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This study highlights the significance of overexpressing 1-deoxy-d-xylulose-5-phosphate synthase (DXS) from the MEP (methylerythritol 4-phosphate) pathway, in addition to short-chain prenyltransferase fusions for the improved production of the diterpene, taxa-4,11-diene, the first committed intermediate in the production of anti-cancer drug paclitaxel. The results showed that the strain which has (i) the taxadiene synthase (txs) gene integrated into the genome, (ii) the MEP pathway genes overexpressed, (iii) the fpps-crtE prenyltransferases fusion protein and (iv) additional expression of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), yielded the highest production of taxa-4,11-diene at 390 mg/L (26 mg/L/OD 600). This represents a thirteen-fold increase compared to the highest reported concentration in B. subtilis. The focus on additional overexpression of DXS and utilizing short-chain prenyltransferase fusions underscores their pivotal role in achieving significant titer improvements in terpene biosynthesis. [Display omitted] • Utilizing Bacillus subtilis as a cell factory for the production of taxadiene. • Additional expression of DXS from MEP pathway can boost taxadiene production. • Application of fused prenyltransferases can enhance the yield of taxadiene. • The highest taxadiene production so far in B. subtilis has been achieved. [ABSTRACT FROM AUTHOR]

Prenylation of peptides is widely observed in the secondary metabolites of diverse organisms, granting peptides unique chemical properties distinct from proteinogenic amino acids. Discovery of prenylated peptide agents has largely relied on isolation or genome mining of naturally occurring molecules. To devise a platform technology for de novo discovery of artificial prenylated peptides targeting a protein of choice, here we have integrated the thioether‐macrocyclic peptide (teMP) library construction/selection technology, so‐called RaPID (Random nonstandard Peptides Integrated Discovery) system, with a Trp‐C3‐prenyltransferase KgpF involved in the biosynthesis of a prenylated natural product. This unique enzyme exhibited remarkably broad substrate tolerance, capable of modifying various Trp‐containing teMPs to install a prenylated residue with tricyclic constrained structure. We constructed a vast library of prenylated teMPs and subjected it to in vitro selection against a phosphoglycerate mutase. This selection platform has led to the identification of a pseudo‐natural prenylated teMP inhibiting the target enzyme with an IC50 of 30 nM. Importantly, the prenylation was essential for the inhibitory activity, enhanced serum stability, and cellular uptake of the peptide, highlighting the benefits of peptide prenylation. This work showcases the de novo discovery platform for pseudo‐natural prenylated peptides, which is readily applicable to other drug targets. [ABSTRACT FROM AUTHOR]

Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, β-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, β-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals. Key points: • The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum. • TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF. • The research offers insights into the potential applications of fungal indole diterpene prenyltransferases. [ABSTRACT FROM AUTHOR]

As the most widely distributed phenolic compounds in the plant kingdom, flavonoids play an integral role in plant reproduction and defense. Also, they represent many important quality traits of edible plants like color and antioxidants, and have a variety of biological activities beneficial to human health. To diversify the functions of synthesized flavonoids, plants have evolved various enzymes to perform structural modifications on different flavonoid backbones. One of these modifications is prenylation, which refers to the attachment of an isoprenoid moiety, most commonly a prenyl (C5) group. Numerous structure-activity analyses of prenylflavonoids have shown that isopentenyl substitutions at specific sites can significantly expand and enhance their chemical properties, bioactivities and potential health benefits. This review summarizes prenylflavonoids reported so far in all plant species and highlights the current knowledge on naturally occurring prenyltransferases from different biological sources that can act on plant flavonoids to synthesize prenylflavonoids. Most of them have strict flavonoid substrate- and regio-specificities, and they provide a valuable gene repository to facilitate the efficient scale-up production of flavonoids with specific prenylation patterns in cell factories. To truly achieve this goal, it is necessary to explore more diversified natural prenyltransferases, and to optimize the bioreactors system such as pathway regulation and modular co-culture engineering in the future. [ABSTRACT FROM AUTHOR]

Fungal hybrid terpenoid saccharides constitute a new and growing family of natural products with significant biomedical and agricultural activities. One representative family is the cosmosporasides, which feature oxidized terpenoid units and saccharide moieties; however, the assembly line of these building blocks has been elusive. Herein, a cos cluster from Fusarium orthoceras was discovered for the synthesis of cosmosporaside C (1) by genome mining. A UbiA family intramembrane prenyltransferase (UbiA‐type PT), a multifunctional cytochrome P450, an α,β‐hydrolase, an acetyltransferase, a dimethylallyl transferase (DMAT‐type PT) and a glycosyltransferase function cooperatively in the assembly of the scaffold of 1 using primary central metabolites. The absolute configuration at C4, C6 and C7 of 1 was also established. Our work clarifies the unexpected functions of UbiA‐type and DMAT‐type PTs and provides an example for understanding the synthetic logic of hybrid terpenoid saccharides in fungi. [ABSTRACT FROM AUTHOR]

The fungal prenyltransferase ShPT from Stereum hirsutum was believed to prenylate 4-hydroxybenzyl alcohol and thereby be involved in the vibralactone biosynthesis. In this study, we demonstrate that hydroxynaphthalenes instead of benzyl alcohol or aldehyde were accepted by ShPT for regular C-prenylation in the presence of both dimethylallyl and geranyl diphosphate. Although the natural substrate of ShPT remains unknown, our results provide one additional prenyltransferase from basidiomycetes, which are less studied, in comparison to those from other sources. Furthermore, this study expands the chemical toolbox for regioselective production of prenylated naphthalene derivatives. Key points: •Basidiomycetous prenyltransferase •Biochemical characterization •A DMATS prenyltransferase prenylating hydroxynaphthalene derivatives [ABSTRACT FROM AUTHOR]

Anhydroicaritin (1a), baohuoside (1b) and icariin (1c) were recognized as major pharmacologically active ingredients of Epimedium plants. Their primary means of acquisition were chemical isolation from plants. However, it suffers from low yield, environmental pollution and shortage of plants. Herein, to remedy these problems, biosynthesis was explored to obtain the three active ingredients. Fortunately, with SfFPT as 8-prenyltransferase, EpPF3RT and Ep7GT as glycosyltransferases, kaempferide (1) was transferred to 1a, 1b and 1c enzymatically. Thus, we report the details of this method. This approach represents a promising environmental friendly alternative for the production of these compounds from an abundant analogue. [ABSTRACT FROM AUTHOR]

Cyanobactin prenyltransferases (PTases) belong to the ABBA-type PTases and commonly adopt a truncated β/α barrel fold, which enables them to accommodate large peptide substrates. The unique architecture makes cyanobactin PTases catalyze a particular prenylation on substrates, with diversified global sequences. Various cyanobactin PTases have been characterized to prenylate various acceptor residues, including Tyr, Trp, Ser, Thr, Arg, His, and peptide N/C termini. The relaxed substrate preference makes cyanobactin PTases extraordinarily suitable for diversity-oriented synthesis of prenylated peptides with therapeutic potential. Prenylation is a post-translational modification (PTM) widely found in primary and secondary metabolism. This modification can enhance the lipophilicity of molecules, enabling them to interact with lipid membranes more effectively. The prenylation of peptides is often carried out by cyanobactin prenyltransferases (PTases) from cyanobacteria. These enzymes are of interest due to their ability to add prenyl groups to unmodified peptides, thus making them more effective therapeutics through the subsequent acquisition of increased membrane permeability and bioavailability. Herein we review the current knowledge of cyanobactin PTases, focusing on their discovery, biochemistry, and bioengineering, and highlight the potential application of them as peptide alkylation biocatalysts to generate peptide therapeutics. [ABSTRACT FROM AUTHOR]

