Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.     

酿酒酵母高效合成萜类化合物的组合调控策略

萜类化合物具有广泛的生理活性与重要的经济价值,利用酿酒酵母进行萜类合成具有低价、高效等优势。然而部分植物源合成萜类的关键酶在酿酒酵母中难表达、产量低,难以工业应用,因此有效的调控策略显得至关重要。本文从萜类化合物在酿酒酵母中的合成途径入手,介绍了关键酶、代谢途径、CRISPR基因编辑系统和人工合成染色体技术4个方面的调控策略在酿酒酵母合成萜类化合物中的应用。阐述了关键酶的筛选、改造,理性与非理性设计,MVA途径、乙酰辅酶A合成途径与亚细胞结构的代谢途径改造的优势。指出了多重调控策略组合调控的方式是实现酿酒酵母高效合成萜类化合物的有效方法。此外,CRISPR基因编辑系统与人工合成染色体技术的快速发展将为酿酒酵母细胞工厂的深入开发与利用提供有力工具。     

Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation

Monoterpenoids are industrially important natural products with applications in the flavours, fragrances, fuels and pharmaceutical industries. Most monoterpenoids are produced by plants, but recently two bacterial monoterpene synthases have been identified, including a cineole synthase (bCinS). Unlike plant cineole synthases, bCinS is capable of producing nearly pure cineole from geranyl diphosphate in a complex cyclisation cascade that is tightly controlled. Here we have used a multidisciplinary approach to show that Asn305 controls water attack on the α-terpinyl cation and subsequent cyclisation and deprotonation of the α-terpineol intermediate, key steps in the cyclisation cascade which direct product formation towards cineole. Mutation of Asn305 results in variants that no longer produce α-terpineol or cineole. Molecular dynamics simulations revealed that water coordination is disrupted in all variants tested. Quantum mechanics calculations indicate that Asn305 is most likely a (transient) proton acceptor for the final deprotonation step. Our synergistic approach gives unique insight into how a single residue, Asn305, tames the promiscuous chemistry of monoterpene synthase cyclisation cascades. It does this by tightly controlling the final steps in cineole formation catalysed by bCinS to form a single hydroxylated monoterpene product.     

Structural Insights into the Interaction of Heme with Protein Tyrosine Kinase JAK2**

Janus kinase 2 (JAK2) is the most important signal-transducing tyrosine kinase in erythropoietic precursor cells. Its malfunction drives several myeloproliferative disorders. Heme is a small metal-ion-carrying molecule that is incorporated into hemoglobin in erythroid precursor cells to transport oxygen. In addition, heme is a signaling molecule and regulator of various biochemical processes. Here, we show that heme exposure leads to hyperphosphorylation of JAK2 in a myeloid cancer cell line. Two peptides identified in JAK2 are heme-regulatory motifs and show low-micromolar affinities for heme. These peptides map to the kinase domain of JAK2, which is essential for downstream signaling. We suggest these motifs to be the interaction sites of heme with JAK2, which drive the heme-induced hyperphosphorylation. The results presented herein could facilitate the development of heme-related pharmacological tools to combat myeloproliferative disorders.     

A Versatile Strategy for the Semisynthetic Production of Ser65 Phosphorylated Ubiquitin and Its Biochemical and Structural Characterisation

Ubiquitin phosphorylation is emerging as an important regulatory layer in the ubiquitin system. This is exemplified by the phosphorylation of ubiquitin on Ser65 by the Parkinson's disease‐associated kinase PINK1, which mediates the activation of the E3 ligase Parkin. Additional phosphorylation sites on ubiquitin might also have important cellular roles. Here we report a versatile strategy for preparing phosphorylated ubiquitin. We biochemically and structurally characterise semisynthetic phospho‐Ser65‐ubiquitin. Unexpectedly, we observed disulfide bond formation between ubiquitin molecules, and hence a novel crystal form. The method outlined provides a direct approach to study the combinatorial effects of phosphorylation on ubiquitin function. Our analysis also suggests that disulfide engineering of ubiquitin could be a useful strategy for obtaining alternative crystal forms of ubiquitin species thereby facilitating structural validation.     

