Efficient microbial production of stylopine using a Pichia pastoris expression system.

Stylopine (tetrahydrocoptisine) is a protoberberine-type benzylisoquinoline alkaloid that is found in Argemone mexicana1. This compound is an intermediate in the biosynthetic pathway of benzophenanthridine alkaloids, such as sanguinarine, and is also a reduced form of coptisine, which is found in Coptis rhizome. Although there have been limited pharmacological studies of coptisine and stylopine compared with those on berberine, the physiological activities of protoberberine alkaloids as modulators of lipid metabolism2,3 have led to an increased demand for methods to prepare more diversified alkaloids such as stylopine and coptisine. However, the presence of two methylene-dioxy rings in the protoberberine skeleton of stylopine makes chemical synthesis difficult. Thus, a method that relies more on a biotechnological approach for production is required. Although stylopine is an intermediate in the benzophenanthridine alkaloid biosynthesis, it shows only scant accumulation in cultured cells compared with the accumulation of coptisine, an oxidized form of stylopine, because stylopine is so easily oxidized4,5. As major parts of the biosynthetic pathway for sanguinarine from tyrosine have been characterized at the molecular level (Fig. 1), there are several possible options for the production of stylopine using biotechnological techniques. For example, the RNA silencing of the gene for an enzyme that is downstream of stylopine in sanguinarine biosynthesis, i.e., tetrahydroprotoberberine N-methyltransferase, is feasible, as shown for the successful accumulation of reticuline in transgenic Eschscholzia californica cells with an RNA interference (RNAi) vector for berberine bridge enzyme (BBE) to convert reticuline to scoulerine6. However, the construction of transgenic plant cells with stable productivity is both time-consuming and difficult due to the instability of cultured cells. Figure 1 Isoquinoline alkaloid biosynthetic pathway. In this study, we examined a much simpler method for producing stylopine from reticuline in microbial cells, as we had recently established an efficient system for producing reticuline from a simple carbon source such as glucose/glycerol in Escherichia coli7. Stylopine is synthesized from (S)-reticuline in a reaction consisting of three steps, i.e., berberine bridge enzyme (BBE)8, cheilanthifoline synthase (e.g., CYP719A5 from E. californica, CHS)9, and stylopine synthase (e.g., CYP719A2/A3 from E. californica, STS)10; however, a previous report indicated that the BBE step was inefficient for expression in a microbial system, especially in yeast11. Although reconstruction of the entire pathway in single cells is preferred due to the simplicity of handling and management, we first examined the stepwise optimization of the pathway to overcome the inefficiency of BBE. Next, we examined the reconstruction of the pathway through the co-culturing of each biosynthetic reaction. We also examined the co-expression of the genes for all of the biosynthetic enzymes in a single cell as a consolidated form and compared the efficiency of the biosynthesis with that in the co-culture (Supplementary Fig. S1). In this study, we examined the efficacy of Pichia cells for the high expression of cytochrome P450s, as the expression level of this membrane protein is usually low and not sufficient for conversion. Fortunately, all three enzymes were successfully expressed in Pichia cells. Thus, both the co-culture of the transformants with each step and the consolidated transformant showed the efficient (more than 80%) conversion of reticuline to stylopine. The advantages of the consolidated transformant, which showed very rapid conversion within 2 hr, are discussed along with those of the co-culture system, which offers greater flexibility for pathway design and the potential to avoid metabolic interference among the biosynthetic enzymes.