Producing Strain Research Articles

Pathway engineering of Escherichia coli for the glycerol-based production of recombinant endoglucanase

BackgroundTechnology platforms developed in biorefinery are featured with low-carbon emission but afflicted by the issue of economic viability. This can be addressed by a cascade process that prioritizes the production of value-added products. In this context, the cost-effective production of endoglucanase (encoded by celA) was illustrated using Escherichia coli with crude glycerol. This renewable and sustainable feedstock appears in abundance in waste streams of the biodiesel process. Moreover, glutamate (Glu) was supplemented to tackle the inconsistent quality of yeast extract. MethodsA new fusion tag was first explored to improve the solubility of CelA. CelA was then produced using a strain quipped with a rewired glycerol metabolism. To improve the CelA production, the producer strain was further re-programmed by manipulation of metabolic pathways involved in oxidative phosphorylation and substrate-level phosphorylation. Significant findingsThe production of CelA in the developed strain positively correlated with glycerol. It was found to increase the CelA production by 90% in the engineered strain when the glycerol level was doubled. In contrast, the parent strain gained merely 20% more production of CelA. The result indicates that the physiological status of the engineered strain is refined to favor functioning of the protein synthesis machinery. In conclusion, the developed strain shows promise in production of recombinant proteins based on crude glycerol.

Structural and compositional diversity of biosurfactants produced by a novel strain of Sporosarcina luteola ME44 from oil reservoir.

The urealytically active microorganism Sporosarcina luteola induces the precipitation of metals, which has attracted attention in biomineralization, bioremediation, and industrial waste recycling. Herein, we report a novel biosurfactant-producing strain of S. luteola ME44 isolated from Chinese Oilfield. The structure, composition, and surface activity of the biosurfactants produced by S. luteola ME44 were investigated by using a combination of the high-performance liquid chromatography, time-of-flight mass spectrometry, and surface tensiometer. The biosurfactant extracted by strain ME44 was identified as surfactin with five variants and the yield was 1010±60mg⋅L-1 . This is the first report on the structural composition and surface activity of biosurfactants isolated from the S. luteola. It extended our knowledge about the role of the species S. luteola in the ecosystem of extreme natural environments such as oil reservoir. In addition, S. luteola ME44 showed bioprecipitation properties for metal ions Cd(II), Cu(II), Zn(II), and Ag(I), which indicated the application potential of S. luteola in the field of bioremediation.

A Study of the Influence of L-Lysine and its Transporter LysP on the Characteristics of the L-Threonine Producer Strain

A Study of the Influence of L-Lysine and its Transporter LysP on the Characteristics of the L-Threonine Producer Strain

Production and characterization of plantaricin Y11 from <i>Lactiplantibacillus Plantarum</i> strain y11 (MW642245.1) isolated from Nigerian food samples and their applications in preserving milk

Bacteriocin from lactic acid bacterium (LAB) was characterized and its biopreservation assay carried out using conventional methods and integrative approaches. About 66.7% (n=180) of the bacterial strains isolated from twelve different fermented/non-fermented food products were potential LAB strains by biochemical characterization. The highest bacteriocin producing strain was selected and confirmed with 16S rRNA sequencing and submitted to GenBank of National Center for Biotechnology Information as Lactiplantibacillus plantarum strain Y11 (Accession number: MW642245.1). Polymerase Chain Reaction (PCR) revealed the presence of virulence genes (cytotoxin K and haemolysin C) in L. plantarum Y11. The bacteriocin produced was labelled as plantaricin Y11 with molecular weight of 1540 Da, amino acid sequence was NSHGTADYCVMWLIXK, and possessed the merits of wide pH stability (2–10), high thermal stability and sensitivity to protease. The bacteriocin showed wide spectrum of activity against bacterial reference strains with bacteriocin activity ranging from 3200Au/mL to 6400Au/mL. Preservative potentials of the bacteriocin compared favourably with sodium benzoate in milk, inhibiting bacterial and fungal load compared to the control. There was log reduction of aerobic mesophilic bacterial count of 8.02±0.15 and 8.43±0.21 Log 10 CFU/mL in milk treated with plantaricin Y11 and sodium benzoate, respectively. The log reductions of fungal count in plantaricin Y11 and sodium benzoate treated milk ≥4.41±0.11 and ≥3.1±0.2 Log 10 CFU/mL respectively. The plantaricin Y11 was then classified as class IIa in bacteriocin classification schemes and it sustained some organoleptic conditions of milk making it a promising candidate for safe bio-preservatives.

