Bacterial diterpene synthases: new opportunities for mechanistic enzymology and engineered biosynthesis

Abstract

Diterpenoid biosynthesis has been extensively studied in plants and fungi, yet cloning and engineering diterpenoid pathways in these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial diterpene synthases are poised to make significant contributions to our understanding of terpenoid biosynthesis. Here we will first survey diterpenoid natural products of bacterial origin and briefly review their biosynthesis with emphasis on diterpene synthases (DTSs) that channel geranylgeranyl diphosphate to various diterpenoid scaffolds. We will then highlight differences of DTSs of bacterial and higher organism origins and discuss the challenges in discovering novel bacterial DTSs. We will conclude by discussing new opportunities for DTS mechanistic enzymology and applications of bacterial DTS in biocatalysis and metabolic pathway engineering.

Biosynthesis of Terpenoid Natural Products in Fungi

“…It is believed that these three domain (γβα) proteins found in fungi and plants are the result of an ancient fusion event of bacterial class I (α) and class II (γβ) terpenoid synthases. In fact, typical bacterial diterpene synthases are not fused, and cyclization involves class II (γβ) and class I (α) terpenoid synthases for this twostep cyclization mechanism [181]. In plants, however, many three-domain (γβα) diterpene synthases are monofunctional and catalyze either a protonation-or an ionization-dependent cyclization reaction (on a bicyclic diphosphate or GGPP) because the other domain is not functional [180].…”

Unique Biosynthesis of Sesquarterpenes (C35Terpenes)

“…34) These results suggest not only that two distinct pathways (Fig. 4, pathways A and B), which use E,E-FPP and E,E,E-GGPP respectively as substrates of Mvan 3822, are utilized to biosynthesize sesquarterpenes (13)(14)(15)(16)(17)(18) in the cells, and also that polyprenyl reductase (PR) can reduce both the Z and the E double bonds at C-14 of compounds 20 and 21. 34) In 2012, a new sesquarterpene, 18, was isolated from M. chlorophenolicum cells grown to stationary phase.…”

“…31) It has been reported that many terpene cyclases have broad substrate specificities, and that other classes of terpenes have been created in vitro. [2][3][4][5][6][7][8][9][10][11][12][13][14][15] A combination of studies identifying enzymatic products biosynthesized from unusual substrates and the analysis of genome sequences should lead not only to the discovery of additional functions of these enzymes in the organism, but also to the identification of novel natural terpenoids that have functions in their producers.…”

“…34) In 2012, a new sesquarterpene, 18, was isolated from M. chlorophenolicum cells grown to stationary phase. 35) The absence of compound 22 suggests that the main sesquarterpenes (13)(14)(15) are biosynthesized only via pathway B in the stationary phase, in contrast to the logarithmic growth phase, in which both pathway A and pathway B are involved. 35) In addition, our findings suggest that stepwise reduction of the polyprenyl group in sesquarterpene biosynthesis might proceed in a different order from that in chlorophyll biosynthesis.…”

“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] These compounds including hormones, vitamins, drugs, flavorings, and pigments, are categorized by number of C 5 isoprene units, as follows: hemi-(C 5 ), 1 unit; mono-(C 10 ), 2 units; sesqui-(C 15 ), 3 units; di-(C 20 ), 4 units; sester-(C 25 ), 5 units; tri-(C 30 ), 6 units; and tetraterpenes (C 40 ), 8 units. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] In contrast, little is known about C 35 terpenes. To the best of my knowledge, only 19 cyclic and eight linear C 35 terpenes have been identified to date, [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33]…”