For many years, aldolases catalysing stereoselective C-C bond formation have been considered essential for synthetic applications.[1] Biocatalysed aldolisation reactions are performed under mild conditions, without any protections and are therefore highly valuable for the development of green synthetic processes. In addition, there is room for new C-C bond forming enzymes to construct more complex molecules since this category of enzymes is underused when compared with other biocatalysts.[2] The classification of aldolases is based on the structure of natural nucleophiles leading to five main classes: dihydroxyacetone phosphate- (DHAP), pyruvate-, ethanal-, dihydroxyacetone- (DHA), and glycine- aldolases.[1a] Concerning their substrate specificity, one generally admitted that if they accept a broad range of aldehydes as electrophiles, most of them are strictly dependent on a sole nucleophile substrate. Our work highlight that aldolases are not always dependent on aldehydes as electrophile or on a sole nucleophile substrates. They complete the recent discoveries reported by us and others on their higher nucleophile tolerance.[3] Recent results in exploring nucleophile and electrophile substrates promiscuity among aldolases from biodiversity (see scheme) will be described. In particular, we have revisited DHAP-dependent aldolases with ketones as electrophiles.[4] We have demonstrated that rhamnulose-1-phosphate aldolases display an unprecedented versatility for activated ketones. We selected and characterized a rhamnulose aldolase from Bacteroides thetaio-taomicron as a proof of concept. DHAP was added to several hydroxylated ketones used as electro-philes. This aldol addition was stereoselective and produced branched-chain monosaccharide adducts with a tertiary alcohol moiety, which is rather difficult to prepare optically pure. Other nucleophiles [5] or electrophiles, with different aldolase classes are currently under investigation in our lab, which would confirmed the unprecedented substrate tolerance among aldolases. References: 1. (a) P. Clapés, X. Garrabou, Adv. Synth. Catal. 2011, 353, 2263-2283; (b) P. Clapés, W. D. Fessner, G. A. Sprenger, A. K. Samland, Curr. Opin. Chem. Biol. 2010, 14, 154-167; (c) M. Müller, Adv. Synth. Catal. 2012, 354, 3161-3174; (d) M. Brovetto, D. Gamenara, P. Mendez, G. Seoane, Chem. Rev. 2011, 111, 4346-4403; (e) A. Bolt, A. Berry, A. Nelson, Arch. Biochem. Biophys. 2008, 474, 318-330; (f) A. K. Samland, G. A. Sprenger, Appl. Microbiol. Biotechnol. 2006, 71, 253-264. 2. N. J. Turner, E. O'Reilly Nature Chem. Biol. 2013, 9, 285–288. 3 (a) R. Roldaú n, K. Hernandez, J. Joglar, J. Bujons, T. Parella, I. Saú nchez-Moreno, V. Hélaine, M. Lemaire, C. Gueú rard-Heú laine, W.-D. Fessner, and P. Clapeú s ACS Catal. 2018, 8, 8804−880. (b) I. Sanchez-Moreno, T. Scheidt, V. Hélaine, M. Lemaire, T. Parella, P. Clapés, W.-D. Fessner, C. Guérard-Hélaine Chem. Eur. J., 2017, 23, 2005-2009. (c) V. de Berardinis, C. Guérard-Hélaine, E. Darii, K. Bastard, V. Hélaine, A. Mariage, J.-L. Petit, N. Poupard, I. Sanchez-Moreno, M. Stam, T. Gefflaut, M. Salanoubat, M. Lemaire Green Chem., 2017, 19, 519-526. 4 (a) V. Laurent, E. Darii, A. Aujon, M. Debacker, J.-L. Petit, V. Hélaine, T. Liptaj, M. Breza, L. Nauton, M. Traïkia, M. Salanoubat, M. Lemaire, C. Guérard-Hélaine, V. de Berardinis Angew. Chem., Int. Ed. Engl., 2018, 57, 5467-5471. (b) M. Salanoubat, M. Lemaire, C. Guérard-Hélaine, V. de Berardinis WO 2018/215476 A1. 5 V. Laurent, A. Uzel, V. Hélaine, L. Nauton, M. Traïkia, T. Gefflaut, M. Salanoubat, V. de Berardinis, M. Lemaire and C. Guérard-Hélaine Adv. Synth. Catal. 2019, accepted (special Biotrans 2019 issue)