Diversity and composition of microbial communities in Jinsha earthen site under different degree of deterioration

Earthen sites are the important cultural heritage that carriers of human civilization and contains abundant history information. Microorganisms are one of important factors causing the deterioration of cultural heritage. However, little attention has been paid to the role of biological factors on the deterioration of earthen sites at present. In this study, microbial communities of Jinsha earthen site soils with different deterioration types and degrees as well as related to environmental factors were analyzed. The results showed that the concentrations of Mg2+ and SO42− were higher in the severe deterioration degree soils than in the minor deterioration degree soils. The Chao1 richness and Shannon diversity indices of bacteria in different type deterioration were higher in the summer than in the winter; the Chao1 and Shannon indices of fungi were lower in the summer. The differences in bacterial and fungal communities were associated with differences in Na+, K+, Mg2+ and Ca2+ contents. Based on both the relative abundances in amplicon sequencing and isolated strains, the bacterial phyla Actinobacteria, Firmicutes and Proteobacteria, and the Ascomycota genera Aspergillus, Cladosporium and Penicillium were common in all soils. The OTUs enriched in the severe deterioration degree soils were mostly assigned to Actinobacteria and Proteobacteria, whereas the Firmicutes OTUs differentially abundant in the severe deterioration degree were all depleted. All bacterial isolates produced alkali, implying that the deterioration on Jinsha earthen site may be accelerated through alkali production. The fungal isolates included both alkali and acid producing strains. The fungi with strong ability to produce acid were mainly from the severe deterioration degree samples and were likely to contribute to the deterioration. Taken together, the interaction between soil microbial communities and environment may affect the soil deterioration, accelerate the deterioration process and threaten the long-term preservation of Jinsha earthen site.

Effects of EPS-producing Leuconostoc mesenteroides XR1 on texture, rheological properties, microstructure and volatile flavor of fermented milk

Leuconostoc mesenteroides XR1, a high-quality exopolysaccharides (EPS) producing strain, was isolated from wild Kefir grains. The effect of strain XR1 as a compound starter on yoghurt fermentation and storage was studied. The EPS content, viable counts, titratable acidity, pH, water holding capacity (WHC), texture, rheological properties, microstructure, volatile flavor compounds and sensory evaluation of yoghurts were determined. The EPS content of the yoghurt with XR1 was shown to be 1.64, 2.14, 1.37, and 1.29 times higher than those of the control group at days 1, 7, 14, and 21 of storage, respectively. The WHC, cohesiveness, gumminess, apparent viscosity and gel strength of the yoghurt containing XR1 were higher than those of the control group (P < 0.05). It is worth noting that supplementation with XR1 improved the acidification of the yoghurt. Scanning electron microscope (SEM) observation confirmed that the microstructure of the yoghurt containing XR1 had stronger compactness. Volatile substance analysis showed that after 7 days of storage, yoghurt supplemented with XR1 had an increased aroma of ketones and alkanes. In terms of texture, flavor and taste, Y-XR1 group obtained higher sensory scores (P < 0.05). These results collectively indicated that XR1 improved the quality of the yoghurt by increasing EPS production, thus reducing the use of chemical thickeners or stabilizers.

