Protease S of entomopathogenic bacterium Photorhabdus laumondii: expression, purification and effect on greater wax moth Galleria mellonella.

BACKGROUND: Protease S (PrtS) from Photorhabdus laumondii belongs to the group of protealysin-like proteases (PLPs), which are understudied factors thought to play a role in the interaction of bacteria with other organisms. Since P. laumondii is an insect pathogen and a nematode symbiont, the analysis of the biological functions of PLPs using the PrtS model provides novel data on diverse types of interactions between bacteria and hosts. METHODS AND RESULTS: Recombinant PrtS was produced in Escherichia coli. Efficient inhibition of PrtS activity by photorin, a recently discovered emfourin-like protein inhibitor from P. laumondii, was demonstrated. The Galleria mellonella was utilized to examine the insect toxicity of PrtS and the impact of PrtS on hemolymph proteins in vitro. The insect toxicity of PrtS is reduced compared to protease homologues from non-pathogenic bacteria and is likely not essential for the infection process. However, using proteomic analysis, potential PrtS targets have been identified in the hemolymph. CONCLUSIONS: The spectrum of identified proteins indicates that the function of PrtS is to modulate the insect immune response. Further studies of PLPs' biological role in the PrtS and P. laumondii model must clarify the details of PrtS interaction with the insect immune system during bacterial infection.

Engineering cascade biocatalysis in whole cells for syringic acid bioproduction.

BACKGROUND: Syringic acid (SA) is a high-value natural compound with diverse biological activities and wide applications, commonly found in fruits, vegetables, and herbs. SA is primarily produced through chemical synthesis, nonetheless, these chemical methods have many drawbacks, such as considerable equipment requirements, harsh reaction conditions, expensive catalysts, and numerous by-products. Therefore, in this study, a novel biotransformation route for SA production was designed and developed by using engineered whole cells. RESULTS: An O-methyltransferase from Desulfuromonas acetoxidans (DesAOMT), which preferentially catalyzes a methyl transfer reaction on the meta-hydroxyl group of catechol analogues, was identified. The whole cells expressing DesAOMT can transform gallic acid (GA) into SA when S-adenosyl methionine (SAM) is used as a methyl donor. We constructed a multi-enzyme cascade reaction in Escherichia coli, containing an endogenous shikimate kinase (AroL) and a chorismate lyase (UbiC), along with a p-hydroxybenzoate hydroxylase mutant (PobA**) from Pseudomonas fluorescens, and DesAOMT; SA was biosynthesized from shikimic acid (SHA) by using whole cells catalysis. The metabolic system of chassis cells also affected the efficiency of SA biosynthesis, blocking the chorismate metabolism pathway improved SA production. When the supply of the cofactor NADPH was optimized, the titer of SA reached 133 µM (26.2 mg/L). CONCLUSION: Overall, we designed a multi-enzyme cascade in E. coli for SA biosynthesis by using resting or growing whole cells. This work identified an O-methyltransferase (DesAOMT), which can catalyze the methylation of GA to produce SA. The multi-enzyme cascade containing four enzymes expressed in an engineered E. coli for synthesizing of SA from SHA. The metabolic system of the strain and biotransformation conditions influenced catalytic efficiency. This study provides a new green route for SA biosynthesis.

Escherichia coli self-organizes developmental rosettes.

Rosettes are self-organizing, circular multicellular communities that initiate developmental processes, like organogenesis and embryogenesis, in complex organisms. Their formation results from the active repositioning of adhered sister cells and is thought to distinguish multicellular organisms from unicellular ones. Though common in eukaryotes, this multicellular behavior has not been reported in bacteria. In this study, we found that Escherichia coli forms rosettes by active sister-cell repositioning. After division, sister cells "fold" to actively align at the 2- and 4-cell stages of clonal division, thereby producing rosettes with characteristic quatrefoil configuration. Analysis revealed that folding follows an angular random walk, composed of ~1 µm strokes and directional randomization. We further showed that this motion was produced by the flagellum, the extracellular tail whose rotation generates swimming motility. Rosette formation was found to require de novo flagella synthesis suggesting it must balance the opposing forces of Ag43 adhesion and flagellar propulsion. We went on to show that proper rosette formation was required for subsequent morphogenesis of multicellular chains, rpoS gene expression, and formation of hydrostatic clonal-chain biofilms. Moreover, we found self-folding rosette-like communities in the standard motility assay, indicating that this behavior may be a general response to hydrostatic environments in E. coli. These findings establish self-organization of clonal rosettes by a prokaryote and have implications for evolutionary biology, synthetic biology, and medical microbiology.

