Proteolysis inside the membrane is a rate-governed reaction not driven by substrate affinity.

Enzymatic cleavage of transmembrane anchors to release proteins from the membrane controls diverse signaling pathways and is implicated in more than a dozen diseases. How catalysis works within the viscous, water-excluding, two-dimensional membrane is unknown. We developed an inducible reconstitution system to interrogate rhomboid proteolysis quantitatively within the membrane in real time. Remarkably, rhomboid proteases displayed no physiological affinity for substrates (K(d) ~190 µM/0.1 mol%). Instead, ~10,000-fold differences in proteolytic efficiency with substrate mutants and diverse rhomboid proteases were reflected in k(cat) values alone. Analysis of gate-open mutant and solvent isotope effects revealed that substrate gating, not hydrolysis, is rate limiting. Ultimately, a single proteolytic event within the membrane normally takes minutes. Rhomboid intramembrane proteolysis is thus a slow, kinetically controlled reaction not driven by transmembrane protein-protein affinity. These properties are unlike those of other studied proteases or membrane proteins but are strikingly reminiscent of one subset of DNA-repair enzymes, raising important mechanistic and drug-design implications.

Polyethylene Glycol Impacts Conformation and Dynamics of Escherichia coli Prolyl-tRNA Synthetase Via Crowding and Confinement Effects.

Polyethylene glycol (PEG) is a flexible, nontoxic polymer commonly used in biological and medical research, and it is generally regarded as biologically inert. PEG molecules of variable sizes are also used as crowding agents to mimic intracellular environments. A recent study with PEG crowders revealed decreased catalytic activity of Escherichia coli prolyl-tRNA synthetase (Ec ProRS), where the smaller molecular weight PEGs had the maximum impact. The molecular mechanism of the crowding effects of PEGs is not clearly understood. PEG may impact protein conformation and dynamics, thus its function. In the present study, the effects of PEG molecules of various molecular weights and concentrations on the conformation and dynamics of Ec ProRS were investigated using a combined experimental and computational approach including intrinsic tryptophan fluorescence spectroscopy, atomic force microscopy, and atomistic molecular dynamic simulations. Results of the present study suggest that lower molecular weight PEGs in the dilute regime have modest effects on the conformational dynamics of Ec ProRS but impact the catalytic function primarily via the excluded volume effect; they form large clusters blocking the active site pocket. In contrast, the larger molecular weight PEGs in dilute to semidilute regimes have a significant impact on the protein's conformational dynamics; they wrap on the protein surface through noncovalent interactions. Thus, lower-molecular-weight PEG molecules impact protein dynamics and function via crowding effects, whereas larger PEGs induce confinement effects. These results have implications for the development of inhibitors for protein targets in a crowded cellular environment.

Production of Tyrian purple indigoid dye from tryptophan in Escherichia coli.

Tyrian purple, mainly composed of 6,6'-dibromoindigo (6BrIG), is an ancient dye extracted from sea snails and was recently demonstrated as a biocompatible semiconductor material. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and the difficulty of regiospecific bromination. Here, we introduce an effective 6BrIG production strategy in Escherichia coli using tryptophan 6-halogenase SttH, tryptophanase TnaA and flavin-containing monooxygenase MaFMO. Since tryptophan halogenases are expressed in highly insoluble forms in E. coli, a flavin reductase (Fre) that regenerates FADH2 for the halogenase reaction was used as an N-terminal soluble tag of SttH. A consecutive two-cell reaction system was designed to overproduce regiospecifically brominated precursors of 6BrIG by spatiotemporal separation of bromination and bromotryptophan degradation. These approaches led to 315.0 mg l-1 6BrIG production from tryptophan and successful synthesis of regiospecifically dihalogenated indigos. Furthermore, it was demonstrated that 6BrIG overproducing cells can be directly used as a bacterial dye.

RNA polymerase clamp conformational dynamics: long-lived states and modulation by crowding, cations, and nonspecific DNA binding.

