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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.

acta naturae

Reconstruction of Purine Metabolism in Bacillus subtilis to Obtain the Strain Producer of AICAR: A New Drug with a Wide Range of Therapeutic Applications