Synthetic pathways for microbial biosynthesis of valuable pyrazine derivatives using genetically modified Pseudomonas putida KT2440

Using engineered microbes for synthesizing high-valued chemicals from renewable sources is a foundation in synthetic biology, however, it is still in its early stages. Here, we present peculiarities and troubleshooting of the construction of novel synthetic metabolic pathways in genetically modified work-horse <i>Pseudomonas putida</i> KT2440. The combination of this microbial host and heterologous expressed non-heme diiron monooxygenases enabled <i>de novo</i> biosynthesis of 2,5-dimethylpyrazine (2,5-DMP) carboxylic acid and <i>N</i>-oxides as target products. A key intermediate, 2,5-DMP, was obtained by using <i>Pseudomonas putida</i> KT2440Δ6 strain containing six gene deletions in the L-threonine pathway, along with the overexpression of <i>thrA</i> ^<i>S345F</i> and <i>tdh</i> from <i>E. coli</i>. Thus, the carbon surplus was redirected from glucose through L-threonine metabolism toward the formation of 2,5-DMP, resulting in a product titre of 106 ± 30 mg L^-1. By introducing two native genes (<i>thrB</i> and <i>thrC</i> from <i>P. putida</i> KT2440) from the L-threonine biosynthesis pathway, the production of 2,5-DMP was increased to 168 ± 20 mg L^-1. The resulting 2,5-DMP was further derivatized through two separate pathways. Recombinant <i>P. putida</i> KT2440 strain harboring xylene monooxygenase (XMO) produced 5-methyl-2-pyrazinecarboxylic acid from glucose as a targeted compound in a product titre of 204 ± 24 mg L^-1. The microbial host containing genes of PmlABCDEF monooxygenase (Pml) biosynthesized <i>N</i>-oxides - 2,5-dimethylpyrazine 1-oxide as a main product, and 2,5-dimethylpyrazine 1,4-dioxide as a minor product, reaching product titres of 82 ± 8 mg L^-1 and 11 ± 2 mg L^-1 respectively.