Genes and expression plasmids

In order to express Route 1 as the target metabolic pathway, the synthetic operon developed for P. putida needed to express genes responsible for the conversion of 3-dehydroshikimate (DHS) to protocatechuate (PCA) and then of PCA to GA (Fig. 1). According to a patent that claimed the bacterial production of muconic acid from PCA (Bui et al. 2013), the 3-dehydroshiquimate dehydratase (QuiC) found in Acinetobacter baumannii had demonstrated a superior performance compared to the orthologous versions of other organisms, such as the one from K. baumannii expressed in E. coli by Kambourakis et al. (2000) (Frost 2000) for the first reaction. However, the conversion of PCA to GA was only possible with a mutant version (Tyr385Phe) of the enzyme p-hydroxybenzoate hydroxylase (PobA*) from Pseudomonas aeruginosa, which hydroxylates PCA instead of 4-hydroxybenzoate (4HB) (Kambourakis et al. 2000; Muir et al. 2011; Moriwaki et al. 2019). These two reactions would be enough for the proposed operon; however, in many organisms the condensation of E4P and PEP in 3-deoxy-D-arabino-heptulosonate 7-phosfate (DAHP) is catalyzed by the enzyme phospho-2-dehydro-3-deoxyheptonate aldolase in different versions that can be inhibited by phenylalanine (AroG), tyrosine (AroF) or tryptophan (AroH). So, to avoid this inhibition, it was also necessary to express a mutant version of the gene aroG, called aroG4 (Pro150Leu), insensitive to phenylalanine (Doroshenko et al. 2010).

The gene quiC was obtained from genomic DNA of A. baumannii ATCC 19,606, pobA was obtained from genomic DNA of P. aeruginosa ATCC 9027 and aroG was obtained from genomic DNA of E. coli K12 MG1655, using Polymerase Chain Reaction (PCR) (Saiki et al. 1988). The genomic DNAs were obtained with the Wizard® Genomic DNA Purification Kit (Promega). All primers used to isolate these genes through PCR are in the Table S4. For the gene quiC, the leading strand primer was designed to contain a conserved Pseudomonas ribosomal binding site (RBS), used in vectors constructed by Silva-Rocha et al. (2013), along with an EcoRI restriction site, while its reverse primer received an SmaI restriction site. To isolate the pobA* gene from a genomic DNA containing its parental strain version, the leading strand primer had the same RBS and a SmaI restriction site, while the reverse primer had the nucleotide exchange (T—> A) that would lead to the desired mutation (Tyr385Phe), in addition to a BcuI restriction site. Thus, both genes were amplified and purified using the Wizard® SV Gel and PCR Clean-Up System (Promega), subjected to the corresponding restriction enzymes FastDigest (Thermo Fischer™) and then ligated to the expression vector pJN105 (named here as J0) (Newman and Fuqua 1999) with the aid of the enzyme T4 DNA Ligase (Thermo Fischer™), starting with the insertion of quiC to form the JQ plasmid and, then, pobA* to form the JQP plasmid (Fig. 2).

Maps of the expression plasmids JQP (A) and JAQP (B) done with SnapGene (www.snapgene.com/). Highlighted are the upstream and downstream homologous sequences constructed to clone the aroG4 gene in the JQP plasmid (H1, in light blue and H2, in dark blue)

To clone aroG4, the One-Step Sequence-and-Ligase-Independent Cloning (SLIC) method was applied (Jeong et al. 2012), in which the E. coli K12 MG1655 aroG gene was isolated in the form of two fragments, thereby using four primers with ends that were homologous so they could anneal with each other and vector JQP, which was digested with the enzyme EcoRI. To create cohesive ends for that, the three fragments were submitted to the exonucleolytic action 3'-5 'of the enzyme T4 DNA Polymerase (New England Biolabs®). The result of this annealing was introduced in E. coli DH10B by thermal shock and the bacterium repair system itself made the necessary covalent bonds to joint all fragments in a final plasmid. Both the reverse primer of the first fragment of aroG, and the primer of the leading strand of the second fragment were designed with the point mutation (C—> T), so the aroG4 version (Pro150Leu) could be inserted in the plasmid JQP, resulting in the JAQP version (Fig. 2). The set of expression plasmids constructed is summarized on Table ​Table11.

All genes were isolated by PCR, using the high-fidelity enzymes Phusion® High-Fidelity DNA Polymerase (NEB) or Q5® High-Fidelity DNA Polymerase (NEB). In addition, PCR tests conducted to verify the cloning process used the GoTaq® Green Master Mix kit (Promega). All amplifications were validated with electrophoresis in 0.7% agarose gel (Figure S1) and after each amplification and digestion all amplicons were purified with the Wizard® SV Gel and PCR Clean-Up System (Promega). Furthermore, all plasmids cloned in E. coli DH10B were purified with Wizard® Plus SV Minipreps DNA Purification System (Promega). All primers and maps were designed using the software SnapGene (www.snapgene.com) and NEB Builder (nebuilder.neb.com). Furthermore, plasmid JAQP was sequenced according to the Colson and Sanger technique (Sanger and Coulson 1975). All primers used for sequencing are in Table S4.