Methylotrophic biomass as 2H-labeled substrate for biosynthesis of inosine

Oleg V. Mosin1

1 M. V. Lomonosov State Academy of Fine Chemical Technology, Vernadskogo Prospect 86, Moscow, 117571

 

Abstract

            It was proposed to use the 2H-labeled hydrolysate of RuMP facultative methylotroph Brevibacterium methylicum, obtained from deuterated salt medium dM9 as a substrate for the growth of inosine producing bacterium Bacillus subtilis. The growth of the bacterim was performed via glucose convertion on specially developed medium dHM with 78.5% (m/m) 2H2O and supplimented with 2.5% (m/m) of 2H-labeled methylotrophic hydrolysate. To evaluate the level of deuterium enrichment FAB MS technique was used after the isolation of 2H-labeled inosine. 2H-labeled inosine obtained from dHM medium represented a mixture of molecular species containing various number of included deuterium atoms with different contribution to the enrichment. The level of enrichmet calculated by the presence of most abandant peak of the molecular ion in cluster ((M+H)+ at m/z 274) was estimated as five deuterium atoms, from which three are attributed to ribose and two to hypoxantine.

Keywords: 2H-labeled growth substrates - Bacillus subtilis  - Biosynthesis - 2H-labeled  inosine

Introduction

Nucleosides labeled with deuterium (2H) and other stable isotopes are becoming an indispensable tool for biomedical diagnostic and the investigation of various aspects of the metabolism [1, 2]. Thus inosine which is known as an important intermediate in the synthesis of inosine monophosphate (IMP) is in the focal point of clinical interest in medical diagnostic of heart deceases and in certain medical cases [3, 4]. 

            There are several approaches reported for the preparation of 2H- nucleosides. Chemical synthesis are usually tedious and inefficient. Only by employing mutant forms of bacteria, which can produce a large quantities of the nucleosides when growing of an organism on media containing deuterated substrates, the desired biochemicals can be obtained both with high yields and enrichments. On the microbial production of inosine, there have been many studies so far [5-7].  .

For instance, a certain adenine, histidine and tyrosine auxotrophic mutants derived from Bacillus subtilis have been found to have a remarkable ability to produce a large amount of inosine in the growth medium, and at the present it may be produced on an industrial scale.

             The major disadvantage of production of 2H-nuclesides is difficulty in obtaining the appropriate deuterated growth substrates. One approach to solve this problem is to use the extracts obtained from microorganisms growing on minimal media with 99,9 at.% 2H2O far [8]. Thus, we recently described a facultative methylotrophic bacterium Brevibacterium methylicum, which seems to be an an ideal source for the preparation of uniformelly labeled growth substrates on the basis of its 2H-biomass prepared from 2H2O and [U -2H]MetOH [9, 10]. In this article, we demonstrate the possibility of using the hydrolysates of 2H-labeled biomass of this bacterium as substrates for growing the inosine producing mutant B. subtillis.

           

           

Materials and methods

 

Chemicals

            2H2O (99.9 at.% 2H[1]) was obtained from Russian Scientific Enterprises, Sanct Petersburg and purified by distillation from alkaline permanganate. [U -2H]methanol (95.7 at.% 2H) was from Biophysic Center, Pushino. All other chemicals were of reagent grade.

To create a high isotopic content in growth medium, 2H2O with trade marked isotopic purity 99.9 at.% 2H, was used. However, the deuterium content of used 2H2O verified by NMR was found to be 97 at.% 2H. The water containing salts were several times preliminarily crystallyzed in pure 2H2O and dried in vacuum before using (the true content of deuterium in growth media after the autoclaving was less smaller on 8-10% then isotopic purity of an initial 2H2O.

The bacterial strain

            Adenine, tyrosine and hystidine auxotroph mutant B. subtilis B -3157 capable to produce and accumulate 17 g/liter of inosine during the growth on protonated medium with glucose and yeast extract was employed. The strain was obtained from Russian State Scientific Center for Genetics and Selection of Industrial Microorganisms GNIIGENETIKA. 

 

Preparation of 2H-labeled growth substrates

            The methylotrophic bacterium B. methylicum # 5662 was grown on salt medium dM9 with 93.5% (m/m) 2H2O and 2% (m/m) [U -2H]MetOH in mass culture [11]. Cells were pelleted by centrifugation (2000 g, 10  min), washed once with 2H2O and stored at -14 0C. Periodically, 10 g (wet weight) portions are thawed, suspended in  0.5 N 2HCl solution (in 2H2O) and autoclaved at 1200C for 30 min. After adjusting pH till 7.0-7.2 with potassium hydroxide, the hydrolysate was used as a mixure of 2H-labeled growth substrates for the growth of inosine producing strain.

Media and growth conditions

            The bacterial growth was carried out on FM medium (m/m.%): glucose 12; yeast extract 2.5; ammonium nitrate 3; magnium sulphate 2; chalk 2. The composition of dHM was as the same as FM except dHM was prepared from 2H2O and the hydrolysate of 2H-labeled methylotrophic biomass was added. The media were sterilized by autoclaving at 1200C for 30 min and cooled. Glucose was sterilized separetely in 2H2O solution, and after that added in growth medium. ............