Investigation of thermal stability characteristic in family A DNA polymerase - A theoretical study.

DNA polymerases create complementary DNA strands in living cells and are crucial to genome transmission and maintenance. These enzymes possess similar human right-handed folds which contain thumb, fingers, and palm subdomains and contribute to polymerization activities. These enzymes are classified into seven evolutionary families, A, B, C, D, X, Y, and RT, based on amino acid sequence analysis and biochemical characteristics. Family A DNA polymerases exist in an extended range of organisms including mesophilic, thermophilic, and hyper-thermophilic bacteria, participate in DNA replication and repair, and have a broad application in molecular biology and biotechnology. In this study, we attempted to detect factors that play a role in the thermostability properties of this family member despite their remarkable similarities in structure and function. For this purpose, similarities and differences in amino acid sequences, structure, and dynamics of these enzymes have been inspected. Our results demonstrated that thermophilic and hyper-thermophilic enzymes have more charged, aromatic, and polar residues than mesophilic ones and consequently show further electrostatic and cation-pi interactions. In addition, in thermophilic enzymes, aliphatic residues tend to position in buried states more than mesophilic enzymes. These residues within their aliphatic parts increase hydrophobic core packing and therefore enhance the thermostability of these enzymes. Furthermore, a decrease in thermophilic cavities volumes assists in the protein compactness enhancement. Moreover, molecular dynamic simulation results revealed that increasing temperature impacts mesophilic enzymes further than thermophilic ones that reflect on polar and aliphatic residues surface area and hydrogen bonds changes.

Escherichia coli tRNA 2-selenouridine synthase SelU selects its prenyl substrate to accomplish its enzymatic function.

Bacterial tRNA 2-selenouridine synthase (SelU) in vitro converts S2U-RNA to its selenium analog (Se2U-RNA) in a two-step process: (i) geranylation of S2U-RNA (with geranyl pyrophosphate, gePP), and (ii) selenation of the resulting geS2U-RNA (with the selenophosphate anion, SePO33-). Using an S2U-containing anticodon stem-loop fragment derived from tRNALys (S2U-RNA) and recombinant SelU with an MBP tag, we found that only geranyl (C10) pyrophosphate is the substrate for this enzyme, while other pyrophosphates such as isopentenyl (C5), dimethylallyl (C5), farnesyl (C15) and geranylgeranyl (C20) are not. Interestingly, methyl (C1)- and C5-, C10-, and C15-prenyl-containing S2U-RNAs (which were chemically obtained) underwent the selenation reaction promoted by SelU, although the Se2U-RNA product was obtained in decreasing yields in the following order: geranyl ≥ farnesyl > dimethylallyl ≫ methyl. Microscale thermophoresis showed an affinity between gePP and SelU in the micromolar range, while the other pyrophosphates tested, such as isopentenyl, dimethylallyl, farnesyl and geranylgeranyl, either did not bind to the protein or their binding affinity was above 1 mM. These results agree well with the in silico analysis, with gePP being the best binding substrate (the lowest relative free energy of binding (ΔG) and a small solvent-accessible surface area (SASA)). These results suggest that SelU has high substrate specificity for the prenylation reaction (only gePP is accepted), whereas there is little discrimination for the selenation reaction. We therefore suggest that only gePP and the geranylated tRNA serve as substrates for the conversion of 2-thio-tRNAs to 2-seleno-tRNAs, as it is found in the bacterial system.

A conserved and seemingly redundant Escherichia coli biotin biosynthesis gene expressed only during anaerobic growth.

