Modular optimization in metabolic engineering.

There is an increasing demand for bioproducts produced by metabolically engineered microbes, such as pharmaceuticals, biofuels, biochemicals and other high value compounds. In order to meet this demand, modular optimization, the optimizing of subsections instead of the whole system, has been adopted to engineer cells to overproduce products. Research into modularity has focused on traditional approaches such as DNA, RNA, and protein-level modularity of intercellular machinery, by optimizing metabolic pathways for enhanced production. While research into these traditional approaches continues, limitations such as scale-up and time cost hold them back from wider use, while at the same time there is a shift to more novel methods, such as moving from episomal expression to chromosomal integration. Recently, nontraditional approaches such as co-culture systems and cell-free metabolic engineering (CFME) are being investigated for modular optimization. Co-culture modularity looks to optimally divide the metabolic burden between different hosts. CFME seeks to modularly optimize metabolic pathways in vitro, both speeding up the design of such systems and eliminating the issues associated with live hosts. In this review we will examine both traditional and nontraditional approaches for modular optimization, examining recent developments and discussing issues and emerging solutions for future research in metabolic engineering.

Structure and Function in Antimicrobial Piscidins: Histidine Position, Directionality of Membrane Insertion, and pH-Dependent Permeabilization.

Piscidins are histidine-enriched antimicrobial peptides that interact with lipid bilayers as amphipathic α-helices. Their activity at acidic and basic pH in vivo makes them promising templates for biomedical applications. This study focuses on p1 and p3, both 22-residue-long piscidins with 68% sequence identity. They share three histidines (H3, H4, and H11), but p1, which is significantly more permeabilizing, has a fourth histidine (H17). This study investigates how variations in amphipathic character associated with histidines affect the permeabilization properties of p1 and p3. First, we show that the permeabilization ability of p3, but not p1, is strongly inhibited at pH 6.0 when the conserved histidines are partially charged and H17 is predominantly neutral. Second, our neutron diffraction measurements performed at low water content and neutral pH indicate that the average conformation of p1 is highly tilted, with its C-terminus extending into the opposite leaflet. In contrast, p3 is surface bound with its N-terminal end tilted toward the bilayer interior. The deeper membrane insertion of p1 correlates with its behavior at full hydration: an enhanced ability to tilt, bury its histidines and C-terminus, induce membrane thinning and defects, and alter membrane conductance and viscoelastic properties. Furthermore, its pH-resiliency relates to the neutral state favored by H17. Overall, these results provide mechanistic insights into how differences in the histidine content and amphipathicity of peptides can elicit different directionality of membrane insertion and pH-dependent permeabilization. This work features complementary methods, including dye leakage assays, NMR-monitored titrations, X-ray and neutron diffraction, oriented CD, molecular dynamics, electrochemical impedance spectroscopy, surface plasmon resonance, and quartz crystal microbalance with dissipation.

Detection of amyloid ß oligomers toward early diagnosis of Alzheimer's disease.

Amyloid ß (Aß) peptide accumulation in the brain is considered to be one of the hallmarks of Alzheimer's disease. Here, we compare two analytical techniques for detecting neurotoxic Aß1-42 oligomers - Quartz Crystal Microbalance with Dissipation (QCM-D) and Single Molecule Array (Simoa). Both detection methods exploit a feature of the monoclonal antibody bapineuzumab, which targets N-terminal residues 1-5 of Aß with high affinity and use it as both a capture and detection reagent. Assays developed with the two methods allow us to specifically recognize neurotoxic Aß1-42 oligomers and higher aggregates such as fibrils but discriminate against Aß1-42 monomer species. We find that for detection of Aß1-42 oligomers, Simoa was roughly 500 times more sensitive than the QCM-D technique with limits of detection of 0.22 nM and 125 nM, respectively.

Structure of an engineered intein reveals thiazoline ring and provides mechanistic insight.