Alkylation of Staurosporine to Derive a Kinase Probe for Fluorescence Applications

The natural product staurosporine is a high‐affinity inhibitor of nearly all mammalian protein kinases. The labelling of staurosporine has proven effective as a means of generating protein kinase research tools. Most tools have been generated by acylation of the 4′‐methylamine of the sugar moiety of staurosporine. Herein we describe the alkylation of this group as a first step to generate a fluorescently labelled staurosporine. Following alkylation, a polyethylene glycol linker was installed, allowing subsequent attachment of fluorescein. We report that this fluorescein–staurosporine conjugate binds to cAMP‐dependent protein kinase in the nanomolar range. Furthermore, its binding can be antagonised with unmodified staurosporine as well as ATP, indicating it targets the ATP binding site in a similar fashion to native staurosporine. This reagent has potential application as a screening tool for protein kinases of interest.     

The Chloroplast of Chlamydomonas reinhardtii as a Testbed for Engineering Nitrogen Fixation into Plants

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N₂) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N₂ by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme’s sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle’s genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an ‘Fe-only’ nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops.     

Reexamination of the Ergothioneine Biosynthetic Methyltransferase EgtD from Mycobacterium tuberculosis as a Protein Kinase Substrate

Ergothioneine has emerged as a crucial cytoprotectant in the pathogenic lifestyle of Mycobacterium tuberculosis. Production of this antioxidant from primary metabolites may be regulated by phosphorylation of Thr213 in the active site of the methyltransferase EgtD. The structure of mycobacterial EgtD suggests that this post-translational modification would require a large-scale change in conformation to make the active-site residue accessible to a protein kinase. In this report, we show that, under in vitro conditions, EgtD is not a substrate of protein kinase PknD.     

Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade

Dimethylallyl diphosphate (DMAPP) is a key intermediate metabolite in the synthesis of isoprenoids and is also the prenyl donor for biosynthesizing prenylated flavonoids. However, it is difficult to prepare DMAPP via chemical and enzymatic methods. In this study, three promiscuous kinases from Shigella flexneri (SfPK), Escherichia coli (EcPK), and Saccharomyces cerevisiae (ScPK) and three isopentenyl phosphate kinases from Methanolobus tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and Arabidopsis thaliana (AtIPK) were cloned and expressed in Escherichia coli. The enzymatic properties of recombinant enzymes were determined. The Kcat/Km value of SfPK for DMA was 6875 s⁻¹ M⁻¹, which was significantly higher than those of EcPK and ScPK. The Kcat/Km value of MtIPK for DMAP was 402.9 s⁻¹ M⁻¹, which was ~400% of that of MthIPK. SfPK was stable at pH 7.0–9.5 and had a 1 h half-life at 65 °C. MtIPK was stable at pH 6.0–8.5 and had a 1 h half-life at 50 °C. The stability of SfPK and MtIPK was better than that of the other enzymes. Thus, SfPK and MtIPK were chosen to develop a one-pot enzymatic cascade for producing DMAPP from DMA because of their catalytic efficiency and stability. The optimal ratio between SfPK and MtIPK was 1:8. The optimal pH and temperature for the one-pot enzymatic cascade were 7.0 and 35 °C, respectively. The optimal concentrations of ATP and DMA were 10 and 80 mM, respectively. Finally, maximum DMAPP production reached 1.23 mM at 1 h under optimal conditions. Therefore, the enzymatic method described herein for the biosynthesis of DMAPP from DMA can be widely used for the synthesis of isoprenoids and prenylated flavonoids.     

Bacterial Cell Display as a Robust and Versatile Platform for Engineering Low-Affinity Ligands and Enzymes

Directed evolution has been remarkably successful at expanding the chemical and functional boundaries of biology. That progress is heavily dependent on the robustness and flexibility of the available selection platforms, given the significant cost to (re)develop a given platform to target a new desired function. Bacterial cell display has a significant track record as a viable strategy for the engineering of mesophilic enzymes, as enzyme activity can be probed directly and free from interference from the cellular milieu, but its adoption has lagged behind other display-based methods. Herein, we report the development of SNAP as a quantitative reporter for bacterial cell display, which enables fast troubleshooting and the systematic development of the display-based selection platform, thus improving its robustness. In addition, we demonstrate that even weak interactions between displayed proteins and nucleic acids can be harnessed for the specific labelling of bacterial cells, allowing functional characterisation of DNA binding proteins and enzymes, thus making it a highly flexible platform for these biochemical functions. Together, this establishes bacterial display as a robust and flexible platform, ideally suited for the systematic engineering of ligands and enzymes needed for XNA molecular biology.     

