Quercetin Glucoside Production by Engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli strains expressing the O-glucosyltransferases UGT73B3 or UGT84B1 were compared for the production of glucosides from quercetin supplied into a defined medium. The formation of quercetin-3-glucoside (Q3G) by UGT73B3 showed a maximum at 33 °C, while the formation of quercetin-7-glucoside by UGT84B1 increased with increasing temperature to 37 °C. The highest concentrations of Q3G were attained by strains having a deletion in the pgi gene-coding phosphoglucose isomerase, which effectively blocked the entry of glucose-6P into the Embden–Meyerhof–Parnas pathway. Formation of Q3G was improved in 1-L controlled bioreactors compared to shake flask cultures, a result attributed to the greater oxygen transfer rate in bioreactors. Under batch conditions with 30 g/L glucose as the sole carbon source, E. coli MEC367 (MG1655 pgi) expressing UGT73B3 generated 3.9 g/L Q3G in 56 h.     

Study of the growth of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis>, <Emphasis Type="Italic">Escherichia coli</Emphasis> and their mixtures by microcalorimetry

In a majority of environments, microbes live as interacting communities. Microbial communities are composed of a mix of microbes with often unknown functions. Polymicrobial diseases represent the clinical and pathological manifestations induced by the presence of multiple infectious agents. These diseases are difficult to diagnose and treat and usually are more severe than monomicrobial infections. The interaction relationship between Enterococcus faecalis and Escherichia coli was researched using a Calvet calorimeter. Three mixtures of both bacteria were prepared in the following proportions: 20 + 80 % (0.2 mL E. faecalis + 0.8 mL E. coli), 50 + 50 % (0.5 mL E. faecalis + 0.5 mL E. coli) and 80 + 20 % (0.8 mL E. faecalis + 0.2 mL E. coli). Experiments were carried out at concentration of 10⁶ CFU mL^?1 and a constant temperature of 309.65 K. The differences in shape of graph of E. faecalis, E. coli and their mixtures were compared. Also, the thermokinetic parameters such as detection time (t _d), growth constant (k), generation time (G) and the amount of heat released (Q) were calculated.     

Solid-phase microbead array for multiplex O-serotyping of <Emphasis Type="Italic">Escherichia coli</Emphasis>

Typing and classification of Escherichia coli (E. coli) according to cell wall components, like polysaccharides, is routinely done by serotyping. Given the presence of 188 known O-antigens, this process is complex. The authors present a proof-of-concept planar microbead array for multiplexed O-serotyping. Ten clinically relevant E. coli serotypes associated with high risk for diarrhea in humans were examined (O26, O55, O78, O118, O124, O127, O128, O142, O145 and O157). Antisera were assigned to specific microbead populations, which can be differentiated by size and fluorescence color. Automatted image processing and data analysis were conducted by a microscopic interpretation platform. Homogenous antiserum coating of the microbeads was demonstrated by an intra-population CV that ranges from 3.3 to 6.3% and by an inter-population CV of 9.5%. Typical detections limits are in the range from 0.31 to 0.71 refMFI. Significantly elevated fluorescence signals revealed that E. coli of a certain serogroup bound specifically to microbeads with the matching antiserum (p < 0.001). In our perception, the method represents a viable diagnostic tool for automated multiplex serotyping of E. coli. It enables simultaneous and high-throughput screening for different O-antigens by a simple staining and binding protocol.

Open image in new window

Graphical abstract Schematic of a planar microbead array for the typing and classification of E. coli according to cell wall components. Based on coated fluorescent microbeads, multiplex O-serotyping of E. coli is accomplished via fluorescence imaging.

     

Production of <Emphasis Type="Italic">R</Emphasis>-Mandelic Acid Using Nitrilase from Recombinant <Emphasis Type="Italic">E. coli</Emphasis> Cells Immobilized with Tris(Hydroxymethyl)Phosphine

Recombinant Escherichia coli cells harboring nitrilase from Alcaligenes faecalis were immobilized using tris(hydroxymethyl)phosphine (THP) as the coupling agent. The optimal pH and temperature of the THP-immobilized cells were determined at pH 8.0 and 55 °C. The half-lives of THP-immobilized cells measured at 35, 40, and 50 °C were 1800, 965, and 163 h, respectively. The concentration of R-mandelic acid (R-MA) reached 358 mM after merely 1-h conversion by the immobilized cells with 500 mM R,S-mandelonitrile (R,S-MN), affording the highest productivity of 1307 g L^?1 day^?1 and the space-time productivity of 143.2 mmol L^?1 h^?1 g^?1. The immobilized cells with granular shape were successfully recycled for 60 batches using 100 mM R,S-MN as substrate at 40 °C with 64% of relative activity, suggesting that the immobilized E. coli cells obtained in this study are promising for the production of R-MA.     

