Transcriptome analysis guided metabolic engineering of Bacillus subtilis for riboflavin production.

A comparative transcriptome profiling between a riboflavin-producing Bacillus subtilis strain RH33 and the wild-type strain B. subtilis 168 was performed, complemented with metabolite pool and nucleotide sequence analysis, to rationally identify new targets for improving riboflavin production. The pur operon (purEKBCSQLFMNHD) together with other PurR-regulated genes (glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO) was all down-regulated in RH33, which consequently limited the supply of the riboflavin precursors. As 5-phospho-ribosyl-1(a)-pyrophosphate (PRPP) strongly inhibits the binding of PurR to its targets, it was inferred that the reduced expression of PurR-regulated genes might be caused by a low PRPP pool, which was subsequently confirmed by metabolite analysis. Thus, we selected and co-overexpressed prs and ywlF genes in RH33, which are involved in the biosynthetic pathway of PRPP from ribulose-5-phosphate. This co-amplification led to an elevated PRPP pool and thus the increased transcript abundances of PurR-regulated genes participated in riboflavin precursor biosynthesis. The riboflavin titer was increased by 25% (up to 15 g l(-1)) in fed-batch fermentation.

[1]  R. Losick,et al.  Bacillus Subtilis and Other Gram-Positive Bacteria: Biochemistry, Physiology, and Molecular Genetics , 1993 .

[2]  H. Yamanaka,et al.  Phosphoribosylpyrophosphate synthetase in human erythrocytes: assay and kinetic studies using high-performance liquid chromatography. , 1991, Clinica chimica acta; international journal of clinical chemistry.

[3]  K. San,et al.  Transcriptional response of the terpenoid indole alkaloid pathway to the overexpression of ORCA3 along with jasmonic acid elicitation of Catharanthus roseus hairy roots over time. , 2009, Metabolic engineering.

[4]  G. Stephanopoulos,et al.  Metabolic Engineering: Principles And Methodologies , 1998 .

[5]  Lisbeth Olsson,et al.  An expanded role for microbial physiology in metabolic engineering and functional genomics: moving towards systems biology. , 2002, FEMS yeast research.

[6]  Konstantinos Konstantopoulos,et al.  Application of microarrays to identify and characterize genes involved in attachment dependence in HeLa cells. , 2007, Metabolic engineering.

[7]  Dirk Weuster-Botz,et al.  Scale-down and parallel operation of the riboflavin production process with Bacillus subtilis , 2007 .

[8]  Nicola Zamboni,et al.  The Bacillus subtilis yqjI Gene Encodes the NADP+-Dependent 6-P-Gluconate Dehydrogenase in the Pentose Phosphate Pathway , 2004, Journal of bacteriology.

[9]  M. Ikeda,et al.  Effect of transketolase modifications on carbon flow to the purine-nucleotide pathway in Corynebacterium ammoniagenes , 2001, Applied Microbiology and Biotechnology.

[10]  Hanne Jarmer,et al.  Definition of the Bacillus subtilisPurR Operator Using Genetic and Bioinformatic Tools and Expansion of the PurR Regulon with glyA, guaC,pbuG, xpt-pbuX, yqhZ-folD, and pbuO , 2001, Journal of bacteriology.

[11]  Radhakrishnan Mahadevan,et al.  Geobacter sulfurreducens strain engineered for increased rates of respiration. , 2008, Metabolic engineering.

[12]  Nicola Zamboni,et al.  Screening of Bacillus subtilis transposon mutants with altered riboflavin production. , 2008, Metabolic engineering.

[13]  J. Pronk,et al.  Engineering of Penicillium chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor. , 2009, Metabolic engineering.

[14]  R. Doi,et al.  Overlapping promoters transcribed by bacillus subtilis sigma 55 and sigma 37 RNA polymerase holoenzymes during growth and stationary phases. , 1984, The Journal of biological chemistry.

[15]  P. Nygaard.  Purine and Pyrimidine Salvage Pathways , 1993 .

[16]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[17]  E. Nudler,et al.  The riboswitch control of bacterial metabolism. , 2004, Trends in biochemical sciences.

[18]  J. Revuelta,et al.  Metabolic Engineering of the Purine Pathway for Riboflavin Production in Ashbya gossypii , 2005, Applied and Environmental Microbiology.

[19]  A. Kornberg,et al.  Enzymatic synthesis and properties of 5-phosphoribosylpyrophosphate. , 1955, The Journal of biological chemistry.

[20]  C. Anagnostopoulos,et al.  REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS , 1961, Journal of bacteriology.

