2.5.1. HPLC

The concentration of furanic intermediates was determined as follows. The supernatants of the culture media were obtained by centrifugation of 1.0 mL of bacterial culture in a microfuge at maximal speed (14,000 rpm) for 10 minutes at room temperature. The supernatants were further filter-sterilized using a 0.2-μm filtration unit and stored at −20 ◦C. Concentrations of furan derivatives were determined from sample supernatants by high-performance liquid chromatography (HPLC) on a HPLC System Gold (Beckman) system. The column used was an Anion Exchange ION-300 (ICE-99-9850) (300 × 7.8 mm, Transgenomic, Omaha, NE, USA) operated at 65 ◦C. As eluent H2SO4 5 mN was used at a flow of 0.6 mL·min−1. Furfural and furoic acid were detected at 278 nm and furfuryl alcohol at 217 nm.

*Glucose concentration* was colorimetrically measured by using a commercially available enzymatic test based on the glucose/peroxidase activities (Biosystems, Barcelona, Spain).

*Protein concentration* was determined by the Bradford procedure [29].

## 2.5.2. DNA Manipulation

DNA was sequenced using services provided by Sistemas Genómicos (Valencia, Spain). Genomic DNA was purified from *P. pseudoalcaligenes* CECT5344 cells grown in liquid LB medium at pH 8.5 using the GNome DNA isolation kit (QBIOgene). Plasmid DNA was purified with the Genopure plasmid Midi Kit (Roche) from *E. coli* XL1 *blue* MRF cells grown in liquid cultures of LB media supplemented with the antibiotic used for selection. The *E. coli* XL1 *blue* MRF strain was used to clone recombinant DNA, performing restriction enzyme digestion and ligation as recommended by the manufacturers (Fermentas and Promega, respectively). Plasmids were introduced into *E. coli* XL1 *blue* MRF and *P. pseudoalcaligenes* CECT5344 cells by electroporation, as described previously [27]. Mutagenesis of *edd* gene (BN5\_3048): To amplify by PCR a section of the *edd* ORF based on the DNA sequence of the *P. pseudoalcaligenes* CECT5344 (GenBank accession no. JN408065), two couples of sets of specific primers flanking the *edd* gene were designed (edd9U /edd730L and edd1140U /edd1737L, Table 1). A BamHI restriction site was incorporated into edd730L and edd1140U primers. The amplified fragments were cloned separately into pGEM-T Easy vector (Promega) generating the p*edd*<sup>1</sup> and the p*edd*<sup>2</sup> plasmids. The p*edd*<sup>2</sup> plasmid was linearized with ApaI and BamHI. The plasmid p*edd*<sup>1</sup> was digested with the same enzymes and the resulting fragment was gel-purified and subcloned into p*edd*<sup>2</sup> to generate p*edd*1-2, thus obtaining a PGEM-TE derivative plasmid with two internal sequences of *edd* gene separated by a BamHI restriction site. On the other hand, a 1.0 kb BamHI fragment from the pMS255 plasmid [30] containing the gentamicin resistance gene (*aacC1*) was cloned into BamHI-digested PGEM-TE plasmid containing the two internal *edd* fragments (Figure S1). Finally, the resulting suicide plasmid was transferred to *P. pseudoalcaligenes* CECT5344 R1D by electroporation. The mutants were selected on gentamicin (10 μg/mL, Sigma-Aldrich) and mutant strains resulting from double homologous recombination were isolated. The insertion of the gentamicin resistance gene and the loss of the plasmid backbone were confirmed by PCR. Mutagenesis *araC* gene (BN5\_2307): Based on the DNA sequence of the *P. pseudoalcaligenes* CECT5344 (GenBank accession no. JN408065), one couple of specific primers (araC157U/araC*823*L, Table 1) flanking the *ara*C gene was designed for amplification of a section of the *ara*C ORF by PCR. PCR was performed and the amplified fragment that contained a KpnI restriction site at position 542 was cloned into pGEM-T Easy vector (Promega), thus obtaining a PGEM-TE derivative plasmid with one internal sequence of *ara*C. The plasmid was digested with KpnI and ligated to a 1.0-kb KpnI fragment containing the gentamicin-resistance gene (*aac*C1) from the pMS255 plasmid [30]. (Figure S2). The resulting plasmid was transferred to *P. pseudoalcaligenes* CECT5344 R1D by electroporation. The mutants were selected on gentamicin (10 μg/mL, Sigma-Aldrich), and mutant strains resulting from double-homologous recombination were isolated. The insertion of the gentamicin resistance gene and the loss of the plasmid backbone were confirmed by PCR.


**Table 1.** Primers utilized in this work.

#### 2.5.3. PCR Reaction Conditions

PCR samples were prepared with the following components: 0.5 ng/μ<sup>L</sup> DNA, 1.0 μM of each primer, 2.0 mM MgCl2, 0.2 mM each deoxynucleoside triphosphate (dNTP), 0.5 U Taq DNA polymerase, and 2 μL buffer as recommended by the manufacturer (Biotools), in a final volume of 20 μL. The PCR conditions were 2 min at 95 ◦C; followed by 30 cycles of 20 s at 95 ◦C, 10 s at 65.6 ◦C (*edd*) or 64 ◦C (*ara*C) and 1 min at 72 ◦C, followed by a 5 min extension at 72 ◦C.
