scholarly journals Mixing and matching methylotrophic enzymes to design a novel methanol utilization pathway in E. coli

2020 ◽  
Author(s):  
A. De Simone ◽  
C.M. Vicente ◽  
C. Peiro ◽  
L. Gales ◽  
F. Bellvert ◽  
...  
Keyword(s):  
Amino Acids ◽  
High Throughput ◽  
In Silico ◽  
Bacterial Production ◽  
Low Cost ◽  
E Coli ◽  
Considerable Research ◽  
One Step ◽  
Utilization Pathway ◽  
Dihydroxyacetone Synthase

ABSTRACTOne-carbon (C1) compounds, such as methanol, have recently gained attention as alternative low-cost and non-food feedstocks for microbial bioprocesses. Considerable research efforts are thus currently focused on the generation of synthetic methylotrophs by transferring methanol assimilation pathways into established bacterial production hosts. In this study, we used an iterative combination of dry and wet approaches to design, implement and optimize this metabolic trait in the most common chassis, E. coli. Through in silico modeling, we designed a new route that “mixed and matched” two methylotrophic enzymes: a bacterial methanol dehydrogenase (Mdh) and a dihydroxyacetone synthase (Das) from yeast. To identify the best combination of enzymes to introduce into E. coli, we built a library of 266 pathway variants containing different combinations of Mdh and Das homologues and screened it using high-throughput 13C-labeling experiments. The highest level of incorporation, 22% of labeled methanol carbon into the multi-carbon compound PEP, was obtained using a variant composed of a Mdh from A. gerneri and a codon-optimized version of P. angusta Das. Finally, the activity of this new synthetic pathway was further improved by engineering strategic metabolic targets identified using omics and modelling approaches. The final synthetic strain had 1.5 to 5.9 times higher methanol assimilation in intracellular metabolites and proteinogenic amino acids than the starting strain did. Broadening the repertoire of methanol assimilation pathways is one step further toward synthetic methylotrophy in E. coli.

scholarly journals Bacteria Getting into Shape: Genetic Determinants of E. coli Morphology

mBio ◽  
2017 ◽  
Vol 8 (2) ◽  
Author(s):  
Shawn French ◽  
Jean-Philippe Côté ◽  
Jonathan M. Stokes ◽  
Ray Truant ◽  
Eric D. Brown
Keyword(s):  
High Throughput ◽  
Genomic Library ◽  
Low Cost ◽  
Genetic Mutation ◽  
Cellular Functions ◽  
E Coli ◽  
Genetic Perturbations ◽  
Screening Platform ◽  
The Impact ◽  
Very High

ABSTRACT Perturbation of cellular processes is a prevailing approach to understanding biology. To better understand the complicated biology that defines bacterial shape, a sensitive, high-content platform was developed to detect multiple morphological defect phenotypes using microscopy. We examined morphological phenotypes across the Escherichia coli K-12 deletion (Keio) collection at the mid-exponential growth phase, revealing 111 deletions perturbing shape. Interestingly, 64% of these were uncharacterized mutants, illustrating the complex nature of shape maintenance and regulation in bacteria. To understand the roles these genes play in defining morphology, 53 mutants with knockouts resulting in abnormal cell shape were crossed with the Keio collection in high throughput, generating 1,373 synthetic lethal interactions across 1.7 million double deletion mutants. This analysis yielded a highly populated interaction network spanning and linking multiple phenotypes, with a preponderance of interactions involved in transport, oxidation-reduction, and metabolic processes. IMPORTANCE Genetic perturbations of cellular functions are a prevailing approach to understanding cell systems, which are increasingly being practiced in very high throughput. Here, we report a high-content microscopy platform tailored to bacteria, which probes the impact of genetic mutation on cell morphology. This has particular utility in revealing elusive and subtle morphological phenotypes associated with blocks in nonessential cellular functions. We report 111 nonessential mutations impacting E. coli morphology, with nearly half of those genes being poorly annotated or uncharacterized. Further, these genes appear to be tightly linked to transport or redox processes within the cell. The screening platform is simple and low cost and is broadly applicable to any bacterial genomic library or chemical collection. Indeed, this is a powerful tool in understanding the biology behind bacterial shape. IMPORTANCE Genetic perturbations of cellular functions are a prevailing approach to understanding cell systems, which are increasingly being practiced in very high throughput. Here, we report a high-content microscopy platform tailored to bacteria, which probes the impact of genetic mutation on cell morphology. This has particular utility in revealing elusive and subtle morphological phenotypes associated with blocks in nonessential cellular functions. We report 111 nonessential mutations impacting E. coli morphology, with nearly half of those genes being poorly annotated or uncharacterized. Further, these genes appear to be tightly linked to transport or redox processes within the cell. The screening platform is simple and low cost and is broadly applicable to any bacterial genomic library or chemical collection. Indeed, this is a powerful tool in understanding the biology behind bacterial shape.

