Characterization of novel mevalonate kinases from the tardigrade Ramazzottius varieornatus and the psychrophilic archaeon Methanococcoides burtonii.

Mevalonate kinase is central to the isoprenoid biosynthesis pathway. Here, high-resolution X-ray crystal structures of two mevalonate kinases are presented: a eukaryotic protein from Ramazzottius varieornatus and an archaeal protein from Methanococcoides burtonii. Both enzymes possess the highly conserved motifs of the GHMP enzyme superfamily, with notable differences between the two enzymes in the N-terminal part of the structures. Biochemical characterization of the two enzymes revealed major differences in their sensitivity to geranyl pyrophosphate and farnesyl pyrophosphate, and in their thermal stabilities. This work adds to the understanding of the structural basis of enzyme inhibition and thermostability in mevalonate kinases.

Non-covalent binding tags for batch and flow biocatalysis.

Enzyme immobilization offers considerable advantage for biocatalysis in batch and continuous flow reactions. However, many currently available immobilization methods require that the surface of the carrier is chemically modified to allow site specific interactions with their cognate enzymes, which requires specific processing steps and incurs associated costs. Two carriers (cellulose and silica) were investigated here, initially using fluorescent proteins as models to study binding, followed by assessment of industrially relevant enzyme performance (transaminases and an imine reductase/glucose oxidoreductase fusion). Two previously described binding tags, the 17 amino acid long silica-binding peptide from the Bacillus cereus CotB protein and the cellulose binding domain from the Clostridium thermocellum, were fused to a range of proteins without impairing their heterologous expression. When fused to a fluorescent protein both tags conferred high avidity specific binding with their respective carriers (low nanomolar Kd values). The CotB peptide (CotB1p) induced protein aggregation in the transaminase and imine reductase/glucose oxidoreductase fusions when incubated with the silica carrier. The Clostridium thermocellum cellulose binding domain (CBDclos) allowed immobilization of all the proteins tested, but immobilization led to loss of enzymatic activity in the transaminases (< 2-fold) and imine reductase/glucose oxidoreductase fusion (> 80%). A transaminase-CBDclos fusion was then successfully used to demonstrate the application of the binding tag in repetitive batch and a continuous-flow reactor.

Production and Purification of Virus-Like Particles by Transient Expression in Plants.

Transient expression in plants has become a useful production system for virus-like particle (VLP) expression. High yields and flexible approaches to assembling complex VLPs, combine with ease of scale-up and inexpensive reagents to provide an attractive method for recombinant protein expression in general. Plants have demonstrated excellent capacity for the assembly and production of protein cages for use in vaccine design and nanotechnology. Furthermore, numerous virus structures have now been determined using plant-expressed VLPs, showing the utility of this approach in structural virology. Transient protein expression in plants uses common microbiology techniques, leading to a straightforward transformation procedure that does not result in stable transgenesis. In this chapter, we aim to provide a generic protocol for transient expression of VLPs in Nicotiana benthamiana using soil-free plant cultivation and a simple vacuum infiltration procedure, along with methodology for purifying VLPs from plant leaves.

Tunable In Vivo Colocalization of Enzymes within P22 Capsid-Based Nanoreactors.

Virus-like particles (VLPs) derived from bacteriophage P22 have been explored as biomimetic catalytic compartments. In vivo colocalization of enzymes within P22 VLPs uses sequential fusion to the scaffold protein, resulting in equimolar concentrations of enzyme monomers. However, control over enzyme stoichiometry, which has been shown to influence pathway flux, is key to realizing the full potential of P22 VLPs as artificial metabolons. We present a tunable strategy for stoichiometric control over in vivo co-encapsulation of P22 cargo proteins, verified for fluorescent protein cargo by Förster resonance energy transfer. This was then applied to a two-enzyme reaction cascade. l-homoalanine, an unnatural amino acid and chiral precursor to several drugs, can be synthesized from the readily available l-threonine by the sequential activity of threonine dehydratase and glutamate dehydrogenase. We found that the loading density of both enzymes influences their activity, with higher activity found at lower loading density implying an impact of molecular crowding on enzyme activity. Conversely, increasing overall loading density by increasing the amount of threonine dehydratase can increase activity from the rate-limiting glutamate dehydrogenase. This work demonstrates the in vivo colocalization of multiple heterologous cargo proteins in a P22-based nanoreactor and shows that controlled stoichiometry of individual enzymes in an enzymatic cascade is required for the optimal design of nanoscale biocatalytic compartments.

