mM MUGlc [32], or 0.5% esculin with 0.02% FeSO4 added in 0.3% agarose [33]. Active protein was detected after incubation for 30 min at 30 °C and visualization under UV light for MUGlc fluorescence and dark coloration for esculin.

Phylogenetic tree construction

The GH3 proteins predicted as β-glucosidases were retrieved from Uniprot (https://www.uniprot.org/, 15th November 2023) [34]. The region of GH3 catalytic domains was determined using HMMER V3.4 program [35]. The phylogenetic tree was constructed using the “one click” default setting on Phylogeny.fr web platform [36] and visualized using iTOL web-server program [37].

Results

Domain Organization and Phylogenetic Relationship of GH3 β-Glucosidases from Aspergillus and Streptomyces

As an initial step in our study, the variation in the multidomain architecture of GH3 proteins was explored within the Aspergillus and Streptomyces genera, being important industrial protein production organisms (Fig. 1). Analysing GH3 sequences from the UniProt database [34] revealed that the majority of Aspergillus β-glucosidases feature a GH3 domain with a C-terminal Fn3 domain, while lacking CBM domains (Fig. 1A). In contrast, Streptomyces GH3 proteins not only showed accessory Fn3 domains but also CBM2, CBM6, CBM9, and CBM11 domains (Fig. 1B). GH3-Fn3 appeared as the predominant domain organization in both genera, constituting 96% and 79% for Aspergillus and Streptomyces, respectively (Fig. 1C).

To examine the relatedness of GH3 catalytic domains across β-glucosidases within Aspergillus and Streptomyces, the phylogenetic tree was constructed based on the GH3 catalytic domain sequences (Fig. 2). The tree demonstrated that GH3 catalytic domains with the same domain architectures from these genera were not clustered together (Fig. 2). In Aspergillus, GH3 sequences from the GH3-Fn3 domain organization emerged from the same internal branch, although the GH3 did not form a single clade (Fig. 2). Conversely, in Streptomyces, two separate groups of GH3-Fn3 emerged from different branches (Fig. 2). Notably, the Streptomyces GH3 carrying CBM2 and CBM11 exhibit more relatedness with Aspergillus GH3-Fn3 than with those from Streptomyces (Fig. 2).

Fig. 1
figure 1

Domain architecture of GH3 β-glucosidases. (A) Aspergillus; (B) Streptomyces. Between brackets is the number of identified protein sequences that are distributed in the various Aspergillus and Streptomyces species. The presence of Fn3 or/and CBM in the domain organization is indicated on the right side. (C) The percentage of GH3 associated with CBM and Fn3 domain in Aspergillus and Streptomyces

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Furthermore, within the tree depicting Fn3 domain sequences, a distinct grouping of Fn3 domains was noted between GH3-Fn3 sequences originating from the Aspergillus and Streptomyces genera (Additional File 1, Fig. S3). In Streptomyces, Fn3 domain from the rare GH3-Fn3-big2-CBM9 multidomain architecture emerged from the same internal branch with Fn3 domains from Aspergillus GH3-Fn3, signifying a more closely relatedness to fungal Fn3 domain than to those of Streptomyces (Additional File 1, Fig. S3), while this feature was not observed in GH3 tree. Moreover, Streptomyces Fn3 domain from GH3-Fn3 were clustered together, while those from GH3-Fn3-CBM6 were grouped in two separate branches (Additional File 1, Fig. S3). This analysis illustrated a divergence in the evolution of GH3 with their accessory domain sequences between Aspergillus and Streptomyces. Although the GH3 and the accessory domain will have originated each from a common ancestor, protein domains can undergo functional divergence over time [38, 39]. This can be influenced by factors such as functional adaptations and varying selection pressures in different microbial environments, leading to sequences with distinct evolutionary trajectories [40, 41]. This finding highlights the variability as well as the relatedness among domain organizations within GH3 enzymes family from these genera. Furthermore, as an initial approach to explore the role of GH3 domain organization, a screening system was developed based on the bglA gene in A. niger.

