Biosynthetic Systems Research Articles

Functional Characterization of a Condensation Domain That Links Nonribosomal Peptide and Pteridine Biosynthetic Machineries in Photorhabdus luminescens.

Nonribosomal peptide synthetases (NRPSs) produce a wide variety of biologically important small molecules. NRPSs can interface with other enzymes to form hybrid biosynthetic systems that expand the structural and functional diversity of their products. The pepteridines are metabolites encoded by an unprecedented pteridine-NRPS-type hybrid biosynthetic gene cluster in Photorhabdus luminescens, but how the distinct enzymatic systems interface to produce these molecules has not been examined at the biochemical level. By an unknown mechanism, the genetic locus can also affect the regulation of other enzymes involved in autoinducer and secondary metabolite biosynthesis. Here, through in vitro protein biochemical assays, we demonstrate that an atypical NRPS condensation (C) domain present in the pathway condenses acyl units derived from α-keto acids onto a free 5,6,7,8-tetrahydropterin core, producing the tertiary cis-amide-containing pepteridines. Solution studies of the chemically synthesized molecules led to the same amide regiochemistries that were observed in the natural products. The biochemical transformations mediated by the C domain destroy the radical scavenging activity of its redox active tetrahydropterin substrate. Secondary metabolite analyses revealed that the pepteridine locus affects select metabolic pathways associated with quorum sensing, antibiosis, and symbiosis. Taken together, the results suggest that the pathway likely regulates cellular redox and specialized metabolic pathways through engagement with the citric acid cycle.

In Vivo Platforms for Terpenoid Overproduction and the Generation of Chemical Diversity.

Terpenoids represent a highly diverse group of natural products with wide applications. Engineering approaches have been used to increase titers of many value-added terpenoids, such as farnesene, taxadiene, lycopene, and astaxanthin. In this chapter, we review the in vitro reconstitution-based targeted engineering of terpenoids, as well as approaches for the mining of terpene cyclases and for increasing the chemical diversity. Information gained from in vitro reconstitution extends our understanding of the mechanisms underlying terpenoid biosynthesis, the contributions of enzymes and cofactors, and key enzymes and rate-limiting steps for the development of an ideal biosynthetic production system. The in vitro reconstitution-based targeted engineering strategy provides a rational and accurate engineering approach for terpenoid overproduction with high efficiency. Furthermore, an efficient terpenoid overproduction platform can accelerate the entire process for the mining of terpene cyclases and the discovery of novel terpenoids and can substantially increase the chemical diversity of these kinds of terpenoids.

Directed production of aurantizolicin and new members based on a YM-216391 biosynthetic system.

Using a highly effective heterologous expression system, the YM-216391 (1) biosynthetic gene cluster was engineered to yield aurantizolicin (2) and the hybrid compound 3. Both of these compounds were isolated and fully structurally characterised and the bioactivity was evaluated. The results indicate that compound 3 exhibits significantly improved antitumor activity.

Conformational changes in the di-domain structure of Arabidopsis phosphoethanolamine methyltransferase leads to active-site formation

Phosphocholine (pCho) is a precursor for phosphatidylcholine and osmoprotectants in plants. In plants, de novo synthesis of pCho relies on the phosphobase methylation pathway. Phosphoethanolamine methyltransferase (PMT) catalyzes the triple methylation of phosphoethanolamine (pEA) to pCho. The plant PMTs are di-domain methyltransferases that divide the methylation of pEA in one domain from subsequent methylations in the second domain. To understand the molecular basis of this architecture, we examined the biochemical properties of three Arabidopsis thaliana PMTs (AtPMT1-3) and determined the X-ray crystal structures of AtPMT1 and AtPMT2. Although each isoform synthesizes pCho from pEA, their physiological roles differ with AtPMT1 essential for normal growth and salt tolerance, whereas AtPMT2 and AtPMT3 overlap functionally. The structures of AtPMT1 and AtPMT2 reveal unique features in each methyltransferase domain, including active sites that use different chemical mechanisms for phosphobase methylation. These structures also show how rearrangements in both the active sites and the di-domain linker form catalytically competent active sites and provide insight on the evolution of the PMTs in plants, nematodes, and apicomplexans. Connecting conformational changes with catalysis in modular enzymes, like the PMT, provides new insights on interdomain communication in biosynthetic systems.

