Improved Expression of His₆‐Tagged Strictosidine Synthase cDNA for Chemo‐Enzymatic Alkaloid Diversification

Abstract

Strictosidine synthase (STR1) catalyzes the stereoselective formation of 3alpha(S)-strictosidine from tryptamine and secologanin. Strictosidine is the key intermediate in the biosynthesis of 2,000 plant monoterpenoid indole alkaloids, and it is a key precursor of enzyme-mediated synthesis of alkaloids. An improved expression system is described which leads to optimized His(6)-STR1 synthesis in Escherichia coli. Optimal production of STR1 was achieved by determining the impact of co-expression of chaperones pG-Tf2 and pG-LJE8. The amount and activity of STR1 was doubled in the presence of chaperone pG-Tf2 alone. His(6)-STR1 immobilized on Ni-NTA can be used for enzymatic synthesis of strictosidines on a preparative scale. With the newly co-expressed His(6)-STR1, novel 3alpha(S)-12-azastrictosidine was obtained by enzymatic catalysis of 7-azatryptamine and secologanin. The results obtained are of significant importance for application to chemo-enzymatic approaches leading to diversification of alkaloids with novel improved structures.

The Pictet–Spengler Reaction in Nature and in Organic Chemistry

“…[48,56] This "one-pot" chemoenzymatic approach has recently been simplified and extended for the generation of the new alkaloid aza-tetrahydroalstonine [56] by using recombinant and His 6 -tagged immobilized STR1 obtained from expression of its cDNA in E. coli. Following optimized overexpression, [57] STR1 was used for the synthesis of azastrictosidine from 7-azatryptamine (Scheme 7). [56,58] Moreover, azastrictosidine has been used for the simple one-step chemical synthesis of a previously unknown lactam of azastrictosidine and for azanacycline under basic and acidic conditions, respectively.…”

A Facile Chemoenzymatic Approach: One‐Step Syntheses of Monoterpenoid Indole Alkaloids

“…The prepurified recombinant STR1-His 6 was immobilized on nickel nitrilotriacetate (Ni-NTA) to get the key intermediates 1 and 2 by enzymatic coupling of secologanin with tryptamine and 7-aza-tryptamine, respectively. [9] The alkaloids (3-8) were simply prepared by a one-step reaction. The lactams 3 [10] and 6 were obtained by conversion of 1 and 2 under mild basic conditions (Scheme 1).…”

“…Previously, we have reported an improved STR1-His 6 expression system that utilizes the co-expression of plasmid pQE-2-STR1 with the molecular chaperone pG-Tf2 in Escherichia coli M15. [9] The immobilized STR1-His 6 exhibits excellent stability (for the C. roseus enzyme, see Ref. [7a]), thus making it a good candidate to enzymatically produce strictosidine (1; Scheme 1) and 12-aza-strictosidine Abstract: Facile chemoenzymatic syntheses of cytotoxic monoterpenoid indole alkaloids with novel skeletons and multiple chiral centers are described.…”

Asymmetric Preparation of prim-, sec-, and tert-Amines Employing Selected Biocatalysts

“…51 A related enzyme catalyzing a Pictet–Spengler reaction is the strictosidine synthase, which seems to be more substrate specific especially regarding the aldehyde. 52 …”