Total biosynthesis of diterpene aphidicolin, a specific inhibitor of DNA polymerase α: heterologous expression of four biosynthetic genes in Aspergillus oryzae

Clustering of biosynthetic genes for producing fungal secondary metabolites, which frequently consist of less than ten genes, has been recognized with numerous genomes. The heterologous expression of whole genes in the clusters will therefore produce various types of natural products when using a suitable fungal host. We introduced the whole gene cluster for the biosynthesis of diterpene aphidicolin into the fungal quadruple auxotrophic host, Aspergillus oryzae, by using four different vectors (pTAex3, pPTRI, pUSA and pAdeA) which harbor a starch-inducible promoter/terminator to examine the expression conditions. The resulting quadruple transformant carrying the genes of geranylgeranyl diphosphate synthase PbGGS, terpene synthase PbACS, and two monooxygenases (PbP450-1 and PbP450-2) produced aphidicolin. The double and triple transformants also respectively produced aphidicolan-16β-ol and 3-deoxyaphidicolin. Alternative host Saccharomyces cerevisiae carrying the genes, PbGGS and PbACS, produced key intermediate aphidicolan-16β-ol. This is the first example of a total biosynthesis of terpenoids using fungal hosts.     

Subcellular localization of sanguinarine biosynthetic enzymes in cultured opium poppy cells

Benzylisoquinoline alkaloids are a large and structurally diverse group of natural plant products that includes many compounds with potent biological activities, including the antimicrobial agent sanguinarine. The putative subcellular localization of the sanguinarine pathway was determined using in-frame N-terminal fusions between cDNAs encoding nine consecutive biosynthetic enzymes and the gene encoding the green fluorescent protein (GFP). Expression constructs were introduced into cultured opium poppy cells by particle bombardment, and the localization of fusion proteins was visualized using epifluorescence microscopy. GFP fusions with two O-methyltransferases and two N-methyltransferases in the sanguinarine pathway all produced non-targeted fluorescence in the cytosol and nucleus. Interspersed between these soluble proteins are five membrane-bound cytochromes P450. Corresponding cDNAs are available for three P450s, all of which produced fluorescence when fused to GFP in association with the endoplasmic reticulum (ER). Two enzymes with suggested or known N-terminal signal peptides were initially associated with the ER, but were subsequently transported to the central vacuole suggesting their occurrence in the ER lumen. The alternating localization of these biosynthetic enzymes to three subcellular compartments indicates extensive trafficking of pathway intermediates across the endomembranes and suggests a key role for compartmentalization in the regulation of sanguinarine metabolism.     

Reconstitution of biosynthetic machinery of fungal polyketides: unexpected oxidations of biosynthetic intermediates by expression host

Reconstitution of whole biosynthetic genes in Aspergillus oryzae has successfully applied for total biosynthesis of various fungal natural products. Heterologous production of fungal metabolites sometimes suffers unexpected side reactions by host enzymes. In the studies on fungal polyketides solanapyrone and cytochalasin, unexpected oxidations of terminal olefin of biosynthetic intermediates were found to give one and four by-products by host enzymes of the transformants harboring biosynthetic genes. In this paper, we reported structure determination of by-products and described a simple solution to avoid the undesired reaction by introducing the downstream gene in the heterologous production of solanapyrone C.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

Frog Virus 3 Open Reading Frame 97R Localizes to the Endoplasmic Reticulum and Induces Nuclear Invaginations

Frog virus 3 (FV3) is the type species of the genus Ranavirus, family Iridoviridae. The genome of FV3 is 105,903 bases in length and encodes 97 open reading frames (ORFs). The FV3 ORF 97R contains a B-cell lymphoma 2 (Bcl-2) homology 1 (BH1) domain and has sequence similarity to the myeloid cell leukemia-1 (Mcl-1) protein, suggesting a potential role in apoptosis. To begin to understand the role of 97R, we characterized 97R through immunofluorescence and mutagenesis. Here we demonstrated that 97R localized to the endoplasmic reticulum (ER) at 24 h posttransfection. However, at 35 h posttransfection, 97R localized to the ER but also began to form concentrated pockets continuous with the nuclear membrane. After 48 h posttransfection, 97R was still localized to the ER, but we began to observe the ER and the outer nuclear membrane invaginating into the nucleus. To further explore 97R targeting to the ER, we created a series of C-terminal transmembrane domain deletion mutants. We found that deletion of 29 amino acids from the C terminus of 97R abolished localization to the ER. In contrast, deletion of 12 amino acids from the C terminus of 97R did not affect 97R localization to the ER. In addition, a hybrid protein containing the 97R C-terminal 33 amino acids was similarly targeted to the ER. These data indicate that the C-terminal 33 amino acids of 97R are necessary and sufficient for ER targeting.     

