Enhancement of antroquinonol production via the overexpression of 4-hydroxybenzoate polyprenyltransferase biosynthesis-related genes in Antrodia cinnamomea

Abstract

Antroquinonol (AQ) as one of the most potent bioactive components in Antrodia cinnamomea (Fomitopsidaceae) shows a broad spectrum of anticancer effects. The lower yield of AQ has hampered its possible clinical application. AQ production may potentially be improved by genetic engineering. In this study, the protoplast-polyethylene glycol method combined with hygromycin as a selection marker was used in the genetic engineering of A. cinnamomea S-29. The optimization of several crucial parameters revealed that the optimal condition for generating maximal viable protoplasts was digestion of 4-day-old germlings with a mixture of enzymes (lysing enzyme, snailase, and cellulase) and 1.0 M MgSO4 for 4 h. The ubiA and CoQ2 genes, which are involved in the synthesis of 4-hydroxybenzoate polyprenyltransferase, were cloned and overexpressed in A. cinnamomea. The results showed that ubiA and CoQ2 overexpression significantly increased AQ production in submerged fermentation. The overexpressing strain produced maximum AQ concentrations of 14.75 ± 0.41 mg/L and 19.25 ± 0.29 mg/L in pCT74-gpd-ubiA and pCT74-gpd-CoQ2 transformants, respectively. These concentrations were 2.00 and 2.61 times greater than those produced by the control, respectively. This research exemplifies how the production of metabolites may be increased by genetic manipulation, and will be invaluable to guide the genetic engineering of other mushrooms that produce medically useful compounds.