High-level production of industrially relevant oxidases by a two-stage fed-batch approach : overcoming catabolite repression in arabinose-inducible Escherichia coli systems
Román, Ramón (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Lončar, Nikola (GECCO Biotech)
Casablancas, Antoni (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Fraaije, Marco (University of Groningen)
González Anadón, Gloria (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)

Fecha:	2020
Resumen:	With the growing interest in enzyme applications there is an urgent demand for economic affordable and flexible enzyme production processes. In the present paper, we developed a high cell density fed-batch process for the production of two cofactor-containing oxidase, 5-hydroxymethylfurfural oxidase (HMFO) and eugenol oxidase (EUGO). The approach involved the arabinose inducible system to drive the expression while using mineral media. In order to overcome a major drawback of arabinose-inducible promoters, carbon catabolite repression, (CCR) by glucose, we developed a High Cell Density Culture (HCDC), two-stage fed-batch protocol allowing us to reach cell densities exceeding 70 g/L of Dry cell weight (DCW) using glucose as carbon source. Then, induction was achieved by adding arabinose, while changing the carbon source to glycerol. This strategy allowed us to obtain an 8-fold increase in recombinant HMFO titer when compared with a reference batch fermentation in Erlenmeyer flasks using terrific broth (TB), typically used with arabinose inducible strains. The optimized protocol was also tested for expression of a structurally unrelated oxidase, EUGO, where a similar yield was achieved. Clearly, this two-step protocol in which a relatively cheap medium (when compared to TB) can be used, reduces costs and provides a way to obtain protein production levels similar to those of IPTG-based systems.
Ayudas:	European Commission 730976
Derechos:	Aquest material està protegit per drets d'autor i/o drets afins. Podeu utilitzar aquest material en funció del que permet la legislació de drets d'autor i drets afins d'aplicació al vostre cas. Per a d'altres usos heu d'obtenir permís del(s) titular(s) de drets.
Lengua:	Anglès
Documento:	Article ; recerca ; Versió acceptada per publicar
Materia:	Arabinose promoter ; Fed-batch ; High cell density culture ; Industrial enzymes ; Oxidases
Publicado en:	Applied microbiology and biotechnology, Vol. 104, issue 12 (June 2020) , p. 5337-5345, ISSN 1432-0614

DOI: 10.1007/s00253-020-10622-y

Postprint 22 p, 1.9 MB

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