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.

Bactericidal effect of tetracycline in E. coli strain ED1a may be associated with ribosome dysfunction.

Ribosomes translate the genetic code into proteins. Recent technical advances have facilitated in situ structural analyses of ribosome functional states inside eukaryotic cells and the minimal bacterium Mycoplasma. However, such analyses of Gram-negative bacteria are lacking, despite their ribosomes being major antimicrobial drug targets. Here we compare two E. coli strains, a lab E. coli K-12 and human gut isolate E. coli ED1a, for which tetracycline exhibits bacteriostatic and bactericidal action, respectively. Using our approach for close-to-native E. coli sample preparation, we assess the two strains by cryo-ET and visualize their ribosomes at high resolution in situ. Upon tetracycline treatment, these exhibit virtually identical drug binding sites, yet the conformation distribution of ribosomal complexes differs. While K-12 retains ribosomes in a translation-competent state, tRNAs are lost in the vast majority of ED1a ribosomes. These structural findings together with the proteome-wide abundance and thermal stability assessments indicate that antibiotic responses are complex in cells and can differ between different strains of a single species, thus arguing that all relevant bacterial strains should be analyzed in situ when addressing antibiotic mode of action.

Profiling expression strategies for a type III polyketide synthase in a lysate-based, cell-free system.

Some of the most metabolically diverse species of bacteria (e.g., Actinobacteria) have higher GC content in their DNA, differ substantially in codon usage, and have distinct protein folding environments compared to tractable expression hosts like Escherichia coli. Consequentially, expressing biosynthetic gene clusters (BGCs) from these bacteria in E. coli often results in a myriad of unpredictable issues with regard to protein expression and folding, delaying the biochemical characterization of new natural products. Current strategies to achieve soluble, active expression of these enzymes in tractable hosts can be a lengthy trial-and-error process. Cell-free expression (CFE) has emerged as a valuable expression platform as a testbed for rapid prototyping expression parameters. Here, we use a type III polyketide synthase from Streptomyces griseus, RppA, which catalyzes the formation of the red pigment flaviolin, as a reporter to investigate BGC refactoring techniques. We applied a library of constructs with different combinations of promoters and rppA coding sequences to investigate the synergies between promoter and codon usage. Subsequently, we assess the utility of cell-free systems for prototyping these refactoring tactics prior to their implementation in cells. Overall, codon harmonization improves natural product synthesis more than traditional codon optimization across cell-free and cellular environments. More importantly, the choice of coding sequences and promoters impact protein expression synergistically, which should be considered for future efforts to use CFE for high-yield protein expression. The promoter strategy when applied to RppA was not completely correlated with that observed with GFP, indicating that different promoter strategies should be applied for different proteins. In vivo experiments suggest that there is correlation, but not complete alignment between expressing in cell free and in vivo. Refactoring promoters and/or coding sequences via CFE can be a valuable strategy to rapidly screen for catalytically functional production of enzymes from BCGs, which advances CFE as a tool for natural product research.

Highly inclined light sheet allows volumetric super-resolution imaging of efflux pumps distribution in bacterial biofilms.

Bacterial biofilms are highly complex communities in which isogenic bacteria display different gene expression patterns and organize in a three-dimensional mesh gaining enhanced resistance to biocides. The molecular mechanisms behind such increased resistance remain mostly unknown, also because of the technical difficulties in biofilm investigation at the sub-cellular and molecular level. In this work we focus on the AcrAB-TolC protein complex, a multidrug efflux pump found in Enterobacteriaceae, whose overexpression is associated with most multiple drug resistance (MDR) phenotypes occurring in Gram-negative bacteria. We propose an optical method to quantify the expression level of the AcrAB-TolC pump within the biofilm volume at the sub-cellular level, with single-molecule sensitivity. Through a combination of super-resolution PALM with single objective light sheet and precision genome editing, we can directly quantify the spatial distribution of endogenous AcrAB-TolC pumps expressed in both planktonic bacteria and, importantly, within the bacterial biofilm volume. We observe a gradient of pump density within the biofilm volume and over the course of biofilm maturation. Notably, we propose an optical method that could be broadly employed to achieve volumetric super-resolution imaging of thick samples.

A production platform for disulfide-bonded peptides in the periplasm of Escherichia coli.