The RNA polymerase (RNAP) clamp, a mobile structural element conserved in RNAP from all domains of life, has been proposed to play critical roles at different stages of transcription. In previous work, we demonstrated using single-molecule Förster resonance energy transfer (smFRET) that RNAP clamp interconvert between three short-lived conformational states (lifetimes ∼ 0.3-0.6 s), that the clamp can be locked into any one of these states by small molecules, and that the clamp stays closed during initial transcription and elongation. Here, we extend these studies to obtain a comprehensive understanding of clamp dynamics under conditions RNAP may encounter in living cells. We find that the RNAP clamp can populate long-lived conformational states (lifetimes > 1.0 s) and can switch between these long-lived states and the previously observed short-lived states. In addition, we find that clamp motions are increased in the presence of molecular crowding, are unchanged in the presence of elevated monovalent-cation concentrations, and are reduced in the presence of elevated divalent-cation concentrations. Finally, we find that RNAP bound to non-specific DNA predominantly exhibits a closed clamp conformation. Our results raise the possibility of additional regulatory checkpoints that could affect clamp dynamics and consequently could affect transcription and transcriptional regulation.

Constructing artificial respiratory chain in polymer compartments: Insights into the interplay between bo3 oxidase and the membrane.

Cytochrome bo3 ubiquinol oxidase is a transmembrane protein, which oxidizes ubiquinone and reduces oxygen, while pumping protons. Apart from its combination with F1Fo-ATPase to assemble a minimal ATP regeneration module, the utility of the proton pump can be extended to other applications in the context of synthetic cells such as transport, signaling, and control of enzymatic reactions. In parallel, polymers have been speculated to be phospholipid mimics with respect to their ability to self-assemble in compartments with increased stability. However, their usability as interfaces for complex membrane proteins has remained questionable. In the present work, we optimized a fusion/electroformation approach to reconstitute bo3 oxidase in giant unilamellar vesicles made of PDMS-g-PEO and/or phosphatidylcholine (PC). This enabled optical access, while microfluidic trapping allowed for online analysis of individual vesicles. The tight polymer membranes and the inward oriented enzyme caused 1 pH unit difference in 30 min, with an initial rate of 0.35 pH·min-1 To understand the interplay in these composite systems, we studied the relevant mechanical and rheological membrane properties. Remarkably, the proton permeability of polymer/lipid hybrids decreased after protein insertion, while the latter also led to a 20% increase of the polymer diffusion coefficient in polymersomes. In addition, PDMS-g-PEO increased the activity lifetime and the resistance to free radicals. These advantageous properties may open diverse applications, ranging from cell-free biotechnology to biomedicine. Furthermore, the presented study serves as a comprehensive road map for studying the interactions between membrane proteins and synthetic membranes, which will be fundamental for the successful engineering of such hybrid systems.

Wzb of Vibrio vulnificus represents a new group of low-molecular-weight protein tyrosine phosphatases with a unique insertion in the W-loop.

Protein tyrosine phosphorylation regulates the production of capsular polysaccharide, an essential virulence factor of the deadly pathogen Vibrio vulnificus. The process requires the protein tyrosine kinase Wzc and its cognate phosphatase Wzb, both of which are largely uncharacterized. Herein, we report the structures of Wzb of V. vulnificus (VvWzb) in free and ligand-bound forms. VvWzb belongs to the low-molecular-weight protein tyrosine phosphatase (LMWPTP) family. Interestingly, it contains an extra four-residue insertion in the W-loop, distinct from all known LMWPTPs. The W-loop of VvWzb protrudes from the protein body in the free structure, but undergoes significant conformational changes to fold toward the active site upon ligand binding. Deleting the four-residue insertion from the W-loop severely impaired the enzymatic activity of VvWzb, indicating its importance for optimal catalysis. However, mutating individual residues or even substituting the whole insertion with four alanine residues only modestly decreased the enzymatic activity, suggesting that the contribution of the insertion to catalysis is not determined by the sequence specificity. Furthermore, inserting the four residues into Escherichia coli Wzb at the corresponding position enhanced its activity as well, indicating that the four-residue insertion in the W-loop can act as a general activity enhancing element for other LMWPTPs. The novel W-loop type and phylogenetic analysis suggested that VvWzb and its homologs should be classified into a new group of LMWPTPs. Our study sheds new insight into the catalytic mechanism and structural diversity of the LMWPTP family and promotes the understanding of the protein tyrosine phosphorylation system in prokaryotes.