Optimization of the Inverted Emulsion Method for High-Yield Production of Biomimetic Giant Unilamellar Vesicles

In the field of bottom-up synthetic biology, lipid vesicles provide an important role in the construction of artificial cells. Giant unilamellar vesicles (GUVs), due to their membrane's similarity to natural biomembranes, have been widely used as cellular mimics. So far, several methods exist for the production of GUVs with the possibility to encapsulate biological macromolecules. The inverted emulsion-based method is one such technique, which has great potential for rapid production of GUVs with high encapsulation efficiencies for large biomolecules. However, the lack of understanding of various parameters that affect production yields has resulted in sparse adaptation within the membrane and bottom-up synthetic biology research communities. Here, we optimize various parameters of the inverted emulsion-based method to maximize the production of GUVs. We demonstrate that the density difference between the emulsion droplets, oil phase, and the outer aqueous phase plays a crucial role in vesicle formation. We also investigated the impact that centrifugation speed/time, lipid concentration, pH, temperature, and emulsion droplet volume has on vesicle yield and size. Compared to conventional electroformation, our preparation method was not found to significantly alter the membrane mechanical properties. Finally, we optimize the parameters to minimize the time from workbench to microscope and in this way open up the possibility of time-sensitive experiments. In conclusion, our findings will promote the usage of the inverted emulsion method for basic membrane biophysics studies as well as the development of GUVs for use as future artificial cells.     

Compendium on Food Crop Plants as a Platform for Pharmaceutical Protein Production

Tremendous advances in crop biotechnology related to the availability of molecular tools and methods developed for transformation and regeneration of specific plant species have been observed. As a consequence, the interest in plant molecular farming aimed at producing the desired therapeutic proteins has significantly increased. Since the middle of the 1980s, recombinant pharmaceuticals have transformed the treatment of many serious diseases and nowadays are used in all branches of medicine. The available systems of the synthesis include wild-type or modified mammalian cells, plants or plant cell cultures, insects, yeast, fungi, or bacteria. Undeniable benefits such as well-characterised breeding conditions, safety, and relatively low costs of production make plants an attractive yet competitive platform for biopharmaceutical production. Some of the vegetable plants that have edible tubers, fruits, leaves, or seeds may be desirable as inexpensive bioreactors because these organs can provide edible vaccines and thus omit the purification step of the final product. Some crucial facts in the development of plant-made pharmaceuticals are presented here in brief. Although crop systems do not require more strictly dedicated optimization of methodologies at any stages of the of biopharmaceutical production process, here we recall the complete framework of such a project, along with theoretical background. Thus, a brief review of the advantages and disadvantages of different systems, the principles for the selection of cis elements for the expression cassettes, and available methods of plant transformation, through to the protein recovery and purification stage, are all presented here. We also outline the achievements in the production of biopharmaceuticals in economically important crop plants and provide examples of their clinical trials and commercialization.     

Increased promiscuity of human galactokinase following alteration of a single amino acid residue distant from the active site

Galactokinase catalyses the site- and stereospecific phosphorylation of galactose at the expense of ATP. The specificity of bacterial galactokinase enzymes can be broadened by alteration of a tyrosine residue to a histidine. The effects of altering the equivalent residue in human galactokinase (Tyr379) were investigated by testing all 19 possible variants. All of these alterations, except Y379P, resulted in soluble protein on expression in Escherichia coli and all the soluble variants could catalyse the phosphorylation of galactose, except Y379A and Y379E. The variants Y379C, Y379K, Y379R, Y379S and Y379W were all able to catalyse the phosphorylation of a variety of monosaccharides, including ones that are not acted on by the wild-type enzyme. Novel substrates for these variant galactokinases included D-mannose and D-fructose. The latter monosaccharide is presumed to react in the pyranose configuration. Molecular modelling suggested that the alterations do not cause changes to the overall structure of the enzyme. However, alteration of Tyr379 increases the flexibility of the peptide backbone in regions surrounding the active site. Therefore, it is proposed that alteration of Tyr379 affects the substrate specificity by the propagation of changes in flexibility to the active site, permitting a broader range of compounds to be accommodated.     

Exploring the Substrate Promiscuity of Drug‐Modifying Enzymes for the Chemoenzymatic Generation of N‐Acylated Aminoglycosides

Aminoglycosides are broad‐spectrum antibiotics commonly used for the treatment of serious bacterial infections. Decades of clinical use have led to the widespread emergence of bacterial resistance to this family of drugs limiting their efficacy in the clinic. Here, we report the development of a methodology that utilizes aminoglycoside acetyltransferases (AACs) and unnatural acyl coenzyme A analogues for the chemoenzymatic generation of N‐acylated aminoglycoside analogues. Generation of N‐acylated aminoglycosides is followed by a simple qualitative test to assess their potency as potential antibacterials. The studied AACs (AAC(6′)‐APH(2′′) and AAC(3)‐IV) show diverse substrate promiscuity towards a variety of aminoglycosides as well as acyl coenzyme A derivatives. The enzymes were also used for the sequential generation of homo‐ and hetero‐di‐N‐acylated aminoglycosides. Following the clinical success of the N‐acylated amikacin and arbekacin, our chemoenzymatic approach offers access to regioselectively N‐acylated aminoglycosides in quantities that allow testing of the antibacterial potential of the synthetic analogues making it possible to decide which molecules will be worth synthesizing on a larger scale.     

