Microbial production of plant benzylisoquinoline alkaloids

Abstract

Benzylisoquinoline alkaloids, such as the analgesic compounds morphine and codeine, and the antibacterial agents berberine, palmatine, and magnoflorine, are synthesized from tyrosine in the Papaveraceae, Berberidaceae, Ranunculaceae, Magnoliaceae, and many other plant families. It is difficult to produce alkaloids on a large scale under the strict control of secondary metabolism in plants, and they are too complex for cost-effective chemical synthesis. By using a system that combines microbial and plant enzymes to produce desired benzylisoquinoline alkaloids, we synthesized (S)-reticuline, the key intermediate in benzylisoquinoline alkaloid biosynthesis, from dopamine by crude enzymes from transgenic Escherichia coli. The final yield of (S)-reticuline was 55 mg/liter within 1 h. Furthermore, we synthesized an aporphine alkaloid, magnoflorine, or a protoberberine alkaloid, scoulerine, from dopamine via reticuline by using different combination cultures of transgenic E. coli and Saccharomyces cerevisiae cells. The final yields of magnoflorine and scoulerine were 7.2 and 8.3 mg/liter culture medium. These results indicate that microbial systems that incorporate plant genes cannot only enable the mass production of scarce benzylisoquinoline alkaloids but may also open up pathways for the production of novel benzylisoquinoline alkaloids.

Fig. 1.

Benzylisoquinoline alkaloid biosynthetic pathway reconstructed in microbes. MAO, MAO from Micrococcus luteus (GenBank accession no. AB010716); NCS, NCS from Coptis japonica (GenBank accession no. AB267399); 6OMT, 6OMT from Coptis japonica (GenBank accession no. D29811); CNMT, CNMT from Coptis japonica (GenBank accession no. AB061863); 4′OMT, 4′OMT from Coptis japonica (GenBank accession no. D29812); CYP80G2, CYP80G2 from Coptis japonica (GenBank accession no. AB288053); BBE, BBE from Coptis japonica.

Fig. 2.

LC-MS analysis of reticuline produced in E. coli (A) or (S)-reticuline synthesized in vitro (B). The selected ion monitoring (SIM) parameters are as follows: m/z = 153 (3,4-DHPAA), m/z = 154 (dopamine), m/z = 288 (norlaudanosoline), m/z = 302 (3′-hydroxycoclaurine), m/z = 316 (3′-hydroxy-N-methylcoclaurine), m/z = 330 (reticuline). The inset shows the time course of reticuline production in E. coli (A) or (S)-reticuline synthesized in vitro (B). Error bars represent the SD of three independent measurements.

Fig. 3.

Production of intermediate alkaloids based on combinations of biosynthetic genes. Reactions were performed in vitro. Th gene combinations are as follows: MAO + NCS, MAO + NCS + CNMT, MAO + NCS + 4′OMT, MAO + NCS + CNMT + 4′OMT (A); MAO + NCS + 6OMT (B); MAO + NCS + 6OMT + CNMT (C); and MAO + NCS + 6OMT + 4′OMT (D). SIM parameters are as follows: m/z = 288 (norlaudanosoline), m/z = 302 (3′-hydroxycoclaurine), m/z = 316 (3′-hydroxy-N-methylcoclaurine or norreticuline), m/z = 330 (reticuline). The products were identified by comparison to authentic chemicals with regard to the fragmentation spectrum in LC-MS/MS (Fig. S4).

Fig. 4.

LC-MS analysis of magnoflorine (A) or scoulerine (B) produced in mixed culture of microbes. SIM parameters are as follows: m/z = 153 (3,4-DHPAA), m/z = 154 (dopamine), m/z = 288 (norlaudanosoline), m/z = 302 (3′-hydroxycoclaurine), m/z = 316 (3′-hydroxy-N-methylcoclaurine), m/z = 328 (corytuberine or scoulerine), m/z = 330 (reticuline), m/z = 342 (magnoflorine or N-methylscoulerine). The inset shows the time course of magnoflorine or scoulerine/N-methylscoulerine production in microbes. E. coli culture, which produced reticuline, was mixed with S. cerevisiae with CYP80G2-CNMT (A) or BBE (B) and cocultured. Cultures were sampled at various intervals and production levels were quantified. The amount of N-methylscoulerine production was calculated by using scoulerine as a standard. The error bars represent the standard deviation of three independent measurements.