Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli

ABSTRACT A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a “top value-added chemical” from biomass.

[1]  K. S. Prabhu,et al.  Molecular cloning, expression, and characterization of myo-inositol oxygenase from mouse, rat, and human kidney. , 2004, Biochemical and biophysical research communications.

[2]  F. Charalampous,et al.  Biochemical studies on inositol , 1966 .

[3]  W. Kilgore,et al.  Purification and properties of uronate dehydrogenase from Pseudomonas syringae. , 1970, Archives of biochemistry and biophysics.

[4]  W. Bentley,et al.  Plasmid‐encoded protein: The principal factor in the “metabolic burden” associated with recombinant bacteria , 1990, Biotechnology and bioengineering.

[5]  J. Szemraj,et al.  d-Glucaric acid content of various fruits and vegetables and cholesterol-lowering effects of dietary d-glucarate in the rat , 1996 .

[6]  M. Dennis.  Outcome after Brain Haemorrhage , 2003, Cerebrovascular Diseases.

[7]  T. Noda,et al.  Inositol monophosphatase activity from the Escherichia coli suhB gene product , 1995, Journal of bacteriology.

[8]  J. W. Frost,et al.  Synthesis of 1,2,3,4-Tetrahydroxybenzene from d-Glucose: Exploiting myo-Inositol as a Precursor to Aromatic Chemicals , 1999 .

[9]  P. Babbitt,et al.  Evolution of enzymatic activities in the enolase superfamily: characterization of the (D)-glucarate/galactarate catabolic pathway in Escherichia coli. , 1998, Biochemistry.

[10]  Tae Seok Moon,et al.  Cloning and Characterization of Uronate Dehydrogenases from Two Pseudomonads and Agrobacterium tumefaciens Strain C58 , 2008, Journal of bacteriology.

[11]  E. Amann,et al.  Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. , 1988, Gene.

[12]  J. Keasling,et al.  Low-copy plasmids can perform as well as or better than high-copy plasmids for metabolic engineering of bacteria. , 2000, Metabolic engineering.

[13]  Linda J. Broadbelt,et al.  Computational discovery of biochemical routes to specialty chemicals , 2004 .

[14]  S. Henry,et al.  Biosynthesis of inositol in yeast. Primary structure of myo-inositol-1-phosphate synthase (EC 5.5.1.4) and functional analysis of its structural gene, the INO1 locus. , 1989, The Journal of biological chemistry.

[15]  Carsten Krebs,et al.  A coupled dinuclear iron cluster that is perturbed by substrate binding in myo-inositol oxygenase. , 2006, Biochemistry.

[16]  Ih Chu,et al.  HPLC determination of D-glucaric acid in human urine. , 1993, Journal of analytical toxicology.

[17]  G R Jacobson,et al.  Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. , 1993, Microbiological reviews.

[18]  J E Bailey,et al.  Plasmid presence changes the relative levels of many host cell proteins and ribosome components in recombinant Escherichia coli , 1991, Biotechnology and bioengineering.

[19]  C. Nakamura,et al.  Metabolic engineering for the microbial production of 1,3-propanediol. , 2003, Current opinion in biotechnology.

[20]  G. A. Hamilton,et al.  myo-Inositol oxygenase from hog kidney. II. Catalytic properties of the homogeneous enzyme. , 1981, Journal of Biological Chemistry.

[21]  A. Majumder,et al.  Chloroplast as a Locale of L-myo-Inositol-1-Phosphate Synthase. , 1987, Plant physiology.

[22]  Jaya Singh,et al.  Induction of apoptosis by calcium D-glucarate in 7,12-dimethyl benz [a] anthracene-exposed mouse skin. , 2007, Journal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer.

[23]  Jaya Singh,et al.  Calcium glucarate prevents tumor formation in mouse skin. , 2003, Biomedical and environmental sciences : BES.

[24]  M. Bagdasarian,et al.  A series of wide-host-range low-copy-number vectors that allow direct screening for recombinants. , 1991, Gene.

[25]  Wei Niu,et al.  Microbial synthesis of the energetic material precursor 1,2,4-butanetriol. , 2003, Journal of the American Chemical Society.

[26]  J. Barnett,et al.  A colorimetric determination of inositol monophosphates as an assay for D-glucose 6-phosphate-1L-myoinositol 1-phosphate cyclase. , 1970, The Biochemical journal.

[27]  G. Wagner,et al.  Uronic acid dehydrogenase from Pseudomonas syringae. Purification and properties. , 1976, European journal of biochemistry.

[28]  Johnathan E. Holladay,et al.  Top Value Added Chemicals From Biomass. Volume 1 - Results of Screening for Potential Candidates From Sugars and Synthesis Gas , 2004 .

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

[30]  Jay D. Keasling,et al.  Metabolic engineering for drug discovery and development , 2003, Nature Reviews Drug Discovery.

[31]  J. T. Thompson,et al.  myo-Inositol oxygenase: molecular cloning and expression of a unique enzyme that oxidizes myo-inositol and D-chiro-inositol. , 2001, The Biochemical journal.

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

[33]  J. Keasling,et al.  Engineering a mevalonate pathway in Escherichia coli for production of terpenoids , 2003, Nature Biotechnology.

[34]  F. Charalampous.  Biochemical studies on inositol. V. Purification and properties of the enzyme that cleaves inositol to D-glucuronic acid. , 1959, The Journal of biological chemistry.

[35]  P. Postma,et al.  Phosphoenolpyruvate:carbohydrate phosphotransferase system of bacteria. , 1985, Microbiological reviews.

[36]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[37]  D. Kiely,et al.  Simple Preparation of Hydroxylated Nylons—Polyamides Derived from Aldaric Acids , 1994 .

[38]  Wendell A Lim,et al.  Synthetic biology: lessons from the history of synthetic organic chemistry , 2007, Nature Chemical Biology.

[39]  C. C. Reddy,et al.  myo-Inositol oxygenase from hog kidney. I. Purification and characterization of the oxygenase and of an enzyme complex containing the oxygenase and D-glucuronate reductase. , 1981, The Journal of biological chemistry.

[40]  Steven A. Benner,et al.  Synthetic biology: Act natural , 2003, Nature.

[41]  F. Charalampous,et al.  Biochemical studies on inositol. IV. Conversion of inositol to glucuronic acid by rat kidney extracts. , 1957, The Journal of biological chemistry.

[42]  M. Mavrovouniotis.  Estimation of standard Gibbs energy changes of biotransformations. , 1991, The Journal of biological chemistry.