Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Reactive oxygen and nitrogen species’ effect on <Emphasis Type="Italic">lux</Emphasis>-biosensors based on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella typhimurium</Emphasis>

The effect of reactive oxygen and nitrogen species on lux-biosensors based on the Escherichia coli K12 MG1655 and Salmonella typhimurium LT2 host strains was investigated. The bioactivity of exogenous free radicals to the constitutively luminescent E. coli strain with plasmid pXen7 decreased in the order H₂O₂ > OCl^– > NO^? > RОO^? > ONOO^–> O₂^?- while the bioluminescence of S. typhimurium strain transformed with this plasmid decreased in the order NO^? > H₂O₂ > ONOO^– > RОO^? > OCl^– > O₂^?- The cross-reactivity of induced lux-biosensors to reactive oxygen and nitrogen species, the threshold sensitivity and the luminescence amplitude dependences from the plasmid specificity and the host strain were indicated. The biosensors with plasmid pSoxS′::lux possessed a wider range of sensitivity, including H₂O₂ and OCl^–, along with O₂^?- and NO^?. Among the used reactive oxygen and nitrogen species, H₂O₂ showed the highest induction activity concerning to the plasmids pKatG′::lux, pSoxS′::lux and pRecA′::lux. The inducible lux-biosensors based on S. typhimurium host strain possessed a higher sensitivity to the reactive oxygen and nitrogen species in comparison with the E. coli lux-biosensors.     

Presence of a [3Fe–4S] cluster in a PsaC variant as a functional component of the photosystem I electron transfer chain in <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

A site-directed C14G mutation was introduced into the stromal PsaC subunit of Synechococcus sp. strain PCC 7002 in vivo in order to introduce an exchangeable coordination site into the terminal F_B [4Fe–4S] cluster of Photosystem I (PSI). Using an engineered PSI-less strain (psaAB deletion), psaC was deleted and replaced with recombinant versions controlled by a strong promoter, and the psaAB deletion was complemented. Modified PSI accumulated at lower levels in this strain and supported slower photoautotrophic growth than wild type. As-isolated PSI complexes containing PsaC_C14G showed resonances with g values of 2.038 and 2.007 characteristic of a [3Fe–4S]¹⁺ cluster. When the PSI complexes were illuminated at 15 K, these resonances partially disappeared and two new sets of resonances appeared. The majority set had g values of 2.05, 1.95, and 1.85, characteristic of F_A ^?, and the minority set had g values of 2.11, 1.90, and 1.88 from F_B′ in the modified site. The S?=?1/2 spin state of the latter implied the presence of a thiolate as the terminal ligand. The [3Fe–4S] clusters could be partially reconstituted with iron, producing a larger population of [4Fe–4S] clusters. Rates of flavodoxin reduction were identical in PSI complexes isolated from wild type and the PsaC_C14G variant strain; this implied equivalent capacity for forward electron transfer in PSI complexes that contained [3Fe–4S] and [4Fe–4S] clusters. The development of this cyanobacterial strain is a first step toward translation of in vitro PSI-based biosolar molecular wire systems in vivo and provides new insights into the formation of Fe/S clusters.     

ISC-like [2Fe–2S] ferredoxin (FdxB) dimer from <Emphasis Type="Italic">Pseudomonas putida</Emphasis> JCM 20004: structural and electron–nuclear double resonance characterization

The crystal structure of the ISC-like [2Fe–2S] ferredoxin (FdxB), probably involved in the de novo iron-sulfur cluster biosynthesis (ISC) system of Pseudomonas putida JCM 20004, was determined at 1.90-Å resolution and displayed a novel tail-to-tail dimeric form. P. putida FdxB lacks the consensus free cysteine usually present near the cluster of ISC-like ferredoxins, indicating its primarily electron transfer role in the iron-sulfur cluster. Orientation-selective electron–nuclear double resonance spectroscopic analysis of reduced FdxB in conjunction with the crystal structure has identified the innermost Fe2 site with a high positive spin population as the nonreducible iron retaining the Fe³⁺ valence and the outermost Fe1 site as the reduced iron with a low negative spin density. The average g _max direction is skewed, forming an angle of about 27.3° (±4°) with the normal of the [2Fe–2S] plane, whereas the g _int and g _min directions are distributed in the cluster plane, presumably tilted by the same angle with respect to this plane. These results are related to those for other [2Fe–2S] proteins in different electron transport chains (e.g. adrenodoxin) and suggest a significant distortion of the electronic structure of the reduced [2Fe–2S] cluster under the influence of the protein environment around each iron site in general.     

