Escherichia coli HGT: Engineered for high glucose throughput even under slowly growing or resting conditions

Aerobic production-scale processes are constrained by the technical limitations of maximum oxygen transfer and heat removal. Consequently, microbial activity is often controlled via limited nutrient feeding to maintain it within technical operability. Here, we present an alternative approach based on a newly engineered Escherichia coli strain. This E. coli HGT (high glucose throughput) strain was engineered by modulating the stringent response regulation program and decreasing the activity of pyruvate dehydrogenase. The strain offers about three-fold higher rates of cell-specific glucose uptake under nitrogen-limitation (0.6 g_Glc g_CDW⁻¹ h⁻¹) compared to that of wild type, with a maximum glucose uptake rate of about 1.8 g_Glc g_CDW⁻¹ h⁻¹ already at a 0.3 h⁻¹ specific growth rate. The surplus of imported glucose is almost completely available via pyruvate and is used to fuel pyruvate and lactate formation. Thus, E. coli HGT represents a novel chassis as a host for pyruvate-derived products.     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from unrelated carbon sources by metabolically engineered Escherichia coli

A metabolically engineered Escherichia coli has been constructed for the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] from unrelated carbon sources. Genes involved in succinate degradation in Clostridium kluyveri and P(3HB) accumulation pathway of Ralstonia eutropha were co-expressed for the synthesis of the above copolyester. E. coli native succinate semialdehyde dehydrogenase genes sad and gabD were both deleted for eliminating succinate formation from succinate semialdehyde, which functioned to enhance the carbon flux to 4HB biosynthesis. The metabolically engineered E. coli produced 9.4 g l^?1 cell dry weight containing 65.5% P(3HB-co-11.1 mol% 4HB) using glucose as carbon source in a 48 h shake flask growth. The presence of 1.5–2 g l^?1 α-ketoglutarate or 1.0 g l^?1 citrate enhanced the 4HB monomer content from 11.1% to more than 20%. In a 6 l fermentor study, a 23.5 g l^?1 cell dry weight containing 62.7% P(3HB-co-12.5 mol% 4HB) was obtained after 29 h of cultivation. To the best of our knowledge, this study reports the highest 4HB monomer content in P(3HB-co-4HB) produced from unrelated carbon sources.     

Succinic acid production from sucrose and molasses by metabolically engineered E. coli using a cell surface display system

To achieve sucrose-metabolizing capability, different sucrose utilization operons have been introduced into E. coli that cannot utilize sucrose. However, these engineered strains still suffer from low growth rates and low sucrose uptake rates. In this study, cell surface display system was adopted in engineered E. coli AFP111 for succinic acid production from sucrose and molasses directly. Invertase (CscA) from E. coli W was successfully anchored to outer membrane by fusion with OmpC anchoring motif, and the displayed CscA showed high extracellular activity. Compared with the sucrose permease system, the cell surface display system consumed less ATP during sucrose metabolism. When less ATP was consumed by AFP111/pTrcC-cscA, the succinic acid productivity from sucrose was 23% higher than that by AFP111/pCR2.1-cscBKA that having the sucrose permease system. As a result, 41 g L⁻¹ and 36.3 g L⁻¹ succinic acid were produced by AFP111/pTrcC-cscA from sucrose and sugarcane molasses respectively at 34 h in 3-L fermentor during dual-phase fermentation. In addition, 79 g L⁻¹ succinic acid was accumulated with recovered AFP111/pTrcC-cscA cells at the end of dual-phase fermentation in 3-L fermentor, and the overall yield was 1.19 mol mol⁻¹ hexose.     

Characterization of a new Cu/Zn-superoxide dismutase from Beauveria bassiana and two site-directed mutations crucial to its antioxidation activity without chaperon

A Cu/Zn-superoxide dismutase (SOD) was characterized for the first time from Beauveria bassiana by gene cloning, heterogeneous expression and function analysis. This 154-aa SOD (BbSod1) was deduced from a 465-bp gene cloned, showing 49–96% sequence identity to Cu/Zn-SODs from other 57 fungi. BbSod1 and its form engineered with two site-directed mutations P143S and P145L (BbSod1-Mut) or a fused copper chaperon Lys7 (BbSod1-Lys7) were expressed well in Escherichia coli. Crude extracts and purified BbSod1-Mut from cell cultures exhibited much higher antioxidation activities than the counterparts of BbSod1-Lys7 whereas BbSod1 showed no substantial activity. The engineered enzymes were best induced by overnight incubation at 20 °C in Luria-Bertani medium including 2.5 mM Cu²⁺, 0.5 mM Zn²⁺ and 0.5 mM isopropyl-d-thiogalactopyranoside after 5-h growth to log-phase at 37 °C. Our results highlight alternative means to producing highly active fungal Cu/Zn-SOD in E. coli by making use of the two site-directed mutations without chaperon.     