Current Metabolic Engineering Strategies for Photosynthetic Bioproduction in Cyanobacteria.

Cyanobacteria are photosynthetic microorganisms capable of using solar energy to convert CO2 and H2O into O2 and energy-rich organic compounds, thus enabling sustainable production of a wide range of bio-products. More and more strains of cyanobacteria are identified that show great promise as cell platforms for the generation of bioproducts. However, strain development is still required to optimize their biosynthesis and increase titers for industrial applications. This review describes the most well-known, newest and most promising strains available to the community and gives an overview of current cyanobacterial biotechnology and the latest innovative strategies used for engineering cyanobacteria. We summarize advanced synthetic biology tools for modulating gene expression and their use in metabolic pathway engineering to increase the production of value-added compounds, such as terpenoids, fatty acids and sugars, to provide a go-to source for scientists starting research in cyanobacterial metabolic engineering.

Rapid Assembly and Prototyping of Biocatalytic Virus-like Particle Nanoreactors.

Protein cages are attractive as molecular scaffolds for the fundamental study of enzymes and metabolons and for the creation of biocatalytic nanoreactors for in vitro and in vivo use. Virus-like particles (VLPs) such as those derived from the P22 bacteriophage capsid protein make versatile self-assembling protein cages and can be used to encapsulate a broad range of protein cargos. In vivo encapsulation of enzymes within VLPs requires fusion to the coat protein or a scaffold protein. However, the expression level, stability, and activity of cargo proteins can vary upon fusion. Moreover, it has been shown that molecular crowding of enzymes inside VLPs can affect their catalytic properties. Consequently, testing of numerous parameters is required for production of the most efficient nanoreactor for a given cargo enzyme. Here, we present a set of acceptor vectors that provide a quick and efficient way to build, test, and optimize cargo loading inside P22 VLPs. We prototyped the system using a yellow fluorescent protein and then applied it to mevalonate kinases (MKs), a key enzyme class in the industrially important terpene (isoprenoid) synthesis pathway. Different MKs required considerably different approaches to deliver maximal encapsulation as well as optimal kinetic parameters, demonstrating the value of being able to rapidly access a variety of encapsulation strategies. The vector system described here provides an approach to optimize cargo enzyme behavior in bespoke P22 nanoreactors. This will facilitate industrial applications as well as basic research on nanoreactor-cargo behavior.

Molecular characterization of cyanobacterial short-chain prenyltransferases and discovery of a novel GGPP phosphatase.

Cyanobacteria are photosynthetic prokaryotes with strong potential to be used for industrial terpenoid production. However, the key enzymes forming the principal terpenoid building blocks, called short-chain prenyltransferases (SPTs), are insufficiently characterized. Here, we examined SPTs in the model cyanobacteria Synechococcus elongatus sp. PCC 7942 and Synechocystis sp. PCC 6803. Each species has a single putative SPT (SeCrtE and SyCrtE, respectively). Sequence analysis identified these as type-II geranylgeranyl pyrophosphate synthases (GGPPSs) with high homology to GGPPSs found in the plastids of green plants and other photosynthetic organisms. In vitro analysis demonstrated that SyCrtE is multifunctional, producing geranylgeranyl pyrophosphate (GGPP; C20 ) primarily but also significant amounts of farnesyl pyrophosphate (FPP, C15 ) and geranyl pyrophosphate (GPP, C10 ); whereas SeCrtE appears to produce only GGPP. The crystal structures were solved to 2.02 and 1.37 Å, respectively, and the superposition of the structures against the GGPPS of Synechococcus elongatus sp. PCC 7002 yield a root mean square deviation of 0.8 Å (SeCrtE) and 1.1 Å (SyCrtE). We also discovered that SeCrtE is co-encoded in an operon with a functional GGPP phosphatase, suggesting metabolic pairing of these two activities and a putative function in tocopherol biosynthesis. This work sheds light on the activity of SPTs and terpenoid synthesis in cyanobacteria. Understanding native prenyl phosphate metabolism is an important step in developing approaches to engineering the production of different chain-length terpenoids in cyanobacteria.

Microbial Production, Extraction, and Quantitative Analysis of Isoprenoids.