SUMMARY: Coumarins are natural products with important medicinal values, and include simple coumarins, furanocoumarins and pyranocoumarins. Female ginseng (Angelica sinensis) is a renowned herb with abundant coumarins, originated in China and known for the treatment of female ailments for thousands of years. The molecular basis of simple coumarin biosynthesis in A. sinensis and the evolutionary history of the genes involved in furanocoumarin biosynthesis are largely unknown. Here, we generated the first chromosome‐scale genome of A. sinensis. It has a genome size of 2.37 Gb, which was generated by combining PacBio and Hi‐C sequencing technologies. The genome was predicted to contain 43 202 protein‐coding genes dispersed mainly on 11 pseudochromosomes. We not only provided evidence for whole‐genome duplication (WGD) specifically occurring in the Apioideae subfamily, but also demonstrated the vital role of tandem duplication for phenylpropanoid biosynthesis in A. sinensis. Combined analyses of transcriptomic and metabolomic data revealed key genes and candidate transcription factors regulating simple coumarin biosynthesis. Furthermore, phylogenomic synteny network analyses suggested prenyltransferase genes involved in furanocoumarin biosynthesis evolved independently in the Moraceae, Fabaceae, Rutaceae and Apiaceae after ζ and ε WGD. Our work sheds light on coumarin biosynthesis, and provides a benchmark for accelerating genetic research and molecular breeding in A. sinensis. Significance Statement: The genomic resources of Angelica sinensis in this study will facilitate efficient germplasm exploration, facilitate comparative and functional genomics studies, and the improvement of A. sinensis for medicinal uses, especially for the specific production of bioactive molecules in planta. [ABSTRACT FROM AUTHOR]

Epimedium pubescens is a species of the family Berberidaceae in the basal eudicot lineage, and a main plant source for the traditional Chinese medicine “Herba Epimedii”. The current study achieved a chromosome-level genome assembly of E. pubescens with the genome size of 3.34 Gb, and the genome guided discovery of a key prenyltransferase (PT) in E. pubescens. Our comparative genomic analyses confirmed the absence of Whole Genome Triplication (WGT-γ) event shared in core eudicots and further revealed the occurrence of an ancient Whole Genome Duplication (WGD) event approximately between 66 and 81 Million Years Ago (MYA). In addition, whole genome search approach was successfully applied to identify 19 potential flavonoid PT genes and an important flavonoid PT (EpPT8) was proven to be an enzyme for the biosynthesis of medicinal compounds, icaritin and its derivatives in E. pubescens. Therefore, our results not only provide a good reference genome to conduct further molecular biological studies in Epimedium genus, but also give important clues for synthetic biology and industrial production of related prenylated flavonoids in future. [ABSTRACT FROM AUTHOR]

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes. [ABSTRACT FROM AUTHOR]

Isobavachalcone (IBC) is a prenylated chalcone mainly distributed in some Fabaceae and Moraceae species. IBC exhibits a wide range of pharmacological properties, including anti-bacterial, anti-viral, anti-inflammatory, and anticancer activities. In this study, we attempted to construct the heterologous biosynthesis pathway of IBC in tobacco (Nicotiana tabacum). Four previously reported prenyltransferases, including GuILDT from Glycyrrhiza uralensis, HlPT1 from Humulus lupulus, and SfILDT and SfFPT from Sophora flavescens, were subjected to an in planta screening to verify their activities for the biosynthesis of IBC, by using tobacco transient expression with exogenous isoliquiritigenin as the substrate. Only SfFPT and HlPT1 could convert isoliquiritigenin to IBC, and the activity of SfFPT was higher than that of HlPT1. By co-expression of GmCHS8 and GmCHR5 from Glycine max, endogenous isoliquiritigenin was generated in tobacco leaves (21.0 mg/g dry weight). After transformation with a multigene vector carrying GmCHS8, GmCHR5, and SfFPT, de novo biosynthesis of IBC was achieved in transgenic tobacco T0 lines, in which the highest amount of IBC was 0.56 mg/g dry weight. The yield of IBC in transgenic plants was nearly equal to that in SfFPT transient expression experiments, in which substrate supplement was sufficient, indicating that low IBC yield was not attributed to the substrate supplement. Our research provided a prospect to produce valuable prenylflavonoids using plant-based metabolic engineering. [ABSTRACT FROM AUTHOR]

Hyperforin is a representative polycyclic polyprenylated acylphloroglucinols (PPAPs) that exerts a variety of pharmacological activities. The complete biosynthesis pathway of hyperforin has not been elucidated due to its complex structure and unclear genetic background of its source plants. This mini-review focuses on the bioactivity and biosynthesis of hyperforin. These analyses can provide useful insights into the biosynthesis investigations of hyperforin and other PPAPs with complex structures. [ABSTRACT FROM AUTHOR]

Terpenoids are the largest class of natural products and are derived from C 5 isoprene units. Recent discoveries of modification enzymes in native isoprene units before cyclization or transfer reactions have revealed that C 5 units with additional carbon atoms are also used to produce terpenoids. These reports indicate that the utilization of these modification enzymes is useful for the enzymatic production of non-natural terpenoids. In this study, we have attempted to produce methylgeranyl polyphenols, which are not observed in nature, by combining a geranyl pyrophosphate C6 methyltransferase, BezA, which was discovered from the benzastatin biosynthetic pathway, and the promiscuous prenyltransferase NphB, which catalyzes prenylation of various flavonoids. We successfully synthesized five methylgeranylated flavonoids from naringenin, apigenin, and genistein. This result demonstrates that BezA is a powerful tool for the synthesis of novel non-natural terpenoids. [ABSTRACT FROM AUTHOR]

Terpenoids are the most diverse class of plant primary and specialized metabolites, and trans-prenyltransferases (trans-PTs) are the first branch point to synthesize precursors of various chain lengths for further metabolism. Whereas the catalytic mechanism of the enzyme is known, there is no reliable method for precisely predicting the functions of trans-PTs. With the exponentially increasing number of available trans-PTs genes in public databases, an in silico functional prediction method for this gene family is urgently needed. Here, we present PTS-Pre, a web tool developed on the basis of the "three floors" model, which shows an overall 86% prediction accuracy for 141 experimentally determined trans-PTs. The method was further validated by in vitro enzyme assays for randomly selected trans-PTs. In addition, using this method, we identified nine new GFPPSs from different plants which are beyond the previously reported Brassicaceae clade, suggesting these genes may have occurred via convergent evolution and are more likely lineage-specific. The high accuracy of our blind prediction validated by enzymatic assays suggests that PTS-Pre provides a convenient and reliable method for genome-wide functional prediction of trans-PTs enzymes and will surely benefit the elucidation and metabolic engineering of terpenoid biosynthetic pathways. [ABSTRACT FROM AUTHOR]

Furanocoumarins (FCs) are widely distributed secondary metabolites found in higher plants, including Apiaceae, Rutaceae, Moraceae, and Fabaceae. They play a crucial role in the physiological functions of plants and are well-known for their diverse pharmacological activities. As a representative plant of the Apiaceae family, Angelica sinensis is highly valued for its medicinal properties and FCs are one of the main ingredients of A. sinensis. However, the biosynthetic mechanism of FCs in A. sinensis remains poorly understood. In this study, we successfully cloned and verified three types of enzymes using genome analysis and in vitro functional verification, which complete the biosynthesis of the FCs core skeleton in A. sinensis. It includes a p -coumaroyl CoA 2′-hydroxylase (AsC2′H) responsible for umbelliferone formation, two UbiA prenyltransferases (AsPT1 and AsPT2) that convert umbelliferone to demethylsuberosin (DMS) and osthenol, respectively, and two CYP736 subfamily cyclases (AsDC and AsOD) that catalyze the formation of FCs core skeleton. Interestingly, AsOD was demonstrated to be a bifunctional cyclase and could catalyze both DMS and osthenol, but had a higher affinity to osthenol. The characterization of these enzymes elucidates the molecular mechanism of FCs biosynthesis, providing new insights and technologies for understanding the diverse origins of FCs biosynthesis. The biosynthetic pathway of furanocoumarins in Angelica sinensis. Simple coumarin (umbelliferone) is transformed into linear FC (marmesin) and angular FC (columbianetin) under the action of a p -coumaroyl CoA 2′-hydroxylase (AsC2′H), two UbiA prenyltransferases (AsPT1 and AsPT2), and two CYP736 subfamily cyclases (AsDC and AsOD). [Display omitted] • The catalytic function of AsC2′H , AsPT1 and AsPT2 were verified successfully. • Two CYP736 subfamily cyclases were verified in Angelica sinensis. • A bifunctional cyclase was discovered for the first time in A. sinensis. [ABSTRACT FROM AUTHOR]