Thermolabile antifreeze protein produced in Escherichia coli for structural analysis

Inclusion bodies of recombinant human growth hormone (r-hGH) were isolated from Escherichia coli, enriched and solubilized in 100 mM Tris buffer containing 6 M n-propanol and 2 M urea. Around 4 mg/ml of r-hGH from inclusion bodies were solubilized in 6 M n-propanol-based buffer containing 2 M urea. Existence of native-like secondary structure of r-hGH in 6 M n-propanol solution was confirmed by CD and fluorescence spectra. Solubilized r-hGH was subsequently refolded by pulsatile dilution, purified to homogeneity and found to be functionally active. Tris buffer containing 6 M n-propanol and 2 M urea also effectively solubilized a number of proteins expressed as inclusion bodies in E. coli. Mild solubilization of inclusion body proteins, chaotropic effect of n-propanol at high concentration and kosmotropic effect at lower concentration helped in improved refolding of the solubilized protein. Around 40% of the r-hGH in the form of inclusion body aggregates was refolded into bioactive form while using n-propanol as solubilization agent. Solubilization with 6 M n-propanol solution thus can be a viable alternative for achieving high throughput recovery of bioactive protein from inclusion bodies of E. coli.     

Moderate hypothermia attenuates α(1)-adrenoceptor-mediated contraction in isolated rat aorta: the role of the endothelium

Moderate hypothermia (25–31 °C) may have a significant influence on vascular tone. We investigated the cellular mechanisms by which moderate hypothermia alters α-adrenoceptor-mediated contraction in rat thoracic aortae. Cyclooxygenase inhibition by indomethacin; nitric oxide (NO) synthase inhibition by l-NAME; potassium channel and endothelium-derived hyperpolarizing factor (EDHF) inhibition by glibenclamide and TEA; G protein inhibition by pertussis toxin; α₂-adrenergic inhibition by yohimbine; and β-adrenergic inhibition by propranolol were assessed for their effect on the contractile response to the α1-adrenoceptor agonist phenylephrine (Phe) in combination with moderate hypothermia (25 °C). Moderate hypothermia produced a shift to the right for the Phe concentration–response curves in endothelium-intact (E+) and endothelium-denuded (E?) aortic rings. The maximal response to Phe in E+ rings was significantly decreased (P < 0.05) at 25 °C compared to 38 °C, whereas there was no significant difference in E? rings. Hypothermia-induced vasorelaxation in E+ rings was attenuated (P < 0.05) following combined pretreatment with l-NAME (10^?4 M) and indomethacin (10^?5 M), whereas other inhibitors had no significant effect. Importantly, the addition of TEA to rings that were pretreated with l-NAME and indomethacin exhibited no further attenuation (P > 0.05) of hypothermia-induced vasorelaxation. The concentrations of cGMP and cAMP, as measured by radioimmunoassay, were significantly increased (P < 0.05) in E+ rings at 25 °C compared to those at 38 °C, whereas there were no significant differences (P > 0.05) in E? rings. The present study demonstrated that rat aortic endothelium is stimulated during moderate hypothermia and that the NO–cGMP and prostacyclin (PGI₂)–cAMP pathways represent endothelium-dependent mechanisms of hypothermia-induced vasorelaxation. In contrast, EDHF may not be associated with hypothermia-induced vasorelaxation.     

Production of d-ribose by metabolically engineered Escherichia coli

Escherichia coli was metabolically engineered for the production of d-ribose, a functional five-carbon sugar, from xylose. For the accumulation of d-ribose, two genes of transketolase catalyzing the conversion of d-ribose-5-phosphate to sedoheptulose-7-phosphate in pentose phosphate pathway were disrupted to create a transketolase-deficient E. coli SGK013. In batch fermentation, E. coli SGK013 grew by utilizing glucose and then started to produce d-ribose from xylose after glucose depletion. E. coli SGK013 produced 0.75 g/L of d-ribose, which was identical to the standard d-ribose as confirmed by HPLC and LC/MS analyses. To improve D-ribose production, the ptsG gene encoding the glucose-specific IICB component was disrupted additionally, resulting in the construction of E. coli SGK015. The carbon catabolite repression-negative E. coli SGK015 utilized xylose and glucose simultaneously and produced up to 3.75 g/L of d-ribose, which is a 5-fold improvement compared to that of E. coli SGK013.     