Immune Response to the Recombinant Apa Protein from Mycobacterium tuberculosis Expressed in Streptomyces lividans After Intranasal Administration in Mice. Induction of Protective Response to Tubercle Bacillus Aerosols Exposure.

Identifying and evaluating potential vaccine candidates has become one of the main objectives to combat tuberculosis. Among them, mannosylated Apa antigen from Mycobacterium tuberculosis and the non-mannosylated protein expressed in Escherichia coli, have been studied. Although both proteins can induce a protective response in mice, it has been considered that native protein can be dispensed. In this work, we study the protective response induced by Apa expressed in E. coli and in Streptomyces lividans. The latter, like native is secreted as a double band of 45/47 kDa, however, only its 47 kDa band is mannosylated. Both antigens and BCG were intranasal administrated in mice, and animals were then challenged by aerosol with M. tuberculosis H37Rv. The results showed that both, Apa from S. lividans and E. coli conferred statistically significantly protection to animals compared to controls. The cytokine immune response was studied by an immunoassay after animals' immunization, revealing that Apa from S. lividans induced a statistically significant proliferation of T cell, as well as the expression of IFN-γ, IL-1ß, IL-17 and IL-10. In contrast, non-proliferation was obtained with non-mannosylated protein, but induction of IL-12 and IL-17 was observed. Together, these results demonstrate that both proteins were able to modulate a specific immune response against M. tuberculosis, that could be driven by different mechanisms possibly associated with the presence or not of mannosylation. Furthermore, stimulation of cells from BCG-vaccinated animals with the proteins could be an important tool, to help define the use of a given subunit-vaccine after BCG vaccination.

Proteomics and metabolic burden analysis to understand the impact of recombinant protein production in E. coli.

The impact of recombinant protein production (RPP) on host cells and the metabolic burden associated with it undermine the efficiency of the production system. This study utilized proteomics to investigate the dynamics of parent and recombinant cells induced at different time points for RPP. The results revealed significant changes in both transcriptional and translational machinery that may have impacted the metabolic burden, growth rate of the culture and the RPP. The timing of protein synthesis induction also played a critical role in the fate of the recombinant protein within the host cell, affecting protein and product yield. The study identified significant differences in the expression of proteins involved in fatty acid and lipid biosynthesis pathways between two E. coli host strains (M15 and DH5⍺), with the E. coli M15 strain demonstrating superior expression characteristics for the recombinant protein. Overall, these findings contribute to the knowledge base for rational strain engineering for optimized recombinant protein production.

Queuosine biosynthetic enzyme, QueE moonlights as a cell division regulator.

In many organisms, stress responses to adverse environments can trigger secondary functions of certain proteins by altering protein levels, localization, activity, or interaction partners. Escherichia coli cells respond to the presence of specific cationic antimicrobial peptides by strongly activating the PhoQ/PhoP two-component signaling system, which regulates genes important for growth under this stress. As part of this pathway, a biosynthetic enzyme called QueE, which catalyzes a step in the formation of queuosine (Q) tRNA modification is upregulated. When cellular QueE levels are high, it co-localizes with the central cell division protein FtsZ at the septal site, blocking division and resulting in filamentous growth. Here we show that QueE affects cell size in a dose-dependent manner. Using alanine scanning mutagenesis of amino acids in the catalytic active site, we pinpoint residues in QueE that contribute distinctly to each of its functions-Q biosynthesis or regulation of cell division, establishing QueE as a moonlighting protein. We further show that QueE orthologs from enterobacteria like Salmonella typhimurium and Klebsiella pneumoniae also cause filamentation in these organisms, but the more distant counterparts from Pseudomonas aeruginosa and Bacillus subtilis lack this ability. By comparative analysis of E. coli QueE with distant orthologs, we elucidate a unique region in this protein that is responsible for QueE's secondary function as a cell division regulator. A dual-function protein like QueE is an exception to the conventional model of "one gene, one enzyme, one function", which has divergent roles across a range of fundamental cellular processes including RNA modification and translation to cell division and stress response.