We have engineered an intein which spontaneously and reversibly forms a thiazoline ring at the native N-terminal Lys-Cys splice junction. We identified conditions to stablize the thiazoline ring and provided the first crystallographic evidence, at 1.54 Å resolution, for its existence at an intein active site. The finding bolsters evidence for a tetrahedral oxythiazolidine splicing intermediate. In addition, the pivotal mutation maps to a highly conserved B-block threonine, which is now seen to play a causative role not only in ground-state destabilization of the scissile N-terminal peptide bond, but also in steering the tetrahedral intermediate toward thioester formation, giving new insight into the splicing mechanism. We demonstrated the stability of the thiazoline ring at neutral pH as well as sensitivity to hydrolytic ring opening under acidic conditions. A pH cycling strategy to control N-terminal cleavage is proposed, which may be of interest for biotechnological applications requiring a splicing activity switch, such as for protein recovery in bioprocessing.

Membrane Filtration with Liquids: A Global Approach with Prior Successes, New Developments and Unresolved Challenges.

After 70 years, modern pressure-driven polymer membrane processes with liquids are mature and accepted in many industries due to their good performance, ease of scale-up, low energy consumption, modular compact construction, and low operating costs compared with thermal systems. Successful isothermal operation of synthetic membranes with liquids requires consideration of three critical aspects or "legs" in order of relevance: selectivity, capacity (i.e. permeation flow rate per unit area) and transport of mass and momentum comprising concentration polarization (CP) and fouling (F). Major challenges remain with respect to increasing selectivity and controlling mass transport in, to and away from membranes. Thus, prediction and control of membrane morphology and a deep understanding of the mechanism of dissolved and suspended solute transport near and in the membrane (i.e. diffusional and convective mass transport) is essential. Here, we focus on materials development to address the relatively poor selectivity of liquid membrane filtration with polymers and discuss the critical aspects of transport limitations. Machine learning could help optimize membrane structure design and transport conditions for improved membrane filtration performance.

Protein Binding Kinetics in Multimodal Systems: Implications for Protein Separations.

In this work, quartz crystal microbalance with dissipation (QCM-D) was employed to study the kinetic processes involved in the interaction of proteins with self-assembled monolayers (SAMs) of multimodal (MM) ligands. SAMs were fabricated to mimic two chromatographic multimodal resins with varying accessibility of the aromatic moiety to provide a well-defined model system. Kinetic parameters were determined for two different proteins in the presence of the arginine and guanidine and a comparison was made with chromatographic retention data. The results indicated that the accessibility of the ligand's aromatic moiety can have an important impact on the kinetics and chromatographic retention behavior. Interestingly, arginine and guanidine had very different effects on the protein adsorption and desorption kinetics in these MM systems. For cytochrome C, arginine resulted in a significant decrease and increase in the adsorption and desorption rates, respectively, while guanidine produced a dramatic increase in the desorption rate, with minimal effect on the adsorption rate. In addition, at different concentrations of arginine, two distinct kinetic scenarios were observed. For α-chymotrypsin, the presence of 0.1 M guanidine in the aromatic exposed ligand system produced an increase in the adsorption rate and only a moderate increase in the desorption rate, which helped to explain the surprising increase in the chromatographic salt elution concentration. These results demonstrate that protein adsorption kinetics in the presence of different mobile phase modifiers and MM ligand chemistries can play an important role in contributing to selectivity in MM chromatography.

Mechanism of Four de Novo Designed Antimicrobial Peptides.

