Matching is the Key Factor to Improve the Production of Patchoulol in the Plant Chassis of Marchantia paleacea

[Image: see text] The valuable terpenoids, such as artemisinin acid, have achieved bioproduction in the chassis of microbes recently. In this study, Marchantia paleacea L, a promising plant synthetic biology chassis, was used to explore the possibility of patchoulol production by constructing a synthetic biology pathway composed of FPS and PTS. The experiment results show that the maximum yields based on the cytoplasm and plastid pathway were 621.56 and 1006.45 μg/g, respectively. However, there is no statistically significant difference in the yield of patchoulol between transformant plants with different subcellular compartment-targeting pathways. However, it was found that the highest yield of patchoulol was achieved in transformant plants with similar transcription levels of FPS and PTS. Also, the optimized transcription ratio between PTS and FPS is determined at 1.12 based on statistical analysis and model simulation. Therefore, two kinds of new optimized pathway vectors were constructed. One is based on the fusion protein method, and the other is based on protein expression individually, in which the same promoter and terminator were used to derive the expression of both FPS and PTS. The effect of pathway optimization was tested by transient and stable transformation. The production of patchoulol in transient transformation was the same for the two abovementioned kinds of matching pathway and higher than that for the original pathway. Also, in stable transformation, the yield of patchoulol reached up to 3250.30 μg/g, being three times the maximum content before optimization. It is suggested that M. paleacea is a powerful plant chassis for terpenoid synthetic biology and the matching between enzymes may be the key factor in determining the metabolic flux of the pathway in the study of synthetic biology.