Enzymatic synthesis of N-succinyl-L-phenylalanine and exploration of its potential as a novel taste enhancer.

N-succinyl-L-phenylalanine (SP) has been identified as a taste-active contributor in an array of foods. Despite its recognized importance, the understanding of its synthesis and taste enhancement properties remains rudimentary. The study examined the enzymatic synthesis of SP with 45.58 ± 1.95% yield. This was achieved under optimized conditions: 0.3 mol/L L-phenylalanine, 0.9 mol/L succinic acid, 30,000 U/L of the AY 50C, pH 4 and 55 °C for 24 h. Sensory evaluation and electronic tongue revealed that the incorporation of a mere 1 mg/L SP substantially increased the kokumi, umami, and saltiness intensities, indicating the potential of SP as a potent taste enhancer. Moreover, time-intensity (TI) results demonstrated a significant increase of umami duration in samples containing 1 mg/L of SP (210.0 ± 0 s), a significant extension compared to the control group (150.0 ± 0 s). Notably, the intensity of umami and saltiness in the SP sample were consistently higher than that of control group. The sigmoid curve analysis further confirmed that SP exhibited a synergistic effect on umami and saltiness perceptions. Moreover, the study also illuminated interaction of SP with T1R1, T1R3, TMC4, TRPV1, and CaSR receptors, resulting in significant enhancement in umami, saltiness, and kokumi.

Engineering of Recombinant Human Papillomavirus 16 L1 Protein for Incorporation with para-Azido-L-Phenylalanine.

Human papillomavirus (HPV) L1 capsid protein were produced in several host systems, but few studies have focused on enhancing the properties of the L1 protein. In this study, we aimed to produce recombinant Human papillomavirus (HPV) L1 capsid protein containing para-azido-L-phenylalanine (pAzF) in Escherichia coli. First, we expressed the maltose-binding protein (MBP)- fused HPV16 L1, and 5 residues in HPV16 L1 protein were selected by the in silico modeling for amber codon substitution. Among the variants of the five locations, we identified a candidate that exhibited significant differences in expression with and without pAzF via genetic code expansion (GCE). The expressed recombinant MBP-HPV16L1 protein was confirmed for incorporation of pAzF and the formation of VLPs was tested in vitro.

Deciphering High-Temperature-Induced Lignin Biosynthesis in Wheat through Comprehensive Transcriptome Analysis.

This study systematically investigated the physiological and molecular responses of the wheat mutant 'XC-MU201' under high-temperature stress through comprehensive transcriptome analysis and physiological measurements. RNA sequencing of 21 samples across seven different treatment groups revealed, through Weighted Gene Co-expression Network Analysis (WGCNA), 13 modules among 9071 genes closely related to high-temperature treatments. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed significant enrichment of lignin biosynthesis-related modules under high-temperature conditions, especially at the H-10DAT, H-20DAT, and H-30DAT time points. Experimental results demonstrated a significant increase in lignin content in high-temperature-treated samples, confirmed by tissue staining methods, indicating wheat's adaptation to heat damage through lignin accumulation. The phenylalanine ammonia-lyase gene (TaPAL33) was significantly upregulated under high-temperature stress, peaking at H-30DAT, suggesting its critical role in cellular defense mechanisms. Overexpression of TaPAL33 in the wheat variety 'Xinchun 11' enhanced lignin synthesis but inhibited growth. Subcellular localization of GFP-labeled TaPAL33 in tobacco cells showed its distribution mainly in the cytoplasm and cell membrane. Transgenic wheat exhibited higher PAL enzyme activity, enhanced antioxidant defense, and reduced oxidative damage under high-temperature stress, outperforming wild-type wheat. These results highlight TaPAL33's key role in improving wheat heat tolerance and provide a genetic foundation for future research and applications.

Poly(phenylalanine) and poly(3,4-dihydroxy-L-phenylalanine): Promising biomedical materials for building stimuli-responsive nanocarriers.