Biotin is an essential metabolic cofactor and de novo biotin biosynthetic pathways are widespread in microorganisms and plants. Biotin synthetic genes are generally found clustered into bio operons to facilitate tight regulation since biotin synthesis is a metabolically expensive process. Dethiobiotin synthetase (DTBS) catalyzes the penultimate step of biotin biosynthesis, the formation of 7,8-diaminononanoate (DAPA). In Escherichia coli, DTBS is encoded by the bio operon gene bioD. Several studies have reported transcriptional activation of ynfK a gene of unknown function, under anaerobic conditions. Alignments of YnfK with BioD have led to suggestions that YnfK has DTBS activity. We report that YnfK is a functional DTBS, although an enzyme of poor activity that is poorly expressed. Supplementation of growth medium with DAPA or substitution of BioD active site residues for the corresponding YnfK residues greatly improved the DTBS activity of YnfK. We confirmed that FNR activates transcriptional level of ynfK during anaerobic growth and identified the FNR binding site of ynfK. The ynfK gene is well conserved in γ-proteobacteria.

Improvement of a synthetic live bacterial therapeutic for phenylketonuria with biosensor-enabled enzyme engineering.

In phenylketonuria (PKU) patients, a genetic defect in the enzyme phenylalanine hydroxylase (PAH) leads to elevated systemic phenylalanine (Phe), which can result in severe neurological impairment. As a treatment for PKU, Escherichia coli Nissle (EcN) strain SYNB1618 was developed under Synlogic's Synthetic Biotic™ platform to degrade Phe from within the gastrointestinal (GI) tract. This clinical-stage engineered strain expresses the Phe-metabolizing enzyme phenylalanine ammonia lyase (PAL), catalyzing the deamination of Phe to the non-toxic product trans-cinnamate (TCA). In the present work, we generate a more potent EcN-based PKU strain through optimization of whole cell PAL activity, using biosensor-based high-throughput screening of mutant PAL libraries. A lead enzyme candidate from this screen is used in the construction of SYNB1934, a chromosomally integrated strain containing the additional Phe-metabolizing and biosafety features found in SYNB1618. Head-to-head, SYNB1934 demonstrates an approximate two-fold increase in in vivo PAL activity compared to SYNB1618.

Influence of Antimicrobial Stewardship and Molecular Rapid Diagnostic Tests on Antimicrobial Prescribing for Extended-Spectrum Beta-Lactamase- and Carbapenemase-Producing Escherichia coli and Klebsiella pneumoniae in Bloodstream Infection.

The objective of this study was to evaluate whether the addition of the Verigene BC-GN molecular rapid diagnostic test to standard antimicrobial stewardship practices (mRDT + ASP) decreased the time to optimal and effective antimicrobial therapy for patients with extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae bloodstream infections (BSI) compared to conventional microbiological methods with ASP (CONV + ASP). This was a multicenter, retrospective cohort study evaluating the time to optimal antimicrobial therapy in 5 years of patients with E. coli or K. pneumoniae BSI determined to be ESBL- or carbapenemase-producing by mRDT and/or CONV. Of the 378 patients included (mRDT + ASP, n = 164; CONV + ASP, n = 214), 339 received optimal antimicrobial therapy (mRDT + ASP, n = 161; CONV + ASP, n = 178), and 360 (mRDT + ASP, n = 163; CONV + ASP, n = 197) received effective antimicrobial therapy. The mRDT + ASP demonstrated a statistically significant decrease in the time to optimal antimicrobial therapy (20.5 h [interquartile range (IQR), 17.0 to 42.2 h] versus 50.1 h [IQR, 27.6 to 77.9 h]; P < 0.001) and the time to effective antimicrobial therapy (15.9 h [IQR, 1.9 to 25.7 h] versus 28.0 h [IQR, 9.5 to 56.7 h]; P < 0.001) compared to CONV + ASP, respectively. IMPORTANCE Our study supports the additional benefit of molecular rapid diagnostic test in combination with timely antimicrobial stewardship program (ASP) intervention on shortening the time to both optimal and effective antimicrobial therapy in patients with ESBL- or carbapenemase-producing Escherichia coli and Klebsiella pneumoniae bloodstream infections, compared to conventional microbiological methods and ASP. Gram-negative infections are associated with significant morbidity and mortality, often resulting in life-threatening organ dysfunction. Both resistance phenotypes confer resistance to many of our first-line antimicrobial agents with carbapenemase-producing Enterobacterales requiring novel beta-lactam and beta-lactamase inhibitor combinations or other susceptible non-beta-lactam antibiotics for treatment. National resistance trends in a cohort of hospitalized patients at U.S. hospitals during our study period demonstrate the increasing incidence of both resistance phenotypes, reinforcing the generalizability and timeliness of such analysis.