scholarly journals Constructing an ethanol utilization pathway in Escherichia coli to produce acetyl-CoA derived compounds

2020 ◽  
Author(s):  
Hong Liang ◽  
Xiaoqiang Ma ◽  
Wenbo Ning ◽  
Yurou Liu ◽  
Anthony J. Sinskey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Carbon Source ◽  
Sole Carbon Source ◽  
Structural Similarity ◽  
List Type ◽  
Acetyl Coa ◽  
E Coli ◽  
Utilization Pathway ◽  
Aspartate Family

AbstractEngineering microbes to utilize non-conventional substrates could create short and efficient pathways to convert substrate into product. In this study, we designed and constructed a two-step heterologous ethanol utilization pathway (EUP) in Escherichia coli by using acetaldehyde dehydrogenase (encoded by ada) from Dickeya zeae and alcohol dehydrogenase (encoded by adh2) from Saccharomyces cerevisiae. This EUP can convert ethanol into acetyl-CoA without ATP consumption, and generate two molecules of NADH per molecule of ethanol. We optimized the expression of these two genes and found that ethanol consumption could be improved by expressing them in a specific order (ada-adh2) with a constitutive promoter (PgyrA). The engineered E. coli strain with EUP consumed approximately 8 g/L of ethanol in 96 hours when it was used as sole carbon source. Subsequently, we combined EUP with the biosynthesis of polyhydroxybutyrate (PHB), a biodegradable polymer derived from acetyl-CoA. The engineered E. coli strain carrying EUP and PHB biosynthetic pathway produced 1.1 g/L of PHB from 10 g/L of ethanol and 1 g/L of aspartate family amino acids in 96 hours. We also engineered E. coli strain to produced 24 mg/L of prenol from 10 g/L of ethanol in 48 hours, supporting the feasibility of converting ethanol into different classes of acetyl-CoA derived compounds.HighlightsEngineered Escherichia coli strains to grow on ethanol as sole carbon sourceDemonstrated that ethanol was converted into acetyl-CoA (AcCoA) through two pathways (acetaldehyde-acetate-AcCoA and acetaldehyde-AcCoA)Converted ethanol into two acetyl-CoA derived products with low structural similarity (polyhydroxybutyrate and prenol)Discovered that supplementation of the aspartate family amino acids can substantially improve cell growth on ethanol

Dual Nickel/Palladium-Catalyzed Reductive Cross-Coupling Reactions Between Two Phenol Derivatives

2020 ◽  
Author(s):  
Baojian Xiong ◽  
Yue Li ◽  
Yin Wei ◽  
Søren Kramer ◽  
Zhong Lian
Keyword(s):  
Functional Group ◽  
Low Cost ◽  
Cross Coupling ◽  
Coupling Reactions ◽  
Phenol Derivatives ◽  
Cross Coupling Reactions ◽  
Palladium Catalyzed ◽  
One Step ◽  
Aryl Tosylates ◽  
Low Sensitivity