Cancer is a serious threat to human health because of its high annual mortality rate. It has attracted significant attention in healthcare, and identifying effective strategies for the treatment and relief of cancer pain requires urgency. Drug delivery systems (DDSs) offer the advantages of excellent efficacy, low cost, and low toxicity for targeting drugs to tumor sites. In recent decades, copolymer carriers based on poly(phenylalanine) (PPhe) and poly(3,4-dihydroxy-L-phenylalanine) (PDopa) have been extensively investigated owing to their good biocompatibility, biodegradability, and controllable stimulus responsiveness, which have resulted in DDSs with loading and targeted delivery capabilities. In this review, we introduce the synthesis of PPhe and PDopa, highlighting the latest proposed synthetic routes and comparing the differences in drug delivery between PPhe and PDopa. Subsequently, we summarize the various applications of PPhe and PDopa in nanoscale-targeted DDSs, providing a comprehensive analysis of the drug release behavior based on different stimulus-responsive carriers using these two materials. In the end, we discuss the challenges and prospects of polypeptide-based DDSs in the field of cancer therapy, aiming to promote their further development to meet the growing demands for treatment.

Co-expression analyses reveal key Cd stress response-related metabolites and transcriptional regulators in Kentucky bluegrass.

Kentucky bluegrass (Poa pratensis) is known for its high cadmium (Cd) tolerance and accumulation, and it is therefore considered to have the potential for phytoremediation of Cd-contaminated soil. However, the mechanisms underlying the accumulation and tolerance of Cd in Kentucky bluegrass are largely unknown. In this study, we examined variances in the transcriptome and metabolome of a Cd-tolerant variety (Midnight, M) and a Cd-sensitive variety (Rugby II, R) to pinpoint crucial regulatory genes and metabolites associated with Cd response. We also validated the role of the key metabolite, l-phenylalanine, in Cd transport and alleviation of Cd stress by applying it to the Cd-tolerant variety M. Metabolites of the M and R varieties under Cd stress were subjected to co-expression analysis. The results showed that shikimate-phenylpropanoid pathway metabolites (phenolic acids, phenylpropanoids, and polyketides) were highly induced by Cd treatment and were more abundant in the Cd-tolerant variety. Gene co-expression network analysis was employed to further identify genes closely associated with key metabolites. The calcium regulatory genes, zinc finger proteins (ZAT6 and PMA), MYB transcription factors (MYB78, MYB62, and MYB33), ONAC077, receptor-like protein kinase 4, CBL-interacting protein kinase 1, and protein phosphatase 2A were highly correlated with the metabolism of phenolic acids, phenylpropanoids, and polyketides. Exogenous l-phenylalanine can significantly increase the Cd concentration in the leaves (22.27%-55.00%) and roots (7.69%-35.16%) of Kentucky bluegrass. The use of 1 mg/L of l-phenylalanine has been demonstrated to lower malondialdehyde levels and higher total phenols, flavonoids, and anthocyanins levels, while also significantly enhancing the uptake of Cd and its translocation from roots to shoots. Our results provide insights into the response mechanisms to Cd stress and offer a novel l-phenylalanine-based phytoremediation strategy for Cd-containing soil.

Cross-protection and cross-feeding between Enterobacter and Comamonas promoting their coexistence and cadmium tolerance in Oryza sativa L.

Metabolic cross-feeding is a pervasive interaction between bacteria to acquire novel phenotypes. However, our current understanding of the survival mechanism for cross-feeding in cocultured bacterial biofilms under heavy-metal conditions remains limited. Herein, we found that Comamonas sp. A23 produces L-phenylalanine to activate the L-phenylalanine degradation pathway in Enterobacter sp. A11, enhancing biofilm formation and cadmium [Cd(II)] immobilization in A11. The genes responsible for L-phenylalanine-degradation (paaK) and cell attachment and aggregation (csgAD) are essential for biofilm formation and Cd(II) immobilization in A11 induced by L-phenylalanine. The augmentation of A11 biofilms, in turn, protects A23 under Cd(II) and H2O2 stresses. The plant-based experiments demonstrate that the induction of two rice Cd(II) transporters, OsCOPT4 and OsBCP1, by A11 and A23 enhances rice resistance against Cd(II) and H2O2 stresses. Overall, our findings unveil the mutual dependence between bacteria and rice on L-phenylalanine cross-feeding for survival under abiotic stress.

Construction of Tough Hydrogel Cross-Linked via Ionic Interaction by Protection Effect of Hydrophobic Domains.