Synthesis of new hydrazones based on <Emphasis Type="Italic">o</Emphasis>- and <Emphasis Type="Italic">p</Emphasis>-hydroxybenzohydrazides

o- and p-Hydroxybenzohydrazides reacted with various unsaturated aromatic aldehydes to give the corresponding N′-(hydroxybenzoyl)hydrazones. Inhibitory activity of the obtained hydrazones against cathepsin E was evaluated.     

Synthesis of oxo derivatives of <Emphasis Type="Italic">N</Emphasis>-(<Emphasis Type="Italic">p</Emphasis>-tolysulfonyl)hexahydrocycloalka[<Emphasis Type="Italic">b</Emphasis>]indoles

Hydroxymercuration-demercuration of N-p-tolysulfonyl-4,4a,9,9a-tetrahydro-3H-carbazoles and N-p-tolyl(or methyl)sulfonyl-1,3a,4,8b-tetrahydrocyclopenta[b]indoles leads to the formation of the corresponding N-p-tolylsulfonyl-2,3,4,4a,9,9a-hexahydro-1H-carbazol-2-ols and N-p-tolyl(or methyl)sulfonyl-1,2,3,3a,4,8b-hexahydrocyclopenta[b]indol-2-ols. The latter are oxidized to 2-oxo derivatives with potassium dichromate. The oxidation of 2-methoxy-8-methyl-N-p-tolylsulfonyl-2,3,4,4a,9,9a-hexahydro-1H-carbazol-1-ol under analogous conditions gives 2-methoxy-8-methyl-N-p-tolysulfonyl-2,3,4,4a,9,9a-hexahydro-1H-carbazol-1-one.     

Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the <Emphasis Type="Italic">Aedes aegypti</Emphasis> Mosquito Utilizing <Emphasis Type="Italic">E. coli</Emphasis> as a host

Background

Studying proteins and enzymes involved in important biological processes in the Aedes aegypti mosquito is limited by the quantity that can be directly isolated from the mosquito. Adding to this difficulty, digestive enzymes (midgut proteases) involved in metabolizing blood meal proteins require a more oxidizing environment to allow proper folding of disulfide bonds. Therefore, recombinant techniques to express foreign proteins in Escherichia coli prove to be effective in producing milligram quantities of the expressed product. However, with the most commonly used strains having a reducing cytoplasm, soluble expression of recombinant proteases is hampered. Fortunately, new E. coli strains with a more oxidizing cytoplasm are now available to ensure proper folding of disulfide bonds.

Results

Utilizing an E. coli strain with a more oxidizing cytoplasm (SHuffle® T7, New England Biolabs) and changes in bacterial growth temperature has resulted in the soluble expression of the four most abundantly expressed Ae. aegypti midgut proteases (AaET, AaSPVI, AaSPVII, and AaLT). A previous attempt of solubly expressing the full-length zymogen forms of these proteases with the leader (signal) sequence and a modified pseudo propeptide with a heterologous enterokinase cleavage site led to insoluble recombinant protein expression. In combination with the more oxidizing cytoplasm, and changes in growth temperature, helped improve the solubility of the zymogen (no leader) native propeptide proteases in E. coli. Furthermore, the approach led to autocatalytic activation of the proteases during bacterial expression and observable BApNA activity. Different time-points after bacterial growth induction were tested to determine the time at which the inactive (zymogen) species is observed to transition to the active form. This helped with the purification and isolation of only the inactive zymogen forms using Nickel affinity.

Conclusions

The difficulty in solubly expressing recombinant proteases in E. coli is caused by the native reducing cytoplasm. However, with bacterial strains with a more oxidizing cytoplasm, recombinant soluble expression can be achieved, but only in concert with changes in bacterial growth temperature. The method described herein should provide a facile starting point to recombinantly expressing Ae. aegypti mosquito proteases or proteins dependent on disulfide bonds utilizing E. coli as a host.

     

<Emphasis Type="Italic">CrMAPK3</Emphasis> regulates the expression of iron-deficiency-responsive genes in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background

Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption. Nevertheless, the response, transmission, and regulation of downstream gene expression in algae cells have not to be investigated. Considering that the MAPK pathway is essential for abiotic stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas.

Results

Arabidopsis MAPK gene sequences were used as entry data to search for homologous genes in Chlamydomonas reinhardtii genome database to investigate the functions of mitogen-activated protein kinase (MAPK) gene family in C. reinhardtii under iron-free conditions. Results revealed 16 C. reinhardtii MAPK genes labeled CrMAPK2–CrMAPK17 with TXY conserved domains and low homology to MAPK in yeast, Arabidopsis, and humans. The expression levels of these genes were then analyzed through qRT-PCR and exposure to high salt (150 mM NaCl), low nitrogen, or iron-free conditions. The expression levels of these genes were also subjected to adverse stress conditions. The mRNA levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were remarkably upregulated under iron-deficient stress. The increase in CrMAPK3 expression was 43-fold greater than that in the control. An RNA interference vector was constructed and transformed into C. reinhardtii 2A38, an algal strain with an exogenous FOX1:ARS chimeric gene, to silence CrMAPK3. After this gene was silenced, the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1 were decreased. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced.

