Jana L. Seymour

Production of 2-Keto-L-Gulonate, an Intermediate in L-Ascorbate Synthesis, by a Genetically Modffied Erwinia herbicola

A new metabolic pathway has been created in the microorganism Erwinia herbicola that gives it the ability to produce 2-keto-L-gulonic acid, an important intermediate in the synthesis of L-ascorbic acid. Initially, a Corynebacterium enzyme that could stereoselectively reduce 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid was identified and purified. DNA probes based on amino acid sequence information from 2,5-diketo-D-gluconic acid reductase were then used to isolate the gene for this enzyme from a Corynebacterium genomic library. The 2,5-diketo-D-gluconic acid reductase coding region was fused to the Escherichia coli trp promoter and a synthetic ribosome binding site and was then introduced into E. herbicola on a multicopy plasmid. Erwinia herbicola naturally produces 2,5-diketo-D-gluconic acid via glucose oxidation, and when recombinant cells expressing the plasmid-encoded reductase were grown in the presence of glucose, 2-keto-L-gulonic acid was made and released into the culture medium. The data demonstrate the feasibility of creating novel in vivo routes for the synthesis of important specialty chemicals by combining useful metabolic traits from diverse sources in a single organism.

Native gel activity stain and preparative electrophoretic method for the detection and purification of pyridine nucleotide-linked dehydrogenases

An activity stain for the detection of pyridine nucleotide-linked dehydrogenases in polyacrylamide gels is described. Following incubation of the gel with substrate and cofactor, bands are visualized under ultraviolet light, where reduced cofactors fluoresce and oxidized cofactors appear black. The methods described are useful for any NAD- or NADP-linked dehydrogenase; the enzymes can be assayed in either the oxidative or the reductive direction. Also described is a preparative polyacrylamide gel system using the activity stain, which can be used as a general purification method for dehydrogenases. The preparative gels are crosslinked with bisacrylylcystamine. These crosslinks can be broken by the addition of thiols after the bands of interest have been located and excised. The protein of interest is then separated from the solubilized acrylamide by adsorption to a suitable resin.

Platelet aggregation inhibitors from the leech

A composition of matter derived from hematophagous leech comprising specified purified amino acid sequences represented by the general formula: CXXXRGDXXXXC(Seq. ID No. 11) and capable of functioning as an antithrombotic by inhibiting the binding of fibrinogen to the platelet glycoprotein IIb IIIa (GP IIb IIIa), a fibrinogen receptor. Methods for the purification of amino acid sequences from leeches, and particularly from leeches of the genus Macrobdella and Placobdella. are provided. Isolated nucleic acid sequences encoding these amino acid sequences; an expression vector containing the isolated nucleic acid; and a cell containing the expression vector are also described. A process for chemical synthesis of the amino acid sequences and a method for reducing platelet aggregation in a mammal by administering a composition containing the amino acid sequences to the mammal in a pharmaceutically effective amount are provided.

Determination of 2-keto-L-gulonic and other ketoaldonic and aldonic acids produced by ketogenic bacterial fermentation.

The quantitative analysis of 2-keto-L-gulonic acid (2-KLG) produced by microbial fermentation is described. 2-KLG is separated from other aldonic and ketoaldonic acids by high-performance liquid chromatography on an Aminex anion exchange column with ammonium formate or potassium phosphate as the eluant. This is a rapid and simple method for routine analysis of a large number of samples generated by fermentation studies. Gas chromatography--mass spectrometry permits the qualitative and quantitative analysis of nanogram levels of 2-keto-L-gulonate in complex media and provides confirmation of the HPLC results. The methodologies presented are useful for the analysis of a number of aldonic and ketoaldonic acids.

Urinary Metabolites of 2, 5, 2′, 5′-Tetrachlorobiphenyl in the Nonhuman Primate:

Summary The metabolism of 2, 5, 2′, 5′- tetrachlorobiphenyl (TCB) in nonhuman primates was found to be different from that previously reported in lower species. Mono-hydroxy TCB (I), the only metabolite in the ether extracts of rat urine, is a minor metabolite in the urine of nonhuman primates. The two major metabolites identified in the urine were dihydroxy TCB (II) and trans-3, 4-dihydro-3, 4-dihydroxy TCB (III). A second minor metabolite was identified as hydroxy-3, 4-dihydro-3, 4-dihydroxy TCB (IV). None of the above mentioned metabolites have been reported in primates and only I and III have been identified in lower animals. It is concluded that a likely mechanism for metabolism of TCB in primates is through arene oxide intermediates. This observation is of particular importance in that these types of intermediates are known to alkylate cellular components causing carcinogenic, mutagenic, necrogenic and teratogenic effects. We thank Dr. Bruce M. Johnson, Department of Clinical Oncology, University of Wisconsin Medical School for the use of the mass spectrometer. This investigation was supported in part by U.S. Public Health Service Grant Nos. ES-00472, HL-10941, CA-13288 and RR-00167 from the National Institutes of Health. Primate Center publication no. 15-007.

A Biocatalytic Approach to Vitamin C Production

Although the primary focus of the biotechnology industry has been on the overproduction of new proteins, primarily for pharmaceutical purposes, there has been growing interest in other fields such as agriculture, diagnostics, and the biocatalytic production of organic chemicals. We have recently been working to develop a novel biosynthetic process for the production of vitamin C (L-ascorbic acid, ASA). This approach has involved the study of the enzymes, coenzymes, and metabolic pathways of different bacteria with the goal of creating new metabolic routes to make a new product (Fig. 6.1). This metabolic pathway engineering approach, which required the identification and characterization of several new enzymes and the cloning and expression of the gene coding for one of these enzymes, has led to a successful one-step bioconversion of D-glucose (G) into 2-keto-L-gulonic acid (2-KLG), a key intermediate in the synthesis of ascorbic acid.