Most hydrogels have poor mechanical properties, severely limiting their potential applications, and numerous approaches have been introduced to fabricate more robust and durable examples. However, these systems consist of nonbiodegradable polymers which limit their application in tissue engineering. Herein, we focus on the fabrication and investigate the influence of hydrophobic segments on ionic cross-linking properties for the construction of a tough, biodegradable hydrogel. A biodegradable, poly(γ-glutamic acid) polymer conjugated with a hydrophobic amino acid, l-phenylalanine ethyl ester (Phe), together with an ionic cross-linking group, alendronic acid (Aln) resulting in γ-PGA-Aln-Phe, was initially synthesized. Rheological assessments through time sweep oscillation testing revealed that the presence of hydrophobic domains accelerated gelation. Comparing gels with and without hydrophobic domains, the compressive strength of γ-PGA-Aln-Phe was found to be six times higher and exhibited longer stability properties in ethylenediaminetetraacetic acid solution, lasting for up to a month. Significantly, the contribution of the hydrophobic domains to the mechanical strength and stability of ionic cross-linking properties of the gel was found to be the dominant factor for the fabrication of a tough hydrogel. As a result, this study provides a new strategy for mechanical enhancement and preserves ionic cross-linked sites by the addition of hydrophobic domains. The development of tough, biodegradable hydrogels reported herein will open up new possibilities for applications in the field of biomaterials.

Boron neutron capture therapy delays the decline in neurological function in a mouse model of metastatic spinal tumors.

Metastatic spinal tumors are increasingly prevalent due to advancements in cancer treatment, leading to prolonged survival rates. This rising prevalence highlights the need for developing more effective therapeutic approaches to address this malignancy. Boron neutron capture therapy (BNCT) offers a promising solution by delivering targeted doses to tumors while minimizing damage to normal tissue. In this study, we evaluated the efficacy and safety of BNCT as a potential therapeutic option for spine metastases in mouse models induced by A549 human lung adenocarcinoma cells. The animal models were randomly allocated into three groups: untreated (n = 10), neutron irradiation only (n = 9), and BNCT (n = 10). Each mouse was administered 4-borono-L-phenylalanine (250 mg/kg) intravenously, followed by measurement of boron concentrations 2.5 h later. Overall survival, neurological function of the hindlimb, and any adverse events were assessed post irradiation. The tumor-to-normal spinal cord and blood boron concentration ratios were 3.6 and 2.9, respectively, with no significant difference observed between the normal and compressed spinal cord tissues. The BNCT group exhibited significantly prolonged survival rates compared with the other groups (vs. untreated, p = 0.0015; vs. neutron-only, p = 0.0104, log-rank test). Furthermore, the BNCT group demonstrated preserved neurological function relative to the other groups (vs. untreated, p = 0.0004; vs. neutron-only, p = 0.0051, multivariate analysis of variance). No adverse events were observed post irradiation. These findings indicate that BNCT holds promise as a novel treatment modality for metastatic spinal tumors.

Enzymatic synthesis and sensory evaluation of the novel kokumi compound N-butyryl phenylalanine.

In this study, food-grade glutamine transaminase (TGase) was utilized for the green-catalyzed preparation of N-butyryl amino acids. For improving the reusability of the enzyme preparation, immobilized TG enzyme (94.23% immobilization rate) was prepared. Furthermore, the yield of N-butyryl phenylalanine (BP) synthesized by TGase was obtained as 20.73% by one-factor experiment. The BP synthesis yield of immobilized TGase was 95.03% of that of TGase and remained above 60% of the initial enzyme activity after five runs. The sensory evaluation and E-tongue results showed that the addition of BP significantly increased the umami, saltiness, and richness intensities of the samples, and decreased the intensities of sourness, bitterness, and aftertaste-B. The molecular docking results indicated that hydrogen bonding dominated the binding of BP to taste receptors in the taste presentation mechanism of BP. These results confirmed the potential of BP as a flavor enhancer with promising applications in the food industry.

Comprehensive investigations of 2-phenylethanol production by the filamentous fungus Annulohypoxylon stygium.