Controlling cell-free metabolism through physiochemical perturbations

Building biosynthetic pathways and engineering metabolic reactions in cells can be time-consuming due to complexities in cellular metabolism. These complexities often convolute the combinatorial testing of biosynthetic pathway designs needed to define an optimal biosynthetic system. To simplify the optimization of biosynthetic systems, we recently reported a new cell-free framework for pathway construction and testing. In this framework, multiple crude-cell extracts are selectively enriched with individual pathway enzymes, which are then mixed to construct full biosynthetic pathways on the time scale of a day. This rapid approach to building pathways aids in the study of metabolic pathway performance by providing a unique freedom of design to modify and control biological systems for both fundamental and applied biotechnology. The goal of this work was to demonstrate the ability to probe biosynthetic pathway performance in our cell-free framework by perturbing physiochemical conditions, using n-butanol synthesis as a model. We carried out three unique case studies. First, we demonstrated the power of our cell-free approach to maximize biosynthesis yields by mapping physiochemical landscapes using a robotic liquid-handler. This allowed us to determine that NAD and CoA are the most important factors that govern cell-free n-butanol metabolism. Second, we compared metabolic profile differences between two different approaches for building pathways from enriched lysates, heterologous expression and cell-free protein synthesis. We discover that phosphate from PEP utilization, along with other physiochemical reagents, during cell-free protein synthesis-coupled, crude-lysate metabolic system operation inhibits optimal cell-free n-butanol metabolism. Third, we show that non-phosphorylated secondary energy substrates can be used to fuel cell-free protein synthesis and n-butanol biosynthesis. Taken together, our work highlights the ease of using cell-free systems to explore physiochemical perturbations and suggests the need for a more controllable, multi-step, separated cell-free framework for future pathway prototyping and enzyme discovery efforts.

Heparan Sulfate Biosynthetic System Is Inhibited in Human Glioma Due to EXT1/2 and HS6ST1/2 Down-Regulation.

Heparan sulfate (HS) is an important component of the extracellular matrix and cell surface, which plays a key role in cell–cell and cell–matrix interactions. Functional activity of HS directly depends on its structure, which determined by a complex system of HS biosynthetic enzymes. During malignant transformation, the system can undergo significant changes, but for glioma, HS biosynthesis has not been studied in detail. In this study, we performed a comparative analysis of the HS biosynthetic system in human gliomas of different grades. RT-PCR analysis showed that the overall transcriptional activity of the main HS biosynthesis-involved genes (EXT1, EXT2, NDST1, NDST2, GLCE, HS2ST1, HS3ST1, HS3ST2, HS6ST1, HS6ST2, SULF1, SULF2, HPSE) was decreased by 1.5–2-fold in Grade II-III glioma (p < 0.01) and by 3-fold in Grade IV glioma (glioblastoma multiforme, GBM) (p < 0.05), as compared with the para-tumourous tissue. The inhibition was mainly due to the elongation (a decrease in EXT1/2 expression by 3–4-fold) and 6-O-sulfation steps (a decrease in 6OST1/2 expression by 2–5-fold) of the HS biosynthesis. Heparanase (HPSE) expression was identified in 50% of GBM tumours by immunostaining, and was characterised by a high intratumoural heterogeneity of the presence of the HPSE protein. The detected disorganisation of the HS biosynthetic system in gliomas might be a potential molecular mechanism for the changes of HS structure and content in tumour microenvironments, contributing to the invasion of glioma cells and the development of the disease.

