Beneventi, Elisa

Bergantino, Elisabetta

Lonigro, Chiara

Zito, Francesca

Elisa Beneventi

Chiara Lonigro

Francesca Zito

Elisabetta Bergantino

Archivio istituzionale della ricerca - Università di Padova

ALGAL CHLOROPLASTS FOR IN VIVO SCREENING AN IN VITROEVOLVED LIBRARY FROM A BACTERIAL FDH:A PROOF-OF-CONCEPT EXPERIMENTElisa Beneventi1,2; Chiara Lonigro1,3; Francesca Zito3; Elisabetta Bergantino1*1 Department of Biology, University of Padova, Viale G.Colombo 3, 35121 Padova, Italy2 present address: Galchimia, S.L. R&D Department, Cebreiro s/n 15823 O Pino, A CorunÞa, Spain3 Institut Biologie Physico-Chimique, UMR7099, CNRS/Universiteì Paris Diderot,13, rue Pierre et Marie Curie, F-75005 Paris, France* Corresponding author: elisabetta.bergantino@unipd.itThe selection of many thousands of different mutants is generally the rate-limiting step of directedevolution, used to produce enzymatic variants capable of better performances in specificbiocatalytic reactions. Even when a direct and simple - e.g. colorimetric - assay is available for thelaboratory practice and applicable in robotic platforms, multiple rounds of high-throughputscreening are often necessary to obtain some, not necessarily extraordinary, improvement. On theother hand, Darwinian evolution teaches us that the natural selection of the best enzymatic variantsis optimally realized by cells and organisms, where the acquired advantage affects growth and/orsurvival. Consequently, if only it can be pursued, an in vivo strategy for screening is predictablymore effective than an in vitro one.We have recently characterized a new FDH, named GraFDH, identified by mining the genome of theextremophile prokaryote Granulicella mallensis MP5ACTX8. The new enzyme displays a valuablestability in the presence of many organic co-solvents as well as double cofactor specificity, withNADP+ preferred over NAD+ at acidic pH values, at which it also shows the highest stability [1]. Thequite low affinities for both cofactors as well as for the substrate formate has prompted us to setexperimental strategies for the optimization of the native enzyme, in view of its possible applicationfor the regeneration of NADPH in biocatalytic reactions.After some experiments of structure based rational design, we moved to an in vitro directedevolution approach combined with an in vivo screening system performed in the chloroplast of thegreen alga Chlamydomonas reinardhtii. The starting point was the following: it has long beenknown that formate inhibits photosynthesis and that this inhibition is reversed by bicarbonate [2],respectively substrate and product of FDHs. Our assumption was that by expressing GraFDH intothe chloroplast of C. reinhardtii the inhibition by formate should be reversed. Moreover, thephotosynthetic process consumes NADPH, which could be regenerated by the recombinant FDH.This implies that, in the chloroplast, the recombinant FDH could establish a virtuous cycle allowingautotrophic growth in the presence of formate. Therefore, the screening system was based on theexpectation that the cells possessing more efficient variants of the enzyme would have showndifferent and more vigorous growth on formate compared to those expressing less performantvariants. We will report on a proof-of-concept experiment carried out to test and validate thedesigned system.[1] Fogal, Beneventi; Cendron, Bergantino Appl Microbiol Biotechnol 2015, 99, 9541-9554[2] Xiong, Minagawa; Crofts, Govindjee Biochim. Biophys. Acta 1998, 1365, 473-491334BioTrans 2017, Budapest P-268

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Algal chloroplasts for in vivo screening an in vitro evolved library from a bacterial FDH: a proof-of-concept experiment.

Algal chloroplasts for in vivo screening an in vitro evolved library from a bacterial FDH: a proof-of-concept experiment.

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Archivio istituzionale della ricerca - Università di Padova