Some enzymes annotated as squalene synthase catalyze the prenylation of carbazole‐3,4‐quinone‐containing substrates in bacterial secondary metabolism. Their reaction mechanisms remain unclear because of their low sequence similarity to well‐characterized aromatic substrate prenyltransferases (PTs). We determined the crystal structures of the carbazole PTs, and these revealed that the overall structure is well superposed on those of squalene synthases. In contrast, the stacking interaction between the prenyl donor and acceptor substrates resembles those observed in aromatic substrate PTs. Structural and mutational analyses suggest that the Ile and Asp residues are essential for the hydrophobic and hydrophilic interactions with the carbazole‐3,4‐quinone moiety of the prenyl acceptor, respectively, and a deprotonation mechanism of an intermediary σ‐complex involving a catalytic triad is proposed. Our results provide a structural basis for a new subclass of aromatic substrate PTs. [ABSTRACT FROM AUTHOR]

Simple Summary: Terpenoid phenazines generally produced in Streptomyces exhibit potential antitumor and antibacterial activities. In this study, we designed and constructed an artificial biosynthetic pathway for the synthesis of terpenoid phenazines in Pseudomonas chlororaphis P3. We successfully synthesized endophenazine A and endophenazine A1 for the first time in Pseudomonas by introducing the prenyltransferase PpzP. Moreover, we revealed the biosynthetic pathway of endophenazine A1 in P. chlororaphis P3. This study enriches the diversity of phenazines in P. chlororaphis P3 and provides a reference for the heterologous synthesis of terpenoid phenazines. Endophenazine A is a terpenoid phenazine with phenazine-1-carboxylic acid (PCA), and dimethylallyl diphosphate (DMAPP) derived from the 2-methyl-D-erythritol-4-phosphate (MEP) pathway as the precursor, which shows good antimicrobial activity against several Gram-positive bacteria and fungi. However, the highest yield of endophenazine A was about 20 mg/L in Streptomyces, limiting its large-scale industrial development. Pseudomonas chlororaphis P3, possessing an efficient PCA synthesis and MEP pathways, is a suitable chassis to synthesize endophenazine A. Herein, we designed an artificial biosynthetic pathway for the synthesis of endophenazine A in P. chlororaphis P3. Primarily, the prenyltransferase PpzP from Streptomyces anulatus 9663 was introduced into P. chlororaphis P3 and successfully synthesized endophenazine A. Another phenazine compound, endophenazine A1, was discovered and identified as a leakage of the intermediate 4-hydroxy-3-methyl-2-butene pyrophosphate (HMBPP). Finally, the yield of endophenazine A reached 279.43 mg/L, and the yield of endophenazine A1 reached 189.2 mg/L by metabolic engineering and medium optimization. In conclusion, we successfully synthesized endophenazine A and endophenazine A1 in P. chlororaphis P3 for the first time and achieved the highest titer, which provides a reference for the heterologous synthesis of terpenoid phenazines. [ABSTRACT FROM AUTHOR]

Dimethylallyltryptophan synthases (DMATSs) catalyze the prenyl transfer reaction from dimethylallyl pyrophosphate (DMAPP) to an indole ring. IptA, a member of the DMATS family, is involved in biosynthesis of 6-dimethylallylindole-3-carbaldehyde in Streptomyces sp. SN-593 and catalyzes the C6-prenylation of l -Trp. The enzyme exhibits prenyl acceptor promiscuity and can accept various Trp derivatives, as observed in several other DMATS family members. Although many crystal structures of DMATS have been determined to date, the structural basis of substrate promiscuity and the acceptance of alternatives to indole-containing natural substrates remain to be clarified. In this study, we determined the crystal structures of the ternary l -Trp derivative (5-methyl-, 6-methyl-, and Nα-methyl- l -Trp) -DMSPP (dimethylallyl S -thiolopyrophosphate; stable analog of DMAPP) -enzyme complex of IptA, in addition to the substrate-free IptA and ternary l -Trp-DMSPP-IptA complex crystal structures. The overall structure of IptA exhibited a typical ABBA-fold, which is commonly found in DMATS family members, while l -Trp and DMSPP are found in a tunnel located inside the ABBA barrel. The crystal structure of the ternary l -Trp-DMSPP-enzyme complex can explain the electrophilic substitution at the C6 atom of l -Trp, which is assisted by Glu84 and His294, as previously suggested for other DMATSs. Although l -Trp snugly fitted into the active site pocket and the unoccupied space around l -Trp is very limited in the l -Trp-DMSPP-IptA complex structure, the enzyme can accommodate 5-methyl- and 6-methyl- l -Trp by slight relocation of the substrate indole ring and adjacent side chain in the active site, resulting in a higher prenylation activity for 5-methyl- l -Trp and C7 prenylation of 6-methyl- l -Trp. Like many other DMATSs, IptA cannot utilize prenyl donors larger than DMAPP. To enlarge the prenyl donor-binding pocket, the W154A mutation was introduced. As expected, this mutant produced prenylated l -Trp from l -Trp and geranyl- and farnesyl pyrophosphate. • IptA, a C6 dimethylallyltryptophan synthase (6-DMATS), can prenylate many Trp- and indole derivatives like several other DMATSs. • We determined five crystal structures of IptA in substrate-free, and Trp- and Trp derivatives-bound forms. • Substrate-bound crystal structures revealed how Trp derivatives are accommodated in IptA. • Substrate-bound structures revealed the structural basis to enhance prenylation activity and prenyl donor selectivity of DMATSs. [ABSTRACT FROM AUTHOR]

Key message: 5-Hydroxyisoflavonoids, no 5-deoxyisoflavonoids, in Lupinus species, are due to lack of CHRs and Type II CHIs, and the key enzymes of isoflavonoid biosynthetic pathway in white lupin were identified. White lupin (Lupinus albus) is used as food ingredients owing to rich protein, low starch, and rich bioactive compounds such as isoflavonoids. The isoflavonoids biosynthetic pathway in white lupin still remains unclear. In this study, only 5-hydroxyisoflavonoids, but no 5-deoxyisoflavonoids, were detected in white lupin and other Lupinus species. No 5-deoxyisoflavonoids in Lupinus species are due to lack of CHRs and Type II CHIs. We further found that the CHI gene cluster containing both Type I and Type II CHIs possibly arose after the divergence of Lupinus with other legume clade. LaCHI1 and LaCHI2 identified from white lupin metabolized naringenin chalcone to naringenin in yeast and tobacco (Nicotiana benthamiana), and were bona fide Type I CHIs. We further identified two isoflavone synthases (LaIFS1 and LaIFS2), catalyzing flavanone naringenin into isoflavone genistein and also catalyzing liquiritigenin into daidzein in yeast and tobacco. In addition, LaG6DT1 and LaG6DT2 prenylated genistein at the C-6 position into wighteone. Two glucosyltransferases LaUGT1 and LaUGT2 metabolized genistein and wighteone into its 7-O-glucosides. Taken together, our study not only revealed that exclusive 5-hydroxyisoflavonoids do exist in Lupinus species, but also identified key enzymes in the isoflavonoid biosynthetic pathway in white lupin. [ABSTRACT FROM AUTHOR]