Spectroscopic evidence for an all-ferrous [4Fe–4S]<Superscript>0</Superscript> cluster in the superreduced activator of 2-hydroxyglutaryl-CoA dehydratase from <Emphasis Type="Italic">Acidaminococcus </Emphasis><Emphasis Type="Italic">fermentans</Emphasis>

The key enzyme of the fermentation of glutamate by Acidaminococcus fermentans, 2-hydroxyglutarylcoenzyme A dehydratase, catalyzes the reversible syn-elimination of water from (R)-2-hydroxyglutaryl-coenzyme A, resulting in (E)-glutaconylcoenzyme A. The dehydratase system consists of two oxygen-sensitive protein components, the activator (HgdC) and the actual dehydratase (HgdAB). Previous biochemical and spectroscopic studies revealed that the reduced [4Fe–4S]⁺ cluster containing activator transfers one electron to the dehydratase driven by ATP hydrolysis, which activates the enzyme. With a tenfold excess of titanium(III) citrate at pH 8.0 the activator can be further reduced, yielding about 50% of a superreduced [4Fe–4S]⁰ cluster in the all-ferrous state. This is inferred from the appearance of a new Mössbauer spectrum with parameters δ = 0.65 mm/s and ΔE _Q = 1.51–2.19 mm/s at 140 K, which are typical of Fe(II)S₄ sites. Parallel-mode electron paramagnetic resonance (EPR) spectroscopy performed at temperatures between 3 and 20 K showed two sharp signals at g = 16 and 12, indicating an integer-spin system. The X-band EPR spectra and magnetic Mössbauer spectra could be consistently simulated by adopting a total spin S _t = 4 for the all-ferrous cluster with weak zero-field splitting parameters D = ?0.66 cm^?1 and E/D = 0.17. The superreduced cluster has apparent spectroscopic similarities with the corresponding [4Fe–4S]⁰ cluster described for the nitrogenase Fe-protein, but in detail their properties differ. While the all-ferrous Fe-protein is capable of transferring electrons to the MoFe-protein for dinitrogen reduction, a similar physiological role is elusive for the superreduced activator. This finding supports our model that only one-electron transfer steps are involved in dehydratase catalysis. Nevertheless we discuss a common basic mechanism of the two diverse systems, which are so far the only described examples of the all-ferrous [4Fe–4S]⁰ cluster found in biology.     

Natively oxidized amino acid residues in the spinach cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript><Emphasis Type="Italic">6</Emphasis></Subscript><Emphasis Type="Italic">f</Emphasis> complex

The cytochrome b ₆ f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b ₆ f is 10–20 fold higher than that observed for the analogous respiratory cytochrome bc₁ complex. The types of ROS produced (O₂^{??, 1}O₂, and, possibly, H₂O₂) and the site(s) of ROS production within the b ₆ f complex have been the subject of some debate. Proposed sources of ROS have included the heme b _p , PQ _p ^?? (possible sources for O₂^??), the Rieske iron–sulfur cluster (possible source of O₂^?? and/or ¹O₂), Chl a (possible source of ¹O₂), and heme c _n (possible source of O₂^?? and/or H₂O₂). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b ₆ f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b ₆ f complex.     