Multiplex suppression of four quadruplet codons via tRNA directed evolution.

Genetic code expansion technologies supplement the natural codon repertoire with assignable variants in vivo, but are often limited by heterologous translational components and low suppression efficiencies. Here, we explore engineered Escherichia coli tRNAs supporting quadruplet codon translation by first developing a library-cross-library selection to nominate quadruplet codon-anticodon pairs. We extend our findings using a phage-assisted continuous evolution strategy for quadruplet-decoding tRNA evolution (qtRNA-PACE) that improved quadruplet codon translation efficiencies up to 80-fold. Evolved qtRNAs appear to maintain codon-anticodon base pairing, are typically aminoacylated by their cognate tRNA synthetases, and enable processive translation of adjacent quadruplet codons. Using these components, we showcase the multiplexed decoding of up to four unique quadruplet codons by their corresponding qtRNAs in a single reporter. Cumulatively, our findings highlight how E. coli tRNAs can be engineered, evolved, and combined to decode quadruplet codons, portending future developments towards an exclusively quadruplet codon translation system.

Synthesis of (-)-deoxypodophyllotoxin and (-)-epipodophyllotoxin via a multi-enzyme cascade in E. coli.

BACKGROUND: The aryltetralin lignan (-)-podophyllotoxin is a potent antiviral and anti-neoplastic compound that is mainly found in Podophyllum plant species. Over the years, the commercial demand for this compound rose notably because of the high clinical importance of its semi-synthetic chemotherapeutic derivatives etoposide and teniposide. To satisfy this demand, (-)-podophyllotoxin is conventionally isolated from the roots and rhizomes of Sinopodophyllum hexandrum, which can only grow in few regions and is now endangered by overexploitation and environmental damage. For these reasons, targeting the biosynthesis of (-)-podophyllotoxin precursors or analogues is fundamental for the development of novel, more sustainable supply routes. RESULTS: We recently established a four-step multi-enzyme cascade to convert (+)-pinoresinol into (-)-matairesinol in E. coli. Herein, a five-step multi-enzyme biotransformation of (-)-matairesinol to (-)-deoxypodophyllotoxin was proven effective with 98 % yield at a concentration of 78 mg/L. Furthermore, the extension of this cascade to a sixth step leading to (-)-epipodophyllotoxin was evaluated. To this end, seven enzymes were combined in the reconstituted pathway involving inter alia three plant cytochrome P450 monooxygenases, with two of them being functionally expressed in E. coli for the first time. CONCLUSIONS: Both, (-)-deoxypodophyllotoxin and (-)-epipodophyllotoxin, are direct precursors to etoposide and teniposide. Thus, the reconstitution of biosynthetic reactions of Sinopodophyllum hexandrum as an effective multi-enzyme cascade in E. coli represents a solid step forward towards a more sustainable production of these essential pharmaceuticals.

Safety and pharmacodynamics of an engineered E. coli Nissle for the treatment of phenylketonuria: a first-in-human phase 1/2a study.

