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.

Figure 1

Bacterial diterpenoid natural products with their diterpenoid carbon scaffolds highlighted in red: (A) the biosynthetic gene clusters for these natural products have been cloned and partially characterized and (B) biosynthesis for these natural products has not been studied.

Figure 2

Bacterial diterpene synthases (DTSs): (A) Mechanisms of type I and type II DTSs and (B) pathways for bacterial diterpenoid natural product biosynthesis, highlighting known bacterial type I (blue) and type II DTSs (red) that convert GGDP to diverse diterpenoid scaffolds en route to the final natural products. See Figure 1 legend for structures of the diterpenoid natural products. See Figure 3 legend for accession numbers of the type I and type II DTSs. DTSs, diterpene synthases; ent-CPP, ent-copalyl diphosphate; GGDP, geranylgeranyl diphosphate.

Figure 3

Minimum evolution trees of primary amino acid sequences from plant (green), fungal (red), and bacterial (blue) type I (A) and type II DTSs (B). The branch lengths illustrate the extent of sequence diversity found in bacterial DTSs compared with plant or fungal enzymes. The trees were constructed in Mega5.0 with a ClustalW-generated primary sequence alignment. Shown in parentheses are accession numbers. Bacterial type I DTSs: Cyc2 (BAB39207), ORF3 (BAD86798), BjKS (BAC47415), PtmT3 (ACO31279), CotB2 (BAI44338), PtmT1 (ACO31274), PtnT1 (ADD83014), Rv3378 (P_217895). Fungal type I DTSs: PaDC1 (BAG30961), ACS (BAB62102), Smcps/ks (CAP07655), Gfcps/ks (Q9UVY5), FCPS/KS (BAA22426), Orf8 (bsc8) (BAI44849), PaFS (BAF45925). Plant type I DTSs: OsKSL6 (ABH10733), OsKSL5 (ABH10732), OsKSL11 (AAZ76733), OsKSL8 (BAD34478), OsKSL10 (ABH10735), OsKSL7 (ABH10734), OsKS1 (AAQ72559), OsKSL4 (AAU05906), CmKSB (AAB39482), AtKS (AAC39443), LsKS (BAB12441), SrKS1-1 (AF097310_1), PgKS (ADB55708), PpCPS/KS (BAF61135), TPS04/GES (NP_564772), RcCS (XP_002513340), SmCPSKSL1 (AEK75338), TDC1 (AAC49310), GbLPS (AAL09965), PaTPS-Iso (AAS47690), AgAS (Q38710), PaTPS-LAS (AAS47691), PtTPS (AAX07435). Bacterial type II DTSs: Rv3377c (NP_217894), BjCPS (BAC47414), PtmT2 (ACO31276), PtnT2 (ADD83015), Orf2 (BAD86797), Cyc1 (BAB39206), Bra4 (BAG16278), PlaT2 (ABB69743). Fungal type II DTSs: FCPS/KS (BAA22426), SmCPS/KS (CAP07655), GfCPS/KS (Q9UVY5). Plant type II DTSs: OsCPS1ent (BAD42449), OsCPS2ent (Q6ET36), ZmCPS1 (AAT49065), ZmCPS2 (ADB55709), HvCPS (BAH56560), TaCPS3 (AAT70083), OsCPS4syn (NP_0010521), TaCPS1 (BAH56558), CcCLS (ADJ93862), CmCPS1 (AAD04292), CmCPS2 (AAD04923), LsCPS (BAB12440), SrCPS (AAB87091), PsCPS (AAB58822), GA1 (AAA53632), PpCPS/KS (BAF61135), PsCPSb (ADB55709), PgCPS (ADB55707), GbLPS (AAL09965), AgAS (Q38710), PtTPS-LAS (AAX07435), PaTPS-ISO (AAS47690), PaTPS-LAS (AAS47691), SmCPSKSL1 (AEK75338), TPS04/GES (NP_564772). DTSs, diterpene synthases.

Figure 3

Minimum evolution trees of primary amino acid sequences from plant (green), fungal (red), and bacterial (blue) type I (A) and type II DTSs (B). The branch lengths illustrate the extent of sequence diversity found in bacterial DTSs compared with plant or fungal enzymes. The trees were constructed in Mega5.0 with a ClustalW-generated primary sequence alignment. Shown in parentheses are accession numbers. Bacterial type I DTSs: Cyc2 (BAB39207), ORF3 (BAD86798), BjKS (BAC47415), PtmT3 (ACO31279), CotB2 (BAI44338), PtmT1 (ACO31274), PtnT1 (ADD83014), Rv3378 (P_217895). Fungal type I DTSs: PaDC1 (BAG30961), ACS (BAB62102), Smcps/ks (CAP07655), Gfcps/ks (Q9UVY5), FCPS/KS (BAA22426), Orf8 (bsc8) (BAI44849), PaFS (BAF45925). Plant type I DTSs: OsKSL6 (ABH10733), OsKSL5 (ABH10732), OsKSL11 (AAZ76733), OsKSL8 (BAD34478), OsKSL10 (ABH10735), OsKSL7 (ABH10734), OsKS1 (AAQ72559), OsKSL4 (AAU05906), CmKSB (AAB39482), AtKS (AAC39443), LsKS (BAB12441), SrKS1-1 (AF097310_1), PgKS (ADB55708), PpCPS/KS (BAF61135), TPS04/GES (NP_564772), RcCS (XP_002513340), SmCPSKSL1 (AEK75338), TDC1 (AAC49310), GbLPS (AAL09965), PaTPS-Iso (AAS47690), AgAS (Q38710), PaTPS-LAS (AAS47691), PtTPS (AAX07435). Bacterial type II DTSs: Rv3377c (NP_217894), BjCPS (BAC47414), PtmT2 (ACO31276), PtnT2 (ADD83015), Orf2 (BAD86797), Cyc1 (BAB39206), Bra4 (BAG16278), PlaT2 (ABB69743). Fungal type II DTSs: FCPS/KS (BAA22426), SmCPS/KS (CAP07655), GfCPS/KS (Q9UVY5). Plant type II DTSs: OsCPS1ent (BAD42449), OsCPS2ent (Q6ET36), ZmCPS1 (AAT49065), ZmCPS2 (ADB55709), HvCPS (BAH56560), TaCPS3 (AAT70083), OsCPS4syn (NP_0010521), TaCPS1 (BAH56558), CcCLS (ADJ93862), CmCPS1 (AAD04292), CmCPS2 (AAD04923), LsCPS (BAB12440), SrCPS (AAB87091), PsCPS (AAB58822), GA1 (AAA53632), PpCPS/KS (BAF61135), PsCPSb (ADB55709), PgCPS (ADB55707), GbLPS (AAL09965), AgAS (Q38710), PtTPS-LAS (AAX07435), PaTPS-ISO (AAS47690), PaTPS-LAS (AAS47691), SmCPSKSL1 (AEK75338), TPS04/GES (NP_564772). DTSs, diterpene synthases.

Figure 4

(A) Modularity of DTS biochemistry demonstrated by conversion of the platencin- producing strain, S. platensis MA7339, into a platensimycin and platencin dual-producing strain, S. platensis SB12604, by genetic engineering [43], and a proposal of producing new analogues by heterologous expression of additional type I DTSs in S. platensis MA7339. (B) Utility of DTS modularity demonstrated through expression of various type I and type II DTSs in a recombinant E. coli strain that has been engineered to produce GGDP to yield eight different diterpene scaffolds, which provide an entry point to engineered production of thousands of diterpenoid natural products. DTSs, diterpene synthases; ent-CPP, ent-copalyl diphosphate; n-CPP, normal-copalyl diphosphate; syn-CPP, syn-copalyl diphosphate; GGDP, geranylgeranyl diphosphate; pGGeC, pGGnC, and pGGsC, three engineered type II DTSs that convert GGDP to ent-CPP, n-CPP, and syn-CPP, respectively; AtKS, rAgAS:D404A, OsKSL4, OsKSL5j, OsKSL6, OsKSL7, OsKSL8, OsKSL10, and OsKSL11, 11 engineered type II DTSs that convert ent-CPP, n-CPP, or syn-CPP to the nine diterpenoid scaffolds, respectively [66].