Mapping the key residues of SufB and SufD essential for biosynthesis of iron-sulfur clusters

Biogenesis of iron-sulfur (Fe-S) clusters is an indispensable process in living cells. In Escherichia coli, the SUF biosynthetic system consists of six proteins among which SufB, SufC and SufD form the SufBCD complex, which serves as a scaffold for the assembly of nascent Fe-S cluster. Despite recent progress in biochemical and structural studies, little is known about the specific regions providing the scaffold. Here we present a systematic mutational analysis of SufB and SufD and map their critical residues in two distinct regions. One region is located on the N-terminal side of the β-helix core domain of SufB, where biochemical studies revealed that Cys254 of SufB (SufBC254) is essential for sulfur-transfer from SufE. Another functional region resides at an interface between SufB and SufD, where three residues (SufBC405, SufBE434, and SufDH360) appear to comprise the site for de novo cluster formation. Furthermore, we demonstrate a plausible tunnel in the β-helix core domain of SufB through which the sulfur species may be transferred from SufBC254 to SufBC405. In contrast, a canonical Fe-S cluster binding motif (CxxCxxxC) of SufB is dispensable. These findings provide new insights into the mechanism of Fe-S cluster assembly by the SufBCD complex.

The Suf Iron-Sulfur Cluster Biosynthetic System Is Essential in Staphylococcus aureus, and Decreased Suf Function Results in Global Metabolic Defects and Reduced Survival in Human Neutrophils.

Staphylococcus aureus remains a causative agent for morbidity and mortality worldwide. This is in part a result of antimicrobial resistance, highlighting the need to uncover novel antibiotic targets and to discover new therapeutic agents. In the present study, we explored the possibility that iron-sulfur (Fe-S) cluster synthesis is a viable antimicrobial target. RNA interference studies established that Suf (sulfur mobilization)-dependent Fe-S cluster synthesis is essential in S. aureus We found that sufCDSUB were cotranscribed and that suf transcription was positively influenced by sigma factor B. We characterized an S. aureus strain that contained a transposon inserted in the intergenic space between sufC and sufD (sufD*), resulting in decreased transcription of sufSUB Consistent with the transcriptional data, the sufD* strain had multiple phenotypes associated with impaired Fe-S protein maturation. They included decreased activities of Fe-S cluster-dependent enzymes, decreased growth in media lacking metabolites that require Fe-S proteins for synthesis, and decreased flux through the tricarboxylic acid (TCA) cycle. Decreased Fe-S cluster synthesis resulted in sensitivity to reactive oxygen and reactive nitrogen species, as well as increased DNA damage and impaired DNA repair. The sufD* strain also exhibited perturbed intracellular nonchelated Fe pools. Importantly, the sufD* strain did not exhibit altered exoprotein production or altered biofilm formation, but it was attenuated for survival upon challenge by human polymorphonuclear leukocytes. The results presented are consistent with the hypothesis that Fe-S cluster synthesis is a viable target for antimicrobial development.

The Iron Chaperone Protein CyaY from Vibrio cholerae Is a Heme-Binding Protein.

CyaY is an iron transport protein for iron-sulfur (Fe-S) cluster biosynthetic systems. It also transports iron to ferrochelatase that catalyzes insertion of Fe2+ into protoporphyrin IX. Here, we find that CyaY has the ability to bind heme as well as iron, exhibiting an apparent dissociation constant for heme of 21 ± 6 nM. Absorption and resonance Raman spectra revealed that both ferric and ferrous forms of heme were bound to an anionic ligand (e.g., tyrosine and/or cysteine). Consistent with this, mutagenesis studies showed that Tyr67 and Cys78 are possible heme ligands of CyaY. The binding of heme to CyaY increased the apparent dissociation constant of CyaY for iron from 65.2 to 87.9 μM. Circular dichroism spectra of CyaY suggested that binding of heme to CyaY induces rearrangement of aromatic residues. Furthermore, size-exclusion column chromatography demonstrated heme-mediated oligomerization of CyaY. These results suggest that heme binding induces conformational changes, including oligomerization of CyaY, that result in a decrease in the affinity of CyaY for iron. Accordingly, the presence of excess heme in cells would lead to modulation of Fe-S cluster or heme biosynthesis. This report provides the first description of heme dependence of iron transport by CyaY.