Isoprenoids, also known as terpenes or terpenoids, are compounds made of one or more isoprene (C5H8) moieties and constitute the largest class of natural products. They play diverse roles in biology and have broad industrial uses as flavors, fragrances, biofuels, polymers, agricultural chemicals, and medicines. Most isoprenoids are secondary plant metabolites and only produced in very low amounts. To make these valuable compounds economically accessible, significant efforts in the culture and engineering of microbial cells for isoprenoid biosynthesis have been made in the last decades. The protocols presented here describe lab-scale cultivation of microbes, either naturally producing or engineered, for isoprenoid production, the extraction of products and their quantification by high-performance liquid chromatography. Examples of isoprenoids covered in this chapter include (C10) mono-, (C15) sesqui-, (C20) di-, (C30) tri-, and (C40) tetraterpenoids. We focus on yeast and cyanobacteria as production systems, but the protocols can be adapted for other organisms.

An in vivo gene amplification system for high level expression in Saccharomyces cerevisiae.

Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L-1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.

The structure of a plant-specific partitivirus capsid reveals a unique coat protein domain architecture with an intrinsically disordered protrusion.

Persistent plant viruses may be the most common viruses in wild plants. A growing body of evidence for mutualism between such viruses and their hosts, suggests that they play an important role in ecology and agriculture. Here we present the capsid structure of a plant-specific partitivirus, Pepper cryptic virus 1, at 2.9 Å resolution by Cryo-EM. Structural features, including the T = 1 arrangement of 60 coat protein dimers, are shared with fungal partitiviruses and the picobirnavirus lineage of dsRNA viruses. However, the topology of the capsid is markedly different with protrusions emanating from, and partly comprising, the binding interface of coat protein dimers. We show that a disordered region at the apex of the protrusion is not required for capsid assembly and represents a hypervariable site unique to, and characteristic of, the plant-specific partitiviruses. These results suggest a structural basis for the acquisition of additional functions by partitivirus coat proteins that enables mutualistic relationships with diverse plant hosts.

Bacterial catabolism of s-triazine herbicides: biochemistry, evolution and application.

The synthetic s-triazines are abundant, nitrogen-rich, heteroaromatic compounds used in a multitude of applications including, herbicides, plastics and polymers, and explosives. Their presence in the environment has led to the evolution of bacterial catabolic pathways in bacteria that allow use of these anthropogenic chemicals as a nitrogen source that supports growth. Herbicidal s-triazines have been used since the mid-twentieth century and are among the most heavily used herbicides in the world, despite being withdrawn from use in some areas due to concern about their safety and environmental impact. Bacterial catabolism of the herbicidal s-triazines has been studied extensively. Pseudomonas sp. strain ADP, which was isolated more than thirty years after the introduction of the s-triazine herbicides, has been the model system for most of these studies; however, several alternative catabolic pathways have also been identified. Over the last five years, considerable detail about the molecular mode of action of the s-triazine catabolic enzymes has been uncovered through acquisition of their atomic structures. These structural studies have also revealed insights into the evolutionary origins of this newly acquired metabolic capability. In addition, s-triazine-catabolizing bacteria and enzymes have been used in a range of applications, including bioremediation of herbicides and cyanuric acid, introducing metabolic resistance to plants, and as a novel selectable marker in fermentation organisms. In this review, we cover the discovery and characterization of bacterial strains, metabolic pathways and enzymes that catabolize the s-triazines. We also consider the evolution of these new enzymes and pathways and discuss the practical applications that have been considered for these bacteria and enzymes. One Sentence Summary: A detailed understanding of bacterial herbicide catabolic enzymes and pathways offer new evolutionary insights and novel applied tools.

The evolving story of AtzT, a periplasmic binding protein.

Atrazine is an s-triazine-based herbicide that is used in many countries around the world in many millions of tons per year. A small number of organisms, such as Pseudomonas sp. strain ADP, have evolved to use this modified s-triazine as a food source, and the various genes required to metabolize atrazine can be found on a single plasmid. The atomic structures of seven of the eight proteins involved in the breakdown of atrazine by Pseudomonas sp. strain ADP have been determined by X-ray crystallography, but the structures of the proteins required by the cell to import atrazine for use as an energy source are still lacking. The structure of AtzT, a periplasmic binding protein that may be involved in the transport of a derivative of atrazine, 2-hydroxyatrazine, into the cell for mineralization, has now been determined. The structure was determined by SAD phasing using an ethylmercury phosphate derivative that diffracted X-rays to beyond 1.9 Šresolution. `Native' (guanine-bound) and 2-hydroxyatrazine-bound structures were also determined to high resolution (1.67 and 1.65 Å, respectively), showing that 2-hydroxyatrazine binds in a similar way to the purportedly native ligand. Structural similarities led to the belief that it may be possible to evolve AtzT from a purine-binding protein to a protein that can bind and detect atrazine in the environment.