Phospholipase A2 catalysis and lipid mediator lipidomics

Phospholipase A₂ (PLA₂) enzymes are the upstream regulators of the eicosanoid pathway liberating free arachidonic acid from the sn-2 position of membrane phospholipids. Free intracellular arachidonic acid serves as a substrate for the eicosanoid biosynthetic enzymes including cyclooxygenases, lipoxygenases, and cytochrome P450s that lead to inflammation. The Group IVA cytosolic (cPLA₂), Group VIA calcium-independent (iPLA₂), and Group V secreted (sPLA₂) are three well-characterized human enzymes that have been implicated in eicosanoid formation. In this review, we will introduce and summarize the regulation of catalytic activity and cellular localization, structural characteristics, interfacial activation and kinetics, substrate specificity, inhibitor binding and interactions, and the downstream implications for eicosanoid biosynthesis of these three important PLA₂ enzymes.     

Endophytic fungus Falciphora oryzae promotes lateral root growth by producing indole derivatives after sensing plant signals

The endophytic fungus Falciphora oryzae was initially isolated from wild rice (Oryza granulata) and colonizes many crop species and promotes plant growth. However, the molecular mechanisms underlying F. oryzae-mediated growth promotion are still unknown. We found that F. oryzae was able to colonize Arabidopsis thaliana. The most dramatic change after F. oryzae inoculation was observed in the root architecture, as evidenced by increased lateral root growth but reduced primary root length, similar to the effect of auxin, a significant plant growth hormone. The expression of genes responsible for auxin biosynthesis, transport, and signalling was regulated in Arabidopsis roots after F. oryzae cocultivation. Indole derivatives were detected at significantly higher levels in liquid media after cocultivation compared with separate cultivation of Arabidopsis and F. oryzae. Consistently, the expression of indole biosynthetic genes was highly upregulated in F. oryzae upon treatment with Arabidopsis exudates. Global analysis of Arabidopsis gene expression at the early stage after F. oryzae cocultivation suggested that signals were exchanged to initiate Arabidopsis–F. oryzae interactions. All these results suggest that signalling molecules from Arabidopsis roots are perceived by F. oryzae and induce the biosynthesis of indole derivatives in F. oryzae, consequently stimulating Arabidopsis lateral root growth.     

Two nucleotide sugar transporters are important for cell wall integrity and full virulence of Magnaporthe oryzae

Cell wall polysaccharides play key roles in fungal development, virulence, and resistance to the plant immune system, and are synthesized from many nucleotide sugars in the endoplasmic reticulum (ER)-Golgi secretory system. Nucleotide sugar transporters (NSTs) are responsible for transporting cytosolic-derived nucleotide sugars to the ER lumen for processing, but their roles in plant-pathogenic fungi remain to be revealed. Here, we identified two important NSTs, NST1 and NST2, in the rice blast fungus Magnaporthe oryzae. Both NSTs were localized in the ER, which was consistent with a function in transporting nucleotide sugar for processing in the ER. Sugar transport property analysis suggested that NST1 is involved in transportation of mannose and glucose, while NST2 is only responsible for mannose transportation. Accordingly, deletion of NSTs resulted in a significant decrease in corresponding soluble saccharides abundance and defect in sugar utilization. Moreover, both NSTs played important roles in cell wall integrity, were involved in asexual development, and were required for full virulence. The NST mutants exhibited decreasing external glycoproteins and exposure of inner chitin, which resulted in activation of the host defence response. Altogether, our results revealed that two sugar transporters are required for fungal cell wall polysaccharides accumulation and full virulence of M. oryzae.     

Localization of plant N‐glycan processing enzymes along the secretory pathway

Abstract

N‐glycosylation is an abundant covalent protein modification in all eukaryotic cells. The biosynthesis and processing of protein N‐linked glycans results from a series of highly co‐ordinated step‐by‐step enzymatic conversions occurring mainly in the endoplasmic reticulum (ER) and Golgi apparatus. N‐glycan processing enzymes are thought to act on cargo glycoproteins in a highly ordered fashion in an assembly line. Thus, the subcellular localization of these enzymes together with their in vivo substrate specificity determines the carbohydrate structures of glycoproteins transported through the secretory pathway. While the substrate specificities of many plant N‐glycan processing enzymes are fairly well characterized, the molecular mechanisms underlying enzyme localization to the ER and Golgi have remained largely elusive so far. This review discusses current data on ER and Golgi localization of plant N‐glycan processing enzymes.     

