Enhancing structural diversity of terpenoids by multisubstrate terpene synthases

Terpenoids are one of the largest class of natural products with diverse structures and activities. This enormous diversity is embedded in enzymes called terpene synthases (TSs), which generate diverse terpene skeletons via sophisticated cyclization cascades. In addition to the many highly selective TSs, there are many promiscuous TSs that accept multiple prenyl substrates, or even noncanonical ones, with 6, 7, 8, 11, and 16 carbon atoms, synthesized via chemical approaches, C-methyltransferases, or engineered lepidopteran mevalonate pathways. The substrate promiscuity of TSs not only expands the structural diversity of terpenes but also highlights their potential for the discovery of novel terpenoids via combinatorial biosynthesis. In this review, we focus on the current knowledge on multisubstrate terpene synthases (MSTSs) and highlight their potential applications.

The reactions of TSs are one of the most important factors contributing to terpene diversity, as they often synthesize multiple products from a single substrate through complex cyclization cascades [4][5][6][7][8][9][10].Based on the mechanism of initial carbocation generation, TSs generally fall into two main classes.Class I TSs generate an allylic cation from a prenyl substrate by depyrophosphorylation, whereas class II TSs utilize a general acid (a key Asp residue) to protonate the terminal C=C bond or epoxide group to yield a tertiary carbocation.The highly reactive carbocation is then converted to different carbocation intermediates, facilitated by the hydrophobic pocket of the TSs, which often results in multiple terpene products from a single prenyl substrate.In addition to their distinct mechanisms, the two major classes of TSs are classified according to their sequences, structures, and functions.For instance, class I TSs often have conserved sequence motifs, DDXXD and NSE/DTE, that bind trinuclear magnesium clusters for diphosphate abstraction, whereas class II TSs have a DXDD motif that acts as the catalytic acid.Recently, several novel unconventional TSs that share low sequence and structural similarities with classical TSs have been discovered and comprehensively reviewed [11,12].
In addition to the capability to generate multiple products using a single substrate, a growing number of TSs called multisubstrate terpene synthases (MSTSs) are capable of utilizing prenyl precursors with different chain lengths or configurations to synthesize diverse terpenoid products.Notably, MSTSs can also convert noncanonical prenyl substrates, including chemically synthesized analogs and bio-originated 6-, 7-, 8-, 11-, and 16-carbon substrates generated by methyltransferases or engineered lepidopteran mevalonate pathways.The multisubstrate features of these enzymes have often been characterized using in vitro assays.The in vivo activities of MSTSs were revealed by the development of an efficient precursor-providing chassis.The inherent features of MSTSs not only increase the structural diversity of terpenoids but also underscore their potential for generating new terpenoids through combinatorial biosynthesis.An important review published previously comprehensively addressed the transformation of synthetic prenyl-substrate analogs by TSs as well as TS-mimicking chemical transformations [13].In this review, we discuss representative MSTSs originating from different species that use canonical prenyl substrates.We also highlight recent advances in the production of novel terpenoids by MSTSs using synthetic prenyl substrates.Finally, we focused on MSTSs that catalyze the transformation of naturally occurring noncanonical prenyl substrates.
Although many MSTSs exhibit a broad substrate scope in vitro, their product profiles may be altered in vivo owing to the subcellular localization of enzymes and the availability of substrates in different intracellular compartments [23].For instance, two nerolidol/linalool synthases from Antirrhinum majus (AmNES/LIS-1, -2) both synthesize 6 and 7 in vitro, but cytosol-localized AmNES/LIS-1 produces only 7, while plastidlocalized AmNES/LIS-2 synthesizes 6 [24].Similarly, in the case of CoTPS5, the transient expressed cytosol CoTPS5 in N. benthamiana only generated 8, while the plastid-localized CoTPS5 yielded 9 and 10 other than 8 (Table 1).These studies indicate that redirecting MSTSs to different subcellular compartments may facilitate the generation of multiple terpenoids in plants.

