Kataoka Naoya, Hirata Kaori, Matsutani Minenosuke, Ano Yoshitaka, Nguyen Thuy Minh, Adachi Osao, Matsushita Kazunobu, Yakushi Toshiharu
Faculty of Agriculture, Yamaguchi University, Yamaguchi, 753-8515, Japan.
Graduate School of Science and Technology for Innovation, Yamaguchi University, Yamaguchi, 753-8515, Japan.
Appl Microbiol Biotechnol. 2021 Feb;105(3):1227-1236. doi: 10.1007/s00253-021-11092-6. Epub 2021 Jan 21.
Dihydroxyacetone (DHA), a chemical suntan agent, is produced by the regiospecific oxidation of glycerol with Gluconobacter thailandicus NBRC3255. However, this microorganism consumes DHA produced in the culture medium. Here, we attempted to understand the pathway for DHA metabolism in NBRC3255 to minimize DHA degradation. The two gene products, NBRC3255_2003 (DhaK) and NBRC3255_3084 (DerK), have been annotated as DHA kinases in the NBRC 3255 draft genome. Because the double deletion derivative for dhaK and derK showed ATP-dependent DHA kinase activity similar to that of the wild type, we attempted to purify DHA kinase from ∆dhaK ∆derK cells to identify the gene for DHA kinase. The identified gene was NBRC3255_0651, of which the product was annotated as glycerol kinase (GlpK). Mutant strains with several combinations of deletions for the dhaK, derK, and glpK genes were constructed. The single deletion strain ∆glpK showed approximately 10% of wild-type activity and grew slower on glycerol than the wild type. The double deletion strain ∆derK ∆glpK and the triple deletion strain ∆dhaK ∆derK ∆glpK showed DHA kinase activity less than a detection limit and did not grow on glycerol. In addition, although ΔderK ΔglpK consumed a small amount of DHA in the late phase of growth, ∆dhaK ΔderK ΔglpK did not show DHA consumption on glucose-glycerol medium. The transformants of the ∆dhaK ΔderK ΔglpK strain that expresses one of the genes from plasmids showed DHA kinase activity. We concluded that all three DHA kinases, DhaK, DerK, and GlpK, are involved in DHA metabolism of G. thailandicus. KEY POINTS: • Dihydroxyacetone (DHA) is produced but degraded by Gluconobacter thailandicus. • Phosphorylation rather than reduction is the first committed step in DHA metabolism. • Three kinases are involved in DHA metabolism with the different properties.
二羟基丙酮（DHA）是一种化学防晒剂，由泰国葡糖杆菌NBRC3255对甘油进行区域特异性氧化产生。然而，这种微生物会消耗培养基中产生的DHA。在此，我们试图了解NBRC3255中DHA的代谢途径，以尽量减少DHA的降解。在NBRC 3255草图基因组中，两个基因产物NBRC3255_2003（DhaK）和NBRC3255_3084（DerK）已被注释为DHA激酶。由于dhaK和derK的双缺失衍生物显示出与野生型相似的ATP依赖性DHA激酶活性，我们试图从∆dhaK ∆derK细胞中纯化DHA激酶，以鉴定DHA激酶的基因。鉴定出的基因是NBRC3255_0651，其产物被注释为甘油激酶（GlpK）。构建了dhaK、derK和glpK基因几种缺失组合的突变菌株。单缺失菌株∆glpK显示出约10%的野生型活性，在甘油上的生长速度比野生型慢。双缺失菌株∆derK ∆glpK和三缺失菌株∆dhaK ∆derK ∆glpK的DHA激酶活性低于检测限，在甘油上无法生长。此外，尽管ΔderK ΔglpK在生长后期消耗少量DHA，但∆dhaK ΔderK ΔglpK在葡萄糖-甘油培养基上未显示出DHA消耗。表达质粒中一个基因的∆dhaK ΔderK ΔglpK菌株的转化体显示出DHA激酶活性。我们得出结论，所有三种DHA激酶DhaK、DerK和GlpK都参与了泰国葡糖杆菌的DHA代谢。要点：• 二羟基丙酮（DHA）由泰国葡糖杆菌产生但会被其降解。• 磷酸化而非还原是DHA代谢的第一个关键步骤。• 三种激酶参与DHA代谢，具有不同特性。
