Biosynthesis of plant tetrahydroisoquinoline alkaloids through an imine reductase route

An artificial approach has been developed for efficient biosynthesis of plant tetrahydroisoquinoline alkaloids from dihydroisoquinoline precursors.


Supplementary
. Kinetic Parameters of IR45 and Its Mutants on the Conversion of 6a. Table S2. Kinetic Parameters of CNMT on the Conversion of 1b-5b.             3

1.1General Materials and Methods
Bacteria strains and plasmids are listed in Table S3. Escherichia coli was cultivated and manipulated according to the standard methods. 1

Expression and Purification of Proteins
Cloning and site-directed mutagenesis. The Coptis japonica Nmethyltransferase (CNMT) gene was codon-optimized based on E. coli bias and synthesized by Genewiz Biotech Co. Ltd (sequence see below). It was further cloned into the NdeI and XhoI sites of the pET28a to yield the plasmid pWHU2491. Mutations in the W191 of IR45 were introduced by PCR using pWHUIR45 2 as the template and primers listed in Table S4. Mutations in the F190 of W191F were introduced by Rolling circle PCR amplification using pWHUIR45-W191F as the template. After PCR amplification 0.5 μL DpnI enzyme was added to the products, and digestion was carried out at 37 °C for 2 hrs before transformation into E. coli DH5α. After verification by DNA sequencing, 4 plasmids containing mutations in W191 or F190 were further transformed into E. coli BL21 (DE3) for overexpression of N-terminal 6×His-tagged fusion proteins.

Synthesis of Substrates
General procedure for synthesis of 1a-5a 1a-5a were prepared according to literature method 3 with slightly modification. 3,4-dimethoxyphenylethylamine (50 mmol, 1.0 equiv) was added in dichloromethane (60 mL) under argon and cool to 0 o C. Triethylamine (TEA, 75 mmol, 1.5 equiv) and arylcarboxyl chloride (50 mmol, 1.0 equiv) were slowly added to the solution. The reaction mixture was stirred at room temperature for 18 hrs and then concentrated under vacuum. The residue was dissolved in EtOAc (40 mL) and washed with 1 M HCl solution (40 mL). The aqueous layer was further extracted with EtOAc (2 × 40 mL). The combined organic extracts were washed with brine (80 mL) and dried over by MgSO4. Solvent was evaporated to give the corresponding amide, which was used in the next step without further purification. 6 The resulting amide (5 mmol, 1 equiv) was then dissolved into a DCM (30 mL) solution of 2-chloropyridine (6.0 mmol, 1.2 equiv), which was added by trifluoromethanesulfonic anhydride (5.5 mmol, 1.1 equiv) via syringe drop and kept at -78 °C to initiate the reaction. Reaction system was stirred at -78 °C for 15 min and slowly warm to 30 °C overnight. The reaction was quenched with saturated NaHCO3 solution and extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with brine, dried over MgSO4, and concentrated under vacuum. The crude product was purified with column chromatography (dichloromethane / methanol) to yield desired product.    Table S5). For achieving accurate value, three repeats were performed and analyzed for each conversion.  Table S5). For achieving accurate value, three repeats were performed and analyzed for each conversion.

Determination of Kinetic Constants
Kinetic analysis of IRED. Kinetic parameters for substrates were determined  Table S5). The conversion was calculated based on product generation.
Standard with different concentrations were prepared to draw the standard curve which was used for products' quantification in the enzymatic reactions.

Chiral HPLC analysis.
For the chiral analysis of the products, two methods are  Table S5C.

Biotransformation with Recombinant E. coli Strains
Co-expression of GDH and IREDs. The GDH gene 9 Tables   Table S1. Kinetic Parameters of IR45 and Its Mutants on the Conversion of 6a

Supplementary
(also see Fig. S3A). [a] W191A has the biggest catalytic efficency (kcat / Km value), however its extremely low Km value makes it readily saturated by substrate when the medium-to-high concentration of substrates was employed. Therefore, its catalytic performance of conversion of 6a is indeed not as good as W191F (see Fig. S2).