A novel NADPH-dependent reductase of <Emphasis Type="Italic">Sulfobacillus acidophilus</Emphasis> TPY phenol hydroxylase: expression,characterization, and functional analysis

The reductase component (MhpP) of the Sulfobacillus acidophilus TPY multicomponent phenol hydroxylase exhibits only 40 % similarity to Pseudomonas sp. strain CF600 phenol hydroxylase reductase. Amino acid sequence alignment analysis revealed that four cysteine residues (Cys-X ₄ -Cys-X ₂ -Cys-X _29-35 -Cys) are conserved in the N terminus of MhpP for [2Fe-2S] cluster binding, and two other motifs (RXYS and GXXS/T) are conserved in the C terminus for binding the isoalloxazine and phosphate groups of flavin adenine dinucleotide (FAD). Two motifs (S/T-R and yXCGp) responsible for binding to reduce nicotinamide adenine dinucleotide phosphate (NADPH) are also conserved in MhpP, although some residues differ. To confirm the function of this reductase, MhpP was heterologously expressed in Escherichia coli BL21(DE3) and purified. UV-visible spectroscopy and electron paramagnetic resonance spectroscopy revealed that MhpP contains a [2Fe-2S] cluster. MhpP mutants in which the four cysteine residues were substituted via site-directed mutagenesis lost the ability to bind the [2Fe-2S] cluster, resulting in a decrease in enzyme-specific oxidation of NADPH. Thin-layer chromatography revealed that MhpP contains FAD. Substrate specificity analyses confirmed that MhpP uses NADPH rather than NADH as an electron donor. MhpP oxidizes NADPH using cytochrome c, potassium ferricyanide, or nitro blue tetrazolium as an electron acceptor, with a specific activity of 1.7 ± 0.36, 0.78 ± 0.13, and 0.16 ± 0.06 U/mg, respectively. Thus, S. acidophilus TPY MhpP is a novel NADPH-dependent reductase component of phenol hydroxylase that utilizes FAD and a [2Fe-2S] cluster as cofactors.     

High-frequency underwater plasma discharge application in antibacterial activity

Plasma discharge is a novel disinfection and effectual inactivation approach to treat microorganisms in aqueous systems. Inactivation of Gram-negative Escherichia coli (E. coli) by generating high-frequency, high-voltage, oxygen (O₂) injected and hydrogen peroxide (H₂O₂) added discharge in water was achieved. The effect of H₂O₂ dose and oxygen injection rate on electrical characteristics of discharge and E. coli disinfection has been reported. Microbial log reduction dependent on H₂O₂ addition with O₂ injection was observed. The time variation of the inactivation efficiency quantified by the log reduction of the initial E. coli population on the basis of optical density measurement was reported. The analysis of emission spectrum recorded after discharge occurrence illustrated the formation of oxidant species (OH^?, H, and O). Interestingly, the results demonstrated that O₂ injected and H₂O₂ added, underwater plasma discharge had fabulous impact on the E. coli sterilization. The oxygen injection notably reduced the voltage needed for generating breakdown in flowing water and escalated the power of discharge pulses. No impact of hydrogen peroxide addition on breakdown voltage was observed. A significant role of oxidant species in bacterial inactivation also has been identified. Furthermore the E. coli survivability in plasma treated water with oxygen injection and hydrogen peroxide addition drastically reduced to zero. The time course study also showed that the retardant effect on E. coli colony multiplication in plasma treated water was favorable, observed after long time. High-frequency underwater plasma discharge based biological applications is technically relevant and would act as baseline data for the development of novel antibacterial processing strategies.     

Production of caffeoylmalic acid from glucose in engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To achieve biosynthesis of caffeoylmalic acid from glucose in engineered Escherichia coli.

Results

We constructed the biosynthetic pathway of caffeoylmalic acid in E. coli by co-expression of heterologous genes RgTAL, HpaBC, At4CL2 and HCT2. To enhance the production of caffeoylmalic acid, we optimized the tyrosine metabolic pathway of E. coli to increase the supply of the substrate caffeic acid. Consequently, an E. coli–E. coli co-culture system was used for the efficient production of caffeoylmalic acid. The final titer of caffeoylmalic acid reached 570.1 mg/L.

Conclusions

Microbial production of caffeoylmalic acid using glucose has application potential. In addition, microbial co-culture is an efficient tool for producing caffeic acid esters.