Genetic and biochemical characterization of the GH72 family of cell wall transglycosylases in Neurospora crassa

The Neurospora crassa genome encodes five GH72 family transglycosylases, and four of these enzymes (GEL-1, GEL-2, GEL-3 and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2 and GEL-5 is required for the growth of vegetative hyphae, while the GEL-1, GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping enzymatic activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of the transglycosylase-deficient mutants demonstrated that the incorporation of cell wall proteins was severely compromised. Interestingly, we found that the transglycosylase-deficient mutant cell walls contained more β-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 transglycosylases are not needed for the incorporation of β-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall.

Profiling influences of gene overexpression on heterologous resveratrol production in Saccharomyces cerevisiae

Metabolic engineering of heterologous resveratrol production in Saccharomyces cerevisiae faces challenges as the precursor l-tyrosine is stringently regulated by a complex biosynthetic system. We overexpressed the main gene targets in the upstream pathways to investigate their influences on the downstream resveratrol production. Single-gene overexpression and DNA assembly-directed multigene overexpression affect the production of resveratrol as well as its precursor p-coumaric acid. Finally, the collaboration of selected gene targets leads to an optimal resveratrol production of 66.14±3.74 mg·L–1, 2.27 times higher than the initial production in YPD medium (4% glucose). The newly discovered gene targets TRP1 expressing phosphoribosylanthranilate isomerase, ARO3 expressing 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, and 4CL expressing 4-coumaryl-CoA ligase show notable positive impacts on resveratrol production in S. cerevisiae. Open image in new window

One-Pot Combinatorial Biosynthesis of Glycosylated Anthracyclines by Cocultivation of Streptomyces Strains Producing Aglycones and Nucleotide Deoxysugars.

Anthracyclines, such as doxorubicin, are effective anticancer drugs composed of a tetracyclic polyketide aglycone and one or more deoxysugar moieties, which play a critical role in their biological activity. A facile one-pot combinatorial biosynthetic system was developed for the generation of a range of glycosylated derivatives of anthracyclines. Cocultivation of Streptomyces venezuelae mutants producing two anthracycline aglycones with eight different nucleotide deoxysugar-producing S. venezuelae mutants that coexpress a substrate-flexible glycosyltransferase led to the generation of 16 aklavinone or ε-rhodomycinone glycosides containing diverse deoxysugar moieties, seven of which are new. This demonstrates the potential of the one-pot combinatorial biosynthetic system based on cocultivation as a facile biological tool capable of combining diverse aglycones and deoxysugars to generate structurally diverse polyketides carrying engineered sugars for drug discovery and development.

Abstract A40: A novel, biosynthetic 3D hydrogel system for breast cancer mechanobiology studies

