Telescoping a Prenyltransferase and a Diterpene Synthase to Transform Unnatural FPP Derivatives to Diterpenoids.

New diterpenoids are accessible from non-natural FPP derivatives as substrates for an enzymatic elongation cyclization cascade using the geranylgeranyl pyrophosphate synthase (GGPPS) from Streptomyces cyaneofuscatus and the spata-13,17-diene synthase (SpS) from Streptomyces xinghaiensis. This approach led to four new biotransformation products including three new cyclododecane cores and a macrocyclic ether. For the first time, a 1,12-terpene cyclization was observed when shifting the central olefinic double bond toward the geminial methyl groups creating a nonconjugated 1,4-diene.

Glucose-oxygen deprivation constrains HMGCR function and Rac1 prenylation and activates the NLRP3 inflammasome in human monocytes.

Hypoxia and low glucose abundance often occur simultaneously at sites of inflammation. In monocytes and macrophages, glucose-oxygen deprivation stimulates the assembly of the NLRP3 inflammasome to generate the proinflammatory cytokine IL-1ß. We found that concomitant glucose deprivation and hypoxia activated the NLRP3 inflammasome by constraining the function of HMG-CoA reductase (HMGCR), the rate-limiting enzyme of the mevalonate kinase pathway. HMGCR is involved in the synthesis of geranylgeranyl pyrophosphate (GGPP), which is required for the prenylation and lipid membrane integration of proteins. Under glucose-oxygen deprivation, GGPP synthesis was decreased, leading to reduced prenylation of the small GTPase Rac1, increased binding of nonprenylated Rac1 to the scaffolding protein IQGAP1, and enhanced activation of the NLRP3 inflammasome. In response to restricted oxygen and glucose supply, patient monocytes with a compromised mevalonate pathway due to mevalonate kinase deficiency or Muckle-Wells syndrome released more IL-1ß than did control monocytes. Thus, reduced GGPP synthesis due to inhibition of HMGCR under glucose-oxygen deprivation results in proinflammatory innate responses, which are normally kept in check by the prenylation of Rac1. We suggest that this mechanism is also active in inflammatory autoimmune conditions.

Discovery of a terpene synthase synthesizing a nearly non-flexible eunicellane reveals the basis of flexibility.

Eunicellane diterpenoids, containing a typical 6,10-bicycle, are bioactive compounds widely present in marine corals, but rarely found in bacteria and plants. The intrinsic macrocycle exhibits innate structural flexibility resulting in dynamic conformational changes. However, the mechanisms controlling flexibility remain unknown. The discovery of a terpene synthase, MicA, that is responsible for the biosynthesis of a nearly non-flexible eunicellane skeleton, enable us to propose a feasible theory about the flexibility in eunicellane structures. Parallel studies of all eunicellane synthases in nature discovered to date, including 2Z-geranylgeranyl diphosphate incubations and density functional theory-based Boltzmann population computations, reveale that a trans-fused bicycle with a 2Z-configuration alkene restricts conformational flexibility resulting in a nearly non-flexible eunicellane skeleton. The catalytic route and the enzymatic mechanism of MicA are also elucidated by labeling experiments, density functional theory calculations, structural analysis of the artificial intelligence-based MicA model, and mutational studies.

A classic antibiotic reimagined: Rationally designed bacitracin variants exhibit potent activity against vancomycin-resistant pathogens.

Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C55PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C55PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C55PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C55PP. The structure-activity insights thus obtained serve to inform the design of C55PP-targeting antibiotics, a key and underexploited antibacterial strategy.

Absence of farnesol salvage in Candida albicans and probably in other fungi.

Farnesol salvage, a two-step pathway converting farnesol to farnesyl pyrophosphate (FPP), occurs in bacteria, plants, and animals. This paper investigates the presence of this pathway in fungi. Through bioinformatics, biochemistry, and physiological analyses, we demonstrate its absence in the yeasts Saccharomyces cerevisiae and Candida albicans, suggesting a likely absence across fungi. We screened 1,053 fungal genomes, including 34 from C. albicans, for potential homologs to four genes (Arabidopsis thaliana AtFOLK, AtVTE5, AtVTE6, and Plasmodium falciparum PfPOLK) known to accomplish farnesol/prenol salvage in other organisms. Additionally, we showed that 3H-farnesol was not converted to FPP or any other phosphorylated prenol, and exogenous farnesol was not metabolized within 90 minutes at any phase of growth and did not rescue cells from the toxic effects of atorvastatin, but it did elevate the levels of intracellular farnesol (Fi). All these experiments were conducted with C. albicans. In sum, we found no evidence for farnesol salvage in fungi. IMPORTANCE: The absence of farnesol salvage constitutes a major difference in the metabolic capabilities of fungi. In terms of fungal physiology, the lack of farnesol salvage pathways relates to how farnesol acts as a quorum-sensing molecule in Candida albicans and why farnesol should be investigated for use in combination with other known antifungal antibiotics. Its absence is essential for a model (K. W. Nickerson et al., Microbiol Mol Biol Rev 88:e00081-22, 2024), wherein protein farnesylation, protein chaperones, and the unfolded protein response are combined under the unifying umbrella of a cell's intracellular farnesol (Fi). In terms of human health, farnesol should have at least two different modes of action depending on whether those cells have farnesol salvage. Because animals have farnesol salvage, we can now see the importance of dietary prenols as well as the potential importance of farnesol in treating neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and multiple sclerosis.