A novel decarboxylating amidohydrolase involved in avoiding metabolic dead ends during cyanuric acid catabolism in Pseudomonas sp. strain ADP.

Cyanuric acid is a common environmental contaminant and a metabolic intermediate in the catabolism of s-triazine compounds, including atrazine and other herbicides. Cyanuric acid is catabolized via a number of bacterial pathways, including one first identified in Pseudomonas sp. strain ADP, which is encoded by a single, five-gene operon (atzDGEHF) found on a self-transmissible plasmid. The discovery of two of the five genes (atzG and atzH) was reported in 2018 and although the function of atzG was determined, the role of atzH was unclear. Here, we present the first in vitro reconstruction of the complete, five-protein cyanuric acid catabolism pathway, which indicates that AtzH may be an amidase responsible for converting 1,3-dicarboxyurea (the AtzE product) to allophanate (the AtzF substrate). We have solved the AtzH structure (a DUF3225 protein from the NTF2 superfamily) and used it to predict the substrate-binding pocket. Site-directed mutagenesis experiments suggest that two residues (Tyr22 and Arg46) are needed for catalysis. We also show that atzH homologs are commonly found in Proteobacteria associated with homologs of the atzG and atzE genes. The genetic context of these atzG-atzE-atzH clusters imply that they have a role in the catabolism of nitrogenous compounds. Moreover, their presence in many genomes in the absence of homologs of atzD and atzF suggests that the atzG-atzE-atzH cluster may pre-date the evolution of the cyanuric acid catabolism operon.

An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme.

Cyanuric acid is a metabolic intermediate of s-triazines, such as atrazine (a common herbicide) and melamine (used in resins and plastics). Cyanuric acid is mineralized to ammonia and carbon dioxide by the soil bacterium Pseudomonas sp. strain ADP via three hydrolytic enzymes (AtzD, AtzE, and AtzF). Here, we report the purification and biochemical and structural characterization of AtzE. Contrary to previous reports, we found that AtzE is not a biuret amidohydrolase, but instead it catalyzes the hydrolytic deamination of 1-carboxybiuret. X-ray crystal structures of apo AtzE and AtzE bound with the suicide inhibitor phenyl phosphorodiamidate revealed that the AtzE enzyme complex consists of two independent molecules in the asymmetric unit. We also show that AtzE forms an α2ß2 heterotetramer with a previously unidentified 68-amino acid-long protein (AtzG) encoded in the cyanuric acid mineralization operon from Pseudomonas sp. strain ADP. Moreover, we observed that AtzG is essential for the production of soluble, active AtzE and that this obligate interaction is a vestige of their shared evolutionary origin. We propose that AtzEG was likely recruited into the cyanuric acid-mineralizing pathway from an ancestral glutamine transamidosome that required protein-protein interactions to enforce the exclusion of solvent from the transamidation reaction.

Structural and biochemical characterization of the biuret hydrolase (BiuH) from the cyanuric acid catabolism pathway of Rhizobium leguminasorum bv. viciae 3841.

Biuret deamination is an essential step in cyanuric acid mineralization. In the well-studied atrazine degrading bacterium Pseudomonas sp. strain ADP, the amidase AtzE catalyzes this step. However, Rhizobium leguminosarum bv. viciae 3841 uses an unrelated cysteine hydrolase, BiuH, instead. Herein, structures of BiuH, BiuH with bound inhibitor and variants of BiuH are reported. The substrate is bound in the active site by a hydrogen bonding network that imparts high substrate specificity. The structure of the inactive Cys175Ser BiuH variant with substrate bound in the active site revealed that an active site cysteine (Cys175), aspartic acid (Asp36) and lysine (Lys142) form a catalytic triad, which is consistent with biochemical studies of BiuH variants. Finally, molecular dynamics simulations highlighted the presence of three channels from the active site to the enzyme surface: a persistent tunnel gated by residues Val218 and Gln215 forming a potential substrate channel and two smaller channels formed by Val28 and a mobile loop (including residues Phe41, Tyr47 and Met51) that may serve as channels for co-product (ammonia) or co-substrate (water).