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 Pseudomonas putida KT2440. The combination of this microbial host and heterologous expressed non-heme diiron monooxygenases enabled de novo biosynthesis of 2,5-dimethylpyrazine (2,5-DMP) carboxylic acid and N-oxides as target products. A key intermediate, 2,5-DMP, was obtained by using Pseudomonas putida KT2440Δ6 strain containing six gene deletions in the L-threonine pathway, along with the overexpression of thrA^S345F and tdh from E. coli. 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 (thrB and thrC from P. putida 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 P. putida 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 N-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.