Escherichia coli tRNA 2-selenouridine synthase SelU selects its prenyl substrate to accomplish its enzymatic function.

Bacterial tRNA 2-selenouridine synthase (SelU) in vitro converts S2U-RNA to its selenium analog (Se2U-RNA) in a two-step process: (i) geranylation of S2U-RNA (with geranyl pyrophosphate, gePP), and (ii) selenation of the resulting geS2U-RNA (with the selenophosphate anion, SePO33-). Using an S2U-containing anticodon stem-loop fragment derived from tRNALys (S2U-RNA) and recombinant SelU with an MBP tag, we found that only geranyl (C10) pyrophosphate is the substrate for this enzyme, while other pyrophosphates such as isopentenyl (C5), dimethylallyl (C5), farnesyl (C15) and geranylgeranyl (C20) are not. Interestingly, methyl (C1)- and C5-, C10-, and C15-prenyl-containing S2U-RNAs (which were chemically obtained) underwent the selenation reaction promoted by SelU, although the Se2U-RNA product was obtained in decreasing yields in the following order: geranyl ≥ farnesyl > dimethylallyl ≫ methyl. Microscale thermophoresis showed an affinity between gePP and SelU in the micromolar range, while the other pyrophosphates tested, such as isopentenyl, dimethylallyl, farnesyl and geranylgeranyl, either did not bind to the protein or their binding affinity was above 1 mM. These results agree well with the in silico analysis, with gePP being the best binding substrate (the lowest relative free energy of binding (ΔG) and a small solvent-accessible surface area (SASA)). These results suggest that SelU has high substrate specificity for the prenylation reaction (only gePP is accepted), whereas there is little discrimination for the selenation reaction. We therefore suggest that only gePP and the geranylated tRNA serve as substrates for the conversion of 2-thio-tRNAs to 2-seleno-tRNAs, as it is found in the bacterial system.

Kinetic Analysis of Transient Intermediates in the Mechanism of Prenyl-Flavin-Dependent Ferulic Acid Decarboxylase.

Ferulic acid decarboxylase catalyzes the decarboxylation of various substituted phenylacrylic acids to their corresponding styrene derivatives and CO2 using the recently discovered cofactor prenylated FMN (prFMN). The mechanism involves an unusual 1,3-dipolar cycloaddition reaction between prFMN and the substrate to generate a cycloadduct capable of undergoing decarboxylation. Using native mass spectrometry, we show the enzyme forms a stable prFMN-styrene cycloadduct that accumulates on the enzyme during turnover. Pre-steady state kinetic analysis of the reaction using ultraviolet-visible stopped-flow spectroscopy reveals a complex pattern of kinetic behavior, best described by a half-of-sites model involving negative cooperativity between the two subunits of the dimeric enzyme. For the reactive site, the cycloadduct of prFMN with phenylacylic acid is formed with a kapp of 131 s-1. This intermediate converts to the prFMN-styrene cycloadduct with a kapp of 75 s-1. Cycloelimination of the prFMN-styrene cycloadduct to generate styrene and free enzyme appears to determine kcat for the overall reaction, which is 11.3 s-1.

A Mechanistic Basis for Phosphoethanolamine Modification of the Cellulose Biofilm Matrix in Escherichia coli.

Biofilms are communities of self-enmeshed bacteria in a matrix of exopolysaccharides. The widely distributed human pathogen and commensal Escherichia coli produces a biofilm matrix composed of phosphoethanolamine (pEtN)-modified cellulose and amyloid protein fibers, termed curli. The addition of pEtN to the cellulose exopolysaccharide is accomplished by the action of the pEtN transferase, BcsG, and is essential for the overall integrity of the biofilm. Here, using the synthetic co-substrates p-nitrophenyl phosphoethanolamine and ß-d-cellopentaose, we demonstrate using an in vitro pEtN transferase assay that full activity of the pEtN transferase domain of BcsG from E. coli (EcBcsGΔN) requires Zn2+ binding, a catalytic nucleophile/acid-base arrangement (Ser278/Cys243/His396), disulfide bond formation, and other newly uncovered essential residues. We further confirm that EcBcsGΔN catalysis proceeds by a ping-pong bisubstrate-biproduct reaction mechanism and displays inefficient kinetic behavior (kcat/KM = 1.81 × 10-4 ± 2.81 × 10-5 M-1 s-1), which is typical of exopolysaccharide-modifying enzymes in bacteria. Thus, the results presented, especially with respect to donor binding (as reflected by KM), have importantly broadened our understanding of the substrate profile and catalytic mechanism of this class of enzymes, which may aid in the development of inhibitors targeting BcsG or other characterized members of the pEtN transferase family, including the intrinsic and mobile colistin resistance factors.

