Design

Recent studies have shown that multiple organisms have the ability of metabolizing 5-hydroxymethylfurfural (HMF). For instance, enzyme able to oxidize HMF into Furan-2,5-dicarboxylic acid (FDCA) has been discovered in fungal species such as Caldariomyces fumago [1] or in prokaryotic species Cupriavidus basilensis (C. basilensis) [2]. and Methylovorus sp. [4].

As we intended to work with Escherichia coli (E. coli), we focused on the enzymes discovered in prokaryotes. Due to glycosylations on the enzymes that has been found in Caldaryomyces fumago and Eukaryotes/Prokaryotes gene compatibility issue, fungal enzymes have been dismissed. The following section describes the enzymes selected to achieve an efficient transformation of HMF into FDCA.

HmfH is an enzyme that has been discovered in C. basilensis. This enzyme is part of a gene cluster implied in furanic compound degradation. HmfH homologues has also been found in several other bacterial species [6]. In a study, HmfH gene has been cloned into Pseudomonas putida in order to enable this bacteria to synthetize FDCA from HMF. HmfH catalyse two successive oxidations that transform HMF into 5-(hydroxymethyl)furoic acid ( HMF acid) and then into FDCA [5] ( fig 2. ). The modified P. putida with HmfH was able to produce FDCA without excessive amount of other furan derivatives. One issue with this enzyme is that the second oxidation of HMF acid into FDCA is slower than the first one. It results in the accumulation of the intermediate product, HMF acid. In fed batch culture the accumulation of intermediate product could lead to an efficient production of high pure FDCA.

One way to compensate for that is to add other enzymes to the artificial metabolic pathway, in order to speed up the second oxidation of HMF acid into FDCA.

An enzyme of interest, HMF dehydrogenase (Aldh1), is found to catalyse identical reaction as HmfH. This enzyme oxidizes HMF into FDCA with HMF acid as a reaction intermediate. The Kcat of this enzyme on 5-HMF has been estimated to 5.1 mM-1.min-1 [8]. By acting synergically with HmfH, Aldh1 could limit the accumulation of HmfAcid.

An other enzyme that may turn worthwhile for the biosynthesis of FDCA is HmfO (5-(hydroxymethyl)furfural Oxydase). This enzyme is present in Methylovorus sp. and in C. basilensis. HmfO belongs to a C. basilensis HMF14 gene cluster involved in Hmf degradation pathway. HmfO and HmfH are homologous, they both belongs to the GMC (glucose-methanol-choline) oxidoreductase proteins family. The N-ter GMC domain bind to FAD, and release H2O2 as a byproduct [4]. However, unlike HmfH , HmfO needs three successive oxidations of HMF to reach FDCA (fig 1.). Thereby two intermediary compounds are formed, 2,5-Furandicarboxaldehyde (furfural) (DFF) and 5-formyl-2-furancarboxylic acid (FFA) [4].

In order to obtain an efficient whole cell biocatalyst genes coding for the three previously described enzymes have to be under control of a strong promoter. To maximize the expression of these proteins it was planned to put them under control of promoter improved by freiburg 2011 iGem team and characterized by Slovenia HS 2015 iGem team (BBa_K608002).

Genes overexpression could be a drag on bacterial growth. In order to able bacterial culture to reach stationary phase quicker it was planned to use an inducible promoter.The part pBAD strong (BBa_K206000) , registered by iGEM09_British_Columbia, offer a high expression level.

In order to study each proteins activity individually, it was planned to clone each one of them in pSB1-C3 under control of BBa_K608002 (“strong promoter strong RBS”). Moreover, to study their joint activity, an operon structure of HmfH, HmfO and Aldh1 under control of an constitutive and inducible promoter has been designed (fig 3).

See standards biobrick constructions below (click to view it fullscreen)

pSB1C3 + Aldh1 (Standard BioBrick)

pSB1C3 + HmfH (Standard BioBrick)

pSB1C3 + HmfO (Standard BioBrick)