Metabolic engineering in Streptomyces albidoflavus for the biosynthesis of the methylated flavonoids sakuranetin, acacetin, and genkwanin

Flavonoids are important plant secondary metabolites showing antioxidant, antitumor, anti-inflammatory, and antiviral activities, among others. Methylated flavonoids are particularly interesting compared to non-methylated ones due to their greater stability and intestinal absorption, which improves their oral bioavailability. In this work we have stablished a metabolic engineered strain of Streptomyces albidoflavus with enhanced capabilities for flavonoid production, achieving a 1.6-fold increase in the biosynthesis of naringenin with respect to the parental strain. This improved strain, S. albidoflavus UO-FLAV-004, has been used for the heterologous biosynthesis of the methylated flavonoids sakuranetin, acacetin and genkwanin. The achieved titers of sakuranetin and acacetin were 8.2 mg/L and 5.8 mg/L, respectively. The genkwanin titers were 0.8 mg/L, with a bottleneck identified in this producing strain. After applying a co-culture strategy, genkwanin production titers reached 3.5 mg/L, which represents a 4.4-fold increase. To our knowledge, this study presents the first biosynthesis of methylated flavonoids in not only any Streptomyces species, but also in any Gram-positive bacteria.

Keratinolytic activity of pigmenting and non-pigmenting soils strains of Trichophyton ajelloi

The aim of the study was to evaluate the keratinolytic activity of 37 Trichophyton ajelloi strains, both pigmenting and non–pigmenting, isolated from loamy soil and chernozem, and to identify strains with the highest efficiency of converting feather waste into easily assimilable forms of nitrogen and sulfur for plants. The study showed that among Trichophyton ajelloi strains isolated from loamy soil, pigmenting strains were more capable of biodegrading native feather keratin, while among the strains isolated from chernozem, non–pigmenting strains showed better abilities. The keratinolytic activity, as measured by feather weight loss after 42 days of culture, was on average higher in Trichophyton ajelloi strains isolated from loamy soil compared to those from chernozem. Extracellular keratinase activity, as well as release of soluble proteins and peptides in cultures of Trichophyton ajelloi strains isolated from loamy soil and chernozem increased throughout the experiment. Protease activity was high during the first 7 days of the experiment and increased again from day 28 of culture. The increase in the pH of the culture fluids was associated with the release of ammonium ions. The maximum release of ammonium and sulfate ions occurred on day 21 of culture. Three pigment–producing strains (No. III, XII, and XIV/XV) were among the most active in releasing large amounts of ammonium and sulfate ions. The biodegradation of feather waste by Trichophyton ajelloi strains can be used for the production of fertilizers suitable for the cultivation of plants, particularly those with high sulfur requirements.

Resistance to antibiotics in clinical isolates of Escherichia coli producing and non-producing Extended Spectrum Betalactamases (ESBL) obtained from urine cultures in patients with urinary tract infection.

Background: Escherichia coli is a bacterium that is part of the normal biota of the human being, but under certain conditions it can produce diseases such as gastrointestinal and urinary tract infections for which is the main responsible. Enterobacteriaceae such as Escherichia are extended spectrum betalactamases (ESBL) producers, which makes their treatment difficult due to a high rate of resistance to antibiotics.Aim of the study: Identify resistance patterns against various antibiotics in Escherichia coli producers and non-producers of extended spectrum beta-lactamases in urinary infections in a population of the Toluca Valley, MexicoMaterial and methods: 155 samples were collected from patients with suspected urinary tract infection without exclusion criteria such as age or gender. Automated equipment was used for the identification of the etiological agent and sensitivity tests. For the determination of ESBL, the double disc technique was used.Results: 35 strains of Escherichia coli were obtained, of which 45.72 % have ESBL, these strains show 100 % resistance to betalactams, as well as high resistance to quinolones and tetracycline ranging from 70 to 100 %. On the other hand, the resistance shown by non-ESBL producing strains is variable both for betalactams and for other antibiotics.Conclusion: Treatment for urinary tract infections has become increasily difficult due to the high rate of resistance to antibiotics, mainly ESBL-producing strains such as the E coli strains in the present study. Where high rates of resistance to different antibiotics used in clinical practice are shown.

