State Research Institute for Genetics and Selection of Industrial Microorganisms.
Acta Naturae. 2011 Apr;3(2):79-89.
AICAR is a natural compound, an analogue and precursor of adenosine. As activator of AMP-activated protein kinase (AMPK), AICAR has a broad therapeutic potential, since it normalizes the carbohydrate and lipid metabolism and inhibits the proliferation of tumor cells. The synthesis of AICAR inBacillus subtiliscells is controlled by the enzymes of purine biosynthesis; their genes constituting purine operon (pur-operon). Reconstruction of purine metabolism inB. subtiliswas performed to achieve overproduction of AICAR. For this purpose, the genepurH, which encodes formyltransferase/IMP-cyclohydrolase, an enzyme that controls the conversion of AICAR to IMP, was removed from theB. subtilisgenome, ensuring the accumulation of AICAR. An insertion inactivating the genepurRthat encodes the negative transcriptional regulator of the purine biosynthesis operon was introduced into theB.subtilischromosome in order to boost the production of AICAR; the transcription attenuator located in the leader sequence ofpur-operon was deleted. Furthermore, the expression integrative vector carrying a strong promoter of therpsFgene encoding the ribosomal protein S6 was designed. The heterologousEscherichia coligenepurFencoding the first enzyme of the biosynthesis of purines with impaired allosteric regulation, as well as the modifiedE.coligeneprsresponsible for the synthesis of the precursor of purines - phosphoribosyl pyrophosphate (PRPP) - was cloned into this vector under the control of therpsFgene promoter. The modifiedpurFandprsgenes were inserted into the chromosome of theB. subtilisstrain.B. subtilisstrain obtained by these genetic manipulations accumulates 11-13 g/L of AICAR in the culture fluid.
AICAR 是一种天然化合物，是腺苷的类似物和前体。作为 AMP 激活的蛋白激酶（AMPK）的激活剂，AICAR 具有广泛的治疗潜力，因为它可以使碳水化合物和脂质代谢正常化，并抑制肿瘤细胞的增殖。枯草芽孢杆菌细胞中 AICAR 的合成受嘌呤生物合成酶的控制；它们的基因构成嘌呤操纵子（pur-operon）。为了实现 AICAR 的过量生产，对枯草芽孢杆菌中的嘌呤代谢进行了重建。为此，从枯草芽孢杆菌基因组中去除了编码甲酰基转移酶/IMP 环化水解酶的基因 purH，该酶控制 AICAR 向 IMP 的转化，从而确保 AICAR 的积累。引入了一个插入失活的基因 purR，该基因编码嘌呤生物合成操纵子的负转录调节剂，以提高 AICAR 的产量；删除了位于 pur-operon 启动子序列中的转录衰减子。此外，设计了携带编码核糖体蛋白 S6 的强启动子 therpsF 基因的表达整合载体。异源大肠杆菌基因 purF 编码嘌呤生物合成的第一酶，其变构调节受损，以及修饰的大肠杆菌基因 prs 负责合成嘌呤的前体 - 磷酸核糖焦磷酸（PRPP）- 在 therpsF 基因启动子的控制下被克隆到该载体中。修饰的 purF 和 prs 基因被插入枯草芽孢杆菌菌株的染色体中。通过这些遗传操作获得的枯草芽孢杆菌菌株在培养液中积累 11-13 g/L 的 AICAR。