BACKGROUND: Recombinant peptide production in Escherichia coli provides a sustainable alternative to environmentally harmful and size-limited chemical synthesis. However, in-vivo production of disulfide-bonded peptides at high yields remains challenging, due to degradation by host proteases/peptidases and the necessity of translocation into the periplasmic space for disulfide bond formation. RESULTS: In this study, we established an expression system for efficient and soluble production of disulfide-bonded peptides in the periplasm of E. coli. We chose model peptides with varying complexity (size, structure, number of disulfide bonds), namely parathyroid hormone 1-84, somatostatin 1-28, plectasin, and bovine pancreatic trypsin inhibitor (aprotinin). All peptides were expressed without and with the N-terminal, low molecular weight CASPON™ tag (4.1 kDa), with the expression cassette being integrated into the host genome. During BioLector™ cultivations at microliter scale, we found that most of our model peptides can only be sufficiently expressed in combination with the CASPON™ tag, otherwise expression was only weak or undetectable on SDS-PAGE. Undesired degradation by host proteases/peptidases was evident even with the CASPON™ tag. Therefore, we investigated whether degradation happened before or after translocation by expressing the peptides in combination with either a co- or post-translational signal sequence. Our results suggest that degradation predominantly happened after the translocation, as degradation fragments appeared to be identical independent of the signal sequence, and expression was not enhanced with the co-translational signal sequence. Lastly, we expressed all CASPON™-tagged peptides in two industry-relevant host strains during C-limited fed-batch cultivations in bioreactors. We found that the process performance was highly dependent on the peptide-host-combination. The titers that were reached varied between 0.6-2.6 g L-1, and exceeded previously published data in E. coli. Moreover, all peptides were shown by mass spectrometry to be expressed to completion, including full formation of disulfide bonds. CONCLUSION: In this work, we demonstrated the potential of the CASPON™ technology as a highly efficient platform for the production of soluble peptides in the periplasm of E. coli. The titers we show here are unprecedented whenever parathyroid hormone, somatostatin, plectasin or bovine pancreatic trypsin inhibitor were produced in E. coli, thus making our proposed upstream platform favorable over previously published approaches and chemical synthesis.

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.

Metagenomic exploration of cold-active enzymes for detergent applications: Characterization of a novel, cold-active and alkali-stable GH8 endoglucanase from ikaite columns in SW Greenland.

Microbial communities from extreme environments are largely understudied, but are essential as producers of metabolites, including enzymes, for industrial processes. As cultivation of most microorganisms remains a challenge, culture-independent approaches for enzyme discovery in the form of metagenomics to analyse the genetic potential of a community are rapidly becoming the way forward. This study focused on analysing a metagenome from the cold and alkaline ikaite columns in Greenland, identifying 282 open reading frames (ORFs) that encoded putative carbohydrate-modifying enzymes with potential applications in, for example detergents and other processes where activity at low temperature and high pH is desired. Seventeen selected ORFs, representing eight enzyme families were synthesized and expressed in two host organisms, Escherichia coli and Aliivibrio wodanis. Aliivibrio wodanis demonstrated expression of a more diverse range of enzyme classes compared to E. coli, emphasizing the importance of alternative expression systems for enzymes from extremophilic microorganisms. To demonstrate the validity of the screening strategy, we chose a recombinantly expressed cellulolytic enzyme from the metagenome for further characterization. The enzyme, Cel240, exhibited close to 40% of its relative activity at low temperatures (4°C) and demonstrated endoglucanase characteristics, with a preference for cellulose substrates. Despite low sequence similarity with known enzymes, computational analysis and structural modelling confirmed its cellulase-family affiliation. Cel240 displayed activity at low temperatures and good stability at 25°C, activity at alkaline pH and increased activity in the presence of CaCl2, making it a promising candidate for detergent and washing industry applications.

Structure and mechanism of the K+/H+ exchanger KefC.

Intracellular potassium (K+) homeostasis is fundamental to cell viability. In addition to channels, K+ levels are maintained by various ion transporters. One major family is the proton-driven K+ efflux transporters, which in gram-negative bacteria is important for detoxification and in plants is critical for efficient photosynthesis and growth. Despite their importance, the structure and molecular basis for K+-selectivity is poorly understood. Here, we report ~3.1 Å resolution cryo-EM structures of the Escherichia coli glutathione (GSH)-gated K+ efflux transporter KefC in complex with AMP, AMP/GSH and an ion-binding variant. KefC forms a homodimer similar to the inward-facing conformation of Na+/H+ antiporter NapA. By structural assignment of a coordinated K+ ion, MD simulations, and SSM-based electrophysiology, we demonstrate how ion-binding in KefC is adapted for binding a dehydrated K+ ion. KefC harbors C-terminal regulator of K+ conductance (RCK) domains, as present in some bacterial K+-ion channels. The domain-swapped helices in the RCK domains bind AMP and GSH and they inhibit transport by directly interacting with the ion-transporter module. Taken together, we propose that KefC is activated by detachment of the RCK domains and that ion selectivity exploits the biophysical properties likewise adapted by K+-ion-channels.

