# From evolved to artificial CAZymes

Régis Fauré

► To cite this version:

Régis Fauré. From evolved to artificial CAZymes. 15th Carbohydrate Bioengineering Meeting, May 2024, Gand, Belgium. hal-04791132

HAL Id: hal-04791132

https://hal.science/hal-04791132v1

Submitted on 11 Feb 2026

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From evolved to artificial CAZymes

Régis Faure¹

¹ Toulouse Biotechnology Institute (TBI), Université de Toulouse, CNRS, INRAE, INSA, Toulouse, France
regis.faure@insa-toulouse.fr

**Keywords:** glycoside hydrolases, protein engineering, enzyme promiscuity, non-canonical amino acid

Carbohydrate-active enzymes, such as glycoside hydrolases (GHs), are highly attractive tools for glycochemists, providing the means to synthesize glycosidic bonds. The vast bio-diversity of GHs constitutes a reservoir of enzyme candidates and the potential to synthesize almost any glycosidic bond. However, most GHs primarily hydrolyze glycosidic bonds, with transglycosylation being a concomitant minority reaction. Fortunately, the innate plasticity of GHs is amenable to engineering aimed at modifying their catalytic potential.

A series of studies by our group has shed light on the transglycosylation/hydrolysis (T/H) equilibrium in retaining GHs and led to the development of a molecular evolution strategy to switch the activity of these hydrolases towards synthetic activity [1]. Using protein engineering it has been possible to drastically diminish hydrolysis, while retaining or even enhancing glycosynthetic activity. Accordingly, evolved transglycosylases (TGs) displaying high both transfer rates and yields were obtained [2,3]. Careful study of tailored TGs has provided deep insight into the determinants of the T/H partition. In turn, this has procured generic engineering rules allowing the creation of TGs starting from any retaining GH [4,5].

Standard protein engineering involving the substitution of one or more amino acids is powerful, but limited by the chemical diversity offered by the 20 natural amino acids. One option to surmount this is site-specific incorporation of non-canonical amino acids [6]. The use of the existing genetic code makes this strategy attractive and amenable for protein engineering aimed at the creation of new catalytic capabilities and novel molecular assemblies. However, the success of this approach is hampered by highly variable and often low amino acid incorporation efficiencies. In this work we present methodological improvements and discuss outlook for the creation of artificial CAZymes displaying new functions or architectures.

[1] Bissaro B, Monsan P, Fauré R and O'Donohue MJ (2015) Glycosynthesis in a Waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases. *Biochem. J.*, **467**(1): 17-35

[2] Bissaro B, Durand J, Biarnés X, Planas A, Monsan P, O'Donohue MJ and Fauré R (2015) Molecular design of non-Leloir furanose-transferring enzymes from an α-L-arabinofuranosidase: a rationale for the engineering of evolved transglycosylases. *ACS Catal.*, **5**(8): 4598-4611

[3] Durand J, Biarnés X, Watterlot L, Bonzom C, Borsenberger V, Planas A, Bozonnet S, O'Donohue MJ and R. Fauré (2016) A single point mutation alters the transglycosylation/hydrolysis partition, significantly enhancing the synthetic capability of an endo-glycoceramidase. *ACS Catal.*, **6**(12): 8264-8275

[4] Zhao J, Tandrup T, Bissaro B, Barbe S, Poulsen JCN, André I, Dumon C, Lo Leggio L, O'Donohue MJ and Fauré R (2021) Probing the determinants of the transglycosylation/hydrolysis partition in a retaining α-L-arabinofuranosidase. *New Biotechnol*, **62**: 68-78

[5] Teze D, Zhao J, Wiemann M, Ara KZG, Lupo R, Zeuner B, Vuillemin M, Rønne ME, Carlström G, Duus JØ, Sanejouand YH, O'Donohue MJ, Nordberg Karlsson E, Fauré R, Stålbrand H and Svensson B (2021) Rational enzyme design without structural knowledge: a sequence-based approach to generate efficient transglycosylases. *Chem. Eur. J.*, **27**: 10323-10334

[6] Shandell MA, Tan Z and Cornish VW (2021) Genetic code expansion: a brief history and perspective. *Biochemistry*, **60**(46): 3455-3469