Conclusion

CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.

     

Death kinetics of <Emphasis Type="Italic">Escherichia coli</Emphasis> in goat milk and <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> in cloudberry jam treated by ohmic heating

The influence of ohmic heating on the death kinetic parameters of Escherichia coli ATCC® 25922 in goat milk and spores of Bacillus licheniformis ATCC® 14580 in cloudberry jam was investigated and compared with that of conventional heating. Ohmic treatment of goat milk shortened the decimal reduction time D in comparison with the D values obtained at conventional treatment. Similarly, the z value, increase of temperature required for a ten-fold reduction of D, was also lower at ohmic treatment. The death kinetics of Bacillus licheniformis ATCC® 14580 spores in cloudberry jam was also studied employing both types of heat treatment. Similar conclusions were obtained for the D values as in the case of goat milk. However, the differences between the z values obtained for ohmic and conventional heating were not significant.     

Synthesis and antibacterial activity of new <Emphasis Type="Italic">N</Emphasis>-substituted 7-amino-4-methyl-2<Emphasis Type="Italic">H</Emphasis>-chromen-2-ones

N-Substituted 7-amino-4-methyl-2H-chromen-2-ones containing one or two functionalized azole or azine moieties were synthesized. The structures of all synthesized compounds were confirmed by IR, ¹H NMR, and ¹³C NMR spectroscopy. Some of the synthesized compounds exhibited weak antibacterial activity against Rhizobium radiobacter, Escherichia coli, and Xanthomonas campestris.     

Synthesis of <Emphasis Type="Italic">ortho</Emphasis>-carboranyl derivatives of (<Emphasis Type="Italic">S</Emphasis>)-asparagine and (<Emphasis Type="Italic">S</Emphasis>)-glutamine

(S)-Asparagine and (S)-glutamine ortho-carboranyl derivatives with free amino and carboxy groups in the α-position were synthesized. By an example of N ^γ-(1,2-dicarba-closo-dodecarboran-3-yl)-(S)-glutamine it was demonstrated that the developed synthetic approach carboranyl derivatives of amino acids allowed the preparation of optically pure isomers.     

Stability of <Emphasis Type="Italic">s</Emphasis>-, <Emphasis Type="Italic">p</Emphasis>-, and <Emphasis Type="Italic">d</Emphasis>-element diphosphates in water

The mean atomic Gibbs energies of formation of (Δ _f ? _at ⁰ ) of s-, p-, and d-element diphosphates have been calculated using ion increments of the Gibbs energy (Δ _f G ⁰). The diphosphate hydrolysis kinetics is considered, and a correlation between the Δ _f ? _at ⁰ values and the hydrolysis rate constants is presented.     

Research on the Solid State Fermentation of Jerusalem Artichoke Pomace for Producing <Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">R</Emphasis>-2,3-Butanediol by <Emphasis Type="Italic">Paenibacillus polymyxa</Emphasis> ZJ-9

R,R-2,3-butanediol (R,R-2,3-BD) was produced by Paenibacillus polymyxa ZJ-9, which was capable of utilizing inulin without previous hydrolysis. The Jerusalem artichoke pomace (JAP) derived from the conversion of Jerusalem artichoke powder into inulin extract, which was usually used for biorefinery by submerged fermentation (SMF), was utilized in solid state fermentation (SSF) to produce R,R-2,3-BD. In this study, the fermentation parameters of SSF were optimized and determined in flasks. A novel bioreactor was designed and assembled for the laboratory scale-up of SSF, with a maximum yield of R,R-2,3-BD (67.90 g/kg (JAP)). This result is a 36.3% improvement compared with the flasks. Based on the same bath of Jerusalem artichoke powder, the total output of R,R-2,3-BD increased by 38.8% for the SSF of JAP combined with the SMF of inulin extraction. Overall, the utilization of JAP for R,R-2,3-BD production was beneficial to the comprehensive utilization of Jerusalem artichoke tuber.     

Comparison of bacteriolytic activity of human interleukin-2 and chicken egg lysozyme on <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

The article continues studies of the recently discovered bacteriolytic activity of interleukin-2. It was detected earlier that interleukin (IL-2) possesses greater substrate specificity in comparison with chicken egg lysozyme. IL-2 disrupted the cell wall of Escherichia coli but did not lyse lysozyme substrates such as the cell walls of Micrococcus luteus and Bacillus subtilis. In the present study it is demonstrated for the first time that both IL-2 and chicken egg lysozyme are capable of lysing Lactobacillus plantarum. The effects of IL-2 and chicken egg lysozyme on Lactobacillus plantarum are compared with those on Escherichia coli. The dependences of the rate of lysis on the concentration of bacteriolytic factors and pH are studied.     

