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.(S)-reticuline ͉ magnoflorine ͉ scoulerine

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…The availability of large numbers of BIA biosynthetic genes from opium poppy and related plants has facilitated the reconstitution of several pathways leading to the production of (S)-reticuline and (R,S)-reticuline in E. coli (Minami et al 2008) and Saccharomyces cerevisiae (Hawkins and Smolke 2008), respectively. Production of (S)-reticuline in E. coli was initially achieved by supplementing the bacterial culture medium with dopamine, some of which was converted to 3,4-dihydroxyphenylacetaldehyde (3,4-DHPAA) by a heterologously expressed bacterial monoamine oxidase (Minami et al 2008). PR10-type NCS from C. japonica catalyzed the condensation of dopamine and 3,4-DHPAA to (S)-norlaudanosoline, which underwent successive methylations via 6OMT, CNMT and 4′OMT to yield (S)-reticuline.…”

“…In turn, E. coli-generated (S)-reticuline was converted to the aporphine alkaloid magnoflorine via co-culture with a strain of S. cerevisiae engineered to express the C. japonica genes encoding corytuberine synthase (CYP80G2) and an N-methyltransferase. Similarly, (S)-scoulerine was produced using a gene encoding BBe (Minami et al 2008). Alternatively, (R,S)-reticuline was produced in S. cerevisiae from (R,S)-norlaudanosoline.…”

Reconstitution of a 10-gene pathway for synthesis of the plant alkaloid dihydrosanguinarine in Saccharomyces cerevisiae

“…Escherichia coli was recently engineered to produce the key BIA intermediate (S)-reticuline from glucose or glycerol 18,19 and co-cultured with S. cerevisiae expressing heterologous enzymes to synthesize (S)-magnoflorine and (S)-scoulerine 20 . While production of the key intermediate (S)-reticuline from simple carbon sources in E. coli is an undeniable success, S. cerevisiae is a more attractive host for BIA synthesis, due to its superior ability to express the cytochrome P450s common in downstream alkaloid synthesis 21 .…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…Instead, dopamine was converted to 3,4-dHPAA by MAO which was further condensed with the remaining dopamine to form S-laudanosoline. [262] Production of dopamine took place by the action of tyrosine/dopa decarboxylase (TYDC). Therefore, expression of TYDC leads to the production of dopamine (from L-DOPA) and tyramine (from tyrosine) at the same time.…”

“…Minami et al reported the very first microbial production of S-reticuline by feeding the precursor, dopamine. [262] Dopamine was then converted to 3,4-dHPAA by monoamine oxidase (MAO) which was further condensed with the remaining dopamine to form S-laudanosoline by norcoclaurine synthase (NCS). Then, by the additional expression of 6-O-methyltransferase (6OMT), coclaurine N-methyltransferase (CNMT), and 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase (4′OMT) in E. coli, S-reticuline was produced with a titer of 11 mg L −1 from 5 × 10 −3 m of exogenously fed dopamine (Figure 2).…”