A Guide to Computational Cotranscriptional Folding Featuring the SRP RNA.

Although RNA molecules are synthesized via transcription, little is known about the general impact of cotranscriptional folding in vivo. We present different computational approaches for the simulation of changing structure ensembles during transcription, including interpretations with respect to experimental data from literature. Specifically, we analyze different mutations of the E. coli SRP RNA, which has been studied comparatively well in previous literature, yet the details of which specific metastable structures form as well as when they form are still under debate. Here, we combine thermodynamic and kinetic, deterministic, and stochastic models with automated and visual inspection of those systems to derive the most likely scenario of which substructures form at which point during transcription. The simulations do not only provide explanations for present experimental observations but also suggest previously unnoticed conformations that may be verified through future experimental studies.

A novel N-acetylglucosamine-6-phosphate deacetylase that is essential for chitin utilization in the chitinolytic bacterium, Chitiniphilus shinanonensis.

AIMS: We aimed to investigate the function of an unidentified gene annotated as a PIG-L domain deacetylase (cspld) in Chitiniphilus shinanonensis SAY3. cspld was identified using transposon mutagenesis, followed by negatively selecting a mutant incapable of growing on chitin, a polysaccharide consisting of N-acetyl-d-glucosamine (GlcNAc). We focused on the physiological role of CsPLD protein in chitin utilization. METHODS AND RESULTS: Recombinant CsPLD expressed in Escherichia coli exhibited GlcNAc-6-phosphate deacetylase (GPD) activity, which is involved in the metabolism of amino sugars. However, SAY3 possesses two genes (csnagA1 and csnagA2) in its genome that code for proteins whose primary sequences are homologous to those of typical GPDs. Recombinant CsNagA1 and CsNagA2 also exhibited GPD activity with 23 and 1.6% of catalytic efficiency (kcat/Km), respectively, compared to CsPLD. The gene-disrupted mutant, Δcspld was unable to grow on chitin or GlcNAc, whereas the three mutants, ΔcsnagA1, ΔcsnagA2, and ΔcsnagA1ΔcsnagA2 grew similarly to SAY3. The determination of GPD activity in the crude extracts of each mutant revealed that CsPLD is a major enzyme that accounts for almost all cellular activities. CONCLUSIONS: Deacetylation of GlcNAc-6P catalyzed by CsPLD (but not by typical GPDs) is essential for the assimilation of chitin and its constituent monosaccharide, GlcNAc, as a carbon and energy source in C. shinanonensis.

Construction of a grpE-based plasmid addiction system in Escherichia coli and its application in phloroglucinol biosynthesis.

AIM: Biotechnical processes in Escherichia coli often operate with artificial plasmids. However, these bioprocesses frequently encounter plasmid loss. To ensure stable expression of heterologous genes in E. coli BL21(DE3), a novel plasmid addiction system (PAS) was developed. METHODS AND RESULTS: This PAS employed an essential gene grpE encoding a cochaperone in the DnaK-DnaJ-GrpE chaperone system as the selection marker, which represented a chromosomal ΔgrpE mutant harboring episomal expression plasmids that carry supplementary grpE alleles to restore the deficiency. To demonstrate the feasibility of this system, it was implemented in phloroglucinol (PG) biosynthesis, manifesting improved host tolerance to PG and increased PG production. Specifically, PG titer significantly improved from 0.78 ± 0.02 to 1.34 ± 0.04 g l-1, representing a 71.8% increase in shake-flask fermentation. In fed-batch fermentation, the titer increased from 3.71 ± 0.11 to 4.54 ± 0.10 g l-1, showing a 22.4% increase. RNA sequencing and transcriptome analysis revealed that the improvements were attributed to grpE overexpression and upregulation of various protective chaperones and the biotin acetyl-CoA carboxylase ligase coding gene birA. CONCLUSION: This novel PAS could be regarded as a typical example of nonanabolite- and nonmetabolite-related PAS. It effectively promoted plasmid maintenance in the host, improved tolerance to PG, and increased the titer of this compound.

