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BTR Research Draws Attention

The August, 2003, Annual Meeting of the Society for Industrial Microbiology in Minneapolis was the venue for presentations by two of BTR's researchers. Dr. Susan Peng, a research scientist who specializes in enzyme characterization and protein purification, reported on her success at purifying the C12-isomerase in her poster entitled, “Purification and Characterization of a Membrane-Bound Linoleic Acid Isomerase.” The abstract is reprinted below.

Research Manager Dr. Ming-De Deng is part of the team that is working on BTR's process for microbial production of glucosamine, leading the metabolic engineering effort. On Thursday afternoon, his audience heard the presentation, “Metabolic Engineering for Industrial Production of Glucosamine and N-Acetylglucosamine in E. coli.” The abstract of his talk also appears below.

Purification and Characterization of a Membrane-Bound Linoleic Acid Isomerase.
Susan Peng, Al Grund and Reinhardt Rosson

Conjugated linoleic acid (CLA, 18:2) has a number of well-documented beneficial effects on animal and human health such as reducing atherosclerosis, modulating carcinogenesis, enhancing immune function and reducing body fat. CLA produced by chemical synthesis is typically a mixture of two and, in some cases, four to twelve different positional and geometric isomers, which are very difficult to separate from each other. The most abundant isomers found in nature are cis-9, trans-11 CLA and trans-10, cis-12 CLA; these isomers have unique but different biological effects.

We have been developing a technology based on an enzymatic process to produce pure single CLA isomers. Toward this goal, we have examined a variety of bacterial strains for linoleic acid isomerases. Strains with high activity were selected for enzyme purification and characterization. A membrane-bound isomerase (C12-isomerase) converting linoleic acid into cis-9, trans-11 CLA has been purified from Clostridium sporogenes.

After successive chromatography by anion exchange, chromatofocusing and size exclusion, an overall purification of 350-fold was achieved, reaching a specific activity of 350 nmol min-1 mg-1 protein. The purified enzyme showed a single band with an estimated molecular weight of about190 and 48 kDa on native gels and denaturing SDS-PAGE, respectively.

The properties of the purified enzyme were studied. The enzyme did not require the addition of external cofactors or energy. Substrate specific evaluations showed that the enzyme had a definite bias toward substrates containing cis double bonds at 9, 12 positions of C18 fatty acids. The optimum pH for isomerization was found to be about 7.5. The Km for linoleic acid was 11.3 µM.

Metabolic Engineering for Industrial Production of Glucosamine and N-Acetylglucosamine in E. coli
Ming-De Deng, Sarah Wassink, Dave Severson, Al Grund, Candice Leanna, Linsheng Song, Jeff Running, Kathy Nielsen, Bonnie Walsh, Brian Huckins, Troy Lutze, Rich Burlingame, and Reinhardt Rosson

D-glucosamine is an amino sugar that is increasingly used as nutraceutical supplement to treat human and animal osteoarthritic conditions. It is currently produced by the acid hydrolysis of chitin, a process limited by the availability of raw materials, such as crab shells. We have developed a novel fermentation process to produce glucosamine by microbial metabolic engineering.

Like other organisms, E. coli contains necessary enzymes for the production of glucosamine, which is vital to cell wall synthesis, but produces little free glucosamine (4 mg/L). Glucosamine over-production strains were engineered by deleting genes that encode enzymes involved in the transport and catabolism of glucosamine, and by over-expressing the glmS gene.

The glmS gene encodes glucosamine-6-phosphate synthase, which converts fructose-6-phosphate and glutamine to glucosamine-6-phosphate. Since the GlmS enzyme is subject to strong product inhibition, mutant glmS variant were created by error prone PCR and screened by plate assays. Over-expression of product resistant GlmS enzymes led to 6 g/L glucosamine in shake flasks.

By further pathway manipulation and fermentation process development, glucosamine production in a simple mineral salt medium with glucose and ammonium feed in fermentors has reached the target for a competitive manufacturing process. We have also developed a process for the production of N-acetylglucosamine, which has numerous pharmaceutical and cosmetic applications.

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