CARBOHYDRASES – 50 YEARS OF RESEARCH AT THE DEPARTMENT OF CHEMICAL ENZYMOLOGY OF LOMONOSOV MOSCOW STATE UNIVERSITY, HISTORY AND PROSPECTS

The review describes the history of the development of research on carbohydrasеs conducted at the Department of Chemical Enzymology from the mid-1970s to the present. The results concerning the study of the mechanism and kinetics of the processes of enzymatic conversion of cellulose and renewable plant raw materials under the action of a multi-enzyme cellulases complexes; the role of individual components of these complexes - basic (endoglucanases and cellobiohydrolases) and auxiliary enzymes (polysaccharide monooxygenase, β-glucosidase, xylanase) and their synergistic interaction. The features of using reactors of various designs for bioconversion of plant raw materials are described: periodic type, continuous column type, reactor for hydrolysis in a constant electric field, reactor with intensive mixing by ferromagnetic particles in magnetic field. The possibilities of increasing the reactivity of plant raw materials using various pretreatment methods, as well as the influence of the structural and physico-chemical properties of cellulose on the efficiency of its enzymatic conversion are discussed. Data on the creation of highly active strains of microscopic fungi-producers of cellulases and other carbohydrases using methods of induced mutagenesis - Trichoderma (Hypocrea), Penicillium (Talaromyces), Aspergillus, Chrysosporium (Myceliophtora) spp., as well as data on the composition of the enzyme complexes produced by them and the properties of the enzymes forming them are presented. It describes the creation of expression systems based on P. canescens and P. verruculosum and the production of recombinant producer strains with their help, which made it possible to obtain enzyme preparations (EP) that ensure high efficiency of bioconversion processes of plant raw materials, as well as to create producers of a wide range of carbohydrases for practical use in various fields of industry and agriculture. A number of industrially important EP obtained using the P. verruculosum expression system are currently being produced at the Agroferment LLC plant.

Preparation, purification, and biochemical of fat-degrading bacterial enzymes from pig carcass compost and its application

BackgroundA lot of kitchen waste oil is produced every day worldwide, leading to serious environmental pollution. As one of the environmental protection methods, microorganisms are widely used treating of various wastes. Lipase, as one of the cleaning agents can effectively degrade kitchen waste oil. The composting process of pig carcasses produces many lipase producing microorganisms, rendering compost products an excellent source for isolating lipase producing microorganisms. To our knowledge, there are no reports isolating of lipase producing strains from the high temperature phase of pig carcass compost.MethodologyLipase producing strains were isolated using a triglyceride medium and identified by 16S rRNA gene sequencing. The optimal fermentation conditions for maximum lipase yield were gradually optimized by single-factor tests. The extracellular lipase was purified by ammonium sulfate precipitation and Sephadex G-75 gel isolation chromatography. Amino acid sequence analysis, structure prediction, and molecular docking of the purified protein were performed. The pure lipase's enzymatic properties and application potential were evaluated by characterizing its biochemical properties.ResultsIn this study, a lipase producing strain of Bacillus sp. ZF2 was isolated from pig carcass compost products, the optimal fermentation conditions of lipase: sucrose 3 g/L, ammonium sulfate 7 g/L, Mn2+ 1.0 mmol/L, initial pH 6, inoculum 5%, temperature 25 ℃, and fermentation time 48 h. After purification, the specific activity of the purified lipase reached 317.59 U/mg, a 9.78-fold improvement. Lipase had the highest similarity to the GH family 46 chitosanase and molecular docking showed that lipase binds to fat via two hydrogen bonds at Gln146 (A) and Glu203 (A). Under different conditions (temperature, metal ions, organic solvents, and surfactants), lipase can maintain enzymatic activity. Under different types of kitchen oils, lipase has low activity only for ‘chicken oil’, in treating other substrates, the enzyme activity can exceed 50%.ConclusionsThis study reveals the potential of lipase for waste oil removal, and future research will be devoted to the application of lipase.

Preliminary X-ray Study of Crystals Obtained by Co-Crystallization of Hypoxanthine‒Guanine Phosphoribosyltransferase from Escherichia coli and Pyrazine-2-Carboxamide Derivatives

A highly effective producer strain Escherichia coli C3030/pET23d+-EcHGPRT, allowing production of recombinant hypoxanthine‒guanine phosphoribosyltransferase from E. coli (EcHGPRT) in a soluble form, has been created. A method for isolating and purifying the recombinant protein has been developed. The specific activity against the natural substrate and pyrazine-2-carboxamide derivatives has been determined. Crystals of the EcHGPRT complexes with 3-hydroxypyrazine-2-carboxamide (T-1105) and 6-fluoro-3-hydroxypyrazine-2-carboxamide (T-705), suitable for X-ray diffraction analysis, have been grown by capillary counter diffusion. X-ray diffraction sets with a resolution of up to 2.4 and 2.5 Å have been collected at the ESRF synchrotron (France, station ID23-1) at a temperature of 100 K. The crystals belong to the sp. gr. P3(1)21; the independent part of the cell contains two enzyme molecules.