Membrane Permeability Dominates over Electrostatic Interactions in Dictating Drug Transport in Osmotically Shocked Escherichia coli.

To combat surging multidrug-resistant Gram-negative bacterial infections, better strategies to improve the efficacy of existing drugs are critical. Because the dual membrane cell envelope is the first line of defense for these bacteria, it is crucial to understand the permeation properties of the drugs through it. Our recent study shows that isosmotic conditions prevent drug permeation inside Gram-negative bacteria, Escherichia coli, while hypoosmotic stress enhances the process. Here, we unravel the reason behind such differential drug penetration. Specifically, we dissect the roles of electrostatic screening and low membrane permeability in the penetration failure of drugs under osmotically balanced conditions. We compare the transport of a quaternary ammonium compound malachite green in the presence of an electrolyte (NaCl) and a wide variety of commonly used organic osmolytes, e.g., sucrose, proline, glycerol, sorbitol, and urea. These osmolytes of different membrane permeability (i.e., nonpermeable sucrose and NaCl, freely permeable urea and glycerol, and partially permeable proline and sorbitol) clarify the role of osmotic stress in cell envelope permeability. The results showcase that under balanced osmotic conditions, drug molecules fail to penetrate inside E. coli cells because of low membrane permeabilities and not because of electrostatic screening imposed by the osmolytes. Contribution of the electrostatic interactions, however, cannot be completely overruled as at osmotically imbalanced conditions, drug transport across the bacterial subcellular compartments is found to be dependent on the osmolytes used.

Antimicrobial resistance pattern of Uro-Pathogens emphasizing non-lactose fermenting gram negative bacilli.

Objectives: To identify various species of non-lactose fermenting gram-negative bacilli involved in urinary tract infections, and to determine their antimicrobial resistance pattern. METHODS: The retrospective, descriptive, cross-sectional study was conducted from January 1 to April 1, 2022, at the Dow University of Health Sciences, Karachi, and comprised data from the institutional diagnostic laboratory that was related to urine samples regardless of age and gender from January 1, 2020, to December 31, 2021. Data was analysed using SPSS version 25. RESULTS: Of the 103,887 urine samples, 41,280(39.7%) were positive, 51,146(49.2%) showed no bacterial growth, 11,000(10.6%) had non-significant bacterial growth and 461(0.4%) had mixed bacterial growth. Of the positive samples, 18359(44.5%) were positive in 2020, and 22,921(55.5%) in 2021. Gram-negative lactose fermenting bacteria included escherichia coli 23,123(22.3%) and klebsiella pneumoniae 2,993(2.9%), gram-negative non-lactose fermenting bacteria included pseudomonas aeruginosa 1,110(1.07%), and gram-positive bacteria included enterococcus 8,008(7.7%). Pseudomonas aeruginosa was most resistant against tobramycin 880(79.3%) and least resistant against piperacillin-tazobactam 146(13%). CONCLUSIONS: Piperacillin-tazobactam was highly sensitive drug against non-lactose fermenting uro-pathogens.

Heteroxylan hydrolysis by a recombinant cellulase-free GH10 xylanase from the alkaliphilic bacterium Halalkalibacterium halodurans C-125.

The search for affordable enzymes with exceptional characteristics is fundamental to overcoming industrial and environmental constraints. In this study, a recombinant GH10 xylanase (Xyn10-HB) from the extremely alkaliphilic bacterium Halalkalibacterium halodurans C-125 cultivated at pH 10 was cloned and expressed in E. coli BL21(DE3). Removal of the signal peptide improved the expression, and an overall activity of 8 U/mL was obtained in the cell-free supernatant. The molecular weight of purified Xyn10-HB was estimated to be 42.6 kDa by SDS-PAGE. The enzyme was active across a wide pH range (5-10) with optimal activity recorded at pH 8.5 and 60 °C. It also presented good stability with a half-life of 3 h under these conditions. Substrate specificity studies showed that Xyn10-HB is a cellulase-free enzyme that conventionally hydrolyse birchwood and oat spelts xylans (Apparent Km of 0.46 mg/mL and 0.54 mg/mL, respectively). HPLC analysis showed that both xylans hydrolysis produced xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from 2 to 9. The conversion yield was 77% after 24 h with xylobiose and xylotriose as the main end-reaction products. When assayed on alkali-extracted wheat straw heteroxylan, the Xyn10-HB produced active XOS with antioxidant activity determined by the DPPH radical scavenging method (IC50 of 0.54 mg/mL after 4 h). Owing to its various characteristics, Xyn10-HB xylanase is a promising candidate for multiple biotechnological applications.

A Thermoplastic Microsystem to Perform Antibiotic Susceptibility Testing by Monitoring Oxygen Consumption.

Antibiotic susceptibility testing (AST) is a routine procedure in diagnostic laboratories to determine pathogen resistance profiles toward antibiotics. The need for fast and accurate resistance results is rapidly increasing with a global rise in pathogen antibiotic resistance over the past years. Microfluidic technologies can enable AST with lower volumes, lower cell numbers, and a reduction in the sample-to-result time compared to state-of-the-art systems. We present a protocol to perform AST on a miniaturized nanoliter chamber array platform. The chambers are filled with antibiotic compounds and oxygen-sensing nanoprobes that serve as a viability indicator. The growth of bacterial cells in the presence of different concentrations of antibiotics is monitored; living cells consume oxygen, which can be observed as an increase of a luminesce signal within the growth chambers. Here, we demonstrate the technique using a quality control Escherichia coli strain, ATCC 35218. The AST requires 20 µL of a diluted bacterial suspension (OD600 = 0.02) and provides resistance profiles about 2-3 h after the inoculation. The microfluidic method can be adapted to other aerobic pathogens and is of particular interest for slow-growing strains.

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.

Features of the DNA Escherichia coli RecN interaction revealed by fluorescence microscopy and single-molecule methods.

The SOS response is a condition that occurs in bacterial cells after DNA damage. In this state, the bacterium is able to reсover the integrity of its genome. Due to the increased level of mutagenesis in cells during the repair of DNA double-strand breaks, the SOS response is also an important mechanism for bacterial adaptation to the antibiotics. One of the key proteins of the SOS response is the SMC-like protein RecN, which helps the RecA recombinase to find a homologous DNA template for repair. In this work, the localization of the recombinant RecN protein in living Escherichia coli cells was revealed using fluorescence microscopy. It has been shown that the RecN, outside the SOS response, is predominantly localized at the poles of the cell, and in dividing cells, also localized at the center. Using in vitro methods including fluorescence microscopy and optical tweezers, we show that RecN predominantly binds single-stranded DNA in an ATP-dependent manner. RecN has both intrinsic and single-stranded DNA-stimulated ATPase activity. The results of this work may be useful for better understanding of the SOS response mechanism and homologous recombination process.

Nonlethal deleterious mutation-induced stress accelerates bacterial aging.