Fig. 2
figure 2

Phylogenetic tree of GH3 catalytic domains. Phylogenetic tree depicting the relationship of GH3 catalytic domains across modular GH3-β-glucosidases in Aspergillus and Streptomyces genera. The protein used in phylogenetic analysis is solely the sequence of GH3 domain (activity domain), excluding the accessory domain sequences. The tree was generated using the “one click” default program on the Phylogeny.fr web server platform (http://www.phylogeny.fr/). This automated pipeline optimized each step of the process for the input sequence data. Specifically, multiple sequence alignment was performed using the MUSCLE program. Subsequently, phylogenetic tree topology was inferred using Phylogenetic Maximum Likelihood (PhyML) computation. The output of the tree was saved in Newick format for further visualized using iTOL (http://itol.embl.de/). Bootstrap values for 100 replicates are shown at the nodes of the tree. The scale bar indicates 40% sequence divergence. The UniProt IDs of the sequences used for constructing the phylogenetic tree are listed in Additional File 2. With an arrow the position of the catalytic domain of the A. niger BGLA protein is indicated

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β-Glucosidases screening system based on a A. niger bglA deletion strain

As part of a β-glucosidases screening system, an expression construct was generated on the basis of the wild type (WT) β-glucosidase gene (An18g03570; bglA). In addition, a screening host strain was developed in which the WT bglA gene was deleted and confirmed by colony PCR (Additional File 1, Fig. S1b). The bglA knockout strain was analysed by zymogram analysis, confirming the major contribution of bglA to esculin-based enzymatic activity (Fig. 3C).

Furthermore, transformants obtained by co-transformation with the bglA (GH3-Fn3) expression construct (Fig. 3A) were screened for β-glucosidase activity using esculin plate assay (Fig. 3B). Several putative co-transformants showed strong activity (Fig. 3B). To conduct more detailed enzyme activity analysis, a β-glucosidase zymogram assay was performed using esculin and MUGlc substrates for activity staining for one of the purified transformants (Fig. 3C), confirming successful expression of the introduced GH3-Fn3 expression construct. The two faint bands observed in the knockout strain, A. niger MGG029 ΔbglA on zymogram (one at the same position as the BGLA protein) suggested background activity from other native β-glucosidase proteins of GH3 or other GH families (Fig. 3C). As it was reported that A. niger NRRL3, the parental strain of A. niger MGG029, harbours multiple GH3-β-glucosidase encoding genes as listed in CAZy [6] these would probably be responsible for this background activity. Furthermore, as expected, both qualitative (Additional File 1, Fig. S4) and quantitative activity assays (Additional File 1, Fig. S5) on spent culture medium showed increased activity in the selected GH3-Fn3 transformant. It should be noted that the relatively limited reduction on β-glucosidase activity toward pNPGlc may also be due to the fact that also exoglucanases show activity towards this artificial substrate [42].

Fig. 3
figure 3

Screening ofA. nigeras transformation host for reintroducing bglAgene (GH3-Fn3). (A) Domain organization of A. niger β-glucosidase comprising GH3-Fn3 domain. (B) GH3-FN3 putative co-transformants screened on MM-esculin plate. Negative control (-C) is A. niger MGG029 ∆bglA, transformant with empty vector. (C) β-glucosidase activities on zymogram from A. niger spent medium. Lane 1: A. niger with WT bglA deletion (∆bglA strain), lane 2: A. niger with WT bglA present (WT strain), lane 3: A. niger co-transformant (strain carrying bglA expression construct). Very weak intensity was observed at BGLA in lane 1 (∆bglA strain), which is also referred to as background activity

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Discussion

Various studies were focused on engineering enzymes by modifying protein domain organization to enhance catalytic efficiency, as demonstrated in amylase [24], cellulases [21] and LPMO [43]. Amylase and cellulases from filamentous microorganisms were reported to exhibited variation in their domain organization [20]. This research applied the similar approach to fungal β-glucosidase, a key enzyme in complete cellulose degradation. The most prevalent domain organization of fungal β-glucosidase, especially those from Aspergillus consists of GH3 domain carrying a C-terminal Fn3 domain. While none of Aspergillus GH3s are associated with canonical CBM domains, in Streptomyces, GH3 in combination with CBMs were commonly identified, most notable with CBM2 domains [34]. It has been reported that the Fn3 domain present in both Aspergillus and Streptomyces GH3 domain organization may act as a linker between two domains facilitating protein-protein interactions [44]. In bglA, Fn3 is located at the C-terminal end, suggesting a role beyond a linker, such as maintaining protein structure, stability, and enzymatic activity. Moreover, the phylogenetic tree reveals that the GH3 catalytic domain in the fungal GH3-Fn3 arrangement forms a separate cluster from fungal GH3 without an accessory domain. This separation implies substantial differences in the sequences of these GH3 domains, which are likely correlated with their function.