Rehmannia glutinosa has important medicinal value; terpenoid is one of the main active components in R. glutinosa. In this study, iTRAQ technique was used to analyze the relative abundance of proteins in roots of R. glutinosa, and 6,752 reliable proteins were quantified. GO enrichment results indicated that most proteins were involved in metabolic process or cellular process, 57.63% proteins had catalytic activity, and 65.80% proteins were enriched in membrane-bounded organelle. In roots of R. glutinosa, there were 38 KEGG enrichments with significance, more DEPs were found in some pathways, especially the proteasome pathway and TCA cycle with 15.0% DEPs between elongation stage and expansion stage of roots. Furthermore, five KEGG pathways of terpenoid synthesis were found. Most prenyltransferases belong to FPP/GGPP synthase family, involved in terpenoid backbone biosynthesis, and all interacted with biotin carboxylase CAC2. Compared with that at the elongation stage, many prenyltransferases exhibited higher expression at the expansion stage or maturation stage of roots. In addition, eight FPP/GGPP synthase encoding genes were cloned from R. glutinosa, namely FPPS, FPPS1, GGPS, GGPS3, GGPS4, GGPS5, GPPS and GPPS2, introns were also found in FPPS, FPPS1, GGPS5 and GGPS2, and FPP/GPP synthases were more conservative in organisms, especially in viridiplantae, in which the co-occurrence of GPPS or GPPS2 was significantly higher in plants. Further analysis found that FPP/GGPP synthases of R. glutinosa were divided into three kinds, GGPS, GPPS and FPPS, and their gene expression was significantly diverse in different varieties, growth periods, or tissues of R. glutinosa. Compared with that of GGPS, the expression of GPPS and FPPS was much higher in R. glutinosa, especially at the expansion stage and maturation stage. Thus, the synthesis of terpenoids in roots of R. glutinosa is intricately regulated and needs to be further studied.

Reconstitution of prenylflavonoids using the flavonoid biosynthetic pathway and prenyltransferases (PTs) in microbes can be a promising attractive alternative to plant-based production or chemical synthesis. Here, we demonstrate that promiscuous microbial PTs can be a substitute for regiospecific but mostly unidentified botanical PTs. To test the prenylations of naringenin, we constructed a yeast strain capable of producing naringenin from l-phenylalanine by genomic integration of six exogenous genes encoding components of the naringenin biosynthetic pathway. Using this platform strain, various microbial PTs were tested for prenylnaringenin production. In vitro screening demonstrated that the fungal AnaPT (a member of the tryptophan dimethylallyltransferase family) specifically catalyzed C-3′ prenylation of naringenin, whereas SfN8DT-1, a botanical PT, specifically catalyzed C-8 prenylation. In vivo, the naringenin-producing strain expressing the microbial AnaPT exhibited heterologous microbial production of 3′-prenylnaringenin (3′-PN), in contrast to the previously reported in vivo production of 8-prenylnaringenin (8-PN) using the botanical SfN8DT-1. These findings provide strategies towards expanding the production of a variety of prenylated compounds, including well-known prenylnaringenins and novel prenylflavonoids. These results also suggest the opportunity for substituting botanical PTs, both known and unidentified, that display relatively strict regiospecificity of the prenyl group transfer.

Legume plants are rich in prenylated flavonoid compounds, which play an important role in plant defense and human health. In the present study, we identified a prenyltransferase (PT) gene, named LaPT2, in white lupin (Lupinus albus), which shows a high identity and close relationship with the other known PT genes involved in flavonoid prenylation in planta. The recombinant LaPT2 protein expressed in yeast cells exhibited a relatively strong activity toward several flavonols (e.g., kaempferol, quercetin, and myricetin) and a relatively weak activity toward flavanone (naringenin). In addition, the recombinant LaPT2 protein was also active toward several other types of flavonoids, including galangin, morin, 5-deoxyquercetin, 4'-O-methylkaempferol, taxifolin, and aromadendrin, with distinct enzymatic affinities. The LaPT2 gene was preferentially expressed in the roots, which is consistent with the presence of prenylated flavonoid kaempferol in the roots. Moreover, we found that the expression level of LaPT2 paralleled with those of LaF3H1 and LaFLS2 genes that were relatively higher in roots and lower in leaves, suggesting that they were essential for the accumulation of prenylated flavonoid kaempferol in roots. The deduced full-length LaPT2 protein and its signal peptide fused with a green fluorescent protein (GFP) are targeted to plastids in the Arabidopsis thaliana protoplast. Our study demonstrated that LaPT2 from white lupin is responsible for the biosynthesis of prenylated flavonoids, in particular flavonols, which could be utilized as phytoalexin for plant defense and bioactive flavonoid compounds for human health.

Rehmannia glutinosa has important medicinal value; terpenoid is one of the main active components in R. glutinosa. In this study, iTRAQ technique was used to analyze the relative abundance of proteins in roots of R. glutinosa , and 6,752 reliable proteins were quantified. GO enrichment results indicated that most proteins were involved in metabolic process or cellular process, 57.63% proteins had catalytic activity, and 65.80% proteins were enriched in membrane-bounded organelle. In roots of R. glutinosa , there were 38 KEGG enrichments with significance, more DEPs were found in some pathways, especially the proteasome pathway and TCA cycle with 15.0% DEPs between elongation stage and expansion stage of roots. Furthermore, five KEGG pathways of terpenoid synthesis were found. Most prenyltransferases belong to FPP/GGPP synthase family, involved in terpenoid backbone biosynthesis, and all interacted with biotin carboxylase CAC2. Compared with that at the elongation stage, many prenyltransferases exhibited higher expression at the expansion stage or maturation stage of roots. In addition, eight FPP/GGPP synthase encoding genes were cloned from R. glutinosa , namely FPPS, FPPS1, GGPS, GGPS3, GGPS4, GGPS5, GPPS and GPPS2 , introns were also found in FPPS, FPPS1, GGPS5 and GGPS2 , and FPP/GPP synthases were more conservative in organisms, especially in viridiplantae, in which the co-occurrence of GPPS or GPPS2 was significantly higher in plants. Further analysis found that FPP/GGPP synthases of R. glutinosa were divided into three kinds, GGPS, GPPS and FPPS, and their gene expression was significantly diverse in different varieties, growth periods, or tissues of R. glutinosa. Compared with that of GGPS , the expression of GPPS and FPPS was much higher in R. glutinosa , especially at the expansion stage and maturation stage. Thus, the synthesis of terpenoids in roots of R. glutinosa is intricately regulated and needs to be further studied. [ABSTRACT FROM AUTHOR]

Summary: Geranylgeranyl diphosphate (GGPP) produced by GGPP synthase (GGPPS) serves as a precursor for many plastidial isoprenoids, including carotenoids. Phytoene synthase (PSY) converts GGPP into phytoene, the first committed intermediate of the carotenoid pathway.Here we used biochemical, molecular, and genetic tools to characterise the plastidial members of the GGPPS family in tomato (Solanum lycopersicum) and their interaction with PSY isoforms.The three tomato GGPPS isoforms found to localise in plastids (SlG1, 2 and 3) exhibit similar kinetic parameters. Gene expression analyses showed a preferential association of individual GGPPS and PSY isoforms when carotenoid biosynthesis was induced during root mycorrhization, seedling de‐etiolation and fruit ripening. SlG2, but not SlG3, physically interacts with PSY proteins. By contrast, CRISPR‐Cas9 mutants defective in SlG3 showed a stronger impact on carotenoid levels and derived metabolic, physiological and developmental phenotypes compared with those impaired in SlG2. Double mutants defective in both genes could not be rescued.Our work demonstrates that the bulk of GGPP production in tomato chloroplasts and chromoplasts relies on two cooperating GGPPS paralogues, unlike other plant species such as Arabidopsis thaliana, rice or pepper, which produce their essential plastidial isoprenoids using a single GGPPS isoform. [ABSTRACT FROM AUTHOR]

