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BTR Staff Scientists Present Research Results

One of BTR's Research Managers, Ming-De Deng, spoke recently at the Annual Meeting of the Great Lakes Section of the Society for Industrial Microbiology, which was held at Notre Dame on October 19, 2002. Dr. Deng presented research on the metabolic engineering and fermentation of E. coli for the industrial production of glucosamine. The abstract follows:

E. coli Metabolic Engineering and Fermentation for Industrial Production of Glucosamine
Ming-De Deng, Sarah Wassink, Al Grund, Dave Severson, Jeff Running, Candice Leanna, Linsheng Song, Kathy Nielsen, Bonnie Walsh, Brian Huckins, Rich Burlingame
D-glucosamine is a nutraceutical compound with applications in the treatment of human 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 are developing a novel fermentation process to produce glucosamine by E. coli metabolic engineering. First generation glucosamine-overproducing strains were generated by deleting genes that encode key enzymes involved in the transport and metabolism of glucosamine and over-expressing the glmS gene. The glmS gene encodes glucosamine-6-phosphate synthase, which converts fructose-6-phosphate and glutamine to glucosamine-6-phosphate. Such strains produced about 0.4 g/L of glucosamine in shake flasks, representing a 100-fold increase with respect to the wild type strain. Since the GlmS enzyme is subject to strong product inhibition, the inserted glmS gene was mutagenized by error prone PCR and mutant variants were screened in a plate assay for glucosamine overproduction. The best variant produced about 6 g/L glucosamine in shake flasks and about 12 g/L in fermentors. By further strain and fermentation process development, glucosamine production in a simple mineral salt medium with glucose and ammonium feed has reached levels approaching the target for a competitive manufacturing process.

Two other staff members, Jeff Running and Dave Severson, presented posters at the Society for Industrial Microbiology's annual meeting held August 11-15, 2002, in Philadelphia. Each focused on a different aspect of the same research project: pathway determination and process development. Their abstracts appear below.

The Biosynthetic Pathway from Glucose to L-Ascorbic Acid in the colorless Microalga Prototheca moriformis.
Jeff Running, Rich Burlingame, Susan Peng, Dave Severson, Alan Berry
Mutants of Prototheca moriformis ATCC 75669 that varied in their abilities to synthesize L-ascorbic acid (LAA) were isolated. Strain UV77-247 fed [1-¹³C] glucose synthesized LAA with about 73% of the label at the C-1 position, with most of the remaining label at C-6, indicating that LAA is synthesized from glucose by a pathway that retains the carbon chain configuration. This pattern is consistent with some labeled glucose proceeding via glycolysis to 3-carbon compounds, and re-forming by gluconeogenesis. Most mutants rapidly converted L-galactose and L-galactono-g-lactone to LAA. All mutants converted D-mannose to LAA at least as fast as they did glucose. Although enzyme activities from fructose-6-phosphate to GDP-D-mannose were identified in all mutants, there was no correlation between any of these activities and the mutants' LAA-synthesizing ability. There was a strong correlation between this ability and the mutants' GDP-D-mannose-3,5-epimerase activity. These results show this enzyme plays a key role in LAA biosynthesis. L-galactose dehydrogenase activity was also detected. A pathway is proposed for the formation of LAA from glucose in these algae.

Fermentation Process Development for the Production of L-Ascorbic Acid by the Microalga Prototheca moriformis.
Dave Severson, Jeff Running

The significance of inorganic nutrient manipulation during fermentation optimization for the production of L-ascorbic acid (LAA) in a one-step biological process from glucose is described. Wild-type cultures of Prototheca moriformis accumulate extracellular LAA during heterotrophic growth in defined minimal salts medium to titers near 160 mg/L at a low cultivation pH between 3 to 4. A classical strain improvement program resulted in a two-fold specific product formation increase in only three generations of mutants. Growth-related production appeared inadequate, so strategies to uncouple growth from production were evaluated. Growth restriction through magnesium limitation resulted in a four-fold improvement in all strains, including the parent. Other nutrient limitations did not achieve the same dramatic effect. Additional manipulation of other specific inorganic medium components affecting the production and/or stability of LAA resulted in further improvements that yielded an overall titer increase of approximately 18 times to near 3 g/L LAA. The strategy of growth restriction also appeared to minimize limitations normally encountered in flask culture by imposing a standardized lower cell density, which resulted in close agreement between specific product formation values in flask and fermentor culture throughout most of a 14-month program.

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