Lower incidence of clinical allergy with PEG-asparaginase upfront versus the sequential use of native E. coli asparaginase followed by PEG-ASP in pediatric patients with acute lymphoblastic leukemia.

Asparaginase (ASP) is an essential component for the acute lymphoblastic leukemia (ALL) treatment, but toxicities, such as allergy, frequently limit its use. Although the potentially lower PEG-ASP formulation immunogenicity, few studies with conflicting results have compared the allergy incidence between Escherichia coli-ASP and PEG-ASP in the same protocol. We aimed at comparing the allergy incidence in children receiving native E. coli-ASP versus PEG-ASP within the same clinical protocol (Spanish Society of Pediatric Hematology and Oncology ALL-SEHOP-PETHEMA 2013). One hundred and twenty-six children (1-19 years) diagnosed with ALL from 2013 to 2020 were included. Patients in group 1 received a sequential scheme of native E. coli-ASP 10,000 IU/m2 intramuscularly (IM) followed by PEG-ASP 1000 IU/m2 IM. Patients in group 2 received PEG-ASP 1000 IU/m2 IM upfront. Clinical allergy incidence was compared between both groups. Serum ASP activity (SAA) was measured in a subgroup of patients, and silent inactivation was recorded. The cumulative incidence of clinical allergy was significantly higher in group 1 (native followed by PEG-ASP) than in group 2 (PEG-ASP upfront), 24.7% versus 4.1% (p = 0.0085). Adequate ASP activity was achieved with PEG-ASP 1000 IU/m2 dose in most patients (median SAA 412.5 and 453.0 IU/L at days 7 and 14). The incidence of silent inactivation in PEG-ASP upfront patients was very low. PEG-ASP-used upfront was associated with a lower incidence of clinical allergy than that observed in the sequential use of native E. coli-ASP followed by PEG-ASP. PEG-ASP at 1000 IU/m2 was effective in achieving enough ASP activity in most patients.

Evaluation of bacterial hosts for conversion of lignin-derived p-coumaric acid to 4-vinylphenol.

Hydroxycinnamic acids such as p-coumaric acid (CA) are chemically linked to lignin in grassy biomass with fairly labile ester bonds and therefore represent a straightforward opportunity to extract and valorize lignin components. In this work, we investigated the enzymatic conversion of CA extracted from lignocellulose to 4-vinylphenol (4VP) by expressing a microbial phenolic acid decarboxylase in Corynebacterium glutamicum, Escherichia coli, and Bacillus subtilis. The performance of the recombinant strains was evaluated in response to the substrate concentration in rich medium or a lignin liquor and the addition of an organic overlay to perform a continuous product extraction in batch cultures. We found that using undecanol as an overlay enhanced the 4VP titers under high substrate concentrations, while extracting > 97% of the product from the aqueous phase. C. glutamicum showed the highest tolerance to CA and resulted in the accumulation of up to 187 g/L of 4VP from pure CA in the overlay with a 90% yield when using rich media, or 17 g/L of 4VP with a 73% yield from CA extracted from lignin. These results indicate that C. glutamicum is a suitable host for the high-level production of 4VP and that further bioprocess engineering strategies should be explored to optimize the production, extraction, and purification of 4VP from lignin with this organism.

DyP-Type Peroxidases: Recent Advances and Perspectives.

