Petkevičius Vytautas, Juknevičiūtė Justė, Mašonis Domas, Meškys Rolandas
Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Saulėtekio 7, Vilnius, LT-10257, Lithuania.
Metab Eng Commun. 2025 Mar 30;20:e00258. doi: 10.1016/j.mec.2025.e00258. eCollection 2025 Jun.
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 KT2440. The combination of this microbial host and heterologous expressed non-heme diiron monooxygenases enabled biosynthesis of 2,5-dimethylpyrazine (2,5-DMP) carboxylic acid and -oxides as target products. A key intermediate, 2,5-DMP, was obtained by using KT2440Δ6 strain containing six gene deletions in the L-threonine pathway, along with the overexpression of and from . 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. By introducing two native genes ( and from KT2440) from the L-threonine biosynthesis pathway, the production of 2,5-DMP was increased to 168 ± 20 mg L. The resulting 2,5-DMP was further derivatized through two separate pathways. Recombinant 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. The microbial host containing genes of PmlABCDEF monooxygenase (Pml) biosynthesized -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 and 11 ± 2 mg L respectively.
利用工程微生物从可再生资源合成高价值化学品是合成生物学的一个基础领域，然而，它仍处于早期阶段。在此，我们介绍了在基因改造的主力菌株KT2440中构建新型合成代谢途径的特点及故障排除方法。这种微生物宿主与异源表达的非血红素双铁单加氧酶的组合使得能够生物合成2,5 - 二甲基吡嗪（2,5 - DMP）羧酸及其氧化物作为目标产物。通过使用在L - 苏氨酸途径中含有六个基因缺失的KT2440Δ6菌株，以及来自[具体来源]的[具体基因]的过表达，获得了关键中间体2,5 - DMP。因此，碳盈余从葡萄糖通过L - 苏氨酸代谢重新导向2,5 - DMP的形成，产物滴度达到106 ± 30 mg/L。通过引入来自L - 苏氨酸生物合成途径的两个天然基因（来自KT2440的[具体基因]），2,5 - DMP的产量增加到168 ± 20 mg/L。所得的2,5 - DMP通过两条独立途径进一步衍生化。携带二甲苯单加氧酶（XMO）的重组KT2440菌株从葡萄糖产生5 - 甲基 - 2 - 吡嗪羧酸作为目标化合物，产物滴度为204 ± 24 mg/L。含有PmlABCDEF单加氧酶（Pml）基因的微生物宿主生物合成了氧化物——以2,5 - 二甲基吡嗪1 - 氧化物为主要产物，2,5 - 二甲基吡嗪1,4 - 二氧化物为次要产物，产物滴度分别达到82 ± 8 mg/L和11 ± 2 mg/L。