Phenylketonuria (PKU) is a rare disease caused by biallelic mutations in the PAH gene that result in an inability to convert phenylalanine (Phe) to tyrosine, elevated blood Phe levels and severe neurological complications if untreated. Most patients are unable to adhere to the protein-restricted diet, and thus do not achieve target blood Phe levels. We engineered a strain of E. coli Nissle 1917, designated SYNB1618, through insertion of the genes encoding phenylalanine ammonia lyase and L-amino acid deaminase into the genome, which allow for bacterial consumption of Phe within the gastrointestinal tract. SYNB1618 was studied in a phase 1/2a randomized, placebo-controlled, double-blind, multi-centre, in-patient study ( NCT03516487 ) in adult healthy volunteers (n = 56) and patients with PKU and blood Phe level ≥600 mmol l-1 (n = 14). Participants were randomized to receive a single dose of SYNB1618 or placebo (part 1) or up to three times per day for up to 7 days (part 2). The primary outcome of this study was safety and tolerability, and the secondary outcome was microbial kinetics. A D5-Phe tracer (15 mg kg-1) was used to study exploratory pharmacodynamic effects. SYNB1618 was safe and well tolerated with a maximum tolerated dose of 2 × 1011 colony-forming units. Adverse events were mostly gastrointestinal and of mild to moderate severity. All participants cleared the bacteria within 4 days of the last dose. Dose-responsive increases in strain-specific Phe metabolites in plasma (trans-cinnamic acid) and urine (hippuric acid) were observed, providing a proof of mechanism for the potential to use engineered bacteria in the treatment of rare metabolic disorders.

A comparison of E. coli susceptibility for amoxicillin/clavulanic acid according to EUCAST and CLSI guidelines.

In our tertiary care center, the reported susceptibility of E. coli blood isolates to amoxicillin/clavulanic acid exceeded 90% in 2005 and showed a progressive decrease to 50% by 2017. In this study, we investigate whether there is a real increase in resistant E. coli strains or if this apparent decline in reported susceptibility might be attributed to the substitution of CLSI by EUCAST guidelines in 2014. We randomly selected 237 E. coli blood isolates (stored at - 80 °C) from 1985 to 2018 and reassessed their MIC values, applying both the CLSI (fixed ratio of clavulanic acid) and EUCAST guidelines (fixed concentration of clavulanic acid). In parallel, the susceptibility of these isolates was retested by disk diffusion, according to the EUCAST guidelines. Whole genome sequencing was successfully performed on 233 of the 237 isolates. In only 130 of the 237 isolates (55.0%), testing according to the EUCAST and CLSI criteria delivered identical MIC values for amoxicillin/clavulanic acid. In 64 of the 237 isolates (27.0%), the MIC values diverged one dilution; in 38 (16.0%), two dilutions; and in five (2.1%), three dilutions. From these 107 discrepant results, testing according to EUCAST methodology revealed more resistant profiles in 93 E. coli strains (94.1%). Also, phenotypical susceptibility testing according to EUCAST guidelines tends to correlate better with the presence of beta-lactamase genes compared to CLSI testing procedure. This study highlights the low agreement between EUCAST and CLSI methodologies when performing MIC testing of amoxicillin/clavulanic acid. More strains are categorized as resistant when EUCAST guidelines are applied. The low agreement between EUCAST and CLSI was confirmed by WGS, since most of EUCAST resistant/CLSI sensitive isolates harbored beta-lactamase genes.

Application of Cell-Free Protein Synthesis System for the Biosynthesis of l-Theanine.

l-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of l-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure l-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is the toxicity of the substrate ethylamine on microbial host cells. In this study, we introduced a cell-free protein synthesis (CFPS) system for l-theanine production. The CFPS expressed l-theanine synthetase 2 from Camellia sinensis (CsTS2) could produce l-theanine at a concentration of 11.31 µM after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for l-theanine biosynthesis. The γ-glutamylcysteine synthetase from Escherichia coli could produce l-theanine at the highest concentration of 302.96 µM after 24 h of reaction. Furthermore, CFPS was used to validate a hypothetical two-step l-theanine biosynthetic pathway consisting of the l-alanine decarboxylase from C. sinensis (CsAD) and multiple l-theanine synthases. Among them, the combination of CsAD and the l-glutamine synthetase from Pseudomonas taetrolens (PtGS) could synthesize l-theanine at the highest concentration of 13.42 µM. Then, we constructed an engineered E. coli strain overexpressed CsAD and PtGS to further confirm the l-theanine biosynthesis ability in living cells. This engineered E. coli strain could convert l-alanine and l-glutamate in the medium to l-theanine at a concentration of 3.82 mM after 72 h of fermentation. Taken together, these results demonstrated that the CFPS system can be used to produce the l-theanine through the two-step l-theanine biosynthesis pathway, indicating the potential application of CFPS for the biosynthesis of other active compounds.