As pathogenic bacteria become resistant to traditional antibiotics, alternate approaches such as designing and testing new potent selective antimicrobial peptides (AMP) are increasingly attractive. However, whereas much is known regarding the relationship between the AMP sequence and potency, less research has focused on developing links between AMP properties, such as design and structure, with mechanisms. Here we use four natural AMPs of varying known secondary structures and mechanisms of lipid bilayer disruption as controls to determine the mechanisms of four rationally designed AMPs with similar secondary structures and rearranged amino acid sequences. Using a Quartz Crystal Microbalance with Dissipation, we were able to differentiate between molecular models of AMP actions such as barrel-stave pore formation, toroidal pore formation, and peptide insertion mechanisms by quantifying differential frequencies throughout an oscillating supported lipid bilayer. Barrel-stave pores were identified by uniform frequency modulation, whereas toroidal pores possessed characteristic changes in oscillation frequency throughout the bilayer. The resulting modes of action demonstrate that rearrangement of an amino acid sequence of the AMP resulted in identical overall mechanisms, and that a given secondary structure did not necessarily predict mechanism. Also, increased mass addition to Gram-positive mimetic membranes from AMP disruption corresponded with lower minimum inhibitory concentrations against the Gram-positive Staphylococcus aureus.

Exploring Intein Inhibition by Platinum Compounds as an Antimicrobial Strategy.

Inteins, self-splicing protein elements, interrupt genes and proteins in many microbes, including the human pathogen Mycobacterium tuberculosis Using conserved catalytic nucleophiles at their N- and C-terminal splice junctions, inteins are able to excise out of precursor polypeptides. The splicing of the intein in the mycobacterial recombinase RecA is specifically inhibited by the widely used cancer therapeutic cisplatin, cis-[Pt(NH3)2Cl2], and this compound inhibits mycobacterial growth. Mass spectrometric and crystallographic studies of Pt(II) binding to the RecA intein revealed a complex in which two platinum atoms bind at N- and C-terminal catalytic cysteine residues. Kinetic analyses of NMR spectroscopic data support a two-step binding mechanism in which a Pt(II) first rapidly interacts reversibly at the N terminus followed by a slower, first order irreversible binding event involving both the N and C termini. Notably, the ligands of Pt(II) compounds that are required for chemotherapeutic efficacy and toxicity are no longer bound to the metal atom in the intein adduct. The lack of ammine ligands and need for phosphine represent a springboard for future design of platinum-based compounds targeting inteins. Because the intein splicing mechanism is conserved across a range of pathogenic microbes, developing these drugs could lead to novel, broad range antimicrobial agents.

A2T and A2V Aß peptides exhibit different aggregation kinetics, primary nucleation, morphology, structure, and LTP inhibition.

The histopathological hallmark of Alzheimer's disease (AD) is the aggregation and accumulation of the amyloid beta peptide (Aß) into misfolded oligomers and fibrils. Here we examine the biophysical properties of a protective Aß variant against AD, A2T, and a causative mutation, A2T, along with the wild type (WT) peptide. The main finding here is that the A2V native monomer is more stable than both A2T and WT, and this manifests itself in different biophysical behaviors: the kinetics of aggregation, the initial monomer conversion to an aggregation prone state (primary nucleation), the abundances of oligomers, and extended conformations. Aggregation reaction modeling of the conversion kinetics from native monomers to fibrils predicts the enhanced stability of the A2V monomer, while ion mobility spectrometry-mass spectrometry measures this directly confirming earlier predictions. Additionally, unique morphologies of the A2T aggregates are observed using atomic force microscopy, providing a basis for the reduction in long term potentiation inhibition of hippocampal cells for A2T compared with A2V and the wild type (WT) peptide. The stability difference of the A2V monomer and the difference in aggregate morphology for A2T (both compared with WT) are offered as alternate explanations for their pathological effects.

Combined Bioinformatic and Rational Design Approach To Develop Antimicrobial Peptides against Mycobacterium tuberculosis.

