Construction of an Aspergillus enzyme library for the developmentof novel materials
Tomohiko MATSUZAWA
National Institute of Advanced
Bioproduction Research Institute

Industrial

ScienceandTechnology,

Research objective
Microorganisms produce many types of enzymes, such as glycosidases, proteases, andlipases, which degrade and assimilate biopolymers, including polysaccharides and proteins.
For example, the genome of Aspergillus oryzae, an industrially important fungus, encodes
more than 300 putative glycosidases. However, only 10%–20% of these putative
glycosidases have been characterized, and most still remain uncharacterized. In previousstudies, we identified and characterized some A. oryzae glycosidases, such as
isoprimeverose-producing oligoxyloglucan hydrolase (IpeA)1 and β-galactosidase (LacA)2.
These glycosidases play vital roles in the degradation of xyloglucan, one of the major
hemicellulosic polysaccharides in plant cell walls and seeds. Our previous research indicatedthat functional prediction of enzymes from amino acid sequences is an effective approach;nevertheless, in vitro assays are necessary to elucidate the actual roles of these enzymes.
In this study, an expression library of putative secretory proteins of A. oryzae wasconstructed to screen for novel enzymes.
Methods
The genome of A. oryzae (coding for approximately 11,000 proteins) was mined forsecretory proteins carrying an N-terminal signal peptide using SignalP(http://www.cbs.dtu.dk/services/SignalP/)3,4. Transmembrane helices in thesecretory
proteins
werepredictedusing
theTMHMM(http://www.cbs.dtu.dk/services/TMHMM/).

putativeserver

The coding sequences of genes encoding putative secretory proteins were amplified bypolymerase chain reaction (PCR) using a cDNA mixture as template. The cDNA mixture
was synthesized from total RNA of A. oryzae cells cultured under various conditions. Eachamplified DNA fragment was cloned into the Saccharomyces cerevisiae 2-micron plasmid,
containing a TDH3 promoter, an N-terminal α-factor secretion signal sequence, and a TDH3terminator. The amplified DNA fragments were cloned in frame with the N-terminal α-factor
secretion signal sequence and expressed as fusion proteins in S. cerevisiae cells.

Results
Approximately 1100 putative secretory proteins, carrying an N-terminal signal peptide forsecretion, were identified in the A. oryzae genome. Proteins that were predicted to havemultiple transmembrane domains were excluded from the analysis. We selected
approximately 550 genes and designed oligo DNA primers to amplify them. Approximately
520 genes were successfully amplified by PCR using a cDNA mixture as template, among
which approximately 500 genes could be cloned into the S. cerevisiae expression vector (Fig.
1).

Figure 1. Procedure for the construction of the A. oryzae secretory protein library andfor the performance of enzymatic assay.
Next, vectors containing the putative secretory protein genes of A. oryzae were introduced
into S. cerevisiae cells. These were cultured in 96-well plates, and the culture supernatants
were used for enzymatic activity assay. In this assay, insoluble chromogenic substrates,
namely AZCL-xyloglucan, AZCL-xylan, and AZCL-arabinoxylan, and soluble chromogenicsubstrates, namely p-nirtophenyl (pNP) β-D-glucopyranoside, pNP β-D-galactopyranoside,pNP β-D-xylopyranoside, pNP α-D-glucopyranoside, pNP α-D-xylopyranoside, pNP
α-L-arabinopyranoside, pNP α-L-fucopyranoside, and pNP α-L-rhamnopyranoside, wereused for endo- and exo-type glycosidases, respectively. I found that some culture
supernatants of S. cerevisiae cells expressing A. oryzae secretory proteins were able todegrade chromogenic substrates (Fig. 2), indicating that these S. cerevisiae cells harboredgenes involved in oligo- and polysaccharide degradation.

Figure 2. Screening of enzymes catalyzing endo-type polysaccharide degradation using
insoluble chromogenic substrates. Culture supernatants of S. cerevisiae cells harboring anA. oryzae secretory protein library were incubated with AZCL-xyloglucan (A) orAZCL-xylan (B). The wells showing degradation activity toward these substrates areincluded in dashed rectangles.
Conclusion
In this study, I constructed a secretory protein library of A. oryzae and screened for novelenzymes. I believe that this library can be useful for protein screening; however, someconcerns about its quality and comprehensiveness still remain. Therefore, our future efforts
will focus on the improvement of this library and a more comprehensive screening of novelenzymes.
References
1) Matsuzawa, T., Mitsuishi, Y., Kameyama, A., and Yaoi, K. (2016) Identification of thegene encoding isoprimeverose-producing oligoxyloglucan hydrolase in Aspergillus
oryzae. J. Biol. Chem. 291:5080-5087.
2) Matsuzawa, T., Watanabe, M., Kameda, T., Kameyama, A., and Yaoi, K. (2019)
Cooperation between β-galactosidase and an isoprimeverose-producing oligoxyloglucanhydrolase is key for xyloglucan degradation in Aspergillus oryzae. FEBS J. 286:3182-3193.
3) Almagro Armenteros, J.J. et al. (2019) SignalP 5.0 improves signal peptide predictions
using deep neural networks. Nat. Biotechnol. 37: 420-423.
4) Umemura, M. (2020) Peptides derived from Kex2-processed repeat proteins are widelydistributed and highly diverse in the Fungi kingdom. Fungal. Biol. Biotechnol. 7: 11.