Allylix Inc. has developed a proprietary technology platform that allows it to reliably produce high-value, natural products called terpenes at low cost and at sustainable commercial levels. The company’s platform also enables the creation of novel compounds.

Allylix’s technology has two key components:

• The ability to engineer enzymes to produce a range of highly pure terpenes

• The ability to produce those terpenes cost effectively using proprietary fermentation processes

Breakthrough biotechnology

The basis for Allylix’s technology began in the 1990s at San Diego’s Salk Institute and the University of Kentucky where the company’s two scientific founders collaborated in terpene research for 12 years. They were among the first to provide a thorough understanding of the molecular structure of a class of enzymes, called terpene synthases, used by plants to produce terpenes. They cloned a terpene synthase gene and determined the first three-dimensional structure of this class of enzyme. As part of this research, they determined which elements of the enzymes were responsible for product specificity and developed technology to alter specificity, selectivity, and catalytic efficiency of the enzymes. The coupling of this protein engineering technology with yeast metabolic engineering for the production of terpenes provides the basis for a powerful platform for the production of a wide range of diverse terpene products.

From gene isolation to commercial scale production

Allylix’s multifaceted technology platform includes all of the molecular biology and engineering elements necessary to go from gene isolation to commercial scale production of terpenes, including gene cloning and expression, metabolic engineering, protein engineering, fermentation development, recovery and purification, and organic chemistry.

Specifically, Allylix’s technology platform consists of:

• Terpene cyclase gene cloning and expression
• Protein engineering to improve specificity and/or activity of terpene cyclases
• Yeast metabolic engineering to produce terpenes
• Fermentation development and recovery processes for the production of sesquiterpene products
• Production of commercially inaccessible or novel sesquiterpenes by chemobiosynthesis of com­binatorial libraries

Reengineering at work

The first step in developing a terpene biosynthetic process is the isolation of a terpene cyclase gene from its natural source, generally a plant. Using genomics, plant science, and genetic engineering techniques, Allylix scientists either isolate the gene of interest from its natural source, or if the gene sequence is known, have it chemically synthesized. The isolated gene is then further engineered to optimize its expression within the yeast host strain.

Sometimes, a natural gene or enzyme, even after engineering for optimal expression, will lack the required catalytic efficiency or product specificity to make it useful for economic production of the product of interest. To overcome these challenges, Allylix scientists alter the specificity of the terpene cyclase using a small number of discreet changes. This is a powerful technique. In a published example, the selectivity of two enzymes was completely changed by exchanging amino acids in and around the active sites of the enzymes. Once they change specificity, Allylix scientists then alter the catalytic efficiency of the gene to increase overall product yield. Using this process, in conjunction with other strain and process development technologies, Allylix scientists have increased production yields of its first products several hundred fold to reach the initial commercial yield targets.

The path to natural terpene production

Once the genes are optimized, they are introduced into yeast host strains that have been optimized for the production of terpenes. For sesquiterpene production, Allylix uses yeast host strains that have been engineered to overproduce the fifteen carbon biosynthetic precursor, farnesyl pyrophosphate (FPP). Allylix continues to improve these host strains through metabolic engineering, further increasing carbon flux to FPP to obtain higher production yields of sesquiterpene products.

Lastly, Allylix has developed proprietary fermentation, recovery and purification processes that maximize sesquiterpene production and allow economic recovery of the product.

The company’s technologies are protected by 62 issued U.S. and foreign patents that provide Allylix with a strong position and protection in key markets, including claims covering structure-based composition of matter, methods, proprietary enzymes and resulting products. Allylix’s intellectual property also includes trade secrets that cover host strains, expression systems and manufacturing processes.

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