High-level production of industrially relevant oxidases by a two-stage fed-batch approach: overcoming catabolite repression in arabinose-inducible Escherichia coli systems

Ramón Román, Nikola Lončar, Antoni Casablancas, Marco W. Fraaije, Glòria Gonzalez

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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.
Títol traduït de la contribucióProducción de oxidasas de relevancia industrial mediante cultivo de alta densidad fedbatch en dos etapas para superar la represión catabólica en sistemas de Escherichia coli inducibles por arabinosa
Idioma originalEnglish
RevistaApplied Microbiology and Biotechnology
DOIs
Estat de la publicacióPublicada - d’abr. 2020

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