Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation.

An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.

[1]  C. Khosla,et al.  Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli. , 2002, Biochemistry.

[2]  I. Blomfield,et al.  Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature‐sensitive pSC101 replicon , 1991, Molecular microbiology.

[3]  Volker F. Wendisch,et al.  Propionate oxidation in Escherichia coli: evidence for operation of a methylcitrate cycle in bacteria , 1997, Archives of Microbiology.

[4]  J. M. Meyer,et al.  The Fluorescent Pigment of Pseudomonas fluorescens : Biosynthesis, Purification and Physicochemical Properties , 1978 .

[5]  R. Müller,et al.  Recent developments towards the heterologous expression of complex bacterial natural product biosynthetic pathways. , 2005, Current opinion in biotechnology.

[6]  R. Thiericke,et al.  The Impact of Natural Products on Drug Discovery , 2000 .

[7]  T. Bobik,et al.  Identification of the Human Methylmalonyl-CoA Racemase Gene Based on the Analysis of Prokaryotic Gene Arrangements , 2001, The Journal of Biological Chemistry.

[8]  S. Hill,et al.  Characterization of the biosynthetic gene cluster for the antifungal polyketide soraphen A from Sorangium cellulosum So ce26. , 2002, Gene.

[9]  M. Marahiel,et al.  Ways of Assembling Complex Natural Products on Modular Nonribosomal Peptide Synthetases , 2002, Chembiochem : a European journal of chemical biology.

[10]  J. Salanitro,et al.  Quantitative method for the gas chromatographic analysis of short-chain monocarboxylic and dicarboxylic acids in fermentation media. , 1975, Applied microbiology.

[11]  R. Müller,et al.  Analysis of myxobacterial secondary metabolism goes molecular , 2006, Journal of Industrial Microbiology and Biotechnology.

[12]  R. Müller,et al.  Posttranslational modification of myxobacterial carrier protein domains in Pseudomonas sp. by an intrinsic phosphopantetheinyl transferase , 2005, Applied Microbiology and Biotechnology.

[13]  F. Peláez.  The historical delivery of antibiotics from microbial natural products--can history repeat? , 2006, Biochemical pharmacology.

[14]  Rolf Müller,et al.  The impact of bacterial genomics on natural product research. , 2005, Angewandte Chemie.

[15]  G. Gottschalk,et al.  Identification and expression of the genes and purification and characterization of the gene products involved in reactivation of coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii. , 2001, European journal of biochemistry.

[16]  C. R. Howell,et al.  Cloning of Genes Involved in the Synthesis of Pyrrolnitrin from Pseudomonas fluorescens and Role of Pyrrolnitrin Synthesis in Biological Control of Plant Disease , 1994, Applied and environmental microbiology.

[17]  Joern Hopke,et al.  Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts , 2004, Applied and Environmental Microbiology.

[18]  T. Schupp,et al.  A Sorangium cellulosum (myxobacterium) gene cluster for the biosynthesis of the macrolide antibiotic soraphen A: cloning, characterization, and homology to polyketide synthase genes from actinomycetes , 1995, Journal of bacteriology.

[19]  R. Larossa,et al.  Chromosomal promoter replacement of the isoprenoid pathway for enhancing carotenoid production in E. coli. , 2006, Metabolic engineering.

[20]  C. Khosla,et al.  Cloning and heterologous expression of the epothilone gene cluster. , 2000, Science.

[21]  U. Gräfe.  Secondary Metabolites: From Past to Present , 2000 .

[22]  A. Demain,et al.  Pharmaceutically active secondary metabolites of microorganisms , 1999, Applied Microbiology and Biotechnology.

[23]  K. Mori,et al.  Characterization, Sequencing, and Expression of the Genes Encoding a Reactivating Factor for Glycerol-inactivated Adenosylcobalamin-dependent Diol Dehydratase* , 1997, The Journal of Biological Chemistry.

[24]  H. Reichenbach,et al.  Biologically active secondary metabolites from myxobacteria. , 1993, Biotechnology advances.

[25]  Yaoquan Liu,et al.  Heterologous production of epothilone C and D in Escherichia coli. , 2006, Biochemistry.

[26]  Rolf Daniel,et al.  The soil metagenome--a rich resource for the discovery of novel natural products. , 2004, Current opinion in biotechnology.

[27]  R. Armstrong,et al.  Mechanistic diversity in a metalloenzyme superfamily. , 2000, Biochemistry.

[28]  Jeffrey H. Miller.  Experiments in molecular genetics , 1972 .

[29]  Frank Buchholz,et al.  A new logic for DNA engineering using recombination in Escherichia coli , 1998, Nature Genetics.

[30]  W. Fenton,et al.  Cloning of full-length methylmalonyl-CoA mutase from a cDNA library using the polymerase chain reaction. , 1989, Genomics.

[31]  B A Pfeifer,et al.  Biosynthesis of Complex Polyketides in a Metabolically Engineered Strain of E. coli , 2001, Science.