Abstract Breast tumor cell-extracellular matrix (ECM) interactions drive malignant transformation, invasion, and metastasis of breast cancer cells and contribute to treatment resistance. The tumor microenvironment, composed of multiple ECM components, activate mechanochemical pathways associated with breast cancer progression and signaling of runt-related transcription factor 2 (Runx2). There is an urgent need to model these interactions in physiologically relevant three-dimensional (3D) models in vitro. We recently demonstrated that we can create 3D PEG hydrogel systems, containing type I collagen, Arg-Gly-Asp (RGD), and hyaluronic acid, whose stiffness can be tuned independent of the number of ECM components incorporated. In this study, we demonstrate that these hydrogels can be utilized to mimic the in vivo tumor microenvironment. We examined the behavior of breast cancer cell lines MDA-MB-231 (invasive) and MCF7 (non-invasive) as well as MCF10A (non-tumorigenic mammary epithelial cells) in our novel, biosynthetic hydrogel systems. We showed that these hydrogels supported growth for each cell line and formation of acinar structures over a 23-day period in vitro. In future studies, we will assess the migratory behaviors of these cell lines in vitro using microfluidic devices as well as Runx2 expression and activity to determine the influence of matrix physiochemical properties on the invasive potential of breast tumor cells. This novel, biosynthetic system represents a reproducible framework where tumor cell behavior may be quantified as a function of a complex system in which combined microenvironmental parameters, including matrix stiffness, ECM composition, and their interactions, are systematically varied. Citation Format: Amy Elissa Blatt, Bita Carrion, Rhima M. Coleman. A novel, biosynthetic 3D hydrogel system for breast cancer mechanobiology studies. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A40.

Selective biomolecular separation system inspired by the nuclear pore complex and nuclear transport

Inspired by selectively filtering system of the nucleus, we developed biosynthetic system that can selectively “catch-trap” biotoxins from aqueous environment.

Lysine Biosynthesis of Thermococcus kodakarensis with the Capacity to Function as an Ornithine Biosynthetic System

We recently discovered a biosynthetic system using a novel amino group carrier protein called LysW for lysine biosynthesis via α-aminoadipate (AAA), and revealed that this system is also utilized in the biosynthesis of arginine by Sulfolobus In the present study, we focused on the biosynthesis of lysine and ornithine in the hyperthermophilic archaeon Thermococcus kodakarensis, and showed that their biosynthesis is accomplished by a single set of metabolic enzymes. We also determined the crystal structure of the LysX family protein from T. kodakarensis, which catalyzes the conjugation of LysW with either AAA or glutamate, in a complex with LysW-γ-AAA. This crystal structure is the first example to show how LysX recognizes AAA as a substrate and provides a structural basis for the bifunctionality of the LysX family protein from T. kodakarensis Based on comparisons with other LysX family proteins, we propose a mechanism for substrate recognition and its relationship with molecular evolution among LysX family proteins, which have different substrate specificities.

Amino-group carrier-protein-mediated secondary metabolite biosynthesis in Streptomyces.

Amino-group carrier proteins (AmCPs) mediate the biosynthesis of lysine and arginine in some bacteria and archaea. Here we demonstrate that an uncharacterized AmCP-mediated biosynthetic system functions to biosynthesize the previously uncharacterized and nonproteinogenic amino acid (2S,6R)-diamino-(5R,7)-dihydroxy-heptanoic acid (DADH) in Streptomyces sp. SANK 60404. DADH is incorporated into a novel peptide metabolite, vazabitide A, featuring an azabicyclo-ring structure, by nonribosomal peptide synthetases and successive modification enzymes in this bacterium. As the AmCP-mediated machinery for DADH biosynthesis is widely distributed in bacteria, further analysis of uncharacterized AmCP-containing gene clusters will lead to the discovery of novel bioactive compounds and novel biosynthetic enzymes.

Sequence homolog-based molecular engineering for shifting the enzymatic pH optimum