Sesquiterpene Cyclase BcBOT2 Promotes the Unprecedented Wagner-Meerwein Rearrangement of the Methoxy Group.

Presilphiperfolan-8ß-ol synthase (BcBOT2), a substrate-promiscuous sesquiterpene cyclase (STC) of fungal origin, is capable of converting two new farnesyl pyrophosphate (FPP) derivatives modified at C7 of farnesyl pyrophosphate (FPP) bearing either a hydroxymethyl group or a methoxymethyl group. These substrates were chosen based on a computationally generated model. Biotransformations yielded five new oxygenated terpenoids. Remarkably, the formation of one of these tricyclic products can only be explained by a cationically induced migration of the methoxy group, presumably via a Meerwein-salt intermediate, unprecedented in synthetic chemistry and biosynthesis. The results show the great principle and general potential of terpene cyclases for mechanistic studies of unusual cation chemistry and for the creation of new terpene skeletons.

Methods for the preparation and analysis of the diterpene cyclase fusicoccadiene synthase.

Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.

In vivo CRISPR screening identifies geranylgeranyl diphosphate as a pancreatic cancer tumor growth dependency.

OBJECTIVE: Cancer cells must maintain lipid supplies for their proliferation and do so by upregulating lipogenic gene programs. The sterol regulatory element-binding proteins (SREBPs) act as modulators of lipid homeostasis by acting as transcriptional activators of genes required for fatty acid and cholesterol synthesis and uptake. SREBPs have been recognized as chemotherapeutic targets in multiple cancers, however it is not well understood which SREBP target genes are essential for tumorigenesis. In this study, we examined the requirement of SREBP target genes for pancreatic ductal adenocarcinoma (PDAC) tumor growth. METHODS: Here we constructed a custom CRISPR knockout library containing known SREBP target genes and performed in vitro 2D culture and in vivo orthotopic xenograft CRISPR screens using a patient-derived PDAC cell line. In vitro, we grew cells in medium supplemented with 10% fetal bovine serum (FBS) or 10% lipoprotein-deficient serum (LPDS) to examine differences in gene essentiality in different lipid environments. In vivo, we injected cells into the pancreata of nude mice and collected tumors after 4 weeks. RESULTS: We identified terpenoid backbone biosynthesis genes as essential for PDAC tumor development. Specifically, we identified the non-sterol isoprenoid product of the mevalonate pathway, geranylgeranyl diphosphate (GGPP), as an essential lipid for tumor growth. Mechanistically, we observed that restricting mevalonate pathway activity using statins and SREBP inhibitors synergistically induced apoptosis and caused disruptions in small G protein prenylation that have pleiotropic effects on cellular signaling pathways. Finally, we demonstrated that geranylgeranyl diphosphate synthase 1 (GGPS1) knockdown significantly reduces tumor burden in an orthotopic xenograft mouse model. CONCLUSIONS: These findings indicate that PDAC tumors selectively require GGPP over other lipids such as cholesterol and fatty acids and that this is a targetable vulnerability of pancreatic cancer cells.

Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

Characterization of a group of germacrene A synthases involved in the biosynthesis of ß-elemene from Atractylodis macrocephala.