Drug-resistant pathogens are a growing problem, and novel strategies are needed to combat this threat. Among the most significant of these resistant pathogens is Mycobacterium tuberculosis, which is an unusually difficult microbial target due to its complex membrane. Here, we design peptides for specific activity against M. tuberculosis using a combination of "database filtering" bioinformatics, protein engineering, and de novo design. Several variants of these peptides are structurally characterized to validate the design process. The designed peptides exhibit potent activity (MIC values as low as 4 µM) against M. tuberculosis and also exhibit broad activity against a host of other clinically relevant pathogenic bacteria such as Gram-positive bacteria (Streptococcus) and Gram-negative bacteria (Escherichia coli). They also display excellent selectivity, with low cytotoxicity against cultured macrophages and lung epithelial cells. These first-generation antimicrobial peptides serve as a platform for the design of antibiotics and for investigating structure-activity relationships in the context of the M. tuberculosis membrane. The antimicrobial peptide design strategy is expected to be generalizable for any pathogen for which an activity database can be created.

Phosphate Ions Affect the Water Structure at Functionalized Membrane Surfaces.

Antifouling surfaces improve function, efficiency, and safety in products such as water filtration membranes, marine vehicle coatings, and medical implants by resisting protein and biofilm adhesion. Understanding the role of water structure at these materials in preventing protein adhesion and biofilm formation is critical to designing more effective coatings. Such fouling experiments are typically performed under biological conditions using isotonic aqueous buffers. Previous studies have explored the structure of pure water at a few different antifouling surfaces, but the effect of electrolytes and ionic strength (I) on the water structure at antifouling surfaces is not well studied. Here sum frequency generation (SFG) spectroscopy is used to characterize the interfacial water structure at poly(ether sulfone) (PES) and two surface-modified PES films in contact with 0.01 M phosphate buffer with high and low salt (Ionic strength, I= 0.166 and 0.025 M, respectively). Unmodified PES, commonly used as a filtration membrane, and modified PES with a hydrophobic alkane (C18) and with a poly(ethylene glycol) (PEG) were used. In the low ionic strength phosphate buffer, water was strongly ordered near the surface of the PEG-modified PES film due to exclusion of phosphate ions and the creation of a surface potential resulting from charge separation between phosphate anions and sodium cations. However, in the high ionic strength phosphate buffer, the sodium and potassium chloride (138 and 3 mM, respectively) in the phosphate buffered saline screened this charge and substantially reduced water ordering. A much smaller water ordering and subsequent reduction upon salt addition was observed for the C18-modified PES, and little water structure change was seen for the unmodified PES. The large difference in water structuring with increasing ionic strength between widely used phosphate buffer and phosphate buffered saline at the PEG interface demonstrates the importance of studying antifouling coatings in the same aqueous environment for which they are designed. These results further suggest that strong long-range water structuring is limited in high ionic strength environments, such as within cells, facilitating chemical and biological reactions and processes.

Alzheimer's protective A2T mutation changes the conformational landscape of the Aß1₋42 monomer differently than does the A2V mutation.

The aggregation of amyloid-ß (Aß) peptides plays a crucial role in the etiology of Alzheimer's disease (AD). Recently, it has been reported that an A2T mutation in Aß can protect against AD. Interestingly, a nonpolar A2V mutation also has been found to offer protection against AD in the heterozygous state, although it causes early-onset AD in homozygous carriers. Since the conformational landscape of the Aß monomer is known to directly contribute to the early-stage aggregation mechanism, it is important to characterize the effects of the A2T and A2V mutations on Aß1₋42 monomer structure. Here, we have performed extensive atomistic replica-exchange molecular dynamics simulations of the solvated wild-type (WT), A2V, and A2T Aß1₋42 monomers. Our simulations reveal that although all three variants remain as collapsed coils in solution, there exist significant structural differences among them at shorter timescales. A2V exhibits an enhanced double-hairpin population in comparison to the WT, similar to those reported in toxic WT Aß1₋42 oligomers. Such double-hairpin formation is caused by hydrophobic clustering between the N-terminus and the central and C-terminal hydrophobic patches. In contrast, the A2T mutation causes the N-terminus to engage in unusual electrostatic interactions with distant residues, such as K16 and E22, resulting in a unique population comprising only the C-terminal hairpin. These findings imply that a single A2X (where X = V or T) mutation in the primarily disordered N-terminus of the Aß1₋42 monomer can dramatically alter the ß-hairpin population and switch the equilibrium toward alternative structures. The atomistically detailed, comparative view of the structural landscapes of A2V and A2T variant monomers obtained in this study can enhance our understanding of the mechanistic differences in their early-stage aggregation.