Cross-coupling between substrates that can be easily derived from phenols is highly attractive due to the abundance and low cost of phenols. Here, we report a dual nickel/palladium-catalyzed reductive cross-coupling between aryl tosylates and aryl triflates; both substrates can be accessed in just one step from readily available phenols. The reaction has a broad functional group tolerance and substrate scope (>60 examples). Furthermore, it displays low sensitivity to steric effects demonstrated by the synthesis of a 2,2’disubstituted biaryl and a fully substituted aryl product. The widespread presence of phenols in natural products and pharmaceuticals allow for straightforward late-stage functionalization, illustrated with examples such as Ezetimibe and tyrosine. NMR spectroscopy and DFT calculations indicate that the nickel catalyst is responsible for activating the aryl triflate, while the palladium catalyst preferentially reacts with the aryl tosylate.

Surface Modification of Poly(Vinylchloride) for Manufacturing Advanced Catheters

2020 ◽  
Vol 27 (10) ◽  
pp. 1616-1633 ◽  
Author(s):  
Oana Cristina Duta ◽  
Aurel Mihail Ţîţu ◽  
Alexandru Marin ◽  
Anton Ficai ◽  
Denisa Ficai ◽  
...  
Keyword(s):  
Surface Modification ◽  
Chemical Modification ◽  
Medical Devices ◽  
Low Cost ◽  
Physical And Mechanical Properties ◽  
Polymeric Materials ◽  
Modern Medicine ◽  
Structural Failure ◽  
Chemical Grafting ◽  
One Step

Polymeric materials, due to their excellent physicochemical properties and versatility found applicability in multiples areas, including biomaterials used in tissue regeneration, prosthetics (hip, artificial valves), medical devices, controlled drug delivery systems, etc. Medical devices and their applications are very important in modern medicine and the need to develop new materials with improved properties or to improve the existent materials is increasing every day. Numerous reasearches are activated in this domain in order to obtain materials/surfaces that does not have drawbacks such as structural failure, calcifications, infections or thrombosis. One of the most used material is poly(vinylchloride) (PVC) due to its unique properties, availability and low cost. The most common method used for obtaining tubular devices that meet the requirements of medical use is the surface modification of polymers without changing their physical and mechanical properties, in bulk. PVC is a hydrophobic polymer and therefore many research studies were conducted in order to increase the hydrophilicity of the surface by chemical modification in order to improve biocompatibility, to enhance wettability, reduce friction or to make lubricious or antimicrobial coatings. Surface modification of PVC can be achieved by several strategies, in only one step or, in some cases, in two or more steps by applying several techniques consecutively to obtain the desired modification / performances. The most common processes used for modifying the surface of PVC devices are: plasma treatment, corona discharge, chemical grafting, electric discharge, vapour deposition of metals, flame treatment, direct chemical modification (oxidation, hydrolysis, etc.) or even some physical modification of the roughness of the surface.

Production and Preliminary In Vivo Evaluations of a Novel in silico-designed L2-based Potential HPV Vaccine

2020 ◽  
Vol 21 (4) ◽  
pp. 316-324
Author(s):  
Manica Negahdaripour ◽  
Navid Nezafat ◽  
Reza Heidari ◽  
Nasrollah Erfani ◽  
Nasim Hajighahramani ◽  
...  
Keyword(s):  
In Silico ◽  
Group Versus ◽  
Tlr Agonists ◽  
E Coli ◽  
Metal Affinity ◽  
Th1 And Th2 Cytokines ◽  
Ifn Γ ◽  
Humoral Responses ◽  
Future Work