Novel Engineered Bacterium/Black Phosphorus Quantum Dot Hybrid System for Hypoxic Tumor Targeting and Efficient Photodynamic Therapy.

Intratumoral hypoxia significantly constrains the susceptibility of solid tumors to oxygen-dependent photodynamic therapy (PDT), and effort to reverse such hypoxia has achieved limited success to date. Herein, we developed a novel engineered bacterial system capable of targeting hypoxic tumor tissues and efficiently mediating the photodynamic treatment of these tumors. For this system, we genetically engineered Escherichia coli to express catalase, after which we explored an electrostatic adsorption approach to link black phosphorus quantum dots (BPQDs) to the surface of these bacteria, thereby generating an engineered E. coli/BPQDs (EB) system. Following intravenous injection, EB was able to target hypoxic tumor tissues. Subsequent 660 nm laser irradiation drove EB to generate reactive oxygen species (ROS) and destroy the membranes of these bacteria, leading to the release of catalase that subsequently degrades hydrogen peroxide to yield oxygen. Increased oxygen levels alleviate intratumoral hypoxia, thereby enhancing BPQD-mediated photodynamic therapy. This system was able to efficiently kill tumor cells in vivo, exhibiting good therapeutic efficacy. In summary, this study is the first to report the utilization of engineered bacteria to facilitate PDT, and our results highlight new avenues for BPQD-mediated cancer treatment.

High prevalence of multidrug resistant ESBL- and plasmid mediated AmpC-producing clinical isolates of Escherichia coli at Maputo Central Hospital, Mozambique.

BACKGROUND: Epidemiological data of cephalosporin-resistant Enterobacterales in Sub-Saharan Africa is still restricted, and in particular in Mozambique. The aim of this study was to detect and characterize extended-spectrum ß-lactamase (ESBL) - and plasmid-mediated AmpC (pAmpC)-producing clinical strains of Escherichia coli at Maputo Central Hospital (MCH), a 1000-bed reference hospital in Maputo, Mozambique. METHODS: A total of 230 clinical isolates of E. coli from urine (n = 199) and blood cultures (n = 31) were collected at MCH during August-November 2015. Antimicrobial susceptibility testing was performed by the disc diffusion method and interpreted according to EUCAST guidelines. Isolates with reduced susceptibility to 3rd generation cephalosporins were examined further; phenotypically for an ESBL-/AmpC-phenotype by combined disc methods and genetically for ESBL- and pAmpC-encoding genes by PCR and partial amplicon sequencing as well as genetic relatedness by ERIC-PCR. RESULTS: A total of 75 isolates with reduced susceptibility to cefotaxime and/or ceftazidime (n = 75) from urine (n = 58/199; 29%) and blood (n = 17/31; 55%) were detected. All 75 isolates were phenotypically ESBL-positive and 25/75 (33%) of those also expressed an AmpC-phenotype. ESBL-PCR and amplicon sequencing revealed a majority of blaCTX-M (n = 58/75; 77%) dominated by blaCTX-M-15. All AmpC-phenotype positive isolates (n = 25/75; 33%) scored positive for one or more pAmpC-genes dominated by blaMOX/FOX. Multidrug resistance (resistance ≥ three antibiotic classes) was observed in all the 75 ESBL-positive isolates dominated by resistance to trimethoprim-sulfamethoxazole, ciprofloxacin and gentamicin. ERIC-PCR revealed genetic diversity among strains with minor clusters indicating intra-hospital spread. CONCLUSION: We have observed a high prevalence of MDR pAmpC- and/or ESBL-producing clinical E. coli isolates with FOX/MOX and CTX-Ms as the major ß-lactamase types, respectively. ERIC-PCR analyses revealed genetic diversity and some clusters indicating within-hospital spread. The overall findings strongly support the urgent need for accurate and rapid diagnostic services to guide antibiotic treatment and improved infection control measures.