2-Phenylethanol (2-PE) is an aromatic compound with a rose-like fragrance that is widely used in food and other industries. Yeasts have been implicated in the biosynthesis of 2-PE; however, few studies have reported the involvement of filamentous fungi. In this study, 2-PE was detected in Annulohypoxylon stygium mycelia grown in both potato dextrose broth (PDB) and sawdust medium. Among the 27 A. stygium strains investigated in this study, the strain "Jinjiling" (strain S20) showed the highest production of 2-PE. Under optimal culture conditions, the concentration of 2-PE was 2.33 g/L. Each of the key genes in Saccharomyces cerevisiae shikimate and Ehrlich pathways was found to have homologous genes in A. stygium. Upon the addition of L-phenylalanine to the medium, there was an upregulation of all key genes in the Ehrlich pathway of A. stygium, which was consistent with that of S. cerevisiae. A. stygium as an associated fungus provides nutrition for the growth of Tremella fuciformis and most spent composts of T. fuciformis contain pure A. stygium mycelium. Our study on the high-efficiency biosynthesis of 2-PE in A. stygium offers a sustainable solution by utilizing the spent compost of T. fuciformis and provides an alternative option for the production of natural 2-PE. KEY POINTS: • Annulohypoxylon stygium can produce high concentration of 2-phenylethanol. • The pathways of 2-PE biosynthesis in Annulohypoxylon stygium were analyzed. • Spent compost of Tremella fuciformis is a potential source for 2-phenylethanol.

Development of orthogonal aminoacyl-tRNA synthetase mutant for incorporating a non-canonical amino acid.

Genetic code expansion involves introducing non-canonical amino acids (ncAAs) with unique functional groups into proteins to broaden their applications. Orthogonal aminoacyl tRNA synthetase (aaRS), essential for genetic code expansion, facilitates the charging of ncAAs to tRNA. In this study, we developed a new aaRS mutant from Methanosaeta concilii tyrosyl-tRNA synthetase (Mc TyrRS) to incorporate para-azido-L-phenylalanine (AzF). The development involved initial site-specific mutations in Mc TyrRS, followed by random mutagenesis. The new aaRS mutant with amber suppression was isolated through fluorescence-activated cell sorting. The M. concilii aaRS mutant structure was further analyzed to interpret the effect of mutations. This research provides a novel orthogonal aaRS evolution pipeline for highly efficient ncAA incorporation that will contribute to developing novel aaRS from various organisms.

Metabolic Engineering of Escherichia coli for High-Level Production of l-Phenylalanine.

l-Phenylalanine (l-Phe) is widely used in the food and pharmaceutical industries. However, the biosynthesis of l-Phe using Escherichia coli remains challenging due to its lower tolerance to high concentration of l-Phe. In this study, to efficiently synthesize l-Phe, the l-Phe biosynthetic pathway was reconstructed by expressing the heterologous genes aroK1, aroL1, and pheA1, along with the native genes aroA, aroC, and tyrB in the shikimate-producing strain E. coli SA09, resulting in the engineered strain E. coli PHE03. Subsequently, adaptive evolution was conducted on E. coli PHE03 to enhance its tolerance to high concentrations of l-Phe, resulting in the strain E. coli PHE04, which reduced the cell mortality to 36.2% after 48 h of fermentation. To elucidate the potential mechanisms, transcriptional profiling was conducted, revealing MarA, a DNA-binding transcriptional dual regulator, as playing a crucial role in enhancing cell membrane integrity and fluidity for improving cell tolerance to high concentrations of l-Phe. Finally, the titer, yield, and productivity of l-Phe with E. coli PHE05 overexpressing marA were increased to 80.48 g/L, 0.27 g/g glucose, and 1.68 g/L/h in a 5-L fed-batch fermentation, respectively.

Biosynthesis of fragrance 2-phenylethanol from sugars by Pseudomonas putida.

