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

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.

Dynamic Control Strategy to Produce Riboflavin with Lignocellulose Hydrolysate in the Thermophile Geobacillus thermoglucosidasius

“…(II) phosphoribosyl pyrophosphate (PRPP) biosynthesis module converts Xu5P to PRPP, in which the gene prs encoding the phosphoribosylpyrophosphate synthetase is regulated by feedback inhibition of downstream metabolites, such as ADP and GDP 35,36 . (III) purine biosynthesis module is responsible for the conversion of PRPP to GTP 17,37 . This module contains the genes of the pur operon and other downstream genes.…”

A dynamic control strategy to produce riboflavin with lignocellulose hydrolysate in the thermophile Geobacillus thermoglucosidasius

“…(II) phosphoribosyl pyrophosphate (PRPP) biosynthesis module converts Xu5P to PRPP, in which the gene prs encoding the phosphoribosylpyrophosphate synthetase is regulated by feedback inhibition of downstream metabolites, such as ADP and GDP 35,36 . (III) purine biosynthesis module is responsible for the conversion of PRPP to GTP 17,37 . This module contains the genes of the pur operon and other downstream genes.…”

“…This module contains the genes of the pur operon and other downstream genes. Particularly, the 12 genes from the pur operon are organized as a cluster, whose transcription is strictly regulated by the repressor PurR [37][38][39][40][41] . (IV) ribo avin biosynthesis module (the rib cluster) that combines Ru5P and GTP into ribo avin is regulated by the downstream product FMN 8,36,42 .…”

Acadesine Triggers Non-apoptotic Death in Tumor Cells

“…Acadesine was obtained at the State Research Institute of Genetics and Selection of Industrial Microorganisms via the microbiological procedure using an original recombinant strain [14]. Moreover, the cytotoxicity of acadesine purchased from Sigma was assessed.…”