MSTSs from fungi
Fungi are also prolific producers of terpenoids with diverse cyclic structures and important biological activities, which are of great interest.However, the number of known fungal MSTSs is currently limited, and researchers have focused on the promiscuity of their products rather than substrates [25][26][27].
Unlike plant MSTSs, fungal MSTSs convert natural substrates into cyclic skeletons.According to a phylogenetic tree constructed using 51 well-characterized class I TSs, clade III is of particular interest because most characterized di-and sester-TSs are enriched in this clade [28].Two clade III TSs, FgMS and FgGS, from Fusarium graminearum J1-012 were characterized as promiscuous TSs with broad substrate specificities both in vitro and in vivo, indicating that TSs in clade III are more likely to be promiscuous.Using an efficient precursor-provid- ing chassis, 50 terpenoids were generated via combinatorial biosynthesis using only two TSs and three PTs to generate 4, 5 or geranylfarnesyl diphosphate (GFPP, 29, Figure 1), representative products 30-33 are shown in Figure 3a [28].Notably, FgMS is a chimeric enzyme (PTTS) consisting of an N-terminal class I TS domain and a C-terminal GFPP synthase domain.Therefore, to block the generation of 29, a variant of FgMS-D510A with an inactive PT domain, rather than wild-type FgMS, was used in combinatorial biosynthesis.Furthermore, critical residues controlling substrate specificity were identified using site-directed mutagenesis.Interestingly, when the aromatic residue Phe65 was replaced with Ala, the resulting variant F65A produced a novel 5/8/6/6 tetracyclic sesterterpene in the presence of 29 [28].Domain swapping is another useful approach for changing the PTTS product profile.For example, EvVS from Emericella variecolor majorly produced diterpene variediene (34) with a minor production of sesterterpene (2E)α-cericerene (33) in vitro (Figure 3a) [29].By replacing the PT domain of EvVS with that of sester-TS EvSS, the resulting variant generated 33, which was not produced in vivo as the major product, both in vitro and in vivo.These studies revealed that altering and enhancing the supply of prenyl substrates can significantly change the product profile of promiscuous TSs, thereby generating terpenes with novel structures.

TSs using noncanonical prenyl diphosphate substrates
Chemically synthesized noncanonical prenyl substrates Noncanonical prenyl diphosphates are analogs of natural prenyl diphosphates.Most noncanonical prenyl diphosphate substrates are chemically synthesized.Classically, these prenyl analogs have been used as co-crystallization ligands [38], inhibitors of specific TSs [39], and tools to study the reaction mechanisms of cyclization cascades [40,41] which have been comprehensively addressed in important previous reviews [8,13].Currently, noncanonical prenyl analogs have been synthesized to act as actual substrates of TSs to generate novel terpene skeletons, introduce reaction handles, and produce value-added compounds.A previous review has covered the advances of TS-cat-alyzed transformations of synthetic substrate analogs up to 2019 [13,42].Here, we provide updated examples on this topic.
These studies demonstrate the potential of TSs to utilize noncanonical synthetic prenyl analogs to yield unusual terpenoid skeletons and new value-added terpenoids.
In addition to expanding the repertoire of terpenoids, the biotransformation of noncanonical prenyl substrates by TSs provides insights into the mechanisms of cyclization reactions.β-Himachalene synthase (HcS) and (Z)-γ-bisabolene synthase (BbS) from Cryptosporangium arvum, and germacrene A synthase (SmTS6) from Streptomyces mobaraensis were chosen to convert four FPP analogs 72-75, which not only generated several new terpenoids (76-79), but also revealed the cyclization mechanisms of selected TSs [40] (Figure 6a).Similarly, two GGPP analogues 80 and 81 with shifted double bonds were synthesized to study the stereochemistry and cyclization mechanism of casbene synthase (CS) from the castor bean (Ricinus communis), which indicated a stereochemical course in accordance with the reported absolute configuration of casbene [41] (Figure 6b).The same GGPP isomers (80, 81) were employed to generate novel diterpene derivatives and revealed the cyclization mechanisms of 12 di-TSs [48].Similarly, dihydro-GGPP (82) and dihydro-GFPP (83) have been synthesized for biotransformation using several di-and sester-TSs.The conversion of analogues 82 and 83 by TSs led to the production of ruptenes including compounds 84-90, which revealed the structure of the proposed intermediates for the cyclization reactions and there-  fore provided important insights into the reaction mechanism [49] (Figure 6c).With the aid of artificial prenyl analogs, a new route was developed to access a pool of unnatural terpenoids.It is worth noting that the rational design and synthesis of analogs play a valuable role in elucidating the cyclization mechanism of TSs, which further broadens our knowledge of the biosynthesis of terpenoids.