Diversifying de novo TIM barrels by hallucination.

De novo protein design expands the protein universe by creating new sequences to accomplish tailor-made enzymes in the future. A promising topology to implement diverse enzyme functions is the ubiquitous TIM-barrel fold. Since the initial de novo design of an idealized four-fold symmetric TIM barrel, the family of de novo TIM barrels is expanding rapidly. Despite this and in contrast to natural TIM barrels, these novel proteins lack cavities and structural elements essential for the incorporation of binding sites or enzymatic functions. In this work, we diversified a de novo TIM barrel by extending multiple ßα-loops using constrained hallucination. Experimentally tested designs were found to be soluble upon expression in Escherichia coli and well-behaved. Biochemical characterization and crystal structures revealed successful extensions with defined α-helical structures. These diversified de novo TIM barrels provide a framework to explore a broad spectrum of functions based on the potential of natural TIM barrels.

Understanding the stability of a plastic-degrading Rieske iron oxidoreductase system.

Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPADO PDB ID 7Q05) is a structurally characterized heterohexameric α3ß3 RO that, with its cognate reductase (TPARED), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPADO/TPARED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPADO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (Tm) of 39.9°C for the monomeric TPARED, while the independent Tm of TPADO is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPADO with Tm values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol-1, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPADO subunits at 53.8°C, followed by higher-temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by ß-ß interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPADO stability, we propose prioritizing the re-engineering of the ß subunit interfaces, with subsequent targeted improvements of the subunits.

Structural and functional insights into transcription activation of the essential LysR-type transcriptional regulators.

The enormous LysR-type transcriptional regulators (LTTRs), which are diversely distributed amongst prokaryotes, play crucial roles in transcription regulation of genes involved in basic metabolic pathways, virulence and stress resistance. However, the precise transcription activation mechanism of these genes by LTTRs remains to be explored. Here, we determine the cryo-EM structure of a LTTR-dependent transcription activation complex comprising of Escherichia coli RNA polymerase (RNAP), an essential LTTR protein GcvA and its cognate promoter DNA. Structural analysis shows two N-terminal DNA binding domains of GcvA (GcvA_DBD) dimerize and engage the GcvA activation binding sites, presenting the -35 element for specific recognition with the conserved σ70R4. In particular, the versatile C-terminal domain of α subunit of RNAP directly interconnects with GcvA_DBD, σ70R4 and promoter DNA, providing more interfaces for stabilizing the complex. Moreover, molecular docking supports glycine as one potential inducer of GcvA, and single molecule photobleaching experiments kinetically visualize the occurrence of tetrameric GcvA-engaged transcription activation complex as suggested for the other LTTR homologs. Thus, a general model for tetrameric LTTR-dependent transcription activation is proposed. These findings will provide new structural and functional insights into transcription activation of the essential LTTRs.

A genetically encoded biosensor to monitor dynamic changes of c-di-GMP with high temporal resolution.

Monitoring changes of signaling molecules and metabolites with high temporal resolution is key to understanding dynamic biological systems. Here, we use directed evolution to develop a genetically encoded ratiometric biosensor for c-di-GMP, a ubiquitous bacterial second messenger regulating important biological processes like motility, surface attachment, virulence and persistence. The resulting biosensor, cdGreen2, faithfully tracks c-di-GMP in single cells and with high temporal resolution over extended imaging times, making it possible to resolve regulatory networks driving bimodal developmental programs in different bacterial model organisms. We further adopt cdGreen2 as a simple tool for in vitro studies, facilitating high-throughput screens for compounds interfering with c-di-GMP signaling and biofilm formation. The sensitivity and versatility of cdGreen2 could help reveal c-di-GMP dynamics in a broad range of microorganisms with high temporal resolution. Its design principles could also serve as a blueprint for the development of similar, orthogonal biosensors for other signaling molecules, metabolites and antibiotics.

Synthetic intrinsically disordered protein fusion tags that enhance protein solubility.

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation. Recombinant fusion of these SynIDPs to three known inclusion body forming proteins rescue their soluble expression and do not impede the activity of the fusion partner, thereby eliminating the need for removal of the SynIDP tag. These findings highlight the utility of SynIDPs as solubility tags, as they promote the soluble expression of proteins in E. coli and are small, unstructured proteins that minimally interfere with the biological activity of the fused protein.

