Hong Wenrong, Yan Lingbin
College of Biological Science and Technology, Fuzhou University, Fujian, China.
J Gen Appl Microbiol. 2012;58(5):349-56. doi: 10.2323/jgam.58.349.
Gentamicin and sisomicin are two different aminoglycoside antibiotics. The comparison of their chemical structure and biosynthetic gene clusters, coupled with bioinformatic analysis, suggested that the gntK gene would be associated with methylation. The gntK gene fragment in M. purpurea G1008 was inactivated by genetic engineering and its mutant strain M. purpurea GK1101 (ΔgntK) was screened. The metabolites of G1008 and GK1101 was analyzed by HPLC-MS, which revealed that GK1101 no longer produced gentamicin C(1) or C(2), while mainly synthesizing gentamicin C(1a), and the production of C(1a) increased significantly. This indicated that the metabolic flow for the gentamicin C(1) and C(2) biosynthesis was blocked by disrupting the gntK gene, which substantiated that the gntK gene encoded the enzyme that catalyzes the methylation of purpurosamine C-6'. The mutant GK1101 has good prospects for industrial application. In addition, our study provides information that can be used to clarify the function of a single gene and simplify the targeted genetic breeding of important pharmaceutical microorganisms.
庆大霉素和西索米星是两种不同的氨基糖苷类抗生素。对它们的化学结构和生物合成基因簇进行比较，并结合生物信息学分析，表明gntK基因与甲基化有关。通过基因工程使紫色链霉菌G1008中的gntK基因片段失活，并筛选出其突变株紫色链霉菌GK1101（ΔgntK）。通过高效液相色谱-质谱联用（HPLC-MS）分析G1008和GK1101的代谢产物，结果显示GK1101不再产生庆大霉素C(1)或C(2)，而是主要合成庆大霉素C(1a)，且C(1a)的产量显著增加。这表明破坏gntK基因阻断了庆大霉素C(1)和C(2)生物合成的代谢流，证实gntK基因编码催化紫霉素胺C-6'甲基化的酶。突变株GK1101具有良好的工业应用前景。此外，我们的研究提供了可用于阐明单个基因功能并简化重要药用微生物靶向遗传育种的信息。