ß-Elemene, an important component of the volatile oil of Atractylodis macrocephala, has been widely utilized as an antitumor drug for over 20 years. However, the germacrene A synthase (GAS) genes responsible for the biosynthesis of ß-elemene in A. macrocephala were previously unidentified. In this study, two new AmGASs were identified from the A. macrocephala transcriptome, demonstrating their capability to convert farnesyl pyrophosphate into germacrene A, which subsequently synthesizes ß-elemene through Cope rearrangement. Additionally, two highly catalytic AmGAS1 mutations, I307A and E392A, resulted in a 2.23-fold and 1.57-fold increase in ß-elemene synthesis, respectively. Furthermore, precursor supply and fed-batch strategies were employed to enhance the precursor supply, resulting in ß-elemene yields of 7.3 mg/L and 33.3 mg/L, respectively. These findings identify a promising candidate GAS for ß-elemene biosynthesis and lay the foundation for further functional studies on terpene synthases in A. macrocephala.

A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.

Efficient production of (S)-limonene and geraniol in Saccharomyces cerevisiae through the utilization of an Erg20 mutant with enhanced GPP accumulation capability.

Monoterpenes and monoterpenoids such as (S)-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. Saccharomyces cerevisiae has proven to be an effective host to produce various terpenes and terpenoids. (S)-limonene and geraniol are produced from geranyl pyrophosphate (GPP) through the enzymatic actions of limonene synthase (LS) and geraniol synthase (GES), respectively. However, a major hurdle in their production arises from the dual functionality of the Erg20, a farnesyl pyrophosphate (FPP) synthase, responsible for generating GPP. Erg20 not only synthesizes GPP by condensing isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate but also catalyzes further condensation of IPP with GPP to produce FPP. In this study, we have tackled this issue by harnessing previously developed Erg20 mutants, Erg20K197G (Erg20G) and Erg20F96W, N127W (Erg20WW), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20WWG mutant with over four times higher GPP accumulating capability than Erg20WW, as observed through geraniol production levels. The Erg20WWG mutant was fused to the LS from Mentha spicata or the GES from Catharanthus roseus for efficient conversion of GPP to (S)-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20WWG-fused enzymes in peroxisomes, while simultaneously downregulating the essential ERG20 gene using the glucose-sensing HXT1 promoter. In the case of (S)-limonene production, additional Erg20WWG-LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of (S)-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20WWG mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids.

Bacillus subtilis uses the SigM signaling pathway to prioritize the use of its lipid carrier for cell wall synthesis.

Peptidoglycan (PG) and most surface glycopolymers and their modifications are built in the cytoplasm on the lipid carrier undecaprenyl phosphate (UndP). These lipid-linked precursors are then flipped across the membrane and polymerized or directly transferred to surface polymers, lipids, or proteins. Despite its essential role in envelope biogenesis, UndP is maintained at low levels in the cytoplasmic membrane. The mechanisms by which bacteria distribute this limited resource among competing pathways is currently unknown. Here, we report that the Bacillus subtilis transcription factor SigM and its membrane-anchored anti-sigma factor respond to UndP levels and prioritize its use for the synthesis of the only essential surface polymer, the cell wall. Antibiotics that target virtually every step in PG synthesis activate SigM-directed gene expression, confounding identification of the signal and the logic of this stress-response pathway. Through systematic analyses, we discovered 2 distinct responses to these antibiotics. Drugs that trap UndP, UndP-linked intermediates, or precursors trigger SigM release from the membrane in <2 min, rapidly activating transcription. By contrasts, antibiotics that inhibited cell wall synthesis without directly affecting UndP induce SigM more slowly. We show that activation in the latter case can be explained by the accumulation of UndP-linked wall teichoic acid precursors that cannot be transferred to the PG due to the block in its synthesis. Furthermore, we report that reduction in UndP synthesis rapidly induces SigM, while increasing UndP production can dampen the SigM response. Finally, we show that SigM becomes essential for viability when the availability of UndP is restricted. Altogether, our data support a model in which the SigM pathway functions to homeostatically control UndP usage. When UndP levels are sufficiently high, the anti-sigma factor complex holds SigM inactive. When levels of UndP are reduced, SigM activates genes that increase flux through the PG synthesis pathway, boost UndP recycling, and liberate the lipid carrier from nonessential surface polymer pathways. Analogous homeostatic pathways that prioritize UndP usage are likely to be common in bacteria.

Engineering Escherichia coli for increased Und-P availability leads to material improvements in glycan expression technology.