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) localizes to endoplasmic reticulum not peroxisomes, and NH2-terminal hydrophobic property determines the subcellular localization of ABC subfamily D proteins

70-kDa peroxisomal membrane protein related protein (P70R/ABCD4) is a member of ATP-binding cassette (ABC) protein subfamily D. ABC subfamily D proteins are also known as peroxisomal ABC proteins. Therefore, P70R is thought to be a peroxisomal membrane protein. However, the subcellular localization of P70R is not extensively investigated. In this study, we transiently expressed P70R in fusion with HA (P70R-HA) in CHO cells and examined subcellular localization by immunofluorescence. Surprisingly, P70R-HA was localized to the endoplasmic reticulum (ER), not to peroxisomes. To examine the ER-targeting property of P70R, we expressed various NH₂-terminal deletion constructs of P70R. Among the NH₂-terminal deletion constructs, mutant proteins starting with hydrophobic transmembrane segment (TMS) were localized to ER, but the ones containing the NH₂-terminal hydrophilic cytosolic domain were not. ABC subfamily D proteins destined for peroxisomes have NH₂-terminal hydrophilic region adjacent to TMS1. However, only P70R lacks the region and is translated with NH₂-terminal hydrophobic TMS1. Furthermore, attachment of the NH₂-terminal hydrophilic domain to the NH₂-terminus of P70R excluded P70R from the ER-targeting pathway. These data suggest that P70R resides in the ER but not the peroxisomal membranes, and the hydrophobic property of NH₂-terminal region determines the subcellular localization of ABC subfamily D proteins.     

Specialized ER membrane domains for lipid metabolism and transport

The endoplasmic reticulum (ER) is a highly organized organelle that performs vital functions including de novo membrane lipid synthesis and transport. Accordingly, numerous lipid biosynthesis enzymes are localized in the ER membrane. However, it is now evident that lipid metabolism is sub-compartmentalized within the ER and that lipid biosynthetic enzymes engage with lipid transfer proteins (LTPs) to rapidly shuttle newly synthesized lipids from the ER to other organelles. As such, intimate relationships between lipid metabolism and lipid transfer pathways exist within the ER network. Notably, certain LTPs enhance the activities of lipid metabolizing enzymes; likewise, lipid metabolism can ensure the specificity of LTP transfer/exchange reactions. Yet, our understanding of these mutual relationships is still emerging. Here, we highlight past and recent key findings on specialized ER membrane domains involved in efficient lipid metabolism and transport and consider unresolved issues in the field.     

Expression and secretion of glycosylated heparin biosynthetic enzymes using Komagataella pastoris

Heparin, an anticoagulant drug, is biosynthesized in selected animal cells. The heparin biosynthetic enzymes mainly consist of sulfotransferases and all are integral transmembrane glycoproteins. These enzymes are generally produced in engineered Escherichia coli as without their transmembrane domains as non-glycosylated fusion proteins. In this study, we used the yeast, Komagataella pastoris, to prepare four sulfotransferases involved in heparin biosynthesis as glycoproteins. While the yields of these yeast-expressed enzymes were considerably lower than E. coli-expressed enzymes, these enzymes were secreted into the fermentation media simplifying their purification and were endotoxin free. The activities of these sulfotransferases, expressed as glycoproteins in yeast, were compared to the bacterially expressed proteins. The yeast-expressed sulfotransferase glycoproteins showed improved kinetic properties than the bacterially expressed proteins.

     

Identification of a cDNA Coding for a Ca-Binding Phosphoprotein (p90), Calnexin, on Melanosomes in Normal and Malignant Human Melanocytes

In order to have a proper biosynthesis and secretion of the melanin-pigment granules (melanosomes) the melanocyte may require a melanosome-associated molecule that provides a signal for assembly and organization of melanogenic enzymes and proteins within the compartment of melanosomes. This study reports the presence of a Ca²⁺-binding phosphoprotein, p90, which can be engaged in such melanogenic function, located on the melanosomal membrane of human melanocytes. A human melanoma cDNA expression library in λ Zap II was screened with a rabbit polyclonal antibody raised against human melanosomes isolated from cultured human melanoma cells, SK MEL 23. A cDNA encoding a melanosomal protein, M_r 90 kDa, was identified through this immunoscreening. A partial sequencing of nucleotides (822 bp from the N-terminal domain) of this clone (3.8 kb) and predicted amino acids showed more than 90% homology with dog calnexin, a previously reported endoplasmic reticulum (ER) transmembrane protein. A fusion protein of this p90 with β-galactosidase expressed in Escherichia coli revealed both the immuno-cross-reactivity with anti-dog calnexin and anti-human melanosome antibodies and the Ca²⁺-binding property. Upon immunohistochemistry, the anti-dog calnexin antibody revealed the positive immunoreactivities with both normal and malignant human melanocytes, showing a much higher expression of antigenic epitope than nonmelanocytic human cells. The laser scanning confocal immunofluorescence, using an anti-body against a human melanosome-specific antigen (HMSA-5), and immunoelectron microscopy, using immunogold, confirmed the major localization of anti-dog calnexin antibody epitope on the melanosomes and ER.     

Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.     

Localization of human (pro)renin receptor lacking the transmembrane domain on budded baculovirus of <Emphasis Type="Italic">Autographa californica</Emphasis> multiple nucleopolyhedrovirus

Human (pro)renin receptor (hPRR), a construct with native transmembrane and cytoplasmic domains (hPRR-wTM), and hPRR lacking both (hPRR-w/oTM) were expressed using insect cells. The hPRR-wTM was expressed in the peripheral domains of the nucleus in infected Sf-9 cells, and its localization was observed in endoplasmic reticulum (ER). However, it could not be extracted from recombinant Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by Triton X-100 treatment at 4°C. In contrast, hPRR-w/oTM was observed in punctate domains in the cytoplasm of infected Sf-9 cells, but intracellular hPRR-w/oTM did not co-localize in the Golgi apparatus and lysosomes. This indicates that hPRR-wTM and hPRR-w/oTM is localized in the ER and cytoplasmic organelles of Sf-9 cell, respectively. Moreover, the localization of hPRR-w/oTM in budded baculovirus of recombinant AcMNPV was confirmed by Western blotting. This is the first finding of the association of a foreign protein lacking a transmembrane domain with a baculovirus. If this finding is available for double displaying system, being capable of expression on the envelope and the capsid of baculovirus, it will lead to new methodology of baculovirus display system for tissue- and cell-specific targeting and intracellular targeting.     

Molecular characterization of Arabidopsis thaliana cDNAs encoding three purine biosynthetic enzymes

Glycinamide ribonucleotide (GAR) synthetase, GAR transformylase and aminoimidazole ribonucleotide (AIR) synthetase are the second, third and fifth enzymes in the 10-step de novo purine biosynthetic pathway. From a cDNA library of Arabidopsis thaliana, cDNAs encoding the above three enzymes were cloned by functional complementation of corresponding Escherichia coli mutants. Each of the cDNAs encode peptides comprising the complete enzymatic domain of either GAR synthetase, GAR transformylase or AIR synthetase. Comparisons of the three Arabidopsis purine biosynthetic enzymes with corresponding enzymes/polypeptide-fragments from procaryotic and eucaryotic sources indicate a high degree of conserved homology at the amino acid level, in particular with procaryotic enzymes. Assays from extracts of E. coli expressing the complementing clones verified the specific enzymatic activity of Arabidopsis GAR synthetase and GAR transformylase. Sequence analysis, as well as Northern blot analysis indicate that Arabidopsis has single and monofunctional enzymes. In this respect the organization of these three plant purine biosynthesis genes is fundamentally different from the multifunctional purine biosynthesis enzymes characteristic of other eucaryotes and instead resembles the one gene, one enzyme relationship found in procaryotes.     

Murine diacylglycerol acyltransferase-2 (DGAT2) can catalyze triacylglycerol synthesis and promote lipid droplet formation independent of its localization to the endoplasmic reticulum

Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by two distinct acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Both DGAT enzymes reside in the endoplasmic reticulum (ER), but DGAT2 also co-localizes with mitochondria and lipid droplets. In this report, we demonstrate that murine DGAT2 is part of a multimeric complex consisting of several DGAT2 subunits. We also identified the region of DGAT2 responsible for its localization to the ER. A DGAT2 mutant lacking both its transmembrane domains, although still associated with membranes, was absent from the ER and instead localized to mitochondria. Unexpectedly, this mutant was still active and capable of interacting with lipid droplets to promote TG storage. Additional experiments indicated that the ER targeting signal was present in the first transmembrane domain (TMD1) of DGAT2. When fused to a fluorescent reporter, TMD1, but not TMD2, was sufficient to target mCherry to the ER. Finally, the interaction of DGAT2 with lipid droplets was dependent on the C terminus of DGAT2. DGAT2 mutants, in which regions of the C terminus were either truncated or specific regions were deleted, failed to co-localize with lipid droplets when cells were oleate loaded to stimulate TG synthesis. Our findings demonstrate that DGAT2 is capable of catalyzing TG synthesis and promote its storage in cytosolic lipid droplets independent of its localization in the ER.     

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C₁₈ chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product’s structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides.