UDP-glucosyltransferase HvUGT13248 confers type II resistance to Fusarium graminearum in barley.

Fusarium head blight (FHB) of barley (Hordeum vulgare) causes yield losses and accumulation of trichothecene mycotoxins (e.g. deoxynivalenol [DON]) in grains. Glucosylation of DON to the nontoxic DON-3-O-glucoside (D3G) is catalyzed by UDP-glucosyltransferases (UGTs), such as barley UGT13248. We explored the natural diversity of UGT13248 in 496 barley accessions and showed that all carried potential functional alleles of UGT13248, as no genotypes showed strongly increased seedling sensitivity to DON. From a TILLING population, we identified 2 mutant alleles (T368I and H369Y) that, based on protein modeling, likely affect the UDP-glucose binding of UGT13248. In DON feeding experiments, DON-to-D3G conversion was strongly reduced in spikes of these mutants compared to controls, and plants overexpressing UGT13248 showed increased resistance to DON and increased DON-to-D3G conversion. Moreover, field-grown plants carrying the T368I or H369Y mutations inoculated with Fusarium graminearum showed increased FHB disease severity and reduced D3G production. Barley is generally considered to have type II resistance that limits the spread of F. graminearum from the infected spikelet to adjacent spikelets. Point inoculation experiments with F. graminearum showed increased infection spread in T368I and H369Y across the spike compared to wild type, while overexpression plants showed decreased spread of FHB symptoms. Confocal microscopy revealed that F. graminearum spread to distant rachis nodes in T368I and H369Y mutants but was arrested at the rachis node of the inoculated spikelet in wild-type plants. Taken together, our data reveal that UGT13248 confers type II resistance to FHB in barley via conjugation of DON to D3G.

Engineering Bacillus subtilis for the formation of a durable living biocomposite material.

Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis is engineered to display SpyTags on polar flagella for cell attachment to SpyCatcher modified secreted scaffolds. We engineer endospore limited B. subtilis cells to become a structural component of the material with spores for long-term storage of genetic programming. Silica biomineralization peptides are screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of cell containing silica material and that new functions can be incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs.

Developing a Protein Scaffolding System for Rapid Enzyme Immobilization and Optimization of Enzyme Functions for Biocatalysis.

Immobilization of enzymes is required for most biocatalytic processes, but chemistries used in enzyme immobilization are limited and can be challenging. Genetically encoded protein-based biomaterials could provide easy-to-use immobilization platforms for biocatalysts. We recently developed a self-assembling protein scaffold that covalently immobilized SpyTagged enzymes by engineering the bacterial microcompartment protein EutM from Salmonella enterica with a SpyCatcher domain. We also identified a range of EutM homologues as robust protein nanostructures with diverse architectures and electrostatic surface properties. In this work, we created a modular immobilization platform with tunable surface properties by developing a toolbox of self-assembling, robust EutM-SpyCatcher scaffolds. Using an alcohol dehydrogenase as model biocatalyst, we show that the scaffolds improve enzyme activity and stability. This work provides a modular, easy-to-use immobilization system that can be tailored for the optimal function of biocatalysts of interest.

Protein-based scaffolds for enzyme immobilization.

Biocatalysis is emerging as an alternative approach to chemical synthesis of industrially relevant complex molecules. To obtain suitable yields of compounds in a cost-effective manner, biocatalytic reaction cascades must be efficient, robust, and self-sufficient. One approach is to immobilize biocatalysts on a solid support, stabilizing the enzymes and providing optimal microenvironments for reaction sequences. Protein-based scaffolds can be designed as immobilization platforms for biocatalysts, enabling the genetically encoded spatial organization of single enzymes and multistep enzyme cascades. Additionally, protein scaffolds are versatile, are easily adapted, and remain robust under different reaction conditions. In this chapter, we describe methods for the design and production of a self-assembling protein scaffold system for in vitro coimmobilization of biocatalytic cascade enzymes. We provide detailed methods for the characterization of the protein scaffolds, as well as approaches to load biocatalytic cargo enzymes and test activity of immobilized cascades. In addition, we also discuss methods for the development of a scaffold building block toolbox with different surface properties, which could be adapted for a diversity of biocatalysts requiring alternative microenvironments for function.

Building a toolbox of protein scaffolds for future immobilization of biocatalysts.

Biological materials that are genetically encoded and can self-assemble offer great potential as immobilization platforms in industrial biocatalysis. Protein-based scaffolds can be used for the spatial organization of enzymes, to stabilize the catalysts and provide optimal microenvironments for reaction sequences. In our previous work, we created a protein scaffold for enzyme localization by engineering the bacterial microcompartment shell protein EutM from Salmonella enterica. Here, we sought to expand this work by developing a toolbox of EutM proteins with different properties, with the potential to be used for future immobilization of enzymes. We describe the bioinformatic identification of hundreds of homologs of EutM from diverse microorganisms. We specifically select 13 EutM homologs from extremophiles for characterization, based on phylogenetic analyses. We synthesize genes encoding the novel proteins, clone and express them in E. coli, and purify the proteins. In vitro characterization shows that the proteins self-assemble into robust nano- and micron-scale architectures including protein nanotubes, filaments, and scaffolds. We explore the self-assembly characteristics from a sequence-based approach and create a synthetic biology platform for the coexpression of different EutM homologs as hybrid scaffolds with integrated enzyme attachment points. This work represents a step towards our goal of generating a modular toolbox for the rapid production of self-assembling protein-based materials for enzyme immobilization.

Structure and Function of the Stressosome Signalling Hub.

The stressosome is a multi-protein signal integration and transduction hub found in a wide range of bacterial species. The role that the stressosome plays in regulating the transcription of genes involved in the general stress response has been studied most extensively in the Gram-positive model organism Bacillus subtilis. The stressosome receives and relays the signal(s) that initiate a complex phosphorylation-dependent partner switching cascade, resulting in the activation of the alternative sigma factor σB. This sigma factor controls transcription of more than 150 genes involved in the general stress response. X-ray crystal structures of individual components of the stressosome and single-particle cryo-EM reconstructions of stressosome complexes, coupled with biochemical and single cell analyses, have permitted a detailed understanding of the dynamic signalling behaviour that arises from this multi-protein complex. Furthermore, bioinformatics analyses indicate that genetic modules encoding key stressosome proteins are found in a wide range of bacterial species, indicating an evolutionary advantage afforded by stressosome complexes. Interestingly, the genetic modules are associated with a variety of signalling modules encoding secondary messenger regulation systems, as well as classical two-component signal transduction systems, suggesting a diversification in function. In this chapter we review the current research into stressosome systems and discuss the functional implications of the unique structure of these signalling complexes.

Genome of Diaporthe sp. provides insights into the potential inter-phylum transfer of a fungal sesquiterpenoid biosynthetic pathway.

Fungi have highly active secondary metabolic pathways which enable them to produce a wealth of sesquiterpenoids that are bioactive. One example is Δ6-protoilludene, the precursor to the cytotoxic illudins, which are pharmaceutically relevant as anticancer therapeutics. To date, this valuable sesquiterpene has only been identified in members of the fungal division Basidiomycota. To explore the untapped potential of fungi belonging to the division Ascomycota in producing Δ6-protoilludene, we isolated a fungal endophyte Diaporthe sp. BR109 and show that it produces a diversity of terpenoids including Δ6-protoilludene. Using a genome sequencing and mining approach 17 putative novel sesquiterpene synthases were identified in Diaporthe sp. BR109. A phylogenetic approach was used to predict which gene encodes Δ6-protoilludene synthase, which was then confirmed experimentally. These analyses reveal that the sesquiterpene synthase and its putative sesquiterpene scaffold modifying cytochrome P450(s) may have been acquired by inter-phylum horizontal gene transfer from Basidiomycota to Ascomycota. Bioinformatic analyses indicate that inter-phylum transfer of these minimal sequiterpenoid secondary metabolic pathways may have occurred in other fungi. This work provides insights into the evolution of fungal sesquiterpenoid secondary metabolic pathways in the production of pharmaceutically relevant bioactive natural products.

Encapsulation of multiple cargo proteins within recombinant Eut nanocompartments.

Spatial organization via encapsulation of enzymes within recombinant nanocompartments may increase efficiency in multienzyme cascades. Previously, we reported the encapsulation of single cargo proteins within nanocompartments in the heterologous host Escherichia coli. This was achieved by coexpression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK, with a signal sequence EutC1-19 cargo protein fusion. Optimization of this system, leading to the targeting of more than one cargo protein, requires an understanding of the encapsulation mechanism. In this work, we report that the signal sequence EutC1-19 targets cargo to the interior of nanocompartments via a hydrophobic interaction with a helix on shell protein EutS. We confirm that EutC1-19 does not interact with other Eut BMC shell proteins, EutMNLK. Furthermore, we show that a second signal sequence EutE1-21 interacts specifically with the same helix on EutS. Both signal sequences appear to compete for the same EutS helix to simultaneously colocalize two cargo proteins to the interior of recombinant nanocompartments. This work offers the first insights into signal sequence-shell protein interactions required for cargo sequestration within Eut BMCs. It also provides a basis for the future engineering of Eut nanocompartments as a platform for the potential colocalization of multienzyme cascades for synthetic biology applications.

Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli.

Compartmentalization of designed metabolic pathways within protein based nanocompartments has the potential to increase reaction efficiency in multi-step biosynthetic reactions. We previously demonstrated proof-of-concept of this aim by targeting a functional enzyme to single cellular protein nanocompartments, which were formed upon recombinant expression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK in Escherichia coli. To optimize this system, increasing overall encapsulated enzyme reaction efficiency, factor(s) required for the production of more than one nanocompartment per cell must be identified. In this work we report that the cupin domain protein EutQ is required for assembly of more than one nanocompartment per cell. Overexpression of EutQ results in multiple nanocompartment assembly in our recombinant system. EutQ specifically interacts with the shell protein EutM in vitro via electrostatic interactions with the putative cytosolic face of EutM. These findings lead to the theory that EutQ could facilitate multiple nanocompartment biogenesis by serving as an assembly hub for shell proteins. This work offers insights into the biogenesis of Eut bacterial microcompartments, and also provides an improved platform for the production of protein based nanocompartments for targeted encapsulation of enzyme pathways.

Moonlighting Metals: Insights into Regulation of Cyclization Pathways in Fungal Δ(6) -Protoilludene Sesquiterpene Synthases.

Fungal 1,11 cyclizing sesquiterpene synthases are product specific under typical reaction conditions. However, in vivo expression of certain Δ(6)-protoilludene synthases results in dual 1,11 and 1,10 cyclization. To determine the factors regulating this mechanistic variation, in-depth in vitro characterization of Δ(6)-protoilludene synthases was conducted. Divalent metal ions determine cyclization specificity and this product variability. Promiscuity in metal binding is mediated by secondary metal-binding sites away from the conserved D(D/E)XX(D/E) motif in sesquiterpene synthases. Phylogenetic analysis revealed a divergent evolution of Basidiomycota trans-humulyl cation producing sesquiterpene synthases, results that indicate a wider diversity in function than previously predicted. This study provides key insights into the function and evolution of 1,11 cyclizing fungal sesquiterpene synthases.

Traversing the fungal terpenome.

Fungi (Ascomycota and Basidiomycota) are prolific producers of structurally diverse terpenoid compounds. Classes of terpenoids identified in fungi include the sesqui-, di- and triterpenoids. Biosynthetic pathways and enzymes to terpenoids from each of these classes have been described. These typically involve the scaffold generating terpene synthases and cyclases, and scaffold tailoring enzymes such as e.g. cytochrome P450 monoxygenases, NAD(P)+ and flavin dependent oxidoreductases, and various group transferases that generate the final bioactive structures. The biosynthesis of several sesquiterpenoid mycotoxins and bioactive diterpenoids has been well-studied in Ascomycota (e.g. filamentous fungi). Little is known about the terpenoid biosynthetic pathways in Basidiomycota (e.g. mushroom forming fungi), although they produce a huge diversity of terpenoid natural products. Specifically, many trans-humulyl cation derived sesquiterpenoid natural products with potent bioactivities have been isolated. Biosynthetic gene clusters responsible for the production of trans-humulyl cation derived protoilludanes, and other sesquiterpenoids, can be rapidly identified by genome sequencing and bioinformatic methods. Genome mining combined with heterologous biosynthetic pathway refactoring has the potential to facilitate discovery and production of pharmaceutically relevant fungal terpenoids.

Designer microbes for biosynthesis.

Microbes have long been adapted for the biosynthetic production of useful compounds. There is increasing demand for the rapid and cheap microbial production of diverse molecules in an industrial setting. Microbes can now be designed and engineered for a particular biosynthetic purpose, thanks to recent developments in genome sequencing, metabolic engineering, and synthetic biology. Advanced tools exist for the genetic manipulation of microbes to create novel metabolic circuits, making new products accessible. Metabolic processes can be optimized to increase yield and balance pathway flux. Progress is being made towards the design and creation of fully synthetic microbes for biosynthetic purposes. Together, these emerging technologies will facilitate the production of designer microbes for biosynthesis.

A tale of two reductases: extending the bacteriochlorophyll biosynthetic pathway in E. coli.

The creation of a synthetic microbe that can harvest energy from sunlight to drive its metabolic processes is an attractive approach to the economically viable biosynthetic production of target compounds. Our aim is to design and engineer a genetically tractable non-photosynthetic microbe to produce light-harvesting molecules. Previously we created a modular, multienzyme system for the heterologous production of intermediates of the bacteriochlorophyll (BChl) pathway in E. coli. In this report we extend this pathway to include a substrate promiscuous 8-vinyl reductase that can accept multiple intermediates of BChl biosynthesis. We present an informative comparative analysis of homologues of 8-vinyl reductase from the model photosynthetic organisms Rhodobacter sphaeroides and Chlorobaculum tepidum. The first purification of the enzymes leads to their detailed biochemical and biophysical characterization. The data obtained reveal that the two 8-vinyl reductases are substrate promiscuous, capable of reducing the C8-vinyl group of Mg protoporphyrin IX, Mg protoporphyrin IX methylester, and divinyl protochlorophyllide. However, activity is dependent upon the presence of chelated Mg(2+) in the porphyrin ring, with no activity against non-Mg(2+) chelated intermediates observed. Additionally, CD analyses reveal that the two 8-vinyl reductases appear to bind the same substrate in a different fashion. Furthermore, we discover that the different rates of reaction of the two 8-vinyl reductases both in vitro, and in vivo as part of our engineered system, results in the suitability of only one of the homologues for our BChl pathway in E. coli. Our results offer the first insights into the different functionalities of homologous 8-vinyl reductases. This study also takes us one step closer to the creation of a nonphotosynthetic microbe that is capable of harvesting energy from sunlight for the biosynthesis of molecules of choice.

Mushroom hunting by using bioinformatics: application of a predictive framework facilitates the selective identification of sesquiterpene synthases in basidiomycota.

The Basidiomycota fungi represent a diverse source of natural products, particularly the sesquiterpenoids. Recently, genome sequencing, genome mining, and the subsequent discovery of a suite of sesquiterpene synthases in Omphalotus olearius was described. A predictive framework was developed to facilitate the discovery of sesquiterpene synthases in Basidiomycota. Phylogenetic analyses indicated a conservation of both sequence and initial cyclization mechanisms used. Here, the first robust application of this predictive framework is reported. It was used to selectively identify sesquiterpene synthases that follow 1,6-, 1,10-, and 1,11-cyclization mechanisms in the crust fungus Stereum hirsutum. The successful identification and characterization of a 1,6- and a 1,10-cyclizing sesquiterpene synthase, as well as three 1,11-cyclizing Δ(6) -protoilludene synthases, is described. This study verifies the accuracy and utility of the predictive framework as a roadmap for the discovery of specific sesquiterpene synthases from Basidiomycota, and thus represents an important step forward in natural product discovery.

Eut bacterial microcompartments: insights into their function, structure, and bioengineering applications.

Bacterial microcompartments (BMCs) are protein-based polyhedral organelles which serve to encapsulate and organize enzymes involved in key metabolic pathways. The sequestration of these pathways not only improves the overall reaction efficiency; it can also harbor toxic or volatile pathway intermediates, which would otherwise be detrimental to the cell. Genomic and phylogenetic analyses reveal the presence of these unique organelles in a diverse range of bacterial species, highlighting their evolutionary importance and the essential role that they play in bacterial cell survival. Functional and structural analyses of BMCs involved in ethanolamine utilization are developing our understanding of the self-assembly and encapsulation mechanisms employed by these protein supercomplexes. This knowledge will open up exciting new avenues of research with a range of potential engineering and biotechnological applications.