In this review, we chart the major milestones in the research progress on the DyP-type peroxidase family over the past decade. Though mainly distributed among bacteria and fungi, this family actually exhibits more widespread diversity. Advanced tertiary structural analyses have revealed common and different features among members of this family. Notably, the catalytic cycle for the peroxidase activity of DyP-type peroxidases appears to be different from that of other ubiquitous heme peroxidases. DyP-type peroxidases have also been reported to possess activities in addition to peroxidase function, including hydrolase or oxidase activity. They also show various cellular distributions, functioning not only inside cells but also outside of cells. Some are also cargo proteins of encapsulin. Unique, noteworthy functions include a key role in life-cycle switching in Streptomyces and the operation of an iron transport system in Staphylococcus aureus, Bacillus subtilis and Escherichia coli. We also present several probable physiological roles of DyP-type peroxidases that reflect the widespread distribution and function of these enzymes. Lignin degradation is the most common function attributed to DyP-type peroxidases, but their activity is not high compared with that of standard lignin-degrading enzymes. From an environmental standpoint, degradation of natural antifungal anthraquinone compounds is a specific focus of DyP-type peroxidase research. Considered in its totality, the DyP-type peroxidase family offers a rich source of diverse and attractive materials for research scientists.

Stable and selective permeable hydrogel microcapsules for high-throughput cell cultivation and enzymatic analysis.

BACKGROUND: Miniaturization of biochemical reaction volumes within artificial microcompartments has been the key driver for directed evolution of several catalysts in the past two decades. Typically, single cells are co-compartmentalized within water-in-oil emulsion droplets with a fluorogenic substrate whose conversion allows identification of catalysts with improved performance. However, emulsion droplet-based technologies prevent cell proliferation to high density and preclude the feasibility of biochemical reactions that require the exchange of small molecule substrates. Here, we report on the development of a high-throughput screening method that addresses these shortcomings and that relies on a novel selective permeable polymer hydrogel microcapsule. RESULTS: Hollow-core polyelectrolyte-coated chitosan alginate microcapsules (HC-PCAMs) with selective permeability were successfully constructed by jet break-up and layer-by-layer (LBL) technology. We showed that HC-PCAMs serve as miniaturized vessels for single cell encapsulation, enabling cell growth to high density and cell lysis to generate monoclonal cell lysate compartments suitable for high-throughput analysis using a large particle sorter (COPAS). The feasibility of using HC-PCAMs as reaction compartments which exchange small molecule substrates was demonstrated using the transpeptidation reaction catalyzed by the bond-forming enzyme sortase F from P. acnes. The polyelectrolyte shell surrounding microcapsules allowed a fluorescently labelled peptide substrate to enter the microcapsule and take part in the transpeptidation reaction catalyzed by the intracellularly expressed sortase enzyme retained within the capsule upon cell lysis. The specific retention of fluorescent transpeptidation products inside microcapsules enabled the sortase activity to be linked with a fluorescent readout and allowed clear separation of microcapsules expressing the wild type SrtF from those expressing the inactive variant. CONCLUSION: A novel polymer hydrogel microcapsule-based method, which allows for high-throughput analysis based on encapsulation of single cells has been developed. The method has been validated for the transpeptidation activity of sortase enzymes and represents a powerful tool for screening of libraries of sortases, other bond-forming enzymes, as well as of binding affinities in directed evolution experiments. Moreover, selective permeable microcapsules encapsulating microcolonies provide a new and efficient means for preparing novel caged biocatalyst and biosensor agents.

Combination of the CRP mutation and ptsG deletion in Escherichia coli to efficiently synthesize xylitol from corncob hydrolysates.

The biotechnology-based production of xylitol has received widespread attention because it can use cheap and renewable lignocellulose as a raw material, thereby decreasing costs and pollution. The simultaneous use of various sugars in lignocellulose hydrolysates is a primary prerequisite for efficient xylitol production. In this study, a ΔptsG and crp* combinatorial strategy was used to generate Escherichia coli W3110 strain IS5-dI, which completely eliminated glucose repression and simultaneously used glucose and xylose. This strain produced 164 g/L xylitol from detoxified corncob hydrolysates during a fed-batch fermentation in a 15-L bioreactor, which was 14.7% higher than the xylitol produced by the starting strain, IS5-d (143 g/L), and the xylitol productivity was 3.04 g/L/h. These results represent the highest xylitol concentration and productivity reported to date for bacteria and hemicellulosic sugars. Additionally, strain IS5-dG, which differs from IS5-dI at CRP amino acid residue 127 (I127G), was tolerant to the toxins in corncob hydrolysates. In a fed-batch fermentation experiment involving a 15-L bioreactor, IS5-dG produced 137 g/L xylitol from non-detoxified corncob hydrolysates, with a productivity of 1.76 g/L/h. On the basis of these results, we believe that IS5-dI and IS5-dG may be useful host strains for the industrial-scale production of xylitol from detoxified or non-detoxified corncob hydrolysates.

