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

K.V. Lobanov, L. Errais Lopes, N.V. Korol’kova, B.V. Tyaglov, A.V. Glazunov, R.S. Shakulov, A.S. Mironov*State Research Institute for Genetics and Selection of Industrial Microorganisms *E-mail: alexmir@genetika.ruReceived 24.02.2011ABSTRACT 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 in Bacillus subtilis cells is con-trolled by the enzymes of purine biosynthesis; their genes constituting purine operon (pur-operon). Reconstruc-tion of purine metabolism in B. subtilis was performed to achieve overproduction of AICAR. For this purpose, the gene purH, which encodes formyltransferase/IMP-cyclohydrolase, an enzyme that controls the conversion of AICAR to IMP, was removed from the B. subtilis genome, ensuring the accumulation of AICAR. An insertion inactivating the gene purR that encodes the negative transcriptional regulator of the purine biosynthesis operon was introduced into the B.subtilis chromosome in order to boost the production of AICAR; the transcription attenuator located in the leader sequence of pur-operon was deleted. Furthermore, the expression integrative vector carrying a strong promoter of the rpsF gene encoding the ribosomal protein S6 was designed. The heter-ologous Escherichia coli gene purF encoding the first enzyme of the biosynthesis of purines with impaired al-losteric regulation, as well as the modified E.coli gene prs responsible for the synthesis of the precursor of pu-rines — phosphoribosyl pyrophosphate (PRPP) — was cloned into this vector under the control of the rpsF gene promoter. The modified purF and prs genes were inserted into the chromosome of the B. subtilis strain. B. subtilis strain obtained by these genetic manipulations accumulates 11–13 g/L of AICAR in the culture fluid. kEYWORDS anticancer agent AICAR; purine metabolism; genome reconstruction; Bacillus subtilis strain - pro-ducer of AICARABBREVIATIONS AICAR – 5-aminoimidazole-4-carboxamide ribofuranoside; AICAR-P – nucleotide AICAR phosphate; IMP – inosine monophosphate; AMP – adenosine monophosphate; АМPK – 5’-adenosine monophos-phate-activated protein kinase; PRPP – phosphoribosyl pyrophosphate; GAR – 5’- phosphoribosyl glycineamide ribonucleotide; GMP – guanosine monophosphate; PCR – polymerase chain reaction; CF – culture fluidINTRODUCTIONDespite the fact that the structural organization of the genes encoding the enzymes of purine nucleotide bi-osynthesis is quite versatile, the biochemistry of the process is conservative for different organisms: the formation of the purine cycle occurs on the basis of a riboso-5-phosphate (all intermediates are nucleotides) using a monocarbon component (formiate and/or n10-formyltetrahydrofolate) [1]. there is demand for mono-carbon compounds at two stages of purine biosynthesis; therefore, the precursors – phosphoribosylglycinea-mide ribonucleoside (GAr) and 5-aminoimidazole-4-carboxamide ribonucleoside (AIcAr-P) can be accu-mulated if there is a deficiency in these compounds. Among them, AIcAr-P occupies a specific place, since its formulation and subsequent cyclization crown the formation of the purine heterocycle that yields inosine monophosphate (IMP) (Fig. 1). the process of conver-sion of AIcAr-P into IMP in prokaryotic cells is con-trolled by the gene purH, which encodes two domains with the activities of AIcAr-P-formyltransferase and IMP cyclohydrolase [2, 3]. Further modifications of IMP yield AMP and GMP.Despite the fact that the structure of purine hetero-cycle is incomplete, AIcAr-P is a natural analogue of AMP, substituting it in certain enzymatic in vitro reac-tions. the possibility that AMP could be substituted in the reactions of activation of AMP-activated proteinki-nase (AMPK) in mammals has been given a significant degree of attention over the past decade. AMPK is