BACKGROUND: Bacterial surface glycans are assembled by glycosyltransferases (GTs) that transfer sugar monomers to long-chained lipid carriers. Most bacteria employ the 55-carbon chain undecaprenyl phosphate (Und-P) to scaffold glycan assembly. The amount of Und-P available for glycan synthesis is thought to be limited by the rate of Und-P synthesis and by competition for Und-P between phosphoglycosyl transferases (PGTs) and GTs that prime glycan assembly (which we collectively refer to as PGT/GTs). While decreasing Und-P availability disrupts glycan synthesis and promotes cell death, less is known about the effects of increased Und-P availability. RESULTS: To determine if cells can maintain higher Und-P levels, we first reduced intracellular competition for Und-P by deleting all known non-essential PGT/GTs in the Gram-negative bacterium Escherichia coli (hereafter called ΔPGT/GT cells). We then increased the rate of Und-P synthesis in ΔPGT/GT cells by overexpressing the Und-P(P) synthase uppS from a plasmid (puppS). Und-P quantitation revealed that ΔPGT/GT/puppS cells can be induced to maintain 3-fold more Und-P than wild type cells. Next, we determined how increasing Und-P availability affects glycan expression. Interestingly, increasing Und-P availability increased endogenous and recombinant glycan expression. In particular, ΔPGT/GT/puppS cells could be induced to express 7-fold more capsule from Streptococcus pneumoniae serotype 4 than traditional E. coli cells used to express recombinant glycans. CONCLUSIONS: We demonstrate that the biotechnology standard bacterium E. coli can be engineered to maintain higher levels of Und-P. The results also strongly suggest that Und-P pathways can be engineered to increase the expression of potentially any Und-P-dependent polymer. Given that many bacterial glycans are central to the production of vaccines, diagnostics, and therapeutics, increasing Und-P availability should be a foremost consideration when designing bacterial glycan expression systems.

Structural analysis of phosphoribosyltransferase-mediated cell wall precursor synthesis in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis, Rv3806c is a membrane-bound phosphoribosyltransferase (PRTase) involved in cell wall precursor production. It catalyses pentosyl phosphate transfer from phosphoribosyl pyrophosphate to decaprenyl phosphate, to generate 5-phospho-ß-ribosyl-1-phosphoryldecaprenol. Despite Rv3806c being an attractive drug target, structural and molecular mechanistic insight into this PRTase is lacking. Here we report cryogenic electron microscopy structures for Rv3806c in the donor- and acceptor-bound states. In a lipidic environment, Rv3806c is trimeric, creating a UbiA-like fold. Each protomer forms two helical bundles, which, alongside the bound lipids, are required for PRTase activity in vitro. Mutational and functional analyses reveal that decaprenyl phosphate and phosphoribosyl pyrophosphate bind the intramembrane and extramembrane cavities of Rv3806c, respectively, in a distinct manner to that of UbiA superfamily enzymes. Our data suggest a model for Rv3806c-catalysed phosphoribose transfer through an inverting mechanism. These findings provide a structural basis for cell wall precursor biosynthesis that could have potential for anti-tuberculosis drug development.

Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

Cloning and Expression Analysis of Key Enzyme Gene CoGPPS Involved in Iridoid Glycoside Synthesis in Cornus officinalis.

Cornus iridoid glycosides (CIGs), including loganin and morroniside, are the main active components of Cornus officinalis. As one of the key enzymes in the biosynthesis of CIGs, geranyl pyrophosphate synthase (GPPS) catalyzes the formation of geranyl pyrophosphate, which is the direct precursor of CIGs. In this study, the C. officinalis geranyl pyrophosphate synthase (CoGPPS) sequence was cloned from C. officinalis and analyzed. The cDNA sequence of the CoGPPS gene was 915 bp (GenBank No. OR725699). Phylogenetic analysis showed that CoGPPS was closely related to the GPPS sequence of Actinidia chinensis and Camellia sinensis, but relatively distantly related to Paeonia lactiflora and Tripterygium wilfordii. Results from the quantitative real-time PCR showed the spatiotemporal expression pattern of CoGPPS; that is, CoGPPS was specifically expressed in the fruits. Subcellular localization assay proved that CoGPPS was specifically found in chloroplasts. Loganin and morroniside contents in the tissues were detected by high-performance liquid chromatography, and both compounds were found to be at higher levels in the fruits than in leaves. Thus, this study laid the foundation for further studies on the synthetic pathway of CIGs.

Terpenoid balance in Aspergillus nidulans unveiled by heterologous squalene synthase expression.