Naturally occurring noncanonical prenyl substrates
Most of the terpene biosynthesis is well defined by the 'isoprene rule' to form natural products by the polymerization of C 5 isoprene.Although terpenes with irregular carbon atoms (C 6 , C 7 , C 11 , C 12 , C 16 , and C 17 ) have been characterized, they are thought to be synthesized by modifications after the formation of the terpene skeletons [50].Recently, additional routes have been discovered for the production of noncanonical terpenoids, whose biosynthesis requires C-methyltransferases from bacteria.IPP/DMAPP methyltransferases have been shown to convert C 5 prenyl substrates to irregular C 6 (91-95), C 7 (96-100), and C 8 diphosphates (101), which could serve as building blocks for the generation of new terpenoids [51] (Figure 7).Furthermore, a series of noncanonical C 11 , C 12 , C 16 , and C 17 prenyl substrates were synthesized in Escherichia coli harboring heterologously expressed IPP methyltransferase (IPPMT) from Streptomyces monomycini.Notably, polymethylated C 41 , C 42 , and C 43 carotenoids were produced by combining the endogenous terpene biosynthesis pathway and IPPMT, demonstrating the potential of this approach to expand the terpene structural space [52].
In addition to methylation of the elongation unit IPP, noncanonical prenyl substrates can also be prepared by modifying the prenyl substrate of TSs.For instance, the heterologous expression of GPP C2-methyltransferases with C 11 -TSs and mevalonate biosynthesis enzymes in E. coli yielded 35 C 11 terpenes and 11 C 16 terpenes [53].By introducing a GPP C2-methyltransferase from Pseudanabaena limnetica to yeast together with an engineered C 11 -specific TS, 40 C 11 terpene scaffolds were produced, which significantly increased the chemical space of terpenoids [54].More recently, the GPP C6-methyltransferase BezA was discovered in Streptomyces coelicolor [55] (Figure 8a).Further structure-based engineering of BezA successfully repurposed it to catalyze the unprecedented C6-methylation of FPP by a single residue substitution in its substrate-binding pocket [55].Moreover, efforts have also been made to engineer the TSs to modulate their product selectivity with the noncanonical prenyl substrates.To enable the biotechnological synthesis of irregular terpenes, the product selectivity of 2-methylenebornane synthase from Pseudomonas fluorescenes was altered using a semi-rational engineering approach [56].
In contrast to GPP methylation, modification of FPP is catalyzed by the C-methyltransferase SpSodMT.In 2018, the bio-synthesis of an unusual homosesquiterpene, sodorifen (102, Figure 1), from Serratia plymuthica 4RX13 was elucidated [57].The in vitro and in vivo results revealed that a SAM-dependent-C-methyltransferase catalyzed methylation and cyclization reactions to form pre-sodorifen (103, Figure 8b), which was subsequently converted to 102 by TS [57].Key residues lining the catalytic cavity of SpSodMT, Q57, F58, N219, V273, and L302, were found to affect product outcomes, and mutagenesis of these residues resulted in new C 16 -prenyl substrates [58].8c (113-122) [58].Notably, the widespread biosynthesis of C 16 terpenoids was reported in a recent study in which biosynthetic gene clusters for C 16 terpenoids were identified and grouped into four types according to the number of MTs and TSs in the gene cluster [59].A subset of methyltransferase genes was functionally characterized using engineered yeast, which has an enhanced supply of 4 (strain AM109) [60] and the main product of these enzymes was compound 103 [59].Subsequently, 35 selective TSs in these gene clusters were characterized using a yeast chassis, and 47 noncanonical terpenoids were produced with 13 of them being elucidated (123-135, Figure 9), which enabled further studies on their functions and prompted the discovery of new types of terpenoids [59].

Conclusion
During terpenoid biosynthesis, most TSs have strict substrate selectivity; nevertheless, some promiscuous TSs accept multiple prenyl substrates and produce various products.In nature, the biosynthesis of prenyl substrates may have subcellular locations, and the available types of prenyl substrates are limited, especially for noncanonical substrates in living cells.Therefore, the potential of TSs to generate terpenoids has been underestimated.With the development of synthetic biology technologies, an efficient precursor-providing chassis was constructed.Together with the accumulation of genome sequencing data, we systematically evaluated the function of TSs and discovered new terpenoids via genome mining.Nevertheless, for drug development, the accumulated terpene skeletons still require further functionalization, which requires additional genome-mining efforts for the discovery of tailored enzymes.
Researchers have successfully expanded the chemical space of terpenoid biosynthesis using noncanonical prenyl substrates, which were synthesized using chemical approaches or via biosynthetic pathways.Many new terpenoids have been derived from chemically prepared prenyl analogs for decades, and until recently, the conversion of enzymatically modified noncanonical substrates has been utilized.New building blocks with irregular carbon numbers broaden the diversity of terpenoid structures.However, more systematic studies on noncanonical terpenoids are needed to study their biological activities.

Figure 3 :
Figure 3: The structure of representative terpene products of MSTSs.a) From fungi: compounds 30-33 are produced by the fungal TS FgMS, 34 is the product of wild-type EvVS, and 33 is a new product of an EvVS variant with a swapped PT domain.b) From bacteria: compound 35 is a representative product of bacteria MSTSs VenA; compouns 36, 37, and 40 are products of two long β-prene TSs BclTS and BalTS.

Figure 8 :
Figure 8: The structure of noncanonical prenyl substrates generated by C-methyltransferases and variants.a) 2Me-GPP and 6Me-GPP are produced by GPP C-methyltransferases. b) Compound 103 is produced by FPP C-methyltransferases. c) Compounds 104-112 are new C 16 building blocks synthesized by SpSodMT variants, and 113-122 are selected typical products yielded by terpentetriene synthase and kolavelool synthase with further modifications by a cytochrome P450 CYP720B1.

Figure 9 :
Figure 9: Structures of C 16 terpenes identified via genome mining of C 16 biosynthetic gene clusters from bacteria.

Figure 10 :
Figure 10: a) Precursors and final products of the MVA pathway and LMVA pathway.b) The structure of C 6 -and C 7 -isoprenols 139 and 140.