Allylix’ 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. Through these phases, the Allylix platform also allows the discovery of novel terpenes.

Specifically, Allylix’ 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.

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