     

Efficient production of indigoidine in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Indigoidine is a bacterial natural product with antioxidant and antimicrobial activities. Its bright blue color resembles the industrial dye indigo, thus representing a new natural blue dye that may find uses in industry. In our previous study, an indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment at 3.93 g/l. To further improve the production of indigoidine, in this work, the direct biosynthetic precursor l-glutamine was fed into the fermentation broth of the engineered E. coli strain harboring Sc-IndC and Sc-IndB. The highest titer of indigoidine reached 8.81 ± 0.21 g/l at 1.46 g/l l-glutamine. Given the relatively high price of l-glutamine, a metabolic engineering technique was used to directly enhance the in situ supply of this precursor. A glutamine synthetase gene (glnA) was amplified from E. coli and co-expressed with Sc-indC and Sc-indB in E. coli BAP1, leading to the production of indigoidine at 5.75 ± 0.09 g/l. Because a nitrogen source is required for amino acid biosynthesis, we then tested the effect of different nitrogen-containing salts on the supply of l-glutamine and subsequent indigoidine production. Among the four tested salts including (NH₄)₂SO₄, NH₄Cl, (NH₄)₂HPO₄ and KNO₃, (NH₄)₂HPO₄ showed the best effect on improving the titer of indigoidine. Different concentrations of (NH₄)₂HPO₄ were added to the fermentation broths of E. coli BAP1/Sc-IndC+Sc-IndB+GlnA, and the titer reached the highest (7.08 ± 0.11 g/l) at 2.5 mM (NH₄)₂HPO₄. This work provides two efficient methods for the production of this promising blue pigment in E. coli.     

Evaluation of microbial globin promoters for oxygen-limited processes using <Emphasis Type="Italic">Escherichia coli</Emphasis>

Oxygen-responsive promoters can be useful for synthetic biology applications, however, information on their characteristics is still limited. Here, we characterized a group of heterologous microaerobic globin promoters in Escherichia coli. Globin promoters from Bacillus subtilis, Campylobacter jejuni, Deinococcus radiodurans, Streptomyces coelicolor, Salmonella typhi and Vitreoscilla stercoraria were used to express the FMN-binding fluorescent protein (FbFP), which is a non-oxygen dependent marker. FbFP fluorescence was monitored online in cultures at maximum oxygen transfer capacities (OTR_max) of 7 and 11 mmol L^?1 h^?1. Different FbFP fluorescence intensities were observed and the OTR_max affected the induction level and specific fluorescence emission rate (the product of the specific fluorescence intensity multiplied by the specific growth rate) of all promoters. The promoter from S. typhi displayed the highest fluorescence emission yields (the quotient of the fluorescence intensity divided by the scattered light intensity at every time-point) and rate, and together with the promoters from D. radiodurans and S. coelicolor, the highest induction ratios. These results show the potential of diverse heterologous globin promoters for oxygen-limited processes using E. coli.     

The protective effect of a novel antioxidant gene from <Emphasis Type="Italic">Mycobacterium avium</Emphasis> against nitrosative and oxidative stress in <Emphasis Type="Italic">E. coli</Emphasis>

The production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) is an important host defense mechanism in response to infection by Mycobacterium tuberculosis. A variety of genes have been implicated in resistance to ROI and RNI, including noxR1. However, studies in Mycobacterium avium, an important pathogen among nontuberculous mycobacteria, are limited. We aim to investigate the role of a novel gene cloned from M. avium with high similarity to noxR1, noA, in resistance against RNI and ROI in M. tuberculosis. After subcloning noA into vector for expression in E. coli, we performed survival rate analysis in the bacteria transformed with noA (pET-noA) and without noA (pET-his) after exposure to nitrosative stresses by S-nitrosoglutathione (GSNO) and sodium nitrite, and oxidative stresses by H₂O₂. Compared with pET-his, the survival rate of pET-noA was 1 log₁₀-fold higher after exposure to GSNO and sodium nitrite. We observed 1 log₁₀-fold, 2 log₁₀-fold and 3 log₁₀-fold higher survival rate in pET-noA than pET-his after exposure to H₂O₂ for 3, 6 and 9 h, respectively. With the combined treatment of H₂O₂ and GSNO, we found more than 2 log₁₀-fold increase in survival rate in pET-noA comparing with pET-his, suggesting a possible synergistic effect. In summary, noA gene cloned from M. avium has been shown to protect E. coli from both RNI and ROI.     

