Rapid identification of enzyme variants for reengineered alkaloid biosynthesis in periwinkle

Abstract

Monoterpene indole alkaloids from Catharanthus roseus (Madagascar periwinkle), such as the anticancer agents vinblastine and vincristine, have important pharmacological activities. Metabolic engineering of alkaloid biosynthesis can provide an efficient and environmentally friendly route to analogs of these synthetically challenging and pharmaceutically valuable natural products. However, the narrow substrate scope of strictosidine synthase, the enzyme at the entry point of the pathway, limits a pathway engineering approach. We demonstrate that with a different expression system and screening method it is possible to rapidly identify strictosidine synthase variants that accept tryptamine analogs not turned over by the wild-type enzyme. The variants are used in stereoselective synthesis of beta-carboline analogs and are assessed for biosynthetic competence within the terpene indole alkaloid pathway. These results present an opportunity to explore metabolic engineering of "unnatural" product production in the plant periwinkle.

Figure 1

Structures of selected medicinal MIAs produced or derived from Madagascar periwinkle (C. roseus). Ajmalicine 1 acts as an antihypertensive agent, serpentine 2 is a topoisomerase II inhibitor, vinblastine 3 and vincristine 4 are anticancer agents and Vinflunine 5 is a modified vinblastine analog with promising bioactivity.

Figure 2

STS-catalyzed formation of 8a via a Pictet-Spengler reaction between 6a and 7. The reaction proceeds via an iminium intermediate, which undergoes electrophilic aromatic substitution and cyclization to give 8a. Deglucosylation by a dedicated β-glucosidase forms an aglucone 9 that rearranges into dehydrogeissochizine 10 and cathenamine 11. The deglucosylated products are then enzymatically converted into the MIA structures observed in periwinkle. However, aglucone 9 can be intercepted by amine nucleophiles to form colored adducts.

Figure 3

Visualization of expression and purification of C. roseus STS and verification of mutagenesis and screening methods. (A) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of STS samples. STS has a calculated molecular weight of ~37 kDa. Lane 1: Relative mass marker. Lane 2: STS in media without FLAG-tag. Lane 3: STS in media with FLAG-tag. Lane 4: Anti-FLAG antibody purified STS from supernatant. FLAG-tag: Asp-Tyr-Lys-Asp₄-Lys. (B) Representative screen of STS activity after randomization of a residue distant from the active site (Tyr167). All wells contain the same assay mixture (2 mM 6a, 1 mM 7, SG and 25 mM sodium phosphate buffer at pH 7.0). The first column contains no-insert and wild-type-insert controls (rows 1–4 and 5–8, respectively). Columns 2–10 (all rows) contain media from mutagenesis libraries.

Figure 4

Tryptamine binding site and tryptamine substrate scope of STS. (A) Tryptamine binding site of STS from R. serpentina (PDB ID: 2FPB, residues are numbered according to the C. roseus peptide sequence). Side-chains interacting with tryptamine are in stick-representation. The protein main-chain is represented in cartoon form. (B) Tryptamine analogs with indole ring substitutions that are accepted by wild-type STS [21].

Figure 5

Expanded substrate scope: tryptamine analogs assayed with STS variants used in this study.

Figure 6

LC-traces of selected alkaloid analogs formed by feeding strictosidine analogs to two-week-old C. roseus hairy root cultures. (A) Formation of putative serpentine or serpentine isomer analog upon feeding of 8o to periwinkle. The two top traces show that no compounds corresponding to m/z 427 and 429 exist in the culture supplemented with tryptamine 6a. The bottom trace shows the elution of an authentic standard of serpentine 2. The remaining two traces show the formation of an alkaloid analog displaying the expected isotopic pattern that could be a brominated MIA analog. (B) Formation of isositsirikine 14m in a feeding study of 8m. The top trace shows the expected mass of the alkaloid analog; the bottom trace shows that no naturally occuring alkaloid of m/z 385 co-elutes with 14m.