Laccase- and chloroperoxidase-nanotube paint composites with bactericidal and sporicidal activity

Laccase and chloroperoxidase (CPO) were separately immobilized onto multi-walled carbon nanotubes (MWNTs) and subsequently mixed with a commercial eco-friendly paint to generate biocatalytic coatings. The laccase-nanotube based paints showed >99% bactericidal activity against Escherichia coli and Staphylococcus aureus (both challenged with 10⁶ CFU/mL) within 30 min and >98% sporicidal activity against Bacillus cereus and Bacillus anthracis-ΔSterne (initially challenged with 10⁴ CFU/mL) within 120 min. The CPO-nanotube based paints also showed >99% antimicrobial activity within 30 min against E. coli and S. aureus (both challenged with 10⁶ CFU/mL). These enzyme-nanotube based formulations provide an eco-friendly route to generate biocidal compounds, which can prevent the growth of a broad spectrum of bacterial pathogens, including spores. These enzyme-containing paints may be envisioned to be applied as self-decontaminating coatings onto a wide range of surfaces, such as hospital infrastructure, medical devices and equipment, food processing and packaging, etc.; in all cases effective killing of a variety of infectious organisms is critical.     

The addition of Co2+ enhances the catalytic efficiency and thermostability of recombinant glucose isomerase from Thermobifida fusca

Glucose isomerase is an important industrial enzyme that catalyzes the reversible isomerization of glucose to fructose. In this study, the effect of cobalt ions (Co²⁺) on the catalytic efficiency and thermostability of recombinant glucose isomerase from Thermobifida fusca was analyzed. The activity of glucose isomerase from engineered Escherichia coli supplemented with 1 mM Co²⁺ (C-GI) reached 41 U/ml, 2.1-fold higher than enzyme prepared from E. coli without additive (GI). The purified C-GI also exhibited an increased specific activity (23.8 U/mg compared to 12.1 U/mg for GI) and a greater thermostability (half-life of 17 h at 75 °C, 11.3-fold higher than GI (1.5 h)). The optimal temperature for C-GI shifted from 80 °C to 85 °C and demonstrated higher activity over pH 7.0–9.0. The k_cat/K_m value of C-GI (89.3 M^?1 s^?1) for the isomerization of glucose to fructose was nearly 1.75-fold higher than that of GI. In addition, the engineered cells were immobilized with the method of flocculation-cross linking. The immobilized cells supplemented with 1 mM Co²⁺ (C-IGI) had a better operational performance than cells without additives (IGI); at the end of 6 cycles, the conversion rate of C-IGI was still 43.1%, meeting the conversion rate requirement.     

Engineering cofactor and transport mechanisms in Saccharomyces cerevisiae for enhanced acetyl-CoA and polyketide biosynthesis

Synthesis of polyketides at high titer and yield is important for producing pharmaceuticals and biorenewable chemical precursors. In this work, we engineered cofactor and transport pathways in Saccharomyces cerevisiae to increase acetyl-CoA, an important polyketide building block. The highly regulated yeast pyruvate dehydrogenase bypass pathway was supplemented by overexpressing a modified Escherichia coli pyruvate dehydrogenase complex (PDHm) that accepts NADP⁺ for acetyl-CoA production. After 24 h of cultivation, a 3.7-fold increase in NADPH/NADP⁺ ratio was observed relative to the base strain, and a 2.2-fold increase relative to introduction of the native E. coli PDH. Both E. coli pathways increased acetyl-CoA levels approximately 2-fold relative to the yeast base strain. Combining PDHm with a ZWF1 deletion to block the major yeast NADPH biosynthesis pathway resulted in a 12-fold NADPH boost and a 2.2-fold increase in acetyl-CoA. At 48 h, only this coupled approach showed increased acetyl-CoA levels, 3.0-fold higher than that of the base strain. The impact on polyketide synthesis was evaluated in a S. cerevisiae strain expressing the Gerbera hybrida 2-pyrone synthase (2-PS) for the production of the polyketide triacetic acid lactone (TAL). Titers of TAL relative to the base strain improved only 30% with the native E. coli PDH, but 3.0-fold with PDHm and 4.4-fold with PDHm in the Δzwf1 strain. Carbon was further routed toward TAL production by reducing mitochondrial transport of pyruvate and acetyl-CoA; deletions in genes POR2, MPC2, PDA1, or YAT2 each increased titer 2–3-fold over the base strain (up to 0.8 g/L), and in combination to 1.4 g/L. Combining the two approaches (NADPH-generating acetyl-CoA pathway plus reduced metabolite flux into the mitochondria) resulted in a final TAL titer of 1.6 g/L, a 6.4-fold increase over the non-engineered yeast strain, and 35% of theoretical yield (0.16 g/g glucose), the highest reported to date. These biological driving forces present new avenues for improving high-yield production of acetyl-CoA derived compounds.     

Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli

In this work, two proteins, Z-domains and bovine casein, were autodisplayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different autodisplay vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-autodisplayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-autodisplayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-autodisplayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-autodisplayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with autodisplayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.     

Modulation of guanosine 5′-diphosphate-d-mannose metabolism in recombinant Escherichia coli for production of guanosine 5′-diphosphate-l-fucose

Guanosine 5’-diphosphate (GDP)-l-fucose, an activated form of a nucleotide sugar, plays an important role in a wide range of biological functions. In this study, the enhancement of GDP-l-fucose production was attempted by supplementation of mannose, which is a potentially better carbon source to be converted into GDP-l-fucose than glucose, and combinatorial overexpression of the genes involved in the biosynthesis of GDP-d-mannose, a precursor of GDP-l-fucose. Supply of a mannose and glucose led to a 1.3-fold-increase in GDP-l-fucose concentration (52.5 ± 0.8 mg l^?1) in a fed-batch fermentation of recombinant E. coli BL21star(DE3) overexpressing the gmd and wcaG genes, compared with the case using glucose as a sole carbon source. A maximum GDP-l-fucose concentration of 170.3 ± 2.3 mg l^?1, corresponding to a 4.4-fold enhancement compared with the control strain overexpressing gmd and wcaG genes only, was achieved in a glucose-limited fed-batch fermentation of a recombinant E. coli BL21star(DE3) strain overexpressing manB, manC, gmd and wcaG genes. Further improvement of GDP-l-fucose production was not obtained by additional overexpression of the manA gene.     

Engineering Escherichia coli to convert acetic acid to free fatty acids

Fatty acids (FAs) are promising precursors of advanced biofuels. This study investigated conversion of acetic acid (HAc) to FAs by an engineered Escherichia coli strain. We combined established genetic engineering strategies including overexpression of acs and tesA genes, and knockout of fadE in E. coli BL21, resulting in the production of ~1 g/L FAs from acetic acid. The microbial conversion of HAc to FAs was achieved with ~20% of the theoretical yield. We cultured the engineered strain with HAc-rich liquid wastes, which yielded ~0.43 g/L FAs using waste streams from dilute acid hydrolysis of lignocellulosic biomass and ~0.17 g/L FAs using effluent from anaerobic-digested sewage sludge. ¹³C-isotopic experiments showed that the metabolism in our engineered strain had high carbon fluxes toward FAs synthesis and TCA cycle in a complex HAc medium. This proof-of-concept work demonstrates the possibility for coupling the waste treatment with the biosynthesis of advanced biofuel via genetically engineered microbial species.     

Prevotella bivia as a source of lipopolysaccharide in the vagina

ObjectivesTo compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA).MethodsVaginal washes obtained from patients with (n = 43) and without (n = 59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts.ResultsThe median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P < 0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06 ± 0.36 vs 5.4 ± 2.2 log₁₀ CFU/ml, respectively, P < 0.001).Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0 ± 306.6 EU/ml) than either E. coli (4679.0 ± 585.3 EU/ml) or G. vaginalis (0.07 ± 0.01 EU/ml of LPS).The loss of DA neuron was 20–27% in cultures treated with vaginal washes from BV-negative patients and 58–97% in cultures treated with vaginal washes from patients with BV.ConclusionP. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.     