Legume plants are rich in prenylated flavonoid compounds, which play an important role in plant defense and human health. In the present study, we identified a prenyltransferase (PT) gene, named LaPT2 , in white lupin (Lupinus albus), which shows a high identity and close relationship with the other known PT genes involved in flavonoid prenylation in planta. The recombinant LaPT2 protein expressed in yeast cells exhibited a relatively strong activity toward several flavonols (e.g., kaempferol, quercetin, and myricetin) and a relatively weak activity toward flavanone (naringenin). In addition, the recombinant LaPT2 protein was also active toward several other types of flavonoids, including galangin, morin, 5-deoxyquercetin, 4'-O-methylkaempferol, taxifolin, and aromadendrin, with distinct enzymatic affinities. The LaPT2 gene was preferentially expressed in the roots, which is consistent with the presence of prenylated flavonoid kaempferol in the roots. Moreover, we found that the expression level of LaPT2 paralleled with those of LaF3H1 and LaFLS2 genes that were relatively higher in roots and lower in leaves, suggesting that they were essential for the accumulation of prenylated flavonoid kaempferol in roots. The deduced full-length LaPT2 protein and its signal peptide fused with a green fluorescent protein (GFP) are targeted to plastids in the Arabidopsis thaliana protoplast. Our study demonstrated that LaPT2 from white lupin is responsible for the biosynthesis of prenylated flavonoids, in particular flavonols, which could be utilized as phytoalexin for plant defense and bioactive flavonoid compounds for human health. [ABSTRACT FROM AUTHOR]

Cyclodipeptide synthases (CDPSs) catalyze the formation of cyclodipeptides using aminoacylated tRNAs as the substrates and have great potential in the production of diverse 2,5-diketopiperazines (2,5-DKPs). Genome mining of Streptomyces leeuwenhoekii NRRL B-24963 revealed a two-gene locus, saz, encoding CDPS SazA and a unique fused enzyme (SazB) harboring two domains: phytoene synthase-like prenyltransferase (PT) and methyltransferase (MT). Heterologous expression of the saz gene(s) in Streptomyces albus J1074 led to the production of four prenylated indole alkaloids, among which streptoazines A to C (compounds 3 to 5) are new compounds. Expression of different gene combinations showed that the SazA catalyzes the formation of cyclo(LTrp-L-Trp) (cWW; compound 1), followed by consecutive prenylation and methylation by SazB. Biochemical assays demonstrated that SazB is a bifunctional enzyme, catalyzing sequential C-3/C-39 prenylation(s) by SazB-PT and N-1/N-19 methylation(s) by SazB-MT. Of note, the substrate selectivity of SazB-PT and SazB-MT was probed, revealing the stringent specificity of SazB-PT but relative flexibility of SazB-MT. [ABSTRACT FROM AUTHOR]

[Display omitted] • A thermostable prenyltransferase (AfPT) from Aspergillus fumigatiaffinis was cloned. • The expression level of AfPT reached 78.5 mU/mL by optimizing the induction conditions. • AfPT was immobilized onto carboxymethyl cellulose magnetic nanoparticles (CMN). • CMN-AfPT exhibits superior thermal stability and specific activity compared to AfPT. • CMN-AfPT maintained 68% of its original activity following 10 cycles of reuse. Prenyltransferases catalyze the synthesis of prenylated flavonoids, providing these with greater lipid solubility, biological activity, and availability. In this study, a thermostable prenyltransferase (AfPT) from Aspergillus fumigatiaffinis was cloned and expressed in Escherichia coli. By optimizing induction conditions, the expression level of AfPT reached 39.3 mU/mL, which was approximately 200 % of that before optimization. Additionally, we determined the enzymatic properties of AfPT. Subsequently, AfPT was immobilized on carboxymethyl cellulose magnetic nanoparticles (CMN) at a maximum load of 0.6 mg/mg. Optimal activity of CMN-AfPT was achieved at pH 8.0 and 55 °C. Thermostability assays showed that the residual activity of CMN-AfPT was greater than 50 % after incubation at 55 °C for 4 h. Km and Vmax of CMN-AfPT for naringenin were 0.082 mM and 5.57 nmol/min/mg, respectively. The Kcat/Km ratio of CMN-AfPT was higher than that of AfPT. Residual prenyltransferase activity of CMN-AfPT remained higher than 70 % even after 30 days of storage. Further, CMN-AfPT retained 68 % of its original activity after 10 cycles of reuse. Compared with free AfPT, CMN-AfPT showed higher catalytic efficiency, thermostability, metal ion tolerance, substrate affinity, storage stability, and reusability. Our study presents a thermostable prenyltransferase and its immobilized form for the production of prenylated flavonoids in vitro. [ABSTRACT FROM AUTHOR]

Aromatic prenyltransferases (PTases) are enzymes that catalyze Friedel–Crafts reactions between aromatic compounds and isoprenoid diphosphates. In hapalindole biosynthesis, the aromatic PTases AmbP1 and AmbP3 exhibit surprisingly plastic selectivities. AmbP1 not only transfers the geranyl group on the C-3 of cis-indolylvinyl isonitrile, but also on the C-2, which is supressed in the presence of Mg2+ ions. AmbP3 transfers the dimethylallyl group on C-2 of hapalindole U in the reverse manner, but on C-2 of its C-10 stereoisomer in the normal manner. This review highlights the molecular bases of the AmbP1 and AmbP3 functions, elucidated through their X-ray crystal structures. The knowledge presented here will contribute to the understanding of aromatic PTase reactions and will enhance their uses as biocatalysts.

Terpenoids are the most diverse class of plant primary and specialized metabolites, and trans-prenyltransferases (trans-PTs) are the first branch point to synthesize precursors of various chain lengths for further metabolism. Whereas the catalytic mechanism of the enzyme is known, there is no reliable method for precisely predicting the functions of trans-PTs. With the exponentially increasing number of available trans-PTs genes in public databases, an in silico functional prediction method for this gene family is urgently needed. Here, we present PTS-Pre, a web tool developed on the basis of the “three floors” model, which shows an overall 86% prediction accuracy for 141 experimentally determined trans-PTs. The method was further validated by in vitro enzyme assays for randomly selected trans-PTs. In addition, using this method, we identified nine new GFPPSs from different plants which are beyond the previously reported Brassicaceae clade, suggesting these genes may have occurred via convergent evolution and are more likely lineage-specific. The high accuracy of our blind prediction validated by enzymatic assays suggests that PTS-Pre provides a convenient and reliable method for genome-wide functional prediction of trans-PTs enzymes and will surely benefit the elucidation and metabolic engineering of terpenoid biosynthetic pathways.