Stability of proteins on hydrophilic surfaces.

The physical and chemical properties of solid substrates or surfaces critically influence the stability and activity of immobilized proteins such as enzymes. Reports of increased stability and activity of enzymes near/on surfaces as compared with those in solution abound; however, a mechanistic understanding is wanting. Simulations and experiments are used here to provide details toward such a mechanistic understanding. Experiments demonstrate increased activity of alcohol dehydrogenase (ADH) inside moderate hydrophilic mesopourous silica (SBA-15) pores but drastically decreased activity inside very hydrophilic NH2-SBA-15 surfaces as compared with that in solution. Also, the temperature stability of ADH was increased over that in solution when immobilized in a cavity with a mildly hydrophilic surface. Simulations confirm these experimental findings. Simulations calculated in the framework of a hydrophobic-polar (H-P) lattice model show increased thermal stability of a model 64-mer peptide on positive and zero curvature surfaces over that in solution. Peptides immobilized inside negative curvature cavities (concave) with hydrophilic surfaces exhibit increased stability only inside pores that are only 3-4 nm larger than the hydrodynamic radius of the peptide. Peptides are destabilized, however, when the surface hydrophilic character inside very small cavities/pores becomes large.

Cosolute effects on amyloid aggregation in a nondiffusion limited regime: intrinsic osmolyte properties and the volume exclusion principle.

The effects of cosolutes on amyloid aggregation kinetics in vivo are critical and not fully understood. To explore the effects of cosolute additives, the in vitro behavior of destabilizing and stabilizing osmolytes with polymer cosolutes on the aggregation of a model amyloid, human insulin, is probed using experiments coupled with an amyloid aggregation reaction model. The destabilizing osmolyte, guanidine hydrochloride (GuHCl), induces biphasic behavior on the amyloid aggregation rate exhibited by an enhancement of the aggregation kinetics at low concentrations of GuHCl (<0.6 M) and a reduction in kinetics at higher GuHCl concentrations. Stabilizing osmolytes, glycerol, sorbitol and trimethylamine N-oxide, slow the rate of aggregation by reducing the rate of monomer unfolding. Polymer cosolutes, polyvinylpyrrolidone 3.5 kDa and 40 kDa, delay amyloid aggregation mainly through a decrease in the nucleation reaction. These results are in good agreement with the volume exclusion principle for polymer crowding and supports the need to include conformational rearrangement of monomers prior to nucleation. Using fluorescence correlation spectroscopy, we demonstrate that amyloid aggregation is nondiffusion limited, except during fibril accumulation in the presence of high concentrations of long chain polymers. Lastly, the neutral surface area of osmolytes correlates well with the time to initiate fibril formation, tlag, which implicates an intrinsic osmolyte property underlying preferential interactions.

Chimera-induced folding: implications for amyloidosis.

The discoveries that non-native proteins have a role in amyloidosis and that multiple protein misfolding diseases can occur concurrently suggest that cross-seeding of amyloidogenic proteins may be central to misfolding. To study this process, a synthetic chimeric amyloidogenic protein (YEHK21-YE8) composed of two components, one that readily folds to form fibrils (YEHK21) and one that does not (YE8), was designed. Secondary structural conformational changes during YEHK21-YE8 aggregation demonstrate that, under the appropriate conditions, YEHK21 is able to induce fibril formation of YE8. The unambiguous demonstration of the induction of folding and fibrillation within a single molecule illuminates the factors controlling this process and hence suggests the importance of those factors in amyloidogenic diseases.