Background: L2-based Human Papillomavirus (HPV) prophylactic vaccines, containing epitopes from HPV minor capsid proteins, are under investigation as second-generation HPV vaccines. No such vaccine has passed clinical trials yet, mainly due to the low immunogenicity of peptide vaccines; so efforts are being continued. A candidate vaccine composed of two HPV16 L2 epitopes, flagellin and a Toll-Like Receptor (TLR) 4 agonist (RS09) as adjuvants, and two universal T-helper epitopes was designed in silico in our previous researches. Methods: The designed vaccine construct was expressed in E. coli BL21 (DE3) and purified through metal affinity chromatography. Following mice vaccination, blood samples underwent ELISA and flow cytometry analyses for the detection of IgG and seven Th1 and Th2 cytokines. Results: Following immunization, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10) type cytokines, as well as IgG, were induced significantly compared with the PBS group. Significant increases in IFN-γ, IL-2, and IL-5 levels were observed in the vaccinated group versus Freund’s adjuvant group. Conclusion: The obtained cytokine induction profile implied both cellular and humoral responses, with a more Th-1 favored trend. However, an analysis of specific antibodies against L2 is required to confirm humoral responses. No significant elevation in inflammatory cytokines, (IL-6 and TNF-α), suggested a lack of unwanted inflammatory side effects despite using a combination of two TLR agonists. The designed construct might be capable of inducing adaptive and innate immunity; nevertheless, comprehensive immune tests were not conducted at this stage and will be a matter of future work.

Acetate Kinase (AcK) is Essential for Microbial Growth and Betel-derived Compounds Potentially Target AcK, PhoP and MDR Proteins in M. tuberculosis, V. cholerae and Pathogenic E. coli: An in silico and in vitro Study

2019 ◽  
Vol 18 (31) ◽  
pp. 2731-2740 ◽  
Author(s):  
Sandeep Tiwari ◽  
Debmalya Barh ◽  
M. Imchen ◽  
Eswar Rao ◽  
Ranjith K. Kumavath ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
In Silico ◽  
Pathogenic Bacteria ◽  
In Vitro Study ◽  
Docking Studies ◽  
Acetate Kinase ◽  
E Coli ◽  
Pathogenic Escherichia Coli ◽  
Novel Target

Background: Mycobacterium tuberculosis, Vibrio cholerae, and pathogenic Escherichia coli are global concerns for public health. The emergence of multi-drug resistant (MDR) strains of these pathogens is creating additional challenges in controlling infections caused by these deadly bacteria. Recently, we reported that Acetate kinase (AcK) could be a broad-spectrum novel target in several bacteria including these pathogens. Methods: Here, using in silico and in vitro approaches we show that (i) AcK is an essential protein in pathogenic bacteria; (ii) natural compounds Chlorogenic acid and Pinoresinol from Piper betel and Piperidine derivative compound 6-oxopiperidine-3-carboxylic acid inhibit the growth of pathogenic E. coli and M. tuberculosis by targeting AcK with equal or higher efficacy than the currently used antibiotics; (iii) molecular modeling and docking studies show interactions between inhibitors and AcK that correlate with the experimental results; (iv) these compounds are highly effective even on MDR strains of these pathogens; (v) further, the compounds may also target bacterial two-component system proteins that help bacteria in expressing the genes related to drug resistance and virulence; and (vi) finally, all the tested compounds are predicted to have drug-like properties. Results and Conclusion: Suggesting that, these Piper betel derived compounds may be further tested for developing a novel class of broad-spectrum drugs against various common and MDR pathogens.

Large-scale High-throughput Screening Revealed 5'-(carbonylamino)-2,3'- bithiophene-4'-carboxylate as Novel Template for Antibacterial Agents

2020 ◽  
Vol 17 (5) ◽  
pp. 716-724
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Antibacterial Agents ◽  
Sos Response ◽  
Luciferase Assay ◽  
Translational Machinery ◽  
E Coli ◽  
New Class