Prenylation of aromatic natural products by membrane-bound prenyltransferases (PTs) is an important biosynthesis step of many bioactive compounds. At present, only a few plant flavonoid-related PT genes have been functionally characterized, mainly due to the difficulties of expressing these membrane proteins. Rapid and effective methods to produce functional plant membrane proteins are thus indispensable. Here, we evaluated expression systems through cell-based and cell-free approaches to express Boesenbergia rotunda BrPT2 encoding a membrane-bound prenyltransferase. We attempted to express BrPT2 in Escherichia coli and tobacco plants but failed to detect this protein using the Western-blot technique, whereas an intact single band of 43 kDa was detected when BrPT2 was expressed using a cell-free protein synthesis system (PURE). Under in vitro enzymatic condition, the synthesized BrPT2 successfully catalyzed pinostrobin chalcone to pinostrobin. Molecular docking analysis showed that pinostrobin chalcone interacts with BrPT2 at two cavities: (1) the main binding site at the central cavity and (2) the allosteric binding site located away from the central cavity. Our findings suggest that cell-free protein synthesis could be an alternative for rapid production of valuable difficult-to-express membrane proteins. [ABSTRACT FROM AUTHOR]

Isoprenoids, also known as terpenes or terpenoids, represent a large family of natural products composed of five‐carbon isopentenyl diphosphate or its isomer dimethylallyl diphosphate as the building blocks. Isoprenoids are structurally and functionally diverse and include dolichols, steroid hormones, carotenoids, retinoids, aromatic metabolites, the isoprenoid side‐chain of ubiquinone, and isoprenoid attached signaling proteins. Productions of isoprenoids are catalyzed by a group of enzymes known as prenyltransferases, such as farnesyltransferases, geranylgeranyltransferases, terpenoid cyclase, squalene synthase, aromatic prenyltransferase, and cis‐ and trans‐prenyltransferases. Because these enzymes are key in cellular processes and metabolic pathways, they are expected to be potential targets in new drug discovery. In this review, six distinct subsets of characterized prenyltransferases are structurally and mechanistically classified, including (1) head‐to‐tail prenyl synthase, (2) head‐to‐head prenyl synthase, (3) head‐to‐middle prenyl synthase, (4) terpenoid cyclase, (5) aromatic prenyltransferase, and (6) protein prenylation. Inhibitors of those enzymes for potential therapies against several diseases are discussed. Lastly, recent results on the structures of integral membrane enzyme, undecaprenyl pyrophosphate phosphatase, are also discussed. [ABSTRACT FROM AUTHOR]

Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs. [ABSTRACT FROM AUTHOR]

Flavonoids and prenylated flavonoids are active components in medicinal plant extracts which exhibit beneficial effects on human health. Prenylated flavonoids consist of a flavonoid core with a prenyl group attached to it. This prenylation process is catalyzed by prenyltranferases (PTs). At present, only a few flavonoid-related PT genes have been identified. In this study, we aimed to investigate the roles of PT in flavonoid production. We isolated a putative PT gene (designated as BrPT2) from a medicinal ginger, Boesenbergia rotunda. The deduced protein sequence shared highest gene sequence homology (81%) with the predicted homogentisate phytyltransferase 2 chloroplastic isoform X1 from Musa acuminata subsp. Malaccensis. We then cloned the BrPT2 into pRI vector and expressed in B. rotunda cell suspension cultures via Agrobacterium-mediated transformation. The BrPT2-expressing cells were fed with substrate, pinostrobin chalcone, and their products were analyzed by liquid chromatography mass spectrometry. We found that the amount of flavonoids, namely alpinetin, pinostrobin, naringenin and pinocembrin, in BrPT2-expressing cells was higher than those obtained from the wild type cells. However, we were unable to detect any targeted prenylated flavonoids. Further in-vitro assay revealed that the reaction containing the BrPT2 protein produced the highest accumulation of pinostrobin from the substrate pinostrobin chalcone compared to the reaction without BrPT2 protein, suggesting that BrPT2 was able to accelerate the enzymatic reaction. The finding of this study implied that the isolated BrPT2 may not be involved in the prenylation of pinostrobin chalcone but resulted in high yield and production of other flavonoids, which is likely related to enzyme promiscuous activities.

Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis. Due to their ability to use the photon energy of sunlight to fix carbon dioxide into biomass, cyanobacteria are promising hosts for the sustainable production of terpenoids, also known as isoprenoids, a diverse class of natural products with potential as advanced biofuels and high-value chemicals. However, the cyanobacterial enzymes involved in the biosynthesis of the terpene precursors needed to make more complicated terpenoids are poorly characterized. Here we show that the predicted type II prenyltransferase CrtE encoded by the model cyanobacterium Synechococcus sp. PCC 7002 is homodimeric and able to synthesize C20-geranylgeranyl pyrophosphate (GGPP) from C5-isopentenyl pyrophosphate (IPP) and C5-dimethylallyl pyrophosphate (DMAPP). The crystal structure of CrtE solved to a resolution of 2.7 Å revealed a strong structural similarity to the large subunit of the heterodimeric geranylgeranyl pyrophosphate synthase 1 from Arabidopsis thaliana with each subunit containing 14 helices. Using mutagenesis, we confirmed that the fourth and fifth amino acids (Met-87 and Ser-88) before the first conserved aspartate-rich motif (FARM) play important roles in controlling chain elongation. While the WT enzyme specifically produced GGPP, variants M87F and S88Y could only generate C15-farnesyl pyrophosphate (FPP), indicating that residues with large side chains obstruct product elongation. In contrast, replacement of M87 with the smaller Ala residue allowed the formation of the longer C25-geranylfarnesyl pyrophosphate (GFPP) product. Overall, our results provide new structural and functional information on the cyanobacterial CrtE enzyme that could lead to the development of improved cyanobacterial platforms for terpenoid production.

Endophenazine A is a terpenoid phenazine with phenazine-1-carboxylic acid (PCA), and dimethylallyl diphosphate (DMAPP) derived from the 2-methyl-D-erythritol-4-phosphate (MEP) pathway as the precursor, which shows good antimicrobial activity against several Gram-positive bacteria and fungi. However, the highest yield of endophenazine A was about 20 mg/L in Streptomyces, limiting its large-scale industrial development. Pseudomonas chlororaphis P3, possessing an efficient PCA synthesis and MEP pathways, is a suitable chassis to synthesize endophenazine A. Herein, we designed an artificial biosynthetic pathway for the synthesis of endophenazine A in P. chlororaphis P3. Primarily, the prenyltransferase PpzP from Streptomyces anulatus 9663 was introduced into P. chlororaphis P3 and successfully synthesized endophenazine A. Another phenazine compound, endophenazine A1, was discovered and identified as a leakage of the intermediate 4-hydroxy-3-methyl-2-butene pyrophosphate (HMBPP). Finally, the yield of endophenazine A reached 279.43 mg/L, and the yield of endophenazine A1 reached 189.2 mg/L by metabolic engineering and medium optimization. In conclusion, we successfully synthesized endophenazine A and endophenazine A1 in P. chlororaphis P3 for the first time and achieved the highest titer, which provides a reference for the heterologous synthesis of terpenoid phenazines.

The biosynthesis of bacterial cell envelope polysaccharides such as peptidoglycan relies on the use of a dedicated carrier lipid both for the assembly of precursors at the cytoplasmic face of the plasma membrane and for the translocation of lipid linked oligosaccharides across the plasma membrane into the periplasmic space. This dedicated carrier lipid, undecaprenyl phosphate, results from the dephosphorylation of undecaprenyl pyrophosphate, which is generated de novo in the cytoplasm by undecaprenyl pyrophosphate synthase and released as a by-product when newly synthesized glycans are incorporated into the existing cell envelope. The de novo synthesis of undecaprenyl pyrophosphate has been thoroughly characterized from a structural and mechanistic standpoint; however, its dephosphorylation to the active carrier lipid form, both in the course of de novo synthesis and recycling, has only been begun to be studied in depth in recent years. This review provides an overview of bacterial carrier lipid synthesis and presents the current state of knowledge regarding bacterial carrier lipid recycling. Unlabelled Image • Biosynthesis of bacterial cell envelope polysaccharides requires a dedicated carrier lipid. • Undecaprenyl pyrophosphate is synthesized in the cytoplasm by a cis -prenyltransferase. • Dephosphorylation of undecaprenyl pyrophosphate is carried out by PAP2 phosphatases or UppP. • UppP shows potential to act as a lipid flippase. [ABSTRACT FROM AUTHOR]

