Artepillin C is a diprenylated phenylpropanoid with various pharmacological benefits for human health. Its natural occurrence is limited to a few Asteraceae plants, such as Baccharis species, necessitating a stable supply through synthetic biology. In Saccharomyces cerevisiae, the utilization of aromatic substrates within the cell was limited, resulting in very low production of artepillin C. In this study, we used AcPT1, a p-coumaric acid (p-CA)-specific diprenyltransferase, in Komagataella phaffii to produce artepillin C. Detailed studies revealed that the critical bottleneck in K. phaffii was the supply of prenyl diphosphates, not phenylpropanoid flux. By enhancing the prenyl substrate pathway through overexpression of isopentenyl diphosphate isomerase and a truncated HMG-CoA reductase, we achieved a strong increase in artepillin C production. A major part of artepillin C was accumulated in yeast cells. One of the advantages of K. phaffii is its superior growth and ability to achieve high cell density cultivation compared to that of S. cerevisiae. Therefore, fed-batch cultivation with glycerol was performed. As a result, the dry cell weight (DCW) reached 61.0 g/L, and the intracellular amount of de novo produced artepillin C reached 187.3 μg/DCW. Analysis of intermediates revealed that the supply of p-CA constituted a bottleneck in artepillin C production in the engineered strain. By enhancing the p-CA supply, the intracellular accumulation of artepillin C reached 1200 μg/DCW even in batch cultivation. Moreover, the total intra- and extracellular amounts of artepillin C reached 12.5 mg/L, marking the highest de novo synthesis amount of artepillin C reported thus far, even under batch cultivation conditions.
Keywords: Komagataella phaffii; artepillin C; high cell density cultivation; metabolic engineering; prenylated phenolic compounds; prenyltransferase.