Kinetics and thermodynamics of thermal inactivation for recombinant Escherichia coli cellulases, cel12B, cel8C, and polygalacturonase, peh28; biocatalysts for biofuel precursor production.

Lignocellulosic biomass conversion using cellulases/polygalacturonases is a process that can be progressively influenced by several determinants involved in cellulose microfibril degradation. This article focuses on the kinetics and thermodynamics of thermal inactivation of recombinant Escherichia coli cellulases, cel12B, cel8C and a polygalacturonase, peh 28, derived from Pectobacterium carotovorum sub sp. carotovorum. Several consensus motifs conferring the enzymes' thermal stability in both cel12B and peh28 model structures have been detailed earlier, which were confirmed for the three enzymes through the current study of their thermal inactivation profiles over the 20-80°C range using the respective activities on carboxymethylcellulose and polygalacturonic acid. Kinetic constants and half-lives of thermal inactivation, inactivation energy, plus inactivation entropies, enthalpies and Gibbs free energies, revealed high stability, less conformational change and protein unfolding for cel12B and peh28 due to thermal denaturation compared to cel8C. The apparent thermal stability of peh28 and cel12B, along with their hydrolytic efficiency on a lignocellulosic biomass conversion as reported previously, makes these enzymes candidates for various industrial applications. Analysis of the Gibbs free energy values suggests that the thermal stabilities of cel12B and peh28 are entropy-controlled over the tested temperature range.

Discovery of an Inhibitor for Bacterial 3-Mercaptopyruvate Sulfurtransferase that Synergistically Controls Bacterial Survival.

H2S-producing enzymes in bacteria have been shown to be closely engaged in the process of microbial survival and antibiotic resistance. However, no inhibitors have been discovered for these enzymes, e.g., 3-mercaptopyruvate sulfurtransferase (MST). In the present study, we identified several classes of inhibitors for Escherichia coli MST (eMST) through high-throughput screening of ∼26,000 compounds. The thiazolidinedione-type inhibitors were found to selectively bind to Arg178 and Ser239 residues of eMST but hardly affected human MST. Moreover, the pioglitazone of this class concentration dependently accumulates the 3-mercaptopyruvate substrate and suppresses the H2S and reactive sulfane sulfur products in bacteria. Importantly, pioglitazone could potentiate the level of reactive oxygen species in cellulo and consequently enhance the antimicrobial effects of gentamicin and macrophages in culture. This study has identified the bioactive inhibitor of eMST, paving the way for the pharmacological targeting of eMST to synergistically control the survival of E. coli.

Antimicrobial resistance and ESBL genes in E. coli isolated in proximity to a sewage treatment plant.

This study was aimed to determine the prevalence of antibiotic-resistant Escherichia coli in the Szreniawa river with detailed aims of: (i) assessment of differences in the number of microbiological indicators of water quality in a diurnal cycle in a vicinity of the sewage treatment plant (STP); (ii) determination of prevalence of antimicrobial resistant E. coli isolated from three sites located at varying locations toward the STP; (iii) evaluation of the presence of extended-spectrum beta lactamase (ESBL)-determining genes in waterborne E. coli isolated from three sites of Szreniawa and (iv) genetic similarity assessment among the E. coli populations. Bacteriological contamination (coliforms, E. coli, E. faecalis) was assessed using membrane filtration. Fifty E. coli strains, the species of which was confirmed by MALDI-TOF analysis, were subjected to antimicrobial resistance tests using standard disk-diffusion method. Double disk synergy test was used to assess the ESBL production and PCR tests were conducted to detect the ESBL-conferring genes and evaluate the genetic diversity. A significant variation in the number of bacteriological indicators was observed both within and between the sampling sites, suggesting the effect of effluent from the STP, point discharge of household sewage and agricultural runoff on the water contamination. The resistance to amoxicillin/clavulanate (90%) and ampicillin (36%) was most prevalent. Multidrug resistance was observed in 40% of strains but no ESBL-producing strains were observed phenotypically. However, the presence of three ESBL-determining genes (TEM, OXA and CTX-M) was detected in 24, 10 and 8% of strains, respectively. A number of factors caused considerable pollution of the river and numerous multidrug resistant E. coli strains were isolated.