Harnessing Enzymatic Promiscuity in Myxochelin Biosynthesis for the Production of 5‐Lipoxygenase Inhibitors

The siderophore myxochelin A is a potent inhibitor of human 5‐lipoxygenase (5‐LO). To clarify whether the iron‐chelating properties of myxochelin A are responsible for this activity, several analogues of this compound were generated in the native producer Pyxidicoccus fallax by precursor‐directed biosynthesis. Testing in a cell‐free assay unveiled three derivatives with bioactivity comparable with that of myxochelin A. Furthermore, it became evident that inhibition of 5‐LO by myxochelins does not correlate with their iron affinities.     

Structural Studies on the Reaction of Isopenicillin N Synthase with a Sterically Demanding Depsipeptide Substrate Analogue

Isopenicillin N synthase (IPNS) is a nonheme iron(II)‐dependent oxidase that catalyses the central step in penicillin biosynthesis, conversion of the tripeptide δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐valine (ACV) to isopenicillin N (IPN). This report describes mechanistic studies using the analogue δ‐(L ‐α‐aminoadipoyl)‐(3S‐methyl)‐L ‐cysteine D ‐α‐hydroxyisovaleryl ester (A^SmCOV), designed to intercept the catalytic cycle at an early stage. A^SmCOV incorporates two modifications from the natural substrate: the second and third residues are joined by an ester, so this analogue lacks the key amide of ACV and cannot form a β‐lactam; and the cysteinyl residue is substituted at its β‐carbon, bearing a (3S)‐methyl group. It was anticipated that this methyl group will impinge directly on the site in which the co‐substrate dioxygen binds. The novel depsipeptide A^SmCOV was prepared in 13 steps and crystallised with IPNS anaerobically. The 1.65 Å structure of the IPNS–Fe^II–A^SmCOV complex reveals that the additional β‐methyl group is not oriented directly into the oxygen binding site, but does increase steric demand in the active site and increases disorder in the position of the isovaleryl side chain. Crystals of IPNS–Fe^II–A^SmCOV were incubated with high‐pressure oxygen gas, driving substrate turnover to a single product, an ene‐thiol/C‐hydroxylated depsipeptide. A mechanism is proposed for the reaction of A^SmCOV with IPNS, linking this result to previous crystallographic studies with related depsipeptides and solution‐phase experiments with cysteine‐methylated tripeptides. This result demonstrates that a (3S)‐methyl group at the substrate cysteinyl β‐carbon is not in itself a block to IPNS activity as previously proposed, and sheds further light on the steric complexities of IPNS catalysis.     

The Quest for Xenobiotic Enzymes: From New Enzymes for Chemistry to a Novel Chemistry of Life

Enzyme engineering has made impressive progress in the past decades, paving the way for the widespread use of enzymes for various purposes. In contrast to “classical” enzyme engineering, which focuses on optimizing specific properties of natural enzymes, a more recent trend towards the creation of artificial enzymes that catalyze fundamentally distinct, new-to-nature reactions is observable. While approaches for creating such enzymes differ significantly, they share the common goal of enabling biocatalytic novelty to broaden the range of applications for enzymes. Although most artificial enzymes reported to date are only moderately active and barely function in vivo, they have the potential to endow cells with capabilities that were previously out of reach and thus herald a new wave of “functional xenobiology”. Herein, we highlight recent developments in the field of artificial enzymes with a particular focus on challenges and opportunities for their use in xenobiology.     

Compartments for Synthetic Cells: Osmotically Assisted Separation of Oil from Double Emulsions in a Microfluidic Chip

Liposomes are used in synthetic biology as cell-like compartments and their microfluidic production through double emulsions allows for efficient encapsulation of various components. However, residual oil in the membrane remains a critical bottleneck for creating pristine phospholipid bilayers. It has been discovered that osmotically driven shrinking leads to detachment of the oil drop. Separation inside a microfluidic chip has been realized to automate the procedure, which allows for controlled continuous production of monodisperse liposomes.