Egg white varnishes on ancient paintings: a molecular connection to amyloid proteins.

For about 400 years, egg white was used to coat and protect paintings without detailed understanding of its molecular properties. A molecular basis is provided for its advantageous properties and one of its protective properties is demonstrated with oxygen transport behavior. Compared to the native secondary structure of ovalbumin in solution of circa 33% α-helix and ß-sheet, attenuated total reflection-FTIR (ATR-FTIR) spectra showed a 73% decrease of α-helix content and a 44% increase of ß-sheet content over eight days. The data suggest that the final coating of dissolved ovalbumin from egg white after long exposure to air, which is hydrophobic, comprises mostly ß-sheet content (ca. 50%), which is predicted to be the lowest-energy structure of proteins and close to that found in amyloid fibrils. Coating a synthetic polytetrafluoroethylene membrane with multiple layers of egg white decreased oxygen diffusion by 50% per layer with a total decrease of almost 100% for four layers.

Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death.

Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.

Tranilast binds to aß monomers and promotes aß fibrillation.

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimer's disease (AD), in which amyloid ß-protein (Aß) plays a key pathogenic role. We used solution NMR to determine that tranilast binds to Aß40 monomers with ∼300 µM affinity. Remarkably, tranilast increases Aß40 fibrillation more than 20-fold in the thioflavin T assay at a 1:1 molar ratio, as well as significantly reducing the lag time. Tranilast likely promotes fibrillation by shifting Aß monomer conformations to those capable of seed formation and fibril elongation. Molecular docking results qualitatively agree with NMR chemical shift perturbation, which together indicate that hydrophobic interactions are the major driving force of the Aß-tranilast interaction. These data suggest that AD may be a potential complication for tranilast usage in elderly patients.

Oriented covalent immobilization of antibodies for measurement of intermolecular binding forces between zipper-like contact surfaces of split inteins.

In order to measure the intermolecular binding forces between two halves (or partners) of naturally split protein splicing elements called inteins, a novel thiol-hydrazide linker was designed and used to orient immobilized antibodies specific for each partner. Activation of the surfaces was achieved in one step, allowing direct intermolecular force measurement of the binding of the two partners of the split intein (called protein trans-splicing). Through this binding process, a whole functional intein is formed resulting in subsequent splicing. Atomic force microscopy (AFM) was used to directly measure the split intein partner binding at 1 µm/s between native (wild-type) and mixed pairs of C- and N-terminal partners of naturally occurring split inteins from three cyanobacteria. Native and mixed pairs exhibit similar binding forces within the error of the measurement technique (~52 pN). Bioinformatic sequence analysis and computational structural analysis discovered a zipper-like contact between the two partners with electrostatic and nonpolar attraction between multiple aligned ion pairs and hydrophobic residues. Also, we tested the Jarzynski's equality and demonstrated, as expected, that nonequilibrium dissipative measurements obtained here gave larger energies of interaction as compared with those for equilibrium. Hence, AFM coupled with our immobilization strategy and computational studies provides a useful analytical tool for the direct measurement of intermolecular association of split inteins and could be extended to any interacting protein pair.

A multi-dimensional approach for fractionating proteins using charged membranes.

In an effort to increase selectivity among proteins with crossflow ultrafiltration, we offer and demonstrate a comprehensive approach to fractionate proteins of similar molecular weight and relatively close pI values. This multidimensional approach involves optimizing membrane charge type and density together with operating conditions such as precise control of pH, ionic strength, and transmembrane pressure for reduced membrane fouling. Each filtration experiment was performed in cross-flow configuration for ∼20 min, allowing fast screening for optimal separation as determined by maximum selectivity, Ψ, and purity, P. Using our comprehensive approach for fractionating mixtures RNase A-lysozyme and BSA-hemoglobin, we obtained values of Ψ = 9.1, P = 95.7%, and Ψ = 6.5, P = 62.1%, respectively.