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SIM Gets Strong Support from BTR Staff

BTR's exhibit booth has long been a fixture at the the annual meeting of the Society for Industrial Microbiology and this year was no exception. What was exceptional were the number of staff members who actively participated in the technical program.

Since this event fills an important niche in the life sciences community, Dr. Reinhardt Rosson (VP and Director of R&D) volunteered to act as Program Chair for the July meeting in Anaheim. He also agreed to act as co-chair for a session on industrial processes. Two other staff members not only volunteered to chair sessions, but also gave talks as well. A session devoted to isoprenoids was lead by Dr. Tom McMullin (Research Manager), while another of our Research Managers, Dr. Ming-De Deng, headed a session on microbial production of nutraceuticals and pharmaceuticals. Dave Severson (Principal Research Scientist) also presented a poster summarizing his team's progress on process development for glucosamine and N-acetylglucosamine production. Abstracts from all three of their presentations are reproduced below.

Isoprenoid Pathway Engineering in Saccharomyces cerevisiae for Sesquiterpene and Diterpene Production.
Tom McMullin, Julie Maurina-Brunker, Sarah Wassink, Linsheng Song, Candice Leanna, Kim Langley, and Phil Olson

Isoprenoids are a very diverse group of chemicals that contribute to the biosynthesis of a variety of commercially important compounds such as carotenoids, CoQ10, tocopherols, sterols, and terpenes. The structural diversity of this class of biochemicals also makes them attractive as candidates for new lead compounds; however, many of the isoprenoids are of very limited availability. Our work has focused on developing strains of Saccharomyces cerevisiae as potential platforms for the production of compounds that involve sesquiterpene and diterpene isoprenoids. We have focused much of our effort on using pathway engineering to develop strains that accumulate the alcohols of specific isoprenoid pathway intermediates, such as farnesol and geranylgeraniol, as a means of understanding the factors that influence carbon flux into the isoprenoid pathway of this organism. We have successfully used gene knock-outs to block sterol formation and increase the accumulation of farnesol and geranylgeraniol and have used gene amplification to identify other steps that can be manipulated to increase carbon flux into the isoprenoid pathway. We have also examined the role of specific phosphatases in isoprenoid metabolism.

From Concept to Process: Metabolic Engineering for Production of Glucosamine and N-Acetylglucosamine.
Ming-De Deng, Dave Severson, Alan Grund, Sarah Wassink, Jeff Running, Candice Leanna, Linsheng Song, Kathy Nielsen, Bonnie Walsh, Brian Huckins, Troy Lutze, and Reinhardt Rosson

Metabolic engineering and microbial fermentation offer advantageous alternatives to production of numerous chemicals which are traditionally manufactured by chemical synthesis or extracted from raw materials. Developing an academically interesting concept into an industrial process is a challenging task. It often requires creative approaches to overcome different bottlenecks. Metabolic engineering for glucosamine production in E. coli first started with a straightforward strategy: inactivating genes involved in glucosamine transporter and catabolism, and over-expressing the biosynthetic gene (glucosamine synthase, GlmS). This resulted in a 15-fold increase in glucosamine concentration, but the titer remained below 100 mg/L. Over-expression of a GlmS mutant resistant to product inhibition led to a level of 18 g/L. Fast degradation of glucosamine and inhibitory effects of glucosamine and its degradation products on host cells limited further titer improvement. N-acetylglucosamine was identified as the alternative fermentation product since it is stable, non-inhibitory and readily hydrolyzed to glucosamine. Therefore, the glucosamine synthesis pathway was extended to N-acetylglucosamine by over-expressing a yeast glucosamine-6-P N-acetyltransferase (GNA1). Using a simple and low-cost fermentation process, strains over-expressing GlmS and GNA1 produced N-acetylglucosamine at concentrations greater than 110 g/L.

Fermentation Process Development for the Production of Glucosamine and N-Acetylglucosamine by Recombinant E. coli.
Dave Severson, Brian Huckins, Troy Lutze, Jeff Running, and Ming-De Deng

Glucosamine (GlcN) is an important nutraceutical used for the treatment of osteoarthritis. It is currently manufactured by acid hydrolysis of chitin from shellfish waste or fungal biomass. A direct microbial process from glucose offers many potential advantages, including higher yields and lower cost. Mutant strains of E. coli were developed to produce GlcN following induction by lactose. Several process factors were determined to be important, including sensitivity to acetate. Methods that reduced acetate accumulation and increased culture robustness included use of glucose-limiting feeds, lower production temperature (25°C), and strict limitation of iron and other trace elements important to respiration. Titers up to 18 g/L were achieved, but further improvement was difficult because GlcN is labile at neutral pH, and also because GlcN and its degradation products are inhibitory. Lowered pH during production offered only marginal benefit. Additional strain development extended the pathway to N-acetylglucosamine (NAG), a stable, non-inhibitory derivative that is easily hydrolyzed to GlcN. This strategy immediately resulted in a titer of 55 g/L NAG, with further fermentation development resulting in titers greater than 110 g/L within 60 hours. The simplified final process employs a defined low salts medium, higher temperature (37°C), and neutral pH. The growth and production phases are separated with induction by a single point addition of lactose after approximately 20 g/L biomass is achieved.

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