Filamentous fungi produce numerous uncharacterized natural products (NPs) that are often challenging to characterize because of cryptic expression in laboratory conditions. Previously, we have successfully isolated novel NPs by expressing fungal artificial chromosomes (FACs) from a variety of fungal species into Aspergillus nidulans. Here, we demonstrate a twist to FAC utility wherein heterologous expression of a Pseudogymnoascus destructans FAC in A. nidulans altered endogenous terpene biosynthetic pathways. In contrast to wild type, the FAC transformant produced increased levels of squalene and aspernidine type compounds, including three new nidulenes (1- 2, and 5), and lost nearly all ability to synthesize the major A. nidulans characteristic terpene, austinol. Deletion of a squalene synthase gene in the FAC restored wild-type chemical profiles. The altered squalene to farnesyl pyrophosphate ratio leading to synthesis of nidulenes and aspernidines at the expense of farnesyl pyrophosphate-derived austinols provides unexpected insight into routes of terpene synthesis in fungi.

Genetic synergy between Acinetobacter baumannii undecaprenyl phosphate biosynthesis and the Mla system impacts cell envelope and antimicrobial resistance.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that poses a major health concern due to increasing multidrug resistance. The Gram-negative cell envelope is a key barrier to antimicrobial entry and includes an inner and outer membrane. The maintenance of lipid asymmetry (Mla) system is the main homeostatic mechanism by which Gram-negative bacteria maintain outer membrane asymmetry. Loss of the Mla system in A. baumannii results in attenuated virulence and increased susceptibility to membrane stressors and some antibiotics. We recently reported two strain variants of the A. baumannii type strain ATCC 17978: 17978VU and 17978UN. Here, ∆mlaF mutants in the two ATCC 17978 strains display different phenotypes for membrane stress resistance, antibiotic resistance, and pathogenicity in a murine pneumonia model. Although allele differences in obgE were previously reported to synergize with ∆mlaF to affect growth and stringent response, obgE alleles do not affect membrane stress resistance. Instead, a single-nucleotide polymorphism (SNP) in the essential gene encoding undecaprenyl pyrophosphate (Und-PP) synthase, uppS, results in decreased enzymatic rate and decrease in total Und-P levels in 17978UN compared to 17978VU. The UppSUN variant synergizes with ∆mlaF to reduce capsule and lipooligosaccharide (LOS) levels, increase susceptibility to membrane stress and antibiotics, and reduce persistence in a mouse lung infection. Und-P is a lipid glycan carrier required for the biosynthesis of A. baumannii capsule, cell wall, and glycoproteins. These findings uncover synergy between Und-P and the Mla system in maintaining the A. baumannii cell envelope and antibiotic resistance.IMPORTANCEAcinetobacter baumannii is a critical threat to global public health due to its multidrug resistance and persistence in hospital settings. Therefore, novel therapeutic approaches are urgently needed. We report that a defective undecaprenyl pyrophosphate synthase (UppS) paired with a perturbed Mla system leads to synthetically sick cells that are more susceptible to clinically relevant antibiotics and show reduced virulence in a lung infection model. These results suggest that targeting UppS or undecaprenyl species and the Mla system may resensitize A. baumannii to antibiotics in combination therapies. This work uncovers a previously unknown synergistic relationship in cellular envelope homeostasis that could be leveraged for use in combination therapy against A. baumannii.

Functional characterization of a geranylgeranyl diphosphate synthase in the leaf beetle Monolepta hieroglyphica.

Geranylgeranyl diphosphate synthase (GGPPS) as the short-chain prenyltransferases for catalyzing the formation of the acyclic precursor (E)-GGPP has been extensively investigated in mammals, plants, and microbes, but its functional plasticity is poorly understood in insect species. Here, a single GGPPS in leaf beetle Monolepta hieroglyphica, MhieGGPPS, was functionally investigated. Phylogenetic analysis showed that MhieGGPPS was clustered in one clade with homologs and had six conserved motifs. Molecular docking results indicated that binding sites of dimethylallyl diphosphate (DMAPP), (E)-geranyl pyrophosphate (GPP), and (E)-farnesyl pyrophosphate (FPP) were in the chain-length determination region of MhieGGPPS, respectively. In vitro, recombiant MhieGGPPS could catalyze the formation of (E)-geranylgeraniol against different combinations of substrates including isopentenyl pyrophosphate (IPP)/DMAPP, IPP/(E)-GPP, and IPP/(E)-FPP, suggesting that MhieGGPPS could not only use (E)-FPP but also (E)-GPP and DMAPP as the allylic cosubstrates. In kinetic analysis, the (E)-FPP was most tightly bound to MhieGGPPS than that of others. It was proposed that MhieGGPPS as a multifunctional enzyme is differentiated from the other GGPPSs in the animals and plants, which only accepted (E)-FPP as the allylic cosubstrate. These findings provide valuable insights into understanding the functional plasticity of GGPPS in M. hieroglyphica and the novel biosynthesis mechanism in the isoprenoid pathway.