BACKGROUND: Petrochemicals contribute to environmental issues, with concerns ranging from energy consumption and carbon emission to pollution. In contrast, microbial biorefineries offer eco-friendly alternatives. The solvent-tolerant Pseudomonas putida DOT-T1E serves as a suitable host for producing aromatic compounds, specifically L-phenylalanine and its derivative, 2-phenylethanol (2-PE), which find widespread applications in various industries. RESULTS: This study focuses on enhancing 2-PE production in two L-phenylalanine overproducing strains of DOT-T1E, namely CM12-5 and CM12-5Δgcd (xylABE), which grow with glucose and glucose-xylose, respectively. To synthesize 2-PE from L-phenylalanine, these strains were transformed with plasmid pPE-1, bearing the Ehrlich pathway genes, and it was found higher 2-PE production with glucose (about 50-60 ppm) than with xylose (< 3 ppm). To understand the limiting factors, we tested the addition of phenylalanine and intermediates from the Ehrlich and shikimate pathways. The results identified intracellular L-phenylalanine as a key limiting factor for 2-PE production. To overcame this limitation, a chorismate mutase/prephenate dehydratase variant-insentive to feedback inhibition by aromatic amino acids-was introduced in the producing strains. This led to increased L-phenylalanine production and subsequently produced more 2-PE (100 ppm). Random mutagenesis of the strains also produced strains with higher L-phenylalanine titers and increased 2-PE production (up to 120 ppm). The improvements resulted from preventing dead-end product accumulation from shikimate and limiting the catabolism of potential pathway intermediates in the Ehrlich pathway. The study explored agricultural waste substrates, such as corn stover, sugarcane straw and corn-syrup as potential C sources. The best results were obtained using 2G substrates at 3% (between 82 and 100 ppm 2-PE), with glucose being the preferred sugar for 2-PE production among the monomeric sugars in these substrates. CONCLUSIONS: The findings of this study offer strategies to enhance phenylalanine production, a key substrate for the synthesis of aromatic compounds. The ability of P. putida DOT-T1E to thrive with various C-sources and its tolerance to substrates, products, and potential toxicants in industrial wastes, are highlighted. The study identified and overcome possible bottlenecks for 2-PE production. Ultimately, the strains have potential to become efficient microbial platforms for synthesizing 2-PE from agro-industrial waste materials.

Plant-Assisted Green Synthesis of MgO Nanoparticles as a Sustainable Material for Bone Regeneration: Spectroscopic Properties.

This work is devoted to magnesium oxide (MgO) nanoparticles (NPs) for their use as additives for bone implants. Extracts from four different widely used plants, including Aloe vera, Echeveria elegans, Sansevieria trifasciata, and Sedum morganianum, were evaluated for their ability to facilitate the "green synthesis" of MgO nanoparticles. The thermal stability and decomposition behavior of the MgONPs were analyzed by thermogravimetric analysis (TGA). Structure characterization was performed by X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), ultraviolet-visible spectroscopy (UV-Vis), dynamic light scattering (DLS), and Raman scattering spectroscopy (RS). Morphology was studied by scanning electron microscopy (SEM). The photocatalytic activity of MgO nanoparticles was investigated based on the degradation of methyl orange (MeO) using UV-Vis spectroscopy. Surface-enhanced Raman scattering spectroscopy (SERS) was used to monitor the adsorption of L-phenylalanine (L-Phe) on the surface of MgONPs. The calculated enhancement factor (EF) is up to 102 orders of magnitude for MgO. This is the first work showing the SERS spectra of a chemical compound immobilized on the surface of MgO nanoparticles.

Interaction of L-phenylalanine with carbonyl groups in mixed lipid membranes.

The interaction of L-Phe with the membrane components, i.e., lipids and proteins, has been discussed in the current literature due to the interest to understand the effect of single amino acids in relation to the formation of amyloid aggregates. In the present work, it is shown that L-Phe interacts with 9:1 DMPC (1,2-dimyristoyl-sn-glycero-3 phosphocholine)/DPPC (1,2-dipalmitoyl-sn-glycero-3 phosphocholine) mixtures but not in the 1:9 one. An important observation is that the interaction disappears when DPPC is replaced by diether PC (2-di-O-hexadecyl-sn-glycero-3-phosphocholine) a lipid lacking carbonyl groups (CO). This denotes that CO groups may interact specifically with L-Phe in accordance with the appearance of a new peak observed by Infrared spectroscopy (FTIR-ATR). The interaction of L-Phe affects the compressibility pattern of the 9:1 DMPC/DPPC mixture which is congruent with the changes observed by Raman spectra. The specific interaction of L-Phe with CO, propagates to phosphate and choline groups in this particular mixture as analyzed by FTIR-ATR spectroscopy and is absent when DMPC is dopped with diether PC.