Purification, crystallization and preliminary X-ray diffraction analysis of Omp6, a protoilludene synthase from Omphalotus olearius.

Basidiomycetes produce a wide range of industrially relevant natural products. One of the main classes of natural products isolated from fungi are terpenoids, a highly diverse group of secondary metabolites, many of which are bioactive and have been adapted for pharmaceutical purposes. The discovery of a suite of novel sesquiterpene synthases from Omphalotus olearius via genome sequencing and bioinformatic analyses has recently been described. Here, the expression, purification and crystallization of one of these enzymes (Omp6), a protoilludene synthase, is reported. A native crystal diffracted to a resolution of 2.9 Šand belonged to space group P21, with unit-cell parameters a = 43.67, b = 76.76, c = 107.22 Å, α = γ = 90, ß = 95°. A diffraction data set was collected on a home-source Rigaku/MSC MicroMax-007 X-ray generator.

Draft genome of Omphalotus olearius provides a predictive framework for sesquiterpenoid natural product biosynthesis in Basidiomycota.

The secondary metabolome of Basidiomycota represents a largely uncharacterized source of pharmaceutically relevant natural products. Terpenoids are the primary class of bioactive compounds isolated from mushrooms. The Jack O'Lantern mushroom Omphalotus olearius was identified 50 years ago as a prolific producer of anticancer illudin sesquiterpenoids; however, to date there have been exceptionally few studies into the biosynthesis of these important compounds. Here, we report the draft genome sequence of O. olearius, which reveals a diverse network of sesquiterpene synthases and two metabolic gene clusters associated with illudin biosynthesis. Characterization of the sesquiterpene synthases enabled a comprehensive survey of all currently available Basidiomycota genomes, thereby creating a predictive resource for terpenoid natural product biosynthesis in these organisms. Our results will facilitate discovery and biosynthetic production of unique pharmaceutically relevant bioactive compounds from Basidiomycota.

Engineered protein nano-compartments for targeted enzyme localization.

Compartmentalized co-localization of enzymes and their substrates represents an attractive approach for multi-enzymatic synthesis in engineered cells and biocatalysis. Sequestration of enzymes and substrates would greatly increase reaction efficiency while also protecting engineered host cells from potentially toxic reaction intermediates. Several bacteria form protein-based polyhedral microcompartments which sequester functionally related enzymes and regulate their access to substrates and other small metabolites. Such bacterial microcompartments may be engineered into protein-based nano-bioreactors, provided that they can be assembled in a non-native host cell, and that heterologous enzymes and substrates can be targeted into the engineered compartments. Here, we report that recombinant expression of Salmonella enterica ethanolamine utilization (eut) bacterial microcompartment shell proteins in E. coli results in the formation of polyhedral protein shells. Purified recombinant shells are morphologically similar to the native Eut microcompartments purified from S. enterica. Surprisingly, recombinant expression of only one of the shell proteins (EutS) is sufficient and necessary for creating properly delimited compartments. Co-expression with EutS also facilitates the encapsulation of EGFP fused with a putative Eut shell-targeting signal sequence. We also demonstrate the functional localization of a heterologous enzyme (ß-galactosidase) targeted to the recombinant shells. Together our results provide proof-of-concept for the engineering of protein nano-compartments for biosynthesis and biocatalysis.

The bacterial stressosome: a modular system that has been adapted to control secondary messenger signaling.

The stressosome complex regulates downstream effectors in response to environmental signals. In Bacillus subtilis, it activates the alternative sigma factor σ(B), leading to the upregulation of the general stress regulon. Herein, we characterize a stressosome-regulated biochemical pathway in Moorella thermoacetica. We show that the presumed sensor, MtR, and the scaffold, MtS, form a pseudo-icosahedral structure like that observed in B. subtilis. The N-terminal domain of MtR is structurally homologous to B. subtilis RsbR, despite low sequence identity. The affinity of the switch kinase, MtT, for MtS decreases following MtS phosphorylation and not because of structural reorganization. Dephosphorylation of MtS by the PP2C type phosphatase MtX permits the switch kinase to rebind the stressosome to reset the response. We also show that MtT regulates cyclic di-GMP biosynthesis through inhibition of a GG(D/E)EF-type diguanylate cyclase, demonstrating that secondary messenger levels are regulated by the stressosome.