NotF from Aspergillus sp. MF297-2 and BrePT from Aspergillus versicolor catalyze a reverse C2-prenylation of brevianamide F in the biosynthetic pathway of brevianamides and notoamides. NotF was reported to use only brevianamide F as substrate while BrePT demonstrated broad substrate promiscuity. With high identity at amino acid level, it is interesting to reinvestigate the catalytic activities of these two prenyltransferases in vitro toward 14 cyclodipeptides. Product identification of the in vitro assays by MS proved that NotF and BrePT share similar catalytic ability and substrate promiscuity. [ABSTRACT FROM AUTHOR]

Prenylation is a common biological reaction in all domains of life wherein prenyl diphosphate donors transfer prenyl groups onto small molecules as well as large proteins. The enzymes that catalyze these reactions are structurally distinct from ubiquitous terpene cyclases that, instead, assemble terpenes via intramolecular rearrangements of a single substrate. Herein, we report the structure and molecular details of a new family of prenyltransferases from marine algae that repurposes the terpene cyclase structural fold for the N-prenylation of glutamic acid during the biosynthesis of the potent neurochemicals domoic acid and kainic acid. We solved the X-ray crystal structure of the prenyltransferase found in domoic acid biosynthesis, DabA, and show distinct active site binding modifications that remodel the canonical magnesium (Mg2+)-binding motif found in terpene cyclases. We then applied our structural knowledge of DabA and a homologous enzyme from the kainic acid biosynthetic pathway, KabA, to reengineer their isoprene donor specificities (geranyl diphosphate [GPP] versus dimethylallyl diphosphate [DMAPP]) with a single amino acid change. While diatom DabA and seaweed KabA enzymes share a common evolutionary lineage, they are distinct from all other terpene cyclases, suggesting a very distant ancestor to the larger terpene synthase family. [ABSTRACT FROM AUTHOR]

Summary: Analysis of the updated reference tomato genome found 34 full‐length TPS genes and 18 TPS pseudogenes.Biochemical analysis has now identified the catalytic activities of all enzymes encoded by the 34 TPS genes: one isoprene synthase, 10 exclusively or predominantly monoterpene synthases, 17 sesquiterpene synthases and six diterpene synthases. Among the monoterpene and sesquiterpene and diterpene synthases, some use trans‐prenyl diphosphates, some use cis‐prenyl diphosphates and some use both. The isoprene synthase is cytosolic; six monoterpene synthases are plastidic, and four are cytosolic; the sesquiterpene synthases are almost all cytosolic, with the exception of one found in the mitochondria; and three diterpene synthases are found in the plastids, one in the cytosol and two in the mitochondria.New trans‐prenyltransferases (TPTs) were characterised; together with previously characterised TPTs and cis‐prenyltransferases (CPTs), tomato plants can make all cis and trans C10, C15 and C20 prenyl diphosphates. Every type of plant tissue examined expresses some TPS genes and some TPTs and CPTs.Phylogenetic comparison of the TPS genes from tomato and Arabidopsis shows expansions in each clade of the TPS gene family in each lineage (and inferred losses), accompanied by changes in subcellular localisations and substrate specificities. [ABSTRACT FROM AUTHOR]

Natural rubber (NR), principally comprising cis-1,4-polyisoprene, is an industrially important natural hydrocarbon polymer because of its unique physical properties, which render it suitable for manufacturing items such as tires. Presently, industrial NR production depends solely on latex obtained from the Pará rubber tree, Hevea brasiliensis. In latex, NR is enclosed in rubber particles, which are specialized organelles comprising a hydrophobic NR core surrounded by a lipid monolayer and membrane-bound proteins. The similarity of the basic carbon skeleton structure between NR and dolichols and polyprenols, which are found in most organisms, suggests that the NR biosynthetic pathway is related to the polyisoprenoid biosynthetic pathway and that rubber transferase, which is the key enzyme in NR biosynthesis, belongs to the cis-prenyltransferase family. Here, we review recent progress in the elucidation of molecular mechanisms underlying NR biosynthesis through the identification of the enzymes that are responsible for the formation of the NR backbone structure. [ABSTRACT FROM AUTHOR]

Cyanobacteria are photosynthetic prokaryotes that perform oxygenic photosynthesis. Due to their ability to use the photon energy of sunlight to fix carbon dioxide into biomass, cyanobacteria are promising hosts for the sustainable production of terpenoids, also known as isoprenoids, a diverse class of natural products with potential as advanced biofuels and high-value chemicals. However, the cyanobacterial enzymes involved in the biosynthesis of the terpene precursors needed to make more complicated terpenoids are poorly characterized. Here we show that the predicted type II prenyltransferase CrtE encoded by the model cyanobacterium Synechococcus sp. PCC 7002 is homodimeric and able to synthesize C20-geranylgeranyl pyrophosphate (GGPP) from C5-isopentenyl pyrophosphate (IPP) and C5-dimethylallyl pyrophosphate (DMAPP). The crystal structure of CrtE solved to a resolution of 2.7 Å revealed a strong structural similarity to the large subunit of the heterodimeric geranylgeranyl pyrophosphate synthase 1 from Arabidopsis thaliana with each subunit containing 14 helices. Using mutagenesis, we confirmed that the fourth and fifth amino acids (Met-87 and Ser-88) before the first conserved aspartate-rich motif (FARM) play important roles in controlling chain elongation. While the WT enzyme specifically produced GGPP, variants M87F and S88Y could only generate C15-farnesyl pyrophosphate (FPP), indicating that residues with large side chains obstruct product elongation. In contrast, replacement of M87 with the smaller Ala residue allowed the formation of the longer C25-geranylfarnesyl pyrophosphate (GFPP) product. Overall, our results provide new structural and functional information on the cyanobacterial CrtE enzyme that could lead to the development of improved cyanobacterial platforms for terpenoid production. [ABSTRACT FROM AUTHOR]

Summary: Furanocoumarins (FCs) are plant‐specialized metabolites with potent allelochemical properties. The distribution of FCs is scattered with a chemotaxonomical tendency towards four distant families with highly similar FC pathways. The mechanism by which this pathway emerged and spread in plants has not been elucidated.Furanocoumarin biosynthesis was investigated in Ficus carica (fig, Moraceae), focusing on the first committed reaction catalysed by an umbelliferone dimethylallyltransferase (UDT). Comparative RNA‐seq analysis among latexes of different fig organs led to the identification of a UDT. The phylogenetic relationship of this UDT to previously reported Apiaceae UDTs was evaluated.The expression pattern of F. carica prenyltransferase 1 (FcPT1) was related to the FC contents in different latexes. Enzymatic characterization demonstrated that one of the main functions of FcPT1 is UDT activity. Phylogenetic analysis suggested that FcPT1 and Apiaceae UDTs are derived from distinct ancestors, although they both belong to the UbiA superfamily. These findings are supported by significant differences in the related gene structures.This report describes the identification of FcPT1 involved in FC biosynthesis in fig and provides new insights into multiple origins of the FC pathway and, more broadly, into the adaptation of plants to their environments. [ABSTRACT FROM AUTHOR]

Prenylated compounds are pharmacologically attractive natural products that are widely distributed in nature. Prenyltransferases (PTs) could catalyze the transfer of prenyl moieties to aromatic acceptors and increase the structural diversity and biological activity of natural products. In this work, a permissive O‐prenyltransferase AcaPT was discovered from Antrodia camphorata, a precious medicinal mushroom in Taiwan. AcaPT exhibited robust O‐prenylation capability toward structurally diverse drug‐like phenolic compounds, including antrodins, flavonoids, and coumarins. In total, 23 O‐prenylated products were obtained by scaled‐up enzymatic synthesis. AcaPT could be used as an efficient biocatalyst to synthesize O‐prenylated derivatives for drug discovery. [ABSTRACT FROM AUTHOR]

