Structural and Biochemical Characterization of Members of the Cannabinoid Biosynthetic Pathway to Inform their Application in Synthetic Biology

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Abstract

Cannabinoids are a unique group of secondary metabolites only found in the plant species Cannabis sativa. Over 70 cannabinoids have been identified; of which the two present at the highest levels in the plant which, due to their psychoactive and therapeutic activities, have attracted the most attention are tetrahydrocannabinol and cannabidiol. In this study, members of the cannabinoid biosynthetic pathway are characterized using the principles of biophysics and biochemistry in order to inform the synthetic biology process of introducing the pathway into a heterogeneous organism. The first enzyme of the pathway – tetraketide synthase – was expressed and purified using an E. coli expression system. The activity of the recombinant tetraketide synthase was tested by LC/MS analysis of the products from a biotransformation reaction. The product profile from which matched that of the native tetraketide synthase from C. sativa, thus, confirming that E. coli can express active forms of this protein. Successful crystallization and subsequent structure determination for the previously uncharacterized tetraketide synthase was achieved. This structure identified the five layered αβαβα homodimer tertiary structure of the enzyme and the catalytic triad of cysteine, asparagine and histidine, both of which are typical features of a type III polyketide synthase. A number of other conserved type III polyketide synthase features were identified in the tetraketide synthase, including phenylalanine gatekeepers to the active site and a methionine important to dimerization. The structure also allowed the design of variants in an attempt to improve the enzymes efficiency, particularly by reducing the level of by-products. Alanine scanning of the residues Ser126, Met130, Asp185, Met187, Ile248, Leu257, Phe259, Leu261, His297, Asn330 and Ser332 was conducted. Mutation of the residues Met130, His297 and Asn330 confirmed their importance to the activity and stability of the protein. The other mutated residues were localized to the active site of the tetraketide synthase to test their possible impact on the proteins activity. Though none of these mutations were successful in producing active enzyme they can inform future design of variants. The second enzyme of the pathway – olivetolic acid cyclase – was also expressed and purified using an E. coli expression system. The previously reported method of attaching a GST tag to the protein was confirmed as an effective approach to overcome its insolubility. Two other soluble tags, namely TRX and NUS, were also tested but showed to be less effective than the GST. A biotransformation reaction with LC/MS analysis of the products also confirmed that the recombinant olivetolic acid cyclase, both with the GST tag present and removed, was active and produced the intermediate of the cannabinoid pathway, olivetolic acid. From the information gained through these studies the first two enzymes can effectively be introduced to an E. coli host as part of a construct to allow efficient cannabinoid production utilizing a synthetic biology approach. Additionally, further attempts to improve the efficiency of these proteins can be made by using the data collected.

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