Translational Detection of Nonproteinogenic Amino Acids Using an Engineered Complementary Cell-Free Protein Synthesis Assay.

We developed a simple and rapid method for analyzing nonproteinogenic amino acids that does not require conventional chromatographic equipment. In this technique, nonproteinogenic amino acids were first converted to a proteinogenic amino acid through in vitro metabolism in a cell extract. The proteinogenic amino acid generated from the nonproteinogenic precursors were then incorporated into a reporter protein using a cell-free protein synthesis system. The titers of the nonproteinogenic amino acids could be readily quantified by measuring the activity of reporter proteins. This method, which combines the enzymatic conversion of target amino acids with translational analysis, makes amino acid analysis more accessible while minimizing the cost and time requirements. We anticipate that the same strategy could be extended to the detection of diverse biochemical molecules with clinical and industrial implications.

Escherichia coli membrane-derived oxygen-reducing enzyme system (Oxyrase) protects bubaline spermatozoa during cryopreservation.

The objective of this study was to determine the effectiveness of deoxygenation of semen extender using Escherichia coli membrane-derived oxygen scavenger (Oxyrase) on post-thaw quality of buffalo (Bubalus bubalis) spermatozoa. Sixteen semen ejaculates, four each from four bulls, were each divided into five equal fractions, diluted using Tris-egg yolk extender supplemented with different concentrations of Oxyrase (0, 0.3, 0.6, 0.9, and 1.2 U/ml), designated as treatments T1, T2, T3, T4, and T5, respectively, and cryopreserved. Immediately after thawing, Oxyrase did not improve sperm kinetics and motility; however, it improved the keeping quality (significantly lower deterioration of post-thaw sperm motility after incubation for 120 min) in T3. Further, T3 reduced (p < .05) cholesterol efflux and protected the intactness of the sperm plasma membrane. Flow cytometry with Fluo-3 AM/propidium iodide (PI) dual staining revealed the highest (p < .05) proportion of live spermatozoa with low intracellular calcium in T3. Oxyrase supplementation protected spermatozoa from premature capacitation which was confirmed by low expression of tyrosine-phosphorylated proteins (32, 75, and 80 kDa) and a relatively lower percentage of F-pattern (uncapacitated spermatozoa) in chlortetracycline assay. Importantly, the Oxyrase fortification decreased superoxide anion in a dose-dependent manner indicating reduced availability of oxygen at sperm mitochondrial level. Similarly, in Oxyrase-fortified sperm, malondialdehyde concentration, an index of lipid peroxidation, is also reduced in a dose-dependent manner. In conclusion, we demonstrate that deoxygenation of buffalo semen by Oxyrase has the potential of improving post-thaw sperm quality by overcoming the problem of cryocapacitation and oxidative damage during cryopreservation process.

The Phospholipid:Diacylglycerol Acyltransferase-Mediated Acyl-Coenzyme A-Independent Pathway Efficiently Diverts Fatty Acid Flux from Phospholipid into Triacylglycerol in Escherichia coli.

