Genetic Engineering & Biotechnology News

DEC 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Genetic Engineering & Biotechnology News | | DECEMBER 2017 | 15 yeast. "The field," he admits, "isn't good at that yet." Gutting It Out with Living Medicines Synlogic scientists re-engineer genetic cir- cuitry to enable bacteria to degrade metabo- lites or synthesize products more efficiently. When the re-engineered circuitry is united with E. coli Nissle, the human gut microbe that serves as Synlogic's chassis, the result is a Synthetic Biotic™, an organism that can generate medicines that can decrease inflam- mation; compensate for inborn genetic er- rors that cause rare metabolic diseases; or treat relatively common diseases such as can- cer or metabolic conditions such as diabetes and obesity. The company's initial clinical programs are focused on Synthetic Biotics to treat or- phan metabolic diseases such as urea cycle disorders and phenylketonuria. Another Synlogic product is a synthetically engi- neered biotic that produces butyrate. "High permeability in gut epithelium is correlated with a deficiency in short-chain fatty acids in the gastrointestinal tract, most notably butyrate," says Paul Miller, Ph.D., Synlogic's chief science officer. "Our goal is to engineer a safe, butyrate-producing pro- biotic to compensate for low production of natural, endogenous butyrate." Normally, butyrate is synthesized in the large intestine by slow-growing anaerobes such as Clostridia as a byproduct of fermen- tation of undigested plant fiber. Butyrate helps regulate fluid transport across gut epi- thelium, reduces mucosal inflammation and oxidative stress, reinforces the gut's epithelial defense barrier, and modulates visceral sen- sitivity and intestinal motility. Studies have indicated that butyrate may help prevent and inhibit colorectal cancer. Synlogic's proprietary platform uses E. coli Nissle as its chassis for butyrate produc- tion because the organism is nonpathogenic and is easy to scale and manipulate. Discov- ered a century earlier by Alfred Nissle, this Gram-negative bacterium was isolated from a hospitalized German soldier who, though dispatched to a region highly contaminated with pathogenic Shigella bacteria, resisted shigellosis. He was found to possess a notice- ably strong intestinal constitution. "To re-engineer the E. coli Nissle chassis for butyrate biosynthesis, we inserted a Clos- tridia-derived biosynthetic operon under the control of a Tet (tetracycline)-inducible pro- moter into it," states Dr. Miller. "Clostridia occur naturally in the gut and correlate with healthy gut flora." In butyrate's biosynthetic pathway, this operon converts acetyl coenzyme A to bu- tyrate as a reduced product, permitting the recycling of NADH+ to NAD. Synlogic sci- entists reduced the Clostridia pathway from eight genes to five by simplifying the NADH recycling process, as well as the final conver- sion of butyryl-coenzyme A to butyrate. The resultant pathway produced high levels of butyrate in response to activation of the Tet promoter. E. coli uses the master transcriptional regulator FNR (fumarate and nitrate reduc- tase) to control anaerobic gene expression. Since this regulatory mechanism could be exploited to activate gene expression in the mammalian gut's anaerobic environment, the modified butyrate cassette was put into a low copy plasmid vector under the control of an FNR-responsive promoter. The resulting modified strain, called SYN363, was tested for butyrate produc- tion in rich media with 0.5% glucose under both aerobic and anaerobic conditions. Un- der anaerobic conditions, SYN363 showed a fivefold increase in butyrate production over aerobic conditions. Further, SYN363 was tested against butyrate-producing Clos- tridia strains in rich media with 0.5% glu- cose. After a 24-hour growth period, the modified strain matched the productivity of the natural butyrate-producing strains (Fig- ure 2). This level of biosynthetic output was demonstrated when carbon sources similar to those in the colon were available, sources such as glucoronate. "Our driving goal was not simply to pro- duce more butyrate per cell than we could with Clostridia, but to ensure that our prod- uct had a biological effect," Dr. Miller states. "In preclinical models, SYN363 showed ef- ficacy in reducing markers of inflammation in a murine model of DSS (dextran sulphate Learn how to accelerate your pilot-scale media manufacturing at *Non-GMP pilot production. Additional time for shipping. © 2017 BD. BD and the BD Logo are trademarks of Becton, Dickinson and Company. MC8392 FOR SCALABLE, ONE-STOP CELL CULTURE MEDIA PRODUCTION, TURN TO BD. BD continually advances solutions to support process development and manufacturing for scientists. BD ™ Rapid Media Solutions delivers a 10-business-day * turnaround on developmental medium production. 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The engineered strain, which Synlogic calls a Synthetic Biotic, rivals the butyrate production of natural Clostridia strains in vitro.

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