Polyprenyl diphosphate synthase (PPS) plays important roles in the biosynthesis of functionally important plastoquinone (PQ) and ubiquinone (UQ). However, only few plant PPS genes have been functionally characterized. Through genome-wide analysis, two PPS genes, termed SmPPS1 and SmPPS2, were identified from Salvia miltiorrhiza, an economically significant Traditional Chinese Medicine material and an emerging model medicinal plant. SmPPS1 and SmPPS2 belonged to different phylogenetic subgroups of plant trans-long-chain prenyltransferases and exhibited differential tissue expression and light-induced expression patterns. Computational prediction and transient expression assays showed that SmPPS1 was localized in the chloroplasts, whereas SmPPS2 was mainly localized in the mitochondria. SmPPS2, but not SmPPS1, could functionally complement the coq1 mutation in yeast cells and catalyzed the production of UQ-9 and UQ-10. Consistently, both UQ-9 and UQ-10 were detected in S. miltiorrhiza plants. Overexpression of SmPPS2 caused significant UQ accumulation in S. miltiorrhiza transgenics, whereas down-regulation resulted in decreased UQ content. Differently, SmPPS1 overexpression significantly elevated PQ-9 content in S. miltiorrhiza. Transgenic lines showing a down-regulation of SmPPS1 expression exhibited decreased PQ-9 level, abnormal chloroplast and trichome development, and varied leaf bleaching phenotypes. These results suggest that SmPPS1 is involved in PQ-9 biosynthesis, whereas SmPPS2 is involved in UQ-9 and UQ-10 biosynthesis.

Plant specialized metabolism emerged from the land colonization by ancient plants, becoming diversified along with plant evolution. To date, more than 1 million metabolites have been predicted to exist in the plant kingdom, and their metabolic processes have been revealed on the molecular level. Previous studies have reported that rates of evolution are greater for genes involved in plant specialized metabolism than in primary metabolism. This perspective introduces topics on the enigmatic molecular evolution of some plant specialized metabolic processes. Two transferase families, BAHD acyltransferases and aromatic prenyltransferases, which are involved in the biosynthesis of paclitaxel and meroterpenes, respectively, have shown apparent expansion. The latter family has been shown to beinvolved in the biosynthesis of a variety of aromatic substances, including prenylated coumarins in citrus plants and shikonin in Lithospermum erythrorhizon. These genes have evolved in the development of each special subfamily within the plant lineage. The broadness of substrate specificity and the exon-intron structure of their genes may provide hints to explain the evolutionary process underlying chemodiversity in plants.

Summary: The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high‐molecular‐weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis‐1,4‐isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis‐prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT‐like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis‐1,4‐isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1‐RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low‐expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS‐based comparison of latex proteomes from TkCPTL1‐RNAi plants and T. koksaghyz wild‐types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell. Significance Statement: Factors and mechanisms related to the biosynthesis of natural rubber are still not fully elucidated. We show that only TkCPTL1 is necessary for rubber synthesis in the Russian dandelion Taraxacum koksaghyz, while its homolog TkCPTL2 is dispensable for generating poly(cis‐1,4‐isoprene). In‐depth characterization of TkCPTL1‐depleted plants revealed further insights into the distribution of carbon between rubber, triterpenes, and inulin as well as provided further understanding of rubber particle function in storing latex metabolites. [ABSTRACT FROM AUTHOR]

Isoprenoids comprise a diverse group of natural products with a broad range of metabolic functions. Isoprenoids are synthesized from prenyl pyrophosphates by prenyltransferases that catalyze the isoprenoid chain-elongation process to different chain lengths. We hereby present the crystal structure of geranylgeranyl pyrophosphate synthase from the marine flavobacterium Nonlabens dokdonensis DSW-6 (Nd GGPPS). Nd GGPPS forms a hexamer composed of homodimeric trimer, and the monomeric structure is composed of 15 α-helices (α1–α15). In this structure, we observed the binding of one pyrophosphate molecule and two glycerol molecules that mimicked substrate binding to the enzyme. The substrate binding site of Nd GGPPS contains large hydrophobic residues such as Phe, His and Tyr, and structural and amino acids sequence analyses thereof suggest that the protein belongs to the short-chain prenyltransferase family. • Crystal structure of GGPPS from Nonlabens dokdonensis DSW-6 was determined. • Nd GGPPS structure is composed 15 α-helices and shows a class I terpenoid synthase fold. • Nd GGPPS might function as a short-chain prenyltransferase. [ABSTRACT FROM AUTHOR]

In addition to the psychoactive constituents that are typically associated with Cannabis sativa L., there exist numerous other specialized metabolites in this plant that are believed to contribute to its medicinal versatility. This study focused on two such compounds, known as cannflavin A and cannflavin B. These prenylated flavonoids specifically accumulate in C. sativa and are known to exhibit potent anti-inflammatory activity in various animal cell models. However, almost nothing is known about their biosynthesis. Using a combination of phylogenomic and biochemical approaches, an aromatic prenyltransferase from C. sativa (CsPT3) was identified that catalyzes the regiospecific addition of either geranyl diphosphate (GPP) or dimethylallyl diphosphate (DMAPP) to the methylated flavone, chrysoeriol, to produce cannflavins A and B, respectively. Further evidence is presented for an O -methyltransferase (CsOMT21) encoded within the C. sativa genome that specifically converts the widespread plant flavone known as luteolin to chrysoeriol, both of which accumulate in C. sativa. These results therefore imply the following reaction sequence for cannflavins A and B biosynthesis: luteolin ► chrysoeriol ► cannflavin A and cannflavin B. Taken together, the identification of these two unique enzymes represent a branch point from the general flavonoid pathway in C. sativa and offer a tractable route towards metabolic engineering strategies that are designed to produce these two medicinally relevant Cannabis compounds. Image 1 • Cannflavin A and B are prenylated flavonoids that are unique to Cannabis sativa. • Cannflavins exhibit anti-inflammatory activity that is thirty times that of aspirin. • Cannflavins are synthesized via a branch point from the phenylpropanoid pathway. • A single prenyltransferase converts chrysoeriol to cannflavin A and B. [ABSTRACT FROM AUTHOR]

Polyprenyl diphosphate synthase (PPS) plays important roles in the biosynthesis of functionally important plastoquinone (PQ) and ubiquinone (UQ). However, only few plant PPS genes have been functionally characterized. Through genome-wide analysis, two PPS genes, termed SmPPS1 and SmPPS2 , were identified from Salvia miltiorrhiza , an economically significant Traditional Chinese Medicine material and an emerging model medicinal plant. SmPPS1 and SmPPS2 belonged to different phylogenetic subgroups of plant trans-long-chain prenyltransferases and exhibited differential tissue expression and light-induced expression patterns. Computational prediction and transient expression assays showed that SmPPS1 was localized in the chloroplasts, whereas SmPPS2 was mainly localized in the mitochondria. SmPPS2, but not SmPPS1, could functionally complement the coq1 mutation in yeast cells and catalyzed the production of UQ-9 and UQ-10. Consistently, both UQ-9 and UQ-10 were detected in S. miltiorrhiza plants. Overexpression of SmPPS2 caused significant UQ accumulation in S. miltiorrhiza transgenics, whereas down-regulation resulted in decreased UQ content. Differently, SmPPS1 overexpression significantly elevated PQ-9 content in S. miltiorrhiza. Transgenic lines showing a down-regulation of SmPPS1 expression exhibited decreased PQ-9 level, abnormal chloroplast and trichome development, and varied leaf bleaching phenotypes. These results suggest that SmPPS1 is involved in PQ-9 biosynthesis, whereas SmPPS2 is involved in UQ-9 and UQ-10 biosynthesis. [ABSTRACT FROM AUTHOR]