Genome-Scale Investigation of the Regulation of azoR Expression in Escherichia coli Using Computational Analysis and Transposon Mutagenesis.

Bacterial azoreductases are enzymes that catalyze the reduction of ingested or industrial azo dyes. Although azoreductase genes have been well identified and characterized, the regulation of their expression has not been systematically investigated. To determine how different factors affect the expression of azoR, we extracted and analyzed transcriptional data from the Gene Expression Omnibus (GEO) resource, then confirmed computational predictions by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results showed that azoR expression was lower with higher glucose concentration, agitation speed, and incubation temperature, but higher at higher culture densities. Co-expression and clustering analysis indicated ten genes with similar expression patterns to azoR: melA, tpx, yhbW, yciK, fdnG, fpr, nfsA, nfsB, rutF, and chrR (yieF). In parallel, constructing a random transposon library in E. coli K-12 and screening 4320 of its colonies for altered methyl red (MR)-decolorizing activity identified another set of seven genes potentially involved in azoR regulation. Among these genes, arsC, relA, plsY, and trmM were confirmed as potential azoR regulators based on the phenotypic decolorization activity of their transposon mutants, and the expression of arsC and relA was confirmed, by qRT-PCR, to significantly increase in E. coli K-12 in response to different MR concentrations. Finally, the significant decrease in azoR transcription upon transposon insertion in arsC and relA (as compared to its expression in wild-type E. coli) suggests their probable involvement in azoR regulation. In conclusion, combining in silico analysis and random transposon mutagenesis suggested a set of potential regulators of azoR in E. coli.

Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies.

Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.

Development of a scalable recombinant system for cyclic beta-1,2-glucans production.

BACKGROUND: Cyclic ß-1,2-glucans (CßG) are bacterial cyclic homopolysaccharides with interesting biotechnological applications. These ring-shaped molecules have a hydrophilic surface that confers high solubility and a hydrophobic cavity able to include poorly soluble molecules. Several studies demonstrate that CßG and many derivatives can be applied in drug solubilization and stabilization, enantiomer separation, catalysis, synthesis of nanomaterials and even as immunomodulators, suggesting these molecules have great potential for their industrial and commercial exploitation. Nowadays, there is no method to produce CßG by chemical synthesis and bacteria that synthesize them are slow-growing or even pathogenic, which makes the scaling up of the process difficult and expensive. Therefore, scalable production and purification methods are needed to afford the demand and expand the repertoire of applications of CßG. RESULTS: We present the production of CßG in specially designed E. coli strains by means of the deletion of intrinsic polysaccharide biosynthetic genes and the heterologous expression of enzymes involved in CßG synthesis, transport and succinilation. These strains produce different types of CßG: unsubstituted CßG, anionic CßG and CßG of high size. Unsubstituted CßG with a degree of polymerization of 17 to 24 glucoses were produced and secreted to the culture medium by one of the strains. Through high cell density culture (HCDC) of that strain we were able to produce 4,5 g of pure unsubstituted CßG /L in culture medium within 48 h culture. CONCLUSIONS: We have developed a new recombinant bacterial system for the synthesis of cyclic ß-1,2-glucans, expanding the use of bacteria as a platform for the production of new polysaccharides with biotechnological applications. This new approach allowed us to produce CßG in E. coli with high yields and the highest volumetric productivity reported to date. We expect this new highly scalable system facilitates CßG availability for further research and the widespread use of these promising molecules across many application fields.

An integrated cofactor and co-substrate recycling pathway for the biosynthesis of 1,5-pentanediol.

BACKGROUND: 1,5-pentanediol (1,5-PDO) is a linear diol with an odd number of methylene groups, which is an important raw material for polyurethane production. In recent years, the chemical methods have been predominantly employed for synthesizing 1,5-PDO. However, with the increasing emphasis on environmentally friendly production, it has been a growing interest in the biosynthesis of 1,5-PDO. Due to the limited availability of only three reported feasible biosynthesis pathways, we developed a new biosynthetic pathway to form a cell factory in Escherichia coli to produce 1,5-PDO. RESULTS: In this study, we reported an artificial pathway for the synthesis of 1,5-PDO from lysine with an integrated cofactor and co-substrate recycling and also evaluated its feasibility in E.coli. To get through the pathway, we first screened aminotransferases originated from different organisms to identify the enzyme that could successfully transfer two amines from cadaverine, and thus GabT from E. coli was characterized. It was then cascaded with lysine decarboxylase and alcohol dehydrogenase from E. coli to achieve the whole-cell production of 1,5-PDO from lysine. To improve the whole-cell activity for 1,5-PDO production, we employed a protein scaffold of EutM for GabT assembly and glutamate dehydrogenase was also validated for the recycling of NADPH and α-ketoglutaric acid (α-KG). After optimizing the cultivation and bioconversion conditions, the titer of 1,5-PDO reached 4.03 mM. CONCLUSION: We established a novel pathway for 1,5-PDO production through two consecutive transamination reaction from cadaverine, and also integrated cofactor and co-substrate recycling system, which provided an alternative option for the biosynthesis of 1,5-PDO.