Modern Approaches to the Genome Editing of Antibiotic Biosynthetic Clusters in Actinomycetes.

Representatives of the phylum Actinomycetota are one of the main sources of secondary metabolites, including antibiotics of various classes. Modern studies using high-throughput sequencing techniques enable the detection of dozens of potential antibiotic biosynthetic genome clusters in many actinomycetes; however, under laboratory conditions, production of secondary metabolites amounts to less than 5% of the total coding potential of producer strains. However, many of these antibiotics have already been described. There is a continuous "rediscovery" of known antibiotics, and new molecules become almost invisible against the general background. The established approaches aimed at increasing the production of novel antibiotics include: selection of optimal cultivation conditions by modifying the composition of nutrient media; co-cultivation methods; microfluidics, and the use of various transcription factors to activate silent genes. Unfortunately, these tools are non-universal for various actinomycete strains, stochastic in nature, and therefore do not always lead to success. The use of genetic engineering technologies is much more efficient, because they allow for a directed and controlled change in the production of target metabolites. One example of such technologies is mutagenesis-based genome editing of antibiotic biosynthetic clusters. This targeted approach allows one to alter gene expression, suppressing the production of previously characterized molecules, and thereby promoting the synthesis of other unknown antibiotic variants. In addition, mutagenesis techniques can be successfully applied both to new producer strains and to the genes of known isolates to identify new compounds.

Designing structure and &lt;i&gt;E. coli&lt;/i&gt; strain-producer bearing SARS-CoV-2 N, S, M, E protein-related sequence antigen

T-cell immune response is extremely important in protecting human body from diverse viral infections. It is known that it can ensure viral clearance and complete recovery in patients with humoral immunodeficiency. COVID-19 patients were found to have T-cell response primarily directed against SARS-CoV-2 structural S, M, N, E proteins, with nucleocapsid protein being most conserved. To assess patients immunity against coronavirus infection and evaluate an effectiveness of vaccine candidates, it is necessary to develop an optimal diagnostic antigen to evaluate arising T-cell response against SARS-CoV-2 antigenic determinants. A diagnostic test to determine host specific susceptibility to SARS-CoV-2 infection should target conserved regions of global SARS-CoV-2 variants. The study was aimed to develop a structure of an antigen bearing conserved and immunogenic sequences derived from SARS-CoV-2 structural proteins and to obtain an Escherichia coli producer strain containing a recombinant protein to be subsequently used for assessing antiviral T-cell immunity. Developing of the antigen was performed in silico: TepiTool and NetMHCIIpan were used to predict and identify high affinity epitopes spanning SARS-CoV-2 E, M, N, S proteins and MHC II binding. Several variants of recombinant antigen proteins were constructed, from which one was selected based on its physicochemical properties: isoelectric point, hydrophobicity index and aliphatic index, as well as 3D representation built by using the I-TASSER. The sequence was synthesized and cloned into the pET24a(+) vector. The resulting plasmid pCorD_PS was transformed into E. coli DH5 followed by Rosetta (DE3). The strain-producer of the recombinant E. coli protein CorD_PS was assessed for the presence and stability of IPTG-induced antigen protein expression and elimination of recombinant coronavirus antigen-bearing plasmid. Based on the study data, an antigen was developed consisting of conserved regions from SARS-CoV-2 S, M, N, E proteins. A 53 kDa recombinant protein was predicted to be stable in aqueous solutions with isoelectric point of 9.56 potentially allowing to simplify protein purification from E. coli cells. Plasmid DNA pCorD_PS (6695 bp) encoding final recombinant coronavirus antigen cloned into pET24a(+) vector was obtained. A stable, productive E. coli CorD_PS strain was obtained. The obtained strain-producer resulting in recombinant E. coli CorD_PS antigen is stable allowing to move on to design antigen purification technique and further develop SARS-CoV-2-specific diagnostic test system.