Structural and mechanistic basis for the inhibition of Escherichia coli RNA polymerase by T7 Gp2.

The T7 phage-encoded small protein Gp2 is a non-DNA-binding transcription factor that interacts with the jaw domain of the Escherichia coli (Ec) RNA polymerase (RNAp) ß' subunit and inhibits transcriptionally proficient promoter-complex (RPo) formation. Here, we describe the high-resolution solution structure of the Gp2-Ec ß' jaw domain complex and show that Gp2 and DNA compete for binding to the ß' jaw domain. We reveal that efficient inhibition of RPo formation by Gp2 requires the amino-terminal σ(70) domain region 1.1 (R1.1), and that Gp2 antagonizes the obligatory movement of R1.1 during RPo formation. We demonstrate that Gp2 inhibits RPo formation not just by steric occlusion of the RNAp-DNA interaction but also through long-range antagonistic effects on RNAp-promoter interactions around the RNAp active center that likely occur due to repositioning of R1.1 by Gp2. The inhibition of Ec RNAp by Gp2 thus defines a previously uncharacterized mechanism by which bacterial transcription is regulated by a viral factor.

Immunotopological Analysis of the Treponema denticola Major Surface Protein (Msp).

Treponema denticola, one of several recognized periodontal pathogens, is a model organism for studying Treponema physiology and host-microbe interactions. Its major surface protein Msp (or MOSP) comprises an oligomeric outer membrane-associated complex that binds fibronectin, has cytotoxic pore-forming activity, and disrupts several intracellular responses. There are two hypotheses regarding native Msp structure and membrane topology. One hypothesis predicts that the entire Msp protein forms a ß-barrel structure similar to that of well-studied outer membrane porins of Gram-negative bacteria. The second hypothesis predicts a bipartite Msp with distinct and separate periplasmic N-terminal and porin-like ß-barrel C-terminal domains. The bipartite model, based on bioinformatic analysis of the orthologous Treponema pallidum Tpr proteins, is supported largely by studies of recombinant TprC and Msp polypeptides. The present study reports immunological studies in both T. denticola and Escherichia coli backgrounds to identify a prominent Msp surface epitope (residues 229 to 251 in ATCC 35405) in a domain that differs between strains with otherwise highly conserved Msps. These results were then used to evaluate a series of in silico structural models of representative T. denticola Msps. The data presented here are consistent with a model of Msp as a large-diameter ß-barrel porin. This work adds to the knowledge regarding the diverse Msp-like proteins in oral treponemes and may contribute to an understanding of the evolutionary and potential functional relationships between Msps of oral Treponema and the orthologous group of Tpr proteins of T. pallidum.IMPORTANCETreponema denticola is among a small subset of the oral microbiota contributing to severe periodontal disease. Due to its relative genetic tractability, T. denticola is a model organism for studying Treponema physiology and host-microbe interactions. T. denticola Msp is a highly expressed outer membrane-associated oligomeric protein that binds fibronectin, has cytotoxic pore-forming activity, and disrupts intracellular regulatory pathways. It shares homology with the orthologous group of T. pallidum Tpr proteins, one of which is implicated in T. pallidum in vivo antigenic variation. The outer membrane topologies of both Msp and the Tpr family proteins are unresolved, with conflicting reports on protein domain localization and function. In this study, we combined empirical immunological data derived both from diverse T. denticola strains and from recombinant Msp expression in E. coli with in silico predictive structural modeling of T. denticola Msp membrane topology, to move toward resolution of this important issue in Treponema biology.

Escherichia coli and Pseudomonas aeruginosa lipopolysaccharide O-antigen ligases share similar membrane topology and biochemical properties.