Systems engineering Escherichia coli for efficient production p-coumaric acid from glucose.

P-coumaric acid (p-CA), a pant metabolite with antioxidant and anti-inflammatory activity, is extensively utilized in biomedicine, food, and cosmetics industry. In this study, a synthetic pathway (PAL) for p-CA was designed, integrating three enzymes (AtPAL2, AtC4H, AtATR2) into a higher l-phenylalanine-producing strain Escherichia coli PHE05. However, the lower soluble expression and activity of AtC4H in the PAL pathway was a bottleneck for increasing p-CA titers. To overcome this limitation, the soluble expression of AtC4H was enhanced through N-terminal modifications. And an optimal mutant, AtC4HL373T/G211H, which exhibited a 4.3-fold higher kcat/Km value compared to the wild type, was developed. In addition, metabolic engineering strategies were employed to increase the intracellular NADPH pool. Overexpression of ppnk in engineered E. coli PHCA20 led to a 13.9-folds, 1.3-folds, and 29.1% in NADPH content, the NADPH/NADP+ ratio and p-CA titer, respectively. These optimizations significantly enhance p-CA production, in a 5-L fermenter using fed-batch fermentation, the p-CA titer, yield and productivity of engineered strain E. coli PHCA20 were 3.09 g/L, 20.01 mg/g glucose, and 49.05 mg/L/h, respectively. The results presented here provide a novel way to efficiently produce the plant metabolites using an industrial strain.

Probing calmodulin-NO synthase interactions via site-specific infrared spectroscopy: an introductory investigation.

Calmodulin (CaM) binds to a linker between the oxygenase and reductase domains of nitric oxide synthase (NOS) to regulate the functional conformational dynamics. Specific residues on the interdomain interface guide the domain-domain docking to facilitate the electron transfer in NOS. Notably, the docking interface between CaM and the heme-containing oxygenase domain of NOS is isoform specific, which is only beginning to be investigated. Toward advancing understanding of the distinct CaM-NOS docking interactions by infrared spectroscopy, we introduced a cyano-group as frequency-resolved vibrational probe into CaM individually and when associated with full-length and a bi-domain oxygenase/FMN construct of the inducible NOS isoform (iNOS). Site-specific, selective labeling with p-cyano-L-phenylalanine (CNF) by amber suppression of CaM bound to the iNOS has been accomplished by protein coexpression due to the instability of recombinant iNOS protein alone. We introduced CNF at residue 108, which is at the putative CaM-heme (NOS) docking interface. CNF was also introduced at residue 29, which is distant from the docking interface. FT IR data show that the 108 site is sensitive to CaM-NOS complex formation, while insensitivity to its association with the iNOS protein or peptide was observed for the 29 site. Moreover, narrowing of the IR bands at residue 108 suggests the C≡N probe experiences a more limited distribution of environments, indicating side chain restriction apparent for the complex with iNOS. This initial work sets the stage for residue-specific characterizations of structural dynamics of the docked states of NOS proteins.

Fluorescent human RPA to track assembly dynamics on DNA.

DNA metabolic processes including replication, repair, recombination, and telomere maintenance occur on single-stranded DNA (ssDNA). In each of these complex processes, dozens of proteins function together on the ssDNA template. However, when double-stranded DNA is unwound, the transiently open ssDNA is protected and coated by the high affinity heterotrimeric ssDNA binding Replication Protein A (RPA). Almost all downstream DNA processes must first remodel/remove RPA or function alongside to access the ssDNA occluded under RPA. Formation of RPA-ssDNA complexes trigger the DNA damage checkpoint response and is a key step in activating most DNA repair and recombination pathways. Thus, in addition to protecting the exposed ssDNA, RPA functions as a gatekeeper to define functional specificity in DNA maintenance and genomic integrity. RPA achieves functional dexterity through a multi-domain architecture utilizing several DNA binding and protein-interaction domains connected by flexible linkers. This flexible and modular architecture enables RPA to adopt a myriad of configurations tailored for specific DNA metabolic roles. To experimentally capture the dynamics of the domains of RPA upon binding to ssDNA and interacting proteins we here describe the generation of active site-specific fluorescent versions of human RPA (RPA) using 4-azido-L-phenylalanine (4AZP) incorporation and click chemistry. This approach can also be applied to site-specific modifications of other multi-domain proteins. Fluorescence-enhancement through non-canonical amino acids (FEncAA) and Förster Resonance Energy Transfer (FRET) assays for measuring dynamics of RPA on DNA are also described. The fluorescent human RPA described here will enable high-resolution structure-function analysis of RPA-ssDNA interactions.