Optimized methyl donor and reduced precursor degradation pathway for seleno-methylselenocysteine production in Bacillus subtilis

BackgroundSeleno-methylselenocysteine (SeMCys) is an effective component of selenium supplementation with anti-carcinogenic potential that can ameliorate neuropathology and cognitive deficits. In a previous study, a SeMCys producing strain of Bacillus subtilis GBACB was generated by releasing feedback inhibition by overexpression of cysteine-insensitive serine O-acetyltransferase, enhancing the synthesis of S-adenosylmethionine as methyl donor by overexpression of S-adenosylmethionine synthetase, and expressing heterologous selenocysteine methyltransferase. In this study, we aimed to improve GBACB SeMCys production by synthesizing methylmethionine as a donor to methylate selenocysteine and by inhibiting the precursor degradation pathway.ResultsFirst, the performance of three methionine S-methyltransferases that provide methylmethionine as a methyl donor for SeMCys production was determined. Integration of the NmMmt gene into GBACB improved SeMCys production from 20.7 to 687.4 μg/L. Next, the major routes for the degradation of selenocysteine, which is the precursor of SeMCys, were revealed by comparing selenocysteine hyper-accumulating and non-producing strains at the transcriptional level. The iscSB knockout strain doubled SeMCys production. Moreover, deleting sdaA, which is responsible for the degradation of serine as a precursor of selenocysteine, enhanced SeMCys production to 4120.3 μg/L. Finally, the culture conditions in the flasks were optimized. The strain was tolerant to higher selenite content in the liquid medium and the titer of SeMCys reached 7.5 mg/L.ConclusionsThe significance of methylmethionine as a methyl donor for SeMCys production in B. subtilis is reported, and enhanced precursor supply facilitates SeMCys synthesis. The results represent the highest SeMCys production to date and provide insight into Se metabolism.

Evaluating the potential of semi-continuous itaconic acid fermentation by Aspergillus terreus: operational profile and experiences

Itaconic acid is an important bio-based chemical. The present study aims to evaluate the applicability of semi-continuous fermentation technique for itaconic acid production by Aspergillus terreus. The fermentation is planned to be connected with bipolar membrane electrodialysis unit for acid recovery. This process allows the reuse of residual glucose from the effluent. Our particular attention was focused on the effect of glucose concentration. Two different glucose supplementation strategies were tested: constant glucose concentration in the refilling medium and adjusted glucose concentration in order to maintain a continuously high – 120 g/L – glucose concentration in the fermentor. The itaconic acid titre, yield and productivity for the 24 h time periods between draining/refilling interventions were investigated. The constantly high glucose concentration in the fermentor resulted in doubled biomass formation. The average itaconic acid titre was 32.9 ± 2.7 g/L. The producing strain formed numerous spores during semi-continuous fermentation that germinated continuously. Yield and volumetric productivity showed a periodic pattern during the procedure.

Biotransformation Of l-Tryptophan To Produce Arcyriaflavin A With Pseudomonas putida KT2440.

Natural products such as indolocarbazoles are a valuable source of highly bioactive compounds with numerous potential applications in the pharmaceutical industry. Arcyriaflavin A, isolated from marine invertebrates and slime molds, is one representative of this group and acts as a cyclin D1-cyclin-dependent kinase 4 inhibitor. To date, access to this compound has mostly relied on multi-step total synthesis. In this study, biosynthetic access to arcyriaflavin A was explored using recombinant Pseudomonas putida KT2440 based on a previously generated producer strain. We used a Design of Experiment approach to analyze four key parameters, which led to the optimization of the bioprocess. By engineering the formation of outer membrane vesicles and using an adsorbent in the culture broth, we succeeded to increase the yield of arcyriaflavin A in the cell-free supernatant, resulting in a nearly eight-fold increase in the overall production titers. Finally, we managed to scale up the bioprocess leading to a final yield of 4.7 mg arcyriaflavin A product isolated from 1 L of bacterial culture. Thus, this study showcases an integrative approach to improve biotransformation and moreover also provides starting points for further optimization of indolocarbazole production in P. putida.