Random mutagenesis, including when it leads to loss of gene function, is a key mechanism enabling microorganisms' long-term adaptation to new environments. However, loss-of-function mutations are often deleterious, triggering, in turn, cellular stress and complex homeostatic stress responses, called "allostasis," to promote cell survival. Here, we characterize the differential impacts of 65 nonlethal, deleterious single-gene deletions on Escherichia coli growth in three different growth environments. Further assessments of select mutants, namely, those bearing single adenosine triphosphate (ATP) synthase subunit deletions, reveal that mutants display reorganized transcriptome profiles that reflect both the environment and the specific gene deletion. We also find that ATP synthase α-subunit deleted (ΔatpA) cells exhibit elevated metabolic rates while having slower growth compared to wild-type (wt) E. coli cells. At the single-cell level, compared to wt cells, individual ΔatpA cells display near normal proliferation profiles but enter a postreplicative state earlier and exhibit a distinct senescence phenotype. These results highlight the complex interplay between genomic diversity, adaptation, and stress response and uncover an "aging cost" to individual bacterial cells for maintaining population-level resilience to environmental and genetic stress; they also suggest potential bacteriostatic antibiotic targets and -as select human genetic diseases display highly similar phenotypes, - a bacterial origin of some human diseases.

Key interactions of RNA polymerase with 6S RNA and secondary channel factors during pRNA synthesis.

Small non-coding 6S RNA mimics DNA promoters and binds to the σ70 holoenzyme of bacterial RNA polymerase (RNAP) to suppress transcription of various genes mainly during the stationary phase of cell growth or starvation. This inhibition can be relieved upon synthesis of short product RNA (pRNA) performed by RNAP from the 6S RNA template. Here, we have shown that pRNA synthesis depends on specific contacts of 6S RNA with RNAP and interactions of the σ finger with the RNA template in the active site of RNAP, and is also modulated by the secondary channel factors. We have adapted a molecular beacon assay with fluorescently labeled σ70 to analyze 6S RNA release during pRNA synthesis. We found the kinetics of 6S RNA release to be oppositely affected by mutations in the σ finger and in the CRE pocket of core RNAP, similarly to the reported role of these regions in promoter-dependent transcription. Secondary channel factors, DksA and GreB, inhibit pRNA synthesis and 6S RNA release from RNAP, suggesting that they may contribute to the 6S RNA-mediated switch in transcription during stringent response. Our results demonstrate that pRNA synthesis depends on a similar set of contacts between RNAP and 6S RNA as in the case of promoter-dependent transcription initiation and reveal that both processes can be regulated by universal transcription factors acting on RNAP.

An In Vitro Hybrid Biocatalytic System Enabled by a Combination of Surface-Displayed, Purified, and Cell-Free Expressed Enzymes.

Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to (S)-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed. To assemble them, we fused two orthogonal peptide-protein pairs (i.e., SpyTag/SpyCatcher and SnoopTag/SnoopCatcher) to the three enzymes, allowing their spatial organization by covalent assembly. By doing this, we constructed a multienzyme complex, which could enhance the production of (S)-1-phenyl-1,2-ethanediol by 3 times compared to the free-floating enzyme system without assembly. After optimization of the reaction system, the final product yield reached 234.6 µM with a substrate conversion rate of 46.9% (based on 0.5 mM styrene). Taken together, our strategy integrates the merits of advanced biochemical engineering techniques, including cellular surface display, spatial enzyme organization, and cell-free expression, which offers a new solution for chemical biosynthesis by enzymatic cascade biotransformation. We, therefore, anticipate that our approach will hold great potential for designing and constructing highly efficient systems to synthesize chemicals of agricultural, industrial, and pharmaceutical significance.

Metabolic Tug-of-War between Glycolysis and Translation Revealed by Biochemical Reconstitution.

Inside cells, various biological systems work cooperatively for homeostasis and self-replication. These systems do not work independently as they compete for shared elements like ATP and NADH. However, it has been believed that such competition is not a problem in codependent biological systems such as the energy-supplying glycolysis and the energy-consuming translation system. In this study, we biochemically reconstituted the coupling system of glycolysis and translation using purified elements and found that the competition for ATP between glycolysis and protein synthesis interferes with their coupling. Both experiments and simulations revealed that this interference is derived from a metabolic tug-of-war between glycolysis and translation based on their reaction rates, which changes the threshold of the initial substrate concentration for the success coupling. By the metabolic tug-of-war, translation energized by strong glycolysis is facilitated by an exogenous ATPase, which normally inhibits translation. These findings provide chemical insights into the mechanism of competition among biological systems in living cells and provide a framework for the construction of synthetic metabolism in vitro.

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.