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

Date Published: , 2011

Publisher: A.I. Gordeyev

Author(s): K.V. Lobanov, L. Errais Lopes, N.V. Korol’kova, B.V. Tyaglov, A.V. Glazunov, R.S. Shakulov, A.S. Mironov.

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Abstract

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.

Partial Text

Despite the fact that the structural organization of the genes encoding the enzymes of purine nucleotide biosynthesis 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 monocarbon compounds at two stages of purine biosynthesis; therefore, the precursors – phosphoribosylglycineamide ribonucleoside (GAR) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR-P) can be accumulated 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 conversion of AICAR-P into IMP in prokaryotic cells is controlled 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.

Bacterial strains and plasmids

Enhancement of the expression of the

The strain-producer of AICAR, a new drug with potentially wide therapeutic applications, was obtained in studies based on B. subtilis bacteria. The strategy obtaining the AICAR producing strain is based on the directed reconstruction of purine metabolism in B. subtilis cells. At the first stage of the study, an insertion was introduced into the purR gene encoding the pur -operon repressor protein, and the transcription attenuator was removed from the leader region of the pur -operon, ensuring maximum derepression of the enzymes of de novo purine biosynthesis. The purH gene encoding formyltransferase/IMP-cyclohydrolase was then removed from the bacterial genome. Inactivation of this enzyme disturbs the reaction of AICAR conversion into IMP and results in its accumulation in the cell. At the next stage, the site-directed mutagenesis of prs and purFE. coli genes encoding the key enzymes of the synthesis of purine precursors was carried out in order to obtain mutant variants of these genes that would not be susceptible to retroinhibition by purine nucleotides. Finally, at the last stage, the modified prs and purFE. coli geneswere integrated into the B. subtilis chromosome under the control of a strong promoter ensuring a high level of expression of these genes in B. subtilis cells. As a result, we obtained a producing strain accumulating 11–13 g/L of AICAR in CL.

 

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