Researchers have long endeavored to accumulate triacylglycerols (TAGs) or their derivatives in easily managed microbes. The attempted production of TAGs in Escherichia coli has revealed barriers to the broad applications of this technology, including low TAG productivity and slow cell growth. We have demonstrated that an acyl-CoA-independent pathway can divert phospholipid flux into TAG formation in E. coli mediated by Chlamydomonas reinhardtii phospholipid:diacylglycerol acyltransferase (CrPDAT) without interfering with membrane functions. We then showed the synergistic effect on TAG accumulation via the acyl-CoA-independent pathway mediated by PDAT and the acyl-CoA-dependent pathway mediated by wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT). Furthermore, CrPDAT led to synchronous TAG accumulation during cell growth, and this could be enhanced by supplementation of arbutin. We also showed that rationally mutated CrPDAT was capable of decreasing TAG lipase activity without impairing PDAT activity. Finally, ScPDAT from Saccharomyces cerevisiae exhibited similar activities as CrPDAT in E. coli Our results suggest that the improvement in accumulation of TAGs and their derivatives can be achieved by fine-tuning of phospholipid metabolism in E. coli Understanding the roles of PDAT in the conversion of phospholipids into TAGs during the logarithmic growth phase may enable a novel strategy for the production of microbial oils.IMPORTANCE Although phospholipid:diacylglycerol acyltransferase (PDAT) activity is presumed to exist in prokaryotic oleaginous bacteria, the corresponding gene has not been identified yet. In this article, we have demonstrated that an acyl-CoA-independent pathway can divert phospholipid flux into TAG formation in Escherichia coli mediated by exogenous CrPDAT from Chlamydomonas reinhardtii without interfering with membrane functions. In addition, the acyl-CoA-independent pathway and the acyl-CoA-dependent pathway had the synergistic effect on TAG accumulation. Overexpression of CrPDAT led to synchronous TAG accumulation during cell growth. In particular, CrPDAT possessed multiple catalytic activities, and the rational mutation of CrPDAT led to the decrease of TAG lipase activity without impairing acyltransferase activity. The present findings suggested that applying PDAT in E. coli or other prokaryotic microbes may be a promising strategy for accumulation of TAGs and their derivatives.

Combinatorial synthetic pathway fine-tuning and cofactor regeneration for metabolic engineering of Escherichia coli significantly improve production of D-glucaric acid.

D-glucaric acid (GA) has been identified as among promising biotechnological alternatives to oil-based chemicals. GA and its derivatives are widely used in food additives, dietary supplements, drugs, detergents, corrosion inhibitors and biodegradable materials. The increasing availability of a GA market is improving the cost-effectiveness and efficiency of various biosynthetic pathways. In this study, an engineered Escherichia coli strain GA10 was constructed by systematic metabolic engineering. This involved redirecting metabolic flux into the GA biosynthetic pathways, blocking the conversion pathways of d-glucuronic acid (GlcA) and GA into by-products, introducing an in situ NAD+ regeneration system and fine-tuning the activity of the key enzyme, myo-inositol oxygenase (Miox). Subsequently, the culture medium was optimized to achieve the best performance of the GA10 strain. GA was produced at 5.35 g/L (extracellular and intracellular), with a maximized yield of ∼0.46 mol/mol on d-glucose and glycerol, by batch fermentation. This work demonstrates efficient biosynthetic pathways of GA in E. coli by metabolic engineering and should accelerate the application of GA biosynthetic pathways in industrial processes.

Enhancement of Production of D-Glucosamine in Escherichia coli by Blocking Three Pathways Involved in the Consumption of GlcN and GlcNAc.

D-Glucosamine is a commonly used dietary supplement that promotes cartilage health in humans. Metabolic flux analysis showed that D-glucosamine production could be increased by blocking three pathways involved in the consumption of glucosamine-6-phosphate and acetylglucosamine-6-phosphate. By homologous single-exchange, two key genes (nanE and murQ) of Escherichia coli BL21 were knocked out, respectively. The D-glucosamine yields of the engineered strains E. coli BL21ΔmurQ and E. coli BL21ΔnanE represented increases by factors of 2.14 and 1.79, respectively. Meanwhile, for bifunctional gene glmU, we only knocked out its glucosamine-1-phosphate acetyltransferase domain by 3D structural analysis to keep the engineered strain E. coli BL21glmU-Δgpa survival, which resulted in an increase in the production of D-glucosamine by a factor of 2.16. Moreover, for further increasing D-glucosamine production, two genes encoding rate-limiting enzymes, named glmS and gna1, were coexpressed by an RBS sequence in those engineered strains. The total concentrations of D-glucosamine in E. coli BL21 glmU-Δgpa', E. coli BL21ΔmurQ', and E. coli BL21ΔnanE' were 2.65 g/L, 1.73 g/L, and 1.38 g/L, which represented increases by factors of 8.83, 5.76, and 3.3, respectively.