Blue light-mediated gene expression as a promising strategy to reduce antibiotic resistance in Escherichia coli.

The discovery of antibiotics has noticeably promoted the development of human civilization; however, antibiotic resistance in bacteria caused by abusing and overusing greatly challenges human health and food safety. Considering the worsening situation, it is an urgent demand to develop emerging nontraditional technologies or methods to address this issue. With the expanding of synthetic biology, optogenetics exhibits a tempting prospect for precisely regulating gene expression in many fields. Consequently, it is attractive to employ optogenetics to reduce the risk of antibiotic resistance. Here, a blue light-controllable gene expression system was established in Escherichia coli based on a photosensitive DNA-binding protein (EL222). Further, this strategy was successfully applied to repress the expression of ß-lactamase gene (bla) using blue light illumination, resulting a dramatic reduction of ampicillin resistance in engineered E. coli. Moreover, blue light was utilized to induce the expression of the mechanosensitive channel of large conductance (MscL), triumphantly leading to the increase of streptomycin susceptibility in engineered E. coli. Finally, the increased susceptibility of ampicillin and streptomycin was simultaneously induced by blue light in the same E. coli cell, revealing the excellent potential of this strategy in controlling multidrug-resistant (MDR) bacteria. As a proof of concept, our work demonstrates that light can be used as an alternative tool to prolong the use period of common antibiotics without developing new antibiotics. And this novel strategy based on optogenetics shows a promising foreground to combat antibiotic resistance in the future.

Improving the production of recombinant L-Asparaginase-II in Escherichia coli by co-expressing catabolite repressor activator (cra) gene.

Identification of a single genetic target for microbial strain improvement is difficult due to the complexity of the genetic regulatory network. Hence, a more practical approach is to identify bottlenecks in the regulatory networks that control critical metabolic pathways. The present work focuses on enhancing cellular physiology by increasing the metabolic flux through the central carbon metabolic pathway. Global regulator cra (catabolite repressor activator), a DNA-binding transcriptional dual regulator was selected for the study as it controls the expression of a large number of operons that modulate central carbon metabolism. To upregulate the activity of central carbon metabolism, the cra gene was co-expressed using a plasmid-based system. Co-expression of cra led to a 17% increase in the production of model recombinant protein L-Asparaginase-II. A pulse addition of 0.36% of glycerol every two hours post-induction, further increased the production of L-Asparaginase-II by 35% as compared to the control strain expressing only recombinant protein. This work exemplifies that upregulating the activity of central carbon metabolism by tuning the expression of regulatory genes like cra can relieve the host from cellular stress and thereby promote the growth as well as expression of recombinant hosts.

A novel GH3-ß-glucosidase from soda lake metagenomic libraries with desirable properties for biomass degradation.

Beta-glucosidases catalyze the hydrolysis of the glycosidic bonds of cellobiose, producing glucose, which is a rate-limiting step in cellulose biomass degradation. In industrial processes, ß-glucosidases that are tolerant to glucose and stable under harsh industrial reaction conditions are required for efficient cellulose hydrolysis. In this study, we report the molecular cloning, Escherichia coli expression, and functional characterization of a ß-glucosidase from the gene, CelGH3_f17, identified from metagenomics libraries of an Ethiopian soda lake. The CelGH3_f17 gene sequence contains a glycoside hydrolase family 3 catalytic domain (GH3). The heterologous expressed and purified enzyme exhibited optimal activity at 50 °C and pH 8.5. In addition, supplementation of 1 M salt and 300 mM glucose enhanced the ß-glucosidase activity. Most of the metal ions and organic solvents tested did not affect the ß-glucosidase activity. However, Cu2+ and Mn2+ ions, Mercaptoethanol and Triton X-100 reduce the activity of the enzyme. The studied ß-glucosidase enzyme has multiple industrially desirable properties including thermostability, and alkaline, salt, and glucose tolerance.