Enhanced synthesis of 1, 3-medium chain-2-long chain triacylglycerols by engineered Saccharomyces cerevisiae

Enzymatic synthesis is currently the primary method for preparing 1,3-medium chain- 2-long-chain triacylglycerols (MLM-TAGs), which serve as both a dietary component and clinical nutrient for specific populations. The application of MLM-TAGs is obviously constrained by the high cost of catalysts. Hence, a novel approach was proposed for MLM-TAGs production by engineered yeast. Overexpressing the mutated fas1^R1834K increased the production of medium-chain fatty acids (C8-C12) and resulted in an MLM content of 0.41 mol% of the total TAGs. The introduction of RnACSM4 enabled the recombinant to produce MLM-TAGs at a level of 4.2 mol% when supplemented with 0.2 mM sodium laurate. Further deletion of GAT2 and LRO1 increased the content of MLM-TAGs to 6.7 mol%. Iterative optimization involving sodium laurate dosage, culture temperature, and amino acid addition elevated the MLM-TAGs content to 34.4 mol%. Under the optimized conditions, the maximum yield of MLM-TAGs reached 18.5 mg/g DCW, representing a 135-fold improvement over the original strain. This research presents a promising and sustainable alternative for MLM-TAGs production and demonstrates the feasibility of tailoring the acyl composition of intracellular TAGs.