Biosynthesis of plant tetrahydroisoquinoline alkaloids through an imine reductase route

Abstract

Herein, we report a biocatalytic approach to synthesize plant tetrahydroisoquinoline alkaloids (THIQAs) from dihydroisoquinoline (DHIQ) precursors using imine reductases and N-methyltransferase (NMT). The imine reductase IR45 was engineered to significantly expand its substrate specificity, enabling efficient and stereoselective conversion of 1-phenyl and 1-benzyl 6,7-dimethoxy-DHIQs into the corresponding (S)-tetrahydroisoquinolines (S-THIQs). Coclaurine N-methyltransferase (CNMT) was able to further efficiently convert these (S)-THIQ intermediates into (S)-THIQAs. By assembling IRED, CNMT, and glucose dehydrogenase (GDH) in one reaction, we effectively constituted two artificial biosynthetic pathways in Escherichia coli and successfully applied them to the production of five (S)-THIQAs. This highly efficient (100% yield from DHIQs) and easily tailorable (adding other genes) biosynthetic approach will be useful for producing a variety of plant THIQAs.

Scheme 1. Representative THIQAs.

Scheme 2. Approaches for plant THIQA biosynthesis.

Scheme 3. DHIQ substrates prepared and used in this study.

Fig. 1. In silico model of IR45 with substrates 1a and 4a. The structure of IR45 is modelled based on Q1EQE0 (PDB: ; 3zhb) and docked with 1a or 4a (grey) and NADPH (pink); critical residues in the binding cavity are labelled. Protein backbones and residues from different subunits are indicated by yellowish brown and green colours. (A) IR45 with 1a; (B) IR45 with 4a.

Fig. 2. Production of THIQAs 1–5 in the IRED-based biosynthetic systems. The retention time of the precursors 1a–5a is indicated by the dashed lines. Samples from the biosynthetic systems are indicated by the solid lines in dark blue. (I) Biosynthesis of 1 from 1a by E. coli with F190M–W191F + GDH + CNMT; (II) biosynthesis of 2 from 2a by the crude enzymes F190L–W191F + GDH and E. coli with CNMT; (III) biosynthesis of 3 from 3a by the crude enzymes F190L–W191F + GDH and E. coli with CNMT; (IV) biosynthesis of 4 from 4a by E. coli with F190M–W191F + GDH + CNMT; (V) biosynthesis of 5 from 5a by E. coli with F190M–W191F + GDH + CNMT.