Background: The key issue in the development of novel antimicrobials is a rapid expansion of new bacterial strains resistant to current antibiotics. Indeed, World Health Organization has reported that bacteria commonly causing infections in hospitals and in the community, e.g. E. Coli, K. pneumoniae and S. aureus, have high resistance vs the last generations of cephalosporins, carbapenems and fluoroquinolones. During the past decades, only few successful efforts to develop and launch new antibacterial medications have been performed. This study aims to identify new class of antibacterial agents using novel high-throughput screening technique. Methods: We have designed library containing 125K compounds not similar in structure (Tanimoto coeff.< 0.7) to that published previously as antibiotics. The HTS platform based on double reporter system pDualrep2 was used to distinguish between molecules able to block translational machinery or induce SOS-response in a model E. coli system. MICs for most active chemicals in LB and M9 medium were determined using broth microdilution assay. Results: In an attempt to discover novel classes of antibacterials, we performed HTS of a large-scale small molecule library using our unique screening platform. This approach permitted us to quickly and robustly evaluate a lot of compounds as well as to determine the mechanism of action in the case of compounds being either translational machinery inhibitors or DNA-damaging agents/replication blockers. HTS has resulted in several new structural classes of molecules exhibiting an attractive antibacterial activity. Herein, we report as promising antibacterials. Two most active compounds from this series showed MIC value of 1.2 (5) and 1.8 μg/mL (6) and good selectivity index. Compound 6 caused RFP induction and low SOS response. In vitro luciferase assay has revealed that it is able to slightly inhibit protein biosynthesis. Compound 5 was tested on several archival strains and exhibited slight activity against gram-negative bacteria and outstanding activity against S. aureus. The key structural requirements for antibacterial potency were also explored. We found, that the unsubstituted carboxylic group is crucial for antibacterial activity as well as the presence of bulky hydrophobic substituents at phenyl fragment. Conclusion: The obtained results provide a solid background for further characterization of the 5'- (carbonylamino)-2,3'-bithiophene-4'-carboxylate derivatives discussed herein as new class of antibacterials and their optimization campaign.

scholarly journals Tailoring a Global Iron Regulon to a Uropathogen

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Rajdeep Banerjee ◽  
Erin Weisenhorn ◽  
Kevin J. Schwartz ◽  
Kevin S. Myers ◽  
Jeremy D. Glasner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Urinary Tract ◽  
Amino Acid ◽  
Regulatory Network ◽  
Iron Homeostasis ◽  
Iron Limitation ◽  
Content Type ◽  
E Coli ◽  
General Stress ◽  
K 12

ABSTRACT Pathogenicity islands and plasmids bear genes for pathogenesis of various Escherichia coli pathotypes. Although there is a basic understanding of the contribution of these virulence factors to disease, less is known about variation in regulatory networks in determining disease phenotypes. Here, we dissected a regulatory network directed by the conserved iron homeostasis regulator, ferric uptake regulator (Fur), in uropathogenic E. coli (UPEC) strain CFT073. Comparing anaerobic genome-scale Fur DNA binding with Fur-dependent transcript expression and protein levels of the uropathogen to that of commensal E. coli K-12 strain MG1655 showed that the Fur regulon of the core genome is conserved but also includes genes within the pathogenicity/genetic islands. Unexpectedly, regulons indicative of amino acid limitation and the general stress response were also indirectly activated in the uropathogen fur mutant, suggesting that induction of the Fur regulon increases amino acid demand. Using RpoS levels as a proxy, addition of amino acids mitigated the stress. In addition, iron chelation increased RpoS to the same levels as in the fur mutant. The increased amino acid demand of the fur mutant or iron chelated cells was exacerbated by aerobic conditions, which could be partly explained by the O2-dependent synthesis of the siderophore aerobactin, encoded by an operon within a pathogenicity island. Taken together, these data suggest that in the iron-poor environment of the urinary tract, amino acid availability could play a role in the proliferation of this uropathogen, particularly if there is sufficient O2 to produce aerobactin. IMPORTANCE Host iron restriction is a common mechanism for limiting the growth of pathogens. We compared the regulatory network controlled by Fur in uropathogenic E. coli (UPEC) to that of nonpathogenic E. coli K-12 to uncover strategies that pathogenic bacteria use to overcome iron limitation. Although iron homeostasis functions were regulated by Fur in the uropathogen as expected, a surprising finding was the activation of the stringent and general stress responses in the uropathogen fur mutant, which was rescued by amino acid addition. This coordinated global response could be important in controlling growth and survival under nutrient-limiting conditions and during transitions from the nutrient-rich environment of the lower gastrointestinal (GI) tract to the more restrictive environment of the urinary tract. The coupling of the response of iron limitation to increased demand for amino acids could be a critical attribute that sets UPEC apart from other E. coli pathotypes.

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