Recombinant Myostatin: Isolation and Use in Vaccines

Protein myostatin is not only a potential target for therapeutic effects in pathologies of the muscular system, but also a tool for biotechnological regulation of the meat productivity of farm animals. The action of myostatin can be blocked by inducing autoantibody synthesis using recombinant myostatin (rMST) as an antigen. E. coli producer strain Bl21/pET28-MSTN was obtained with a high level of recombinant protein expression in inclusion bodies. Purified rMST was used to immunize sheep and rabbits. Using the created ELISA test system for the detection of antibodies to myostatin in the blood of sheep, it was shown that rMST is immunogenic (the working titer of sera is 1/400–1/800). As a result of vaccination of rabbits over a monthly period, the average body weight and average daily weight gain in animals of the experimental groups was observed to increase compared to the control group.

2-Hydroxyisovalerate production by Klebsiella pneumoniae

2-Hydroxyisovalerate is a valuable chemical that can be used in the production of biodegradable polyesters. In nature, it was only produced at a very low level by Lactococcus lactis. 2-Ketoisovalerate is an intermediate metabolite of the branched-chain amino acid biosynthesis pathway, and Klebsiella pneumoniae ΔbudAΔldhA (Kp ΔbudAΔldhA) was a 2-ketoisovalerate producing strain. In this research, 2-hydroxyisovalerate was identified as a metabolite of Kp ΔbudAΔldhA, and its synthesis pathway was revealed. It was found that 2-ketoisovalerate and 2-hydroxyisovalerate were produced by Kp ΔbudA and Kp ΔbudAΔldhA, but not by Kp ΔbudAΔldhAΔilvD in which the 2-ketoisovalerate synthesis was blocked. budA, ldhA, and ilvD encode α-acetolactate decarboxylase, lactate dehydrogenase, and dihydroxy acid dehydratase, respectively. Thus, it was deduced that 2-hydroxyisovalerate was synthesized from 2-ketoisovalerate. Isoenzymes of ketopantoate reductase PanE, PanE2, and IlvC were suspected of being responsible for this reaction. Kinetic parameters of these enzymes were detected, and they all hold the 2-ketoisovalerate reductase activities. PanE and PanE2 use both NADH and NADPH as co-factors. While IlvC only uses NADH as a co-factor. Over-expression of panE, panE2, or ilvC in Kp ΔbudAΔldhA all enhanced the production of 2-hydroxyisovalerate. Accordingly, 2-hydroxyisovalerate levels were reduced by knocking out panE or panE2. In fed-batch fermentation, 14.41 g/L of 2-hydroxyisovalerate was produced by Kp ΔbudAΔldhA-panE, with a substrate conversion ratio of 0.13 g/g glucose.

Isolation, identification, and functional analysis of quorum sensing producing strain Pectobacterium atrosepticum associated with blackleg disease in potatoes

Potato is an important food and cash crop that production losses have a double effect on society, causing both a food security and financial crisis. The significant problem in the potato fields is blackleg of potato caused by Pectobacterium atrosepticum (Pa). The research concentrates on the isolation and characterization of P. atrosepticum, as one of the major causal organisms of the potato blackleg disease, in particular its prevalence in the Pakistani regions. Differential diagnosis by using biochemical methods and Koch’s Postulates, the etiological agent was identified. The involvement of N-Acyl Homoserine Lactones (NAHLs)-based quorum sensing (QS) in modulating Pa behaviors was clarified by the investigations. Novel QS inhibitors showing non-lethal quorum quenching capacity surfaced, however the long-term possibilities could be further improved. Initial data reflect a strong drop of virulence markers up to Day 16, which demonstrates the importance of increasing the knowledge of limiting influence arising from characteristics of bioactive substances or intrinsic ecological dynamics. Overall, this research contributes significantly to comprehending P. atrosepticum-associated threats and lays foundation for devising eco-friendly intervention tactics centered on quorum quenching paradigms.