Abstract Description

Sapienic acid (cis-6-hexadecenoic acid) is a rare monounsaturated fatty acid and is not readily available commercially at feasible prices. The two known sources are human sebum, where sapienic acid is the primary monounsaturated fatty acid, and Thunbergia alata endosperm, which contains up to 85 % sapienic acid in the seed oil. To produce sapienic acid in the native hosts, palmitic acid is desaturated between the 6th and 7th carbon atoms. In humans, the process is catalysed by fatty acid desaturase 2, a ∆6-desaturase, and in T. alata, by a ∆6-16:0-acyl carrier protein (ACP) desaturase. In T. alata, the desaturation reaction requires ferredoxin, an enzyme that mediates electron transfer from NADPH to the desaturase. Sapienic acid, among various other endogenous lipid groups found on the skin, mucosal surfaces, and saliva, has recently garnered interest for its ability to function as an antimicrobial agent. Numerous studies have demonstrated its potency against a wide variety of Gram-positive bacteria. However, limited research still exists regarding its antimicrobial properties, which may be due to its prohibitive cost. In this project, we aimed to heterologously produce the enzymes required for sapienic acid production, namely T. alata ∆6-16:0-ACP desaturase, ferredoxin from Impatiens balsamina, and vegetative Anabaena sp. strain PCC 7120, in Escherichia coli and Yarrowia lipolytica. Each gene was expressed from the pET28a-(+) vector in E. coli, and ∆6-16:0-ACP desaturase was co-expressed with either ferredoxin by using the pETDUET-1 vector. The ∆6-16:0-ACP desaturase gene was sub-cloned into the pKM177 yeast expression vector and integrated into Y. lipolytica via a CRISPR-Cas9 recombination platform. Protein production in the total and soluble fractions was evaluated by SDS-PAGE. All three enzymes were produced in the total fraction by E. coli; however, no soluble protein expression has been observed. Optimisation of expression was performed by altering the media composition, growth conditions, addition of metal cofactors, adding chemical inducers and co-expression with GroES/EL, which is currently underway. To conclude, producing sapienic acid with cost-effective microbial systems will enable further research on its antimicrobial activity, which may lead to the development of novel therapies in the future.