Cell-free synthetic biology system organizes multiple enzymes (parts) from different sources to implement unnatural catalytic functions. Highly adaption between the catalytic parts is crucial for building up efficient artificial biosynthetic systems. Protein engineering is a powerful technology to tailor various enzymatic properties including catalytic efficiency, substrate specificity, temperature adaptation and even achieve new catalytic functions. However, altering enzymatic pH optimum still remains a challenging task. In this study, we proposed a novel sequence homolog-based protein engineering strategy for shifting the enzymatic pH optimum based on statistical analyses of sequence-function relationship data of enzyme family. By two statistical procedures, artificial neural networks (ANNs) and least absolute shrinkage and selection operator (Lasso), five amino acids in GH11 xylanase family were identified to be related to the evolution of enzymatic pH optimum. Site-directed mutagenesis of a thermophilic xylanase from Caldicellulosiruptor bescii revealed that four out of five mutations could alter the enzymatic pH optima toward acidic condition without compromising the catalytic activity and thermostability. Combination of the positive mutants resulted in the best mutant M31 that decreased its pH optimum for 1.5 units and showed increased catalytic activity at pH < 5.0 compared to the wild-type enzyme. Structure analysis revealed that all the mutations are distant from the active center, which may be difficult to be identified by conventional rational design strategy. Interestingly, the four mutation sites are clustered at a certain region of the enzyme, suggesting a potential “hot zone” for regulating the pH optima of xylanases. This study provides an efficient method of modulating enzymatic pH optima based on statistical sequence analyses, which can facilitate the design and optimization of suitable catalytic parts for the construction of complicated cell-free synthetic biology systems.

Nitric Oxide Synthase and Cyclooxygenase Pathways: A Complex Interplay in Cellular Signaling.

The cellular reaction to external challenges is a tightly regulated process consisting of integrated processes mediated by a variety of signaling molecules, generated as a result of modulation of corresponding biosynthetic systems. Both, nitric oxide synthase (NOS) and cyclooxygenase (COX) systems, consist of constitutive forms (NOS1, NOS3 and COX-1), which are mostly involved in housekeeping tasks, and inducible forms (NOS2 and COX-2), which shape the cellular response to stress and variety of bioactive agents. The complex interplay between NOS and COX pathways can be observed at least at three levels. Firstly, products of NOS and Cox systems can mediate the regulation and the expression of inducible forms (NOS2 and COX-2) in response of similar and dissimilar stimulus. Secondly, the reciprocal modulation of cyclooxygenase activity by nitric oxide and NOS activity by prostaglandins at the posttranslational level has been shown to occur. Mechanisms by which nitric oxide can modulate prostaglandin synthesis include direct S-nitrosylation of COX and inactivation of prostaglandin I synthase by peroxynitrite, product of superoxide reaction with nitric oxide. Prostaglandins, conversely, can promote an increased association of dynein light chain (DLC) (also known as protein inhibitor of neuronal nitric oxide synthase) with NOS1, thereby reducing its activity. The third level of interplay is provided by intracellular crosstalk of signaling pathways stimulated by products of NOS and COX which contributes significantly to the complexity of cellular signaling. Since modulation of COX and NOS pathways was shown to be principally involved in a variety of pathological conditions, the dissection of their complex relationship is needed for better understanding of possible therapeutic strategies. This review focuses on implications of interplay between NOS and COX for cellular function and signal integration.

Manipulation of ubiquitin/SUMO pathways in human herpesviruses infection.

Post-translational modification of proteins with ubiquitin/small ubiquitin-like modifier (SUMO) molecules triggers multiple signaling pathways that are critical for many aspects of cellular physiology. Given that viruses hijack the biosynthetic and degradative systems of their host, it is not surprising that viruses encode proteins to manipulate the host's cellular machinery for ubiquitin/SUMO modification at multiple levels. Infection with a herpesvirus, among the most ubiquitous human DNA viruses, has been linked to many human diseases, including cancers. The interplay between human herpesviruses and the ubiquitylation/SUMOylation modification system has been extensively investigated in the past decade. In this review, we present an overview of recent advances to address how the ubiquitin/SUMO-modified system alters the latency and lytic replication of herpesvirus and how herpesviruses usurp the ubiquitin/SUMO pathways against the host's intrinsic and innate immune response to favor their pathogenesis.

Evaluation of Biosynthetic Pathway and Engineered Biosynthesis of Alkaloids.

Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid), saframycin (tetrahydroisoquinoline alkaloid), strictosidine (monoterpene indole alkaloid), ergotamine (ergot alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details.