[32]  B. Barrell,et al.  Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2) , 2002, Nature.

[33]  J. Gerlt,et al.  Discovering new enzymes and metabolic pathways: conversion of succinate to propionate by Escherichia coli. , 2000, Biochemistry.

[34]  Mihail Sarov,et al.  An improved recombineering approach by adding RecA to λ Red recombination , 2006 .

[35]  H. Reichenbach,et al.  Characterisation, genome size and genetic manipulation of the myxobacterium Sorangium cellulosum So ce56 , 2002, Archives of Microbiology.

[36]  J. R. Sokatch,et al.  Properties of purified methylmalonate semialdehyde dehydrogenase of Pseudomonas aeruginosa. , 1970, The Journal of biological chemistry.

[37]  Yoshiyuki Sakaki,et al.  Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis , 2003, Nature Biotechnology.

[38]  A. Stewart,et al.  Reconstitution of the Myxothiazol Biosynthetic Gene Cluster by Red/ET Recombination and Heterologous Expression in Myxococcus xanthus , 2006, Applied and Environmental Microbiology.

[39]  A. Leiser,et al.  Cloning, sequencing, and expression of the gene encoding methylmalonyl-coenzyme A mutase from Streptomyces cinnamonensis , 1993, Journal of bacteriology.

[40]  P. Leadlay,et al.  Subunit interactions in Propionibacterium shermanii methylmalonyl-CoA mutase studied by analytical ultracentrifugation. , 1989, The Biochemical journal.

[41]  P. Leadlay,et al.  Cloning and structural characterization of the genes coding for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. , 1989, The Biochemical journal.

[42]  H. Blöcker,et al.  New Lessons for Combinatorial Biosynthesis from Myxobacteria , 1999, The Journal of Biological Chemistry.

[43]  M. Lidstrom,et al.  MeaB Is a Component of the Methylmalonyl-CoA Mutase Complex Required for Protection of the Enzyme from Inactivation* , 2004, Journal of Biological Chemistry.

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

[45]  Jonathan Kennedy,et al.  Metabolic engineering of Escherichia coli for improved 6-deoxyerythronolide B production , 2003, Journal of Industrial Microbiology and Biotechnology.

[46]  M. Marahiel,et al.  Characterization of a New Type of Phosphopantetheinyl Transferase for Fatty Acid and Siderophore Synthesis in Pseudomonas aeruginosa * , 2002, The Journal of Biological Chemistry.

[47]  E. Stadtman,et al.  ON THE MECHANISM OF THE COBAMIDE COENZYME DEPENDENT ISOMERIZATION OF METHYLMALONYL COA TO SUCCINYL CoA , 1960 .

[48]  P. Long,et al.  Mining the microbial metabolome: a new frontier for natural product lead discovery. , 2003, Drug discovery today.

[49]  Rolf Müller,et al.  Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering. , 2005, Chemistry & biology.

[50]  A. C. Chang,et al.  Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid , 1978, Journal of bacteriology.

[51]  M. Marahiel,et al.  A new enzyme superfamily - the phosphopantetheinyl transferases. , 1996, Chemistry & biology.

[52]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[53]  J. Keasling,et al.  Metabolic Engineering of a Novel Propionate-Independent Pathway for the Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) in Recombinant Salmonella enterica Serovar Typhimurium , 2002, Applied and Environmental Microbiology.

[54]  J. Staunton,et al.  Polyketide biosynthesis: a millennium review. , 2001, Natural product reports.

[55]  R. Müller,et al.  Myxobacteria: proficient producers of novel natural products with various biological activities--past and future biotechnological aspects with the focus on the genus Sorangium. , 2003, Journal of biotechnology.

[56]  J M Ligon,et al.  The biosynthetic gene cluster for the microtubule-stabilizing agents epothilones A and B from Sorangium cellulosum So ce90. , 2000, Chemistry & biology.

[57]  Patricia C. Babbitt,et al.  Understanding Enzyme Superfamilies , 1997, The Journal of Biological Chemistry.

[58]  A. Crane,et al.  Primary structure and activity of mouse methylmalonyl-CoA mutase. , 1990, The Biochemical journal.

[59]  H. Reichenbach,et al.  Studies on the biosynthesis of epothilones: the biosynthetic origin of the carbon skeleton. , 2000, The Journal of antibiotics.

[60]  Giuseppe Testa,et al.  DNA cloning by homologous recombination in Escherichia coli , 2000, Nature Biotechnology.

[61]  K. Timmis,et al.  Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. , 1981, Gene.

[62]  A. Stewart,et al.  ET-cloning: think recombination first. , 2000, Genetic engineering.

[63]  H. Reichenbach,et al.  Antibiotika aus Gleitenden Bakterien, XXVIII1) Biosynthese des Myxothiazols in Myxococcus fulvus Mx f16 , 1986 .

[64]  Ralf Thiericke,et al.  Drug Discovery from Nature , 2000 .