Pig farm environment as a source of beta-lactamase or AmpC-producing <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis>

The present study was undertaken to detect the occurrence of beta-lactamase-/AmpC-producing Klebsiella and Escherichia coli in healthy pigs, feed, drinking water, and pen floor or surface soil. The study also intended to detect the clonal relationship between the environmental and porcine isolates to confirm the route of transmission. Rectal swabs and environmental samples were collected from apparently healthy pigs kept in organized or backyard farms in India. The pigs had no history of antibiotic intake. Production of phenotypical beta-lactamase, associated genes, and class I integron gene was detected in E. coli and Klebsiella isolates. The phylogenetic relationship among the isolates was established on the basis of Random amplification of polymorphic DNA banding pattern. Beta-lactamase-producing Klebsiella were isolated from healthy pigs (20.0%), pen floor swabs/surface soil swabs (14.0%), and drinking water (100%). Escherichia coli isolated from healthy pigs (14.4%), pen floor/surface soil (8.0%), and drinking water (33.3%) were detected as beta-lactamase producers. Majority of beta-lactamase-producing isolates possessed bla_CTX-M-9. Further, 35 (81%) Klebsiella and all the E. coli isolates were detected as AmpC beta-lactamase ACBL producers and possessed bla_AmpC. Sixteen beta-lactamase-producing Klebsiella (37.20%) and 13 E. coli (86.67%) possessed class I integron. Few resistant isolates from environmental sources (surface soil swab and drinking water) and the studied pigs were detected within the same cluster of the dendrogram representing their similarities. The study indicated about the possible role of contaminated environment as a source of beta-lactamase/AmpC-producing Klebsiella and E. coli in pigs.     

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically-engineered <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To engineer Escherichia coli for the heterologous production of di-rhamnolipids, which are important biosurfactants but mainly produced by opportunistic pathogen Pseudomonas aeruginosa.

Results

The codon-optimized rhlAB and rhlC genes originating from P. aeruginosa and Burkholderia pseudomallei were combinatorially expressed in E. coli to produce di-rhamnolipids with varied congeners compositions. Genes involved in endogenous upstream pathways (rhamnose and fatty acids synthesis) were co-overexpressed with rhlAB–rhlC, resulting in variations of rhamnolipids production and congeners compositions. Under the shake-flask condition, co-overexpression of rfbD with rhlAB–rhlC increased rhamnolipids production (0.64 ± 0.02 g l^?1) than that in strain only expressing rhlAB–rhlC (0.446 ± 0.009 g l^?1), which was mainly composed of di-rhamnolipids congeners Rha–Rha–C₁₀–C₁₀.

Conclusion

Biosynthesis of di-rhamnolipids and variations of congeners composition in genetically engineered E. coli strains were achieved via combiniations of mono-/di-rhamnolipids synthesis modules and endogenous upstream modules.

     

Construction of a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/A-68 for production of carboxymethylcellulase and comparison of its production with its wild type, <Emphasis Type="Italic">Bacillus velezensis</Emphasis> A-68 in a pilot-scale bioreactor

A gene encoding carboxymethylcellulase (CMCase) of Bacillus velezensis A-68 had been cloned in Escherichia coli JM109. Based on productivity and economic aspect, rice bran and ammonium chloride were chosen to be optimal carbon and nitrogen sources for production of CMCase by E. coli JM109/A-68. The optimal conditions for rice bran, ammonium chloride, and initial pH of medium for production of CMCase were established by the response surface methodology (RSM). The concentrations of four salts in the medium, K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄, for production of CMCase also were optimized. The optimal temperatures for cell growth and production of CMCase were 37°C. The maximal production of CMCase by E. coli JM109/A-68 was 880.2 U/mL, which was 10.5 time higher than its wild type, B. velezensis A-68. The production of CMCase by E. coli JM109/A-68 was compared with that by B. velezensis A-68 in a 100 L pilot-scale bioreactor under the optimized conditions. The production of CMCase by E. coli JM109/A-68 was found to be the mixed-growth associated unlike the growthassociated production of CMCase by B. velezensis A-68.     