Systematic engineering enables efficient biosynthesis of L-phenylalanine in E. coli from inexpensive aromatic precursors.

BACKGROUND: L-phenylalanine is an essential amino acid with various promising applications. The microbial pathway for L-phenylalanine synthesis from glucose in wild strains involves lengthy steps and stringent feedback regulation that limits the production yield. It is attractive to find other candidates, which could be used to establish a succinct and cost-effective pathway for L-phenylalanine production. Here, we developed an artificial bioconversion process to synthesize L-phenylalanine from inexpensive aromatic precursors (benzaldehyde or benzyl alcohol). In particular, this work opens the possibility of L-phenylalanine production from benzyl alcohol in a cofactor self-sufficient system without any addition of reductant. RESULTS: The engineered L-phenylalanine biosynthesis pathway comprises two modules: in the first module, aromatic precursors and glycine were converted into phenylpyruvate, the key precursor for L-phenylalanine. The highly active enzyme combination was natural threonine aldolase LtaEP.p and threonine dehydratase A8HB.t, which could produce phenylpyruvate in a titer of 4.3 g/L. Overexpression of gene ridA could further increase phenylpyruvate production by 16.3%, reaching up to 5 g/L. The second module catalyzed phenylpyruvate to L-phenylalanine, and the conversion rate of phenylpyruvate was up to 93% by co-expressing PheDH and FDHV120S. Then, the engineered E. coli containing these two modules could produce L-phenylalanine from benzaldehyde with a conversion rate of 69%. Finally, we expanded the aromatic precursors to produce L-phenylalanine from benzyl alcohol, and firstly constructed the cofactor self-sufficient biosynthetic pathway to synthesize L-phenylalanine without any additional reductant such as formate. CONCLUSION: Systematical bioconversion processes have been designed and constructed, which could provide a potential bio-based strategy for the production of high-value L-phenylalanine from low-cost starting materials aromatic precursors.

Investigation of intermolecular interactions of l-Valine and l-Phenylalanine with muscle relaxant drug mephenesin molecule prevalent in aqueous solution by various physico-chemical methods at T=298.15K-313.15K.

A complete chemical analysis of significant intermolecular interactions of l-Valine (L-Val) and l-Phenylalanine (L-Phe) with Mephenesin (MEPN) molecules in aqueous solution has been studied by different physicochemical methodologies at various temperatures (T = 298.15 K-313.15 K at an interval of 5 K) and concentrations (0.001 mol kg-1, 0.003 mol kg-1, 0.005 mol kg-1) of aqueous MEPN solution. The limiting apparent molar volume (φV0) and experimental slope (SV*) values are found from the equation of Masson, viscosity A and B-coefficient determined using the equation of Jones-Doles, molar refraction (RM) and limiting molar refraction (RM0) derived by the Lorentz-Lorenz equation, express that in our experimental solution of amino acids (AAs) in aqueous MEPN, the solute-solvent interaction predominates over the solute-solute and solvent-solvent interactions for these ternary solutions. These are also justified by the measurement of various thermodynamic parameters, free energy of activation of viscous flow per mole of solvent(Δµ1°#) and solute (Δµ2°#), activation of viscous flow of enthalpies (ΔH°#) and entropies (ΔS°#). The characteristics of structure-breaking of solutes in the aqueous drug solution have been identified by Hepler's method and dB/dT value. The spectroscopic methods like UV-visible and proton-NMR studies help to explicate the strong AA-MEPN interactions in the solution phase and obtain a good correlation with theoretical studies. Theoretical investigations are checked to authenticate the experimental observations and according to both studies, L-Phe-MEPN interaction is greater than L-Val-MEPN interaction. The experimental and correlated research data are useful for the development of model combinations of AAs with drug molecules in pharmaceutical and medicinal chemistry.