[21]  Volker F. Wendisch,et al.  Improving lysine production by Corynebacterium glutamicum through DNA microarray-based identification of novel target genes , 2007, Applied Microbiology and Biotechnology.

[22]  O. Schilling,et al.  Characterization of Riboflavin (Vitamin B2) Transport Proteins from Bacillus subtilis and Corynebacterium glutamicum , 2007, Journal of bacteriology.

[23]  Xueming Zhao,et al.  Over-expression of glucose dehydrogenase improves cell growth and riboflavin production in Bacillus subtilis , 2006, Biotechnology Letters.

[24]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[25]  J. Revuelta,et al.  Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production , 2000, Applied Microbiology and Biotechnology.

[26]  Masaru Tomita,et al.  Dynamic responses of the intracellular metabolite concentrations of the wild type and pykA mutant Escherichia coli against pulse addition of glucose or NH3 under those limiting continuous cultures , 2005 .

[27]  B. Hove-Jensen,et al.  Escherichia coli rpiA gene encoding ribose phosphate isomerase A , 1993, Journal of bacteriology.

[28]  A. Bacher,et al.  GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase are rate-limiting enzymes in riboflavin synthesis of an industrial Bacillus subtilis strain used for riboflavin production , 1999, Journal of Industrial Microbiology and Biotechnology.

[29]  J. Bailey,et al.  Toward a science of metabolic engineering , 1991, Science.

[30]  U. Sauer,et al.  Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis , 1996, Applied and environmental microbiology.

[31]  B. Magasanik,et al.  Synthesis of oxaloacetate in Bacillus subtilis mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex , 1984, Journal of bacteriology.

[32]  Nicola Zamboni,et al.  Reducing maintenance metabolism by metabolic engineering of respiration improves riboflavin production by Bacillus subtilis. , 2003, Metabolic engineering.

[33]  J. Revuelta,et al.  Purine Biosynthesis, Riboflavin Production, and Trophic-Phase Span Are Controlled by a Myb-Related Transcription Factor in the Fungus Ashbya gossypii , 2006, Applied and Environmental Microbiology.

[34]  S. Lee,et al.  Metabolic engineering of Escherichia coli for the production of l-valine based on transcriptome analysis and in silico gene knockout simulation , 2007, Proceedings of the National Academy of Sciences.

[35]  U. Sauer,et al.  The phosphoenolpyruvate carboxykinase also catalyzes C3 carboxylation at the interface of glycolysis and the TCA cycle of Bacillus subtilis. , 2004, Metabolic engineering.

[36]  S. Teshiba,et al.  Identification and characterization of the tktB gene encoding a second transketolase in Escherichia coli K-12 , 1993, Journal of bacteriology.

[37]  J. Perkins,et al.  Riboflavin-overproducing strain of bacterium , 1990 .

[38]  Tao Chen,et al.  Optimization of riboflavin production by recombinant Bacillus subtilis RH44 using statistical designs , 2007, Applied Microbiology and Biotechnology.

[39]  J. Revuelta,et al.  Disruption of the SHM2 gene, encoding one of two serine hydroxymethyltransferase isoenzymes, reduces the flux from glycine to serine in Ashbya gossypii. , 2003, The Biochemical journal.

[40]  J. Revuelta,et al.  Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in Ashbya gossypii , 2008, BMC biotechnology.

[41]  S. Saha,et al.  RNA Expression Analysis Using an AntisenseBacillus subtilis Genome Array , 2001, Journal of bacteriology.

[42]  N. Sandoval,et al.  Parallel mapping of genotypes to phenotypes contributing to overall biological fitness. , 2008, Metabolic engineering.

[43]  H. Zalkin,et al.  Identification of the Bacillus subtilis pur operon repressor. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[44]  A. Goffeau,et al.  The complete genome sequence of the Gram-positive bacterium Bacillus subtilis , 1997, Nature.

[45]  A. Demain,et al.  Biology of industrial microorganisms , 1985 .

[46]  Johannes H. de Winde,et al.  Transcriptome Analysis of a Phenol-Producing Pseudomonas putida S 12 Construct : Genetic and Physiological Basis for Improved Production † , 2007 .

[47]  A. Creuger.  Biotechnology : A Textbook of Industrial Microbiology , 2001 .

[48]  Xin-gang Li,et al.  Strain improvement of Rhizopus oryzae for over-production of l(+)-lactic acid and metabolic flux analysis of mutants , 2004 .

[49]  Seok Jae Lee,et al.  Enhanced Production of Insulin-Like Growth Factor I Fusion Protein in Escherichia coli by Coexpression of the Down-Regulated Genes Identified by Transcriptome Profiling , 2003, Applied and Environmental Microbiology.