Antigenic polysaccharides of bacteria: 41. Structures of the O-specific polysaccharides of <Emphasis Type="Italic">Shigella dysenteriae</Emphasis> types 4 and 5 revised by NMR spectroscopy

The earlier established structures of the acidic O-specific polysaccharides from two typical strains of the Shigella dysenteriae bacterium were revised using modern NMR spectroscopy techniques. In particular, the configurations of the glycosidic linkages of GlcNAc (S. dysenteriae type 4) and mannose (S. dysenteriae type 5) residues were corrected. In addition, the location of the sites of non-stoichiometric O-acetylation in S. dysenteriae type 4 was determined: the lateral fucose residue was shown to be occasionally O-acetylated; also, theposition of the O-acetyl group present at the stoichiometric quantity in S. dysenteriae type 5 was corrected. The revised structures of the polysaccharides studied are shown below. The known identity of the O-specific polysaccharide structures of S. dysenteriae type 5 and Escherichia coli O58 was confirmed by ¹³C NMR spectroscopy and, hence, the structure of the E. coli O58 polysaccharide should be revised in the same manner.

Open image in new window

where L-Rhap3Rlac2Ac is 2-O-acetyl-3-O-[(R-1-carboxyethyl]-L-rhamnose

     

Conjugative plasmid transfer from <Emphasis Type="Italic">Escherichia coli</Emphasis> is a versatile approach for genetic transformation of thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Geobacillus</Emphasis> species

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli–Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1 × 10^?7 and 3.8 × 10^?2/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.     

Biosynthesis of two quercetin <Emphasis Type="Italic">O</Emphasis>-diglycosides in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Various flavonoid glycosides are found in nature, and their biological activities are as variable as their number. In some cases, the sugar moiety attached to the flavonoid modulates its biological activities. Flavonoid glycones are not easily synthesized chemically. Therefore, in this study, we attempted to synthesize quercetin 3-O-glucosyl (1→2) xyloside and quercetin 3-O-glucosyl (1→6) rhamnoside (also called rutin) using two uridine diphosphate-dependent glycosyltransferases (UGTs) in Escherichia coli. To synthesize quercetin 3-O-glucosyl (1→2) xyloside, sequential glycosylation was carried out by regulating the expression time of the two UGTs. AtUGT78D2 was subcloned into a vector controlled by a Tac promoter without a lacI operator, while AtUGT79B1 was subcloned into a vector controlled by a T7 promoter. UDP-xyloside was supplied by concomitantly expressing UDP-glucose dehydrogenase (ugd) and UDP-xyloside synthase (UXS) in the E. coli. Using these strategies, 65.0 mg/L of quercetin 3-O-glucosyl (1→2) xyloside was produced. For the synthesis of rutin, one UGT (BcGT1) was integrated into the E. coli chromosome and the other UGT (Fg2) was expressed in a plasmid along with RHM2 (rhamnose synthase gene 2). After optimization of the initial cell concentration and incubation temperature, 119.8 mg/L of rutin was produced. The strategies used in this study thus show promise for the synthesis of flavonoid diglucosides in E. coli.     

The initial analysis of a serine proteinase gene (<Emphasis Type="Italic">AccSp10</Emphasis>) from <Emphasis Type="Italic">Apis cerana cerana</Emphasis>: possible involvement in pupal development,innate immunity and abiotic stress responses

Serine proteinases play important roles in innate immunity and insect development. We isolated a serine proteinase gene, designated AccSp10, from the Chinese honeybees (Apis cerana cerana). RT-qPCR and a Western blot analysis at different pupal development stages indicated that AccSp10 might be involved in melanin formation in pupae and promote pupal development. In adult workers, the expression of AccSp10 was upregulated by treatments mimicking harmful environments such as the presence of Bacillus bombysepticus, different temperatures (4, 24 and 42 °C), HgCl₂, H₂O₂ and paraquat; the exception was treatment with VC (vitamin C), which did not upregulate AccSp10 expression. Western blot confirmed the results. A disc diffusion assay indicated that recombinant AccSp10 accelerated E. coli cell death during stimulation with harmful substances (HgCl₂, paraquat and cumene hydroperoxide). These findings suggest that AccSp10 may be involved in the pupal development of Chinese honeybees and protection against microorganisms and abiotic harms.