WaaL is an inner membrane glycosyltransferase that catalyzes the transfer of O-antigen polysaccharide from its lipid-linked intermediate to a terminal sugar of the lipid A-core oligosaccharide, a conserved step in lipopolysaccharide biosynthesis. Ligation occurs at the periplasmic side of the bacterial cell membrane, suggesting the catalytic region of WaaL faces the periplasm. Establishing the membrane topology of the WaaL protein family will enable understanding its mechanism and exploit it as a potential antimicrobial target. Applying oxidative labeling of native methionine/cysteine residues, we previously validated a topological model for Escherichia coli WaaL, which differs substantially from the reported topology of the Pseudomonas aeruginosa WaaL, derived from the analysis of truncated protein reporter fusions. Here, we examined the topology of intact E. coli and P. aeruginosa WaaL proteins by labeling engineered cysteine residues with the membrane-impermeable sulfhydryl reagent polyethylene glycol maleimide (PEG-Mal). The accessibility of PEG-Mal to targeted engineered cysteine residues in both E. coli and P. aeruginosa WaaL proteins demonstrates that both ligases share similar membrane topology. Further, we also demonstrate that P. aeruginosa WaaL shares similar functional properties with E. coli WaaL and that E. coli WaaL may adopt a functional dimer conformation.

A Retro-biosynthesis-Based Route to Generate Pinene-Derived Polyesters.

Significantly increased production of biobased polymers is a prerequisite to replace petroleum-based materials towards reaching a circular bioeconomy. However, many renewable building blocks from wood and other plant material are not directly amenable for polymerization, due to their inert backbones and/or lack of functional group compatibility with the desired polymerization type. Based on a retro-biosynthetic analysis of polyesters, a chemoenzymatic route from (-)-α-pinene towards a verbanone-based lactone, which is further used in ring-opening polymerization, is presented. Generated pinene-derived polyesters showed elevated degradation and glass transition temperatures, compared with poly(ϵ-decalactone), which lacks a ring structure in its backbone. Semirational enzyme engineering of the cyclohexanone monooxygenase from Acinetobacter calcoaceticus enabled the biosynthesis of the key lactone intermediate for the targeted polyester. As a proof of principle, one enzyme variant identified from screening in a microtiter plate was used in biocatalytic upscaling, which afforded the bicyclic lactone in 39 % conversion in shake flask scale reactions.

Synthetic metabolic pathway for the production of 1-alkenes from lignin-derived molecules.

BACKGROUND: Integration of synthetic metabolic pathways to catabolically diverse chassis provides new opportunities for sustainable production. One attractive scenario is the use of abundant waste material to produce a readily collectable product, which can reduce the production costs. Towards that end, we established a cellular platform for the production of semivolatile medium-chain α-olefins from lignin-derived molecules: we constructed 1-undecene synthesis pathway in Acinetobacter baylyi ADP1 using ferulate, a lignin-derived model compound, as the sole carbon source for both cell growth and product synthesis. RESULTS: In order to overcome the toxicity of ferulate, we first applied adaptive laboratory evolution to A. baylyi ADP1, resulting in a highly ferulate-tolerant strain. The adapted strain exhibited robust growth in 100 mM ferulate while the growth of the wild type strain was completely inhibited. Next, we expressed two heterologous enzymes in the wild type strain to confer 1-undecene production from glucose: a fatty acid decarboxylase UndA from Pseudomonas putida, and a thioesterase 'TesA from Escherichia coli. Finally, we constructed the 1-undecene synthesis pathway in the ferulate-tolerant strain. The engineered cells were able to produce biomass and 1-undecene solely from ferulate, and excreted the product directly to the culture headspace. CONCLUSIONS: In this study, we employed a bacterium Acinetobacter baylyi ADP1 to integrate a natural aromatics degrading pathway to a synthetic production route, allowing the upgradation of lignin derived molecules to value-added products. We developed a highly ferulate-tolerant strain and established the biosynthesis of an industrially relevant chemical, 1-undecene, solely from the lignin-derived model compound. This study reports the production of alkenes from lignin derived molecules for the first time and demonstrates the potential of lignin as a sustainable resource in the bio-based synthesis of valuable products.