Metabolic Engineering of the Purine Biosynthetic Pathway in Corynebacterium Glutamicum Results in Increased Intracellular Pool Sizes of IMP and Hypoxanthine

Overview

Journal Microb Cell Fact

Publisher Biomed Central

Specialties Biotechnology
Microbiology

Date 2012 Oct 25

PMID 23092390

Citations 15

Authors

Susanne Peifer

Tobias Barduhn

Sarah Zimmet

Dietrich A Volmer

Elmar Heinzle

Konstantin Schneider

Affiliations

Soon will be listed here.

Abstract

Background: Purine nucleotides exhibit various functions in cellular metabolism. Besides serving as building blocks for nucleic acid synthesis, they participate in signaling pathways and energy metabolism. Further, IMP and GMP represent industrially relevant biotechnological products used as flavor enhancing additives in food industry. Therefore, this work aimed towards the accumulation of IMP applying targeted genetic engineering of Corynebacterium glutamicum.

Results: Blocking of the degrading reactions towards AMP and GMP lead to a 45-fold increased intracellular IMP pool of 22 μmol g(CDW)⁻¹. Deletion of the pgi gene encoding glucose 6-phosphate isomerase in combination with the deactivated AMP and GMP generating reactions, however, resulted in significantly decreased IMP pools (13 μmol g(CDW)⁻¹). Targeted metabolite profiling of the purine biosynthetic pathway further revealed a metabolite shift towards the formation of the corresponding nucleobase hypoxanthine (102 μmol g(CDW)⁻¹) derived from IMP degradation.

Conclusions: The purine biosynthetic pathway is strongly interconnected with various parts of the central metabolism and therefore tightly controlled. However, deleting degrading reactions from IMP to AMP and GMP significantly increased intracellular IMP levels. Due to the complexity of this pathway further degradation from IMP to the corresponding nucleobase drastically increased suggesting additional targets for future strain optimization.

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References

Jager W, Schafer A, Puhler A, Labes G, Wohlleben W . Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans. J Bacteriol. 1992; 174(16):5462-5. PMC: 206388. DOI: 10.1128/jb.174.16.5462-5465.1992. View

Wittmann C, Heinzle E . Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria. Appl Environ Microbiol. 2002; 68(12):5843-59. PMC: 134428. DOI: 10.1128/AEM.68.12.5843-5859.2002. View

Li H, Zhang G, Deng A, Chen N, Wen T . De novo engineering and metabolic flux analysis of inosine biosynthesis in Bacillus subtilis. Biotechnol Lett. 2011; 33(8):1575-80. DOI: 10.1007/s10529-011-0597-5. View

Kamada N, Yasuhara A, Takano Y, Nakano T, Ikeda M . Effect of transketolase modifications on carbon flow to the purine-nucleotide pathway in Corynebacterium ammoniagenes. Appl Microbiol Biotechnol. 2001; 56(5-6):710-7. DOI: 10.1007/s002530100738. View

Yang T, Frick O, Heinzle E . Hybrid optimization for 13C metabolic flux analysis using systems parametrized by compactification. BMC Syst Biol. 2008; 2:29. PMC: 2333969. DOI: 10.1186/1752-0509-2-29. View

Wittmann C . Fluxome analysis using GC-MS. Microb Cell Fact. 2007; 6:6. PMC: 1805451. DOI: 10.1186/1475-2859-6-6. View

Marx A, De Graaf A, Wiechert W, Eggeling L, Sahm H . Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing. Biotechnol Bioeng. 1996; 49(2):111-29. DOI: 10.1002/(SICI)1097-0290(19960120)49:2<111::AID-BIT1>3.0.CO;2-T. View

Messenger L, ZALKIN H . Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties. J Biol Chem. 1979; 254(9):3382-92. View

Furuya A, Abe S, Kinoshita S . Production of nucleic acid-related substances by fermentation processes. 33. Accumulation of inosine by a mutant of Brevibacterium ammoniagenes. Appl Microbiol. 1970; 20(2):263-70. PMC: 376913. DOI: 10.1128/am.20.2.263-270.1970. View

10.

Guimaraes P, Londesborough J . The adenylate energy charge and specific fermentation rate of brewer's yeasts fermenting high- and very high-gravity worts. Yeast. 2007; 25(1):47-58. DOI: 10.1002/yea.1556. View

11.

Halpern B . What's in a name? Are MSG and umami the same?. Chem Senses. 2002; 27(9):845-6. DOI: 10.1093/chemse/27.9.845. View

12.

Stephanopoulos G, Vallino J . Network rigidity and metabolic engineering in metabolite overproduction. Science. 1991; 252(5013):1675-81. DOI: 10.1126/science.1904627. View

13.

Mascarenhas D, Ashworth D, Chen C . Deletion of pgi alters tryptophan biosynthesis in a genetically engineered strain of Escherichia coli. Appl Environ Microbiol. 1991; 57(10):2995-9. PMC: 183910. DOI: 10.1128/aem.57.10.2995-2999.1991. View

14.

Becker J, Klopprogge C, Zelder O, Heinzle E, Wittmann C . Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources. Appl Environ Microbiol. 2005; 71(12):8587-96. PMC: 1317465. DOI: 10.1128/AEM.71.12.8587-8596.2005. View

15.

van Winden W, Wittmann C, Heinzle E, Heijnen J . Correcting mass isotopomer distributions for naturally occurring isotopes. Biotechnol Bioeng. 2002; 80(4):477-9. DOI: 10.1002/bit.10393. View

16.

Villas-Boas S, Hojer-Pedersen J, Akesson M, Smedsgaard J, Nielsen J . Global metabolite analysis of yeast: evaluation of sample preparation methods. Yeast. 2005; 22(14):1155-69. DOI: 10.1002/yea.1308. View

17.

Zhou G, Smith J, ZALKIN H . Binding of purine nucleotides to two regulatory sites results in synergistic feedback inhibition of glutamine 5-phosphoribosylpyrophosphate amidotransferase. J Biol Chem. 1994; 269(9):6784-9. View

18.

Matsui H, Kawasaki H, Shimaoka M, Kurahashi O . Investigation of various genotype characteristics for inosine accumulation in Escherichia coli W3110. Biosci Biotechnol Biochem. 2001; 65(3):570-8. DOI: 10.1271/bbb.65.570. View

19.

Kromer J, Fritz M, Heinzle E, Wittmann C . In vivo quantification of intracellular amino acids and intermediates of the methionine pathway in Corynebacterium glutamicum. Anal Biochem. 2005; 340(1):171-3. DOI: 10.1016/j.ab.2005.01.027. View

20.

Matsui H, Shimaoka M, Takenaka Y, Kawasaki H, Kurahashi O . gsk disruption leads to guanosine accumulation in Escherichia coli. Biosci Biotechnol Biochem. 2001; 65(5):1230-5. DOI: 10.1271/bbb.65.1230. View