Synthesis of <Emphasis Type="Italic">N</Emphasis>-and <Emphasis Type="Italic">C</Emphasis>-trimethylsilyl-substituded anililes

N-Metallation of bromoanilines with ethylmagnesium bromide followed by a reaction with trimethylchlorosilane provided N-mono and N-bis(trimethylsilyl)bromoanilines depending on the structure of substrate. The metallation of bissilylated bromoanilines with butyllithium permitted the introduction of a trimethylsilyl substituent in the aromatic ring. Previously unknown 2-bromo-N,N-bis(trimethylsilyl)aniline, 2,6-dibromo-N-trimethylsilylaniline, 2,6-dibromo-N,N-bis(trimethylsilyl)aniline, 2-bromo-6-trimethylsilylaniline, 2-bromo-6-trimethylsilyl-N,N-bis(trimethylsilyl)aniline, 2-bromo-6-trimethylsilyl-N-trimethylsilylaniline, 2,4,6-tribromo-N-trimethylsilylaniline, and 2,4,6-tribromo-N,N-bis(trimethylsilyl)aniline were prepared. The structures of the compounds obtained were established by the chromato-mass spectrometry and ¹H, ¹³C, and ²⁹Si NMR spectroscopy.     

Cycloaminomethylation of dihydric phenols catalyzed by <Emphasis Type="Italic">d</Emphasis>- and <Emphasis Type="Italic">f</Emphasis>-metal compounds

An efficient method has been developed for the synthesis of 7,16,25-triaryl-7,8,16,17,25,26-hexahydro-6H,15H,24H-tribenzo[f,m,t][1,5,8,12,15,19,3,10,17]hexaoxatriazacyclohenicosines, 3,8-diaryl-2,3,4,7,8,9-hexahydrobenzo[1,2-e:4,3-e′]bis[1,3]oxazines, 3,9-bis(chlorophenyl)-3,4,9,10-tetrahydro-2H,8H-benzo[1,2-e:3,4-e′]bis[1,3]oxazines, and 2,9-bis(chlorophenyl)-1,2,3,8,9,10-hexahydrobenzo[1,2-e:6,5-e′]bis-[1,3]oxazines via cycloaminomethylation of pyrocatechol, resorcinol, and hydroquinone with N,N-bis(methoxymethyl) anilines in the presence of samarium catalysts.     

Synthesis of ethyl (2<Emphasis Type="Italic">E</Emphasis>,4<Emphasis Type="Italic">E</Emphasis>)- and (2<Emphasis Type="Italic">E</Emphasis>,4<Emphasis Type="Italic">Z</Emphasis>)-5-chloropenta-2,4-dienoates

An efficient synthetic approach to 2E,4E and 2E,4Z isomers of ethyl 5-chloropenta-2,4-dienoate has been developed on the basis of one-pot oxidation–olefination of readily accessible (E)- and (Z)-3-chloroprop-2-en-1-ols by the action of barium manganate and ethyl (triphenyl-λ⁵-phosphanylidene)acetate.     

Copolymer of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride with maleic acid as drug carrier

A copolymer of N,N-diallyl-N,N-dimethylammonium chloride with maleic acid of constant composition was prepared under the conditions of radical initiation. The possibility of the functionalization of the copolymer with drugs containing amino groups by polymer-analogous transformations was examined. Conditions were found for preparing conjugates of the copolymer with isoniazid. The structures and the quantitative compositions of the conjugates were determined by ¹³С NMR spectroscopy, and the possibility of preparing conjugates with controlled drug content was demonstrated.     

Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60 °C was 30.4 U/mg, obviously higher than those in P. pastoris (22.7 U/mg) and E. coli (19.7 U/mg). Moreover, after 10 min incubation at 80 °C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70 % of the activity at pH 7.0 and 37 °C, whereas the values were only about 25 and 50 % when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.     

Thermooxidative decomposition of heterocyclic <Emphasis Type="Italic">N</Emphasis>-oxides

Thermooxidative decomposition of pyridine N-oxide, 4-(4-dimethylaminostyryl)pyridine N-oxide, 4-(4-methoxystyryl)pyridine N-oxide, quinoline N-oxide, 2-methylquinoline N-oxide, 4-chloroquinoline N-oxide, 2-styrylquinoline N-oxide, and 2-(4-dimethylaminostyryl)quinoline N-oxide was studied. The kinetic parameters of the thermooxidative processes were calculated according to three independent procedures. The relation between the nature of heterocyclic N-oxide and its stability to thermal oxidation was analyzed.