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

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.