In this study, we also developed a screening system in A. niger for further exploration the β-glucosidase domain structure to contribute to our understanding of the intricate relationship between protein domains. A. niger has been renowned for its robust secretion system in filamentous fungi that allows it to efficiently secrete proteins [45, 46]. Hence, this system was developed based on Aspergillus β-glucosidase as a target protein, offering a platform to explore the improvement of β-glucosidase enzymes through protein domain modification. In the screening system, the native β-glucosidase in A. niger was deleted to create a host with reduced background activity. Successful overexpression of the native GH3-Fn3 was used to demonstrate the usefulness of this system. Looking ahead, this research can serve as a basis for exploring other β-glucosidase domain organizations including heterologous chimeric variants. This holds the prospect for unravelling new insights into protein domain organization and optimizing enzyme functionality.

Conclusion

This study has elucidated the modular domain architecture within GH3 β-glucosidase family from filamentous microorganisms. Additionally, we demonstrated the use of the developed screening system by introducing the complete fungal GH3-Fn3 gene encoded by A. niger bglA in a bglA deletion strain resulting in production of an active β-glucosidase. To explore the potential improvement of β-glucosidase through protein domain modification, this system can now be used for expression of chimeric β-glucosidase gene variants including but not limited to the accessory domains found in Streptomyces.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

Fn3:

Fibronectin type III

GH3:

Glycoside hydrolase family

CBM:

Carbohydrate Binding Module

pNPGlc:

p-Nitrophenyl β-D-glucopyranoside

MUGlc:

4-methylumbeliferyl-β-d-glucopyranoside

A. niger bglA:

Aspergillus niger β-glucosidase I

hyg:

Hygromycin

pNP:

p-nitrophenol

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

PCR:

Polymerase chain reaction

WT:

Wild type

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Funding

This study was supported by a scholarship of the Indonesia Endowment Fund for Education (LPDP) from the Ministry of Finance, Indonesia (20160422026103) to AS and by a grant of the Dutch National Organization for Scientific Research NWO, in the framework of an ERA-IB project FilaZyme (053.80.721/EIB.14.021) to EV, GV, and PP.

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Authors and Affiliations

  1. Institute of Biology Leiden, Fungal Genetics and Biotechnology, Leiden University, Leiden, The Netherlands

    Andika Sidar, Gerben P. Voshol, Mark Arentshorst, Arthur F.J. Ram, Erik Vijgenboom & Peter J. Punt

  2. Department of Food and Agricultural Product Technology, Gadjah Mada University, Yogyakarta, Indonesia

    Andika Sidar

  3. Genomescan, Leiden, The Netherlands

    Gerben P. Voshol

  4. Ginkgo Bioworks, Zeist, The Netherlands

    Peter J. Punt

Authors
  1. Andika Sidar
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  2. Gerben P. Voshol
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  5. Erik Vijgenboom
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Contributions

AS wrote the original draft, performed the experiment, data & formal analysis. GV was involved in the design of experiments, software and resources. MA participated in the knockout strain-based split marker approach. AFR verified the bglA gene, presented ideas, review and editing. EV and PJP were involved in the conceptualization, presents ideas, planning, supervision, validation, manuscript outline, writing, review and editing. All authors reviewed and approved the final version of manuscript.

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Correspondence to Andika Sidar or Peter J. Punt.

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Sidar, A., Voshol, G.P., Arentshorst, M. et al. Deciphering domain structures of Aspergillus and Streptomyces GH3-β-Glucosidases: a screening system for enzyme engineering and biotechnological applications. BMC Res Notes 17, 257 (2024). https://doi.org/10.1186/s13104-024-06896-4

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BMC Research Notes

ISSN: 1756-0500

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