Molecular cloning of kman coding for mannanase from Klebsiella oxytoca KUB-CW2-3 and its hybrid mannanase characters

Gene encoding for β-mannanase (E.C 3.2.1.78) from Klebsiella oxytoca KUB-CW2-3 was cloned and expressed by an E. coli system resulting in 400 times higher mannanase activities than the wild type. A 3314 bp DNA fragment obtained revealed an open reading frame of 1164 bp, namely kman-2, which encoded for 387 amino acids with an estimated molecular weight of 43.2 kDa. It belonged to the glycosyl hydrolase family 26 (GH26) exhibited low similarity of 50–71% to β-mannanase produced by other microbial sources. Interestingly, the enzyme had a broad range of substrate specificity of homopolymer of ivory nut mannan (6%), carboxymethyl cellulose (30.6%) and avicel (5%), and heteropolymer of konjac glucomannan (100%), locust bean gum (92.6%) and copra meal (non-defatted 5.3% and defatted 7%) which would be necessary for in vivo feed digestion. The optimum temperature and pH were 30–50 °C and 4–6, respectively. The enzyme was still highly active over a low temperature range of 10–40 °C and over a wide pH range of 4–10. The hydrolysates of konjac glucomannan (H-KGM), locust bean gum (H-LBG) and defatted copra meal (H-DCM) composed of compounds which were different in their molecular weight range from mannobiose to mannohexaose and unknown oligosaccharides indicating the endo action of mannanase. Both H-DCM and H-LBG enhanced the growth of lactic acid bacteria and some pathogens except Escherichia coli E010 with a specific growth rate of 0.36–0.83 h⁻¹. H-LBG was more specific to 3 species of Weissella confusa JCM 1093, Lactobacillus reuteri KUB-AC5, Lb salivarius KL-D4 and E. coli E010 while both H-KGM and H-DCM were to Lb. reuteri KUB-AC5 and Lb. johnsonii KUNN19-2. Based on the nucleotide sequence of kman-2 containing two open reading frames of 1 and 2 at 5′ end of the +1 and +43, respectively, removal of the first open reading frame provided the recombinant clone E. coli KMAN-3 resulting in the mature protein of mannanase composing of 345 amino acid residues confirmed by 3D structure analysis and amino acid sequence at N-terminal namely KMAN (GenBank accession number KM100456). It exhibited 10 times higher extracellular and periplasmic total activities of 17,600 and 14,800 units than E. coli KMAN-2. With its low similarity to mannanases previously proposed, wide range of homo- and hetero-polysaccharide specificity, negative effect to E. coli and most importance of high production, it would be proposed as a novel mannanase source for application in the future.     

Direct bioconversion of d-xylose to 1,2,4-butanetriol in an engineered Escherichia coli

The compound 1,2,4-butanetriol (BT) is a valuable chemical used in the production of plasticizers, polymers, cationic lipids and other medical applications, and is conventionally produced via hydrogenation of malate. In this report, BT is biosynthesized by an engineered Escherichia coli from d-xylose. The pathway: d-xylose → d-xylonate → 2-keto-3-deoxy-d-xylonate → 3,4-dihydroxybutanal → BT, was constructed in E. coli by recruiting a xylose dehydrogenase and a keto acid decarboxylase from Caulobacter crescentus and Pseudomonas putida, respectively. Authentic BT was detected from cultures of the engineered strain. Further improvement on the strain was performed by blocking the native d-xylose and d-xylonate metabolic pathways which involves disruption of xylAB, yjhH and yagE genes in the host chromosome. The final construct produced 0.88 g L⁻¹ BT from 10 g L⁻¹ d-xylose with a molar yield of 12.82%. By far, this is the first report on the direct production of BT from d-xylose by a single microbial host. This may serve as a starting point for further metabolic engineering works to increase the titer of BT toward industrial scale viability.     

The D-galactose specific lectin of field bean (Dolichos lablab) seed binds sugars with extreme negative cooperativity and half-of-the-sites binding

The field bean (Dolichos lablab) lectin designated as PPO-haemagglutinin (DLL-II) is bifunctional, exhibiting both polyphenol oxidase and haemagglutinating activity. The lectin is unusual in that it binds galactose (Gal), lactose (Lac) and N-acetylgalactosamine (GalNAc) only in the presence of (NH₄)₂SO₄ and exhibits negative cooperativity and half-of-the-sites binding. Circular dichroism, isothermal titration calorimetry and fluorescence quenching were used to assess the sugar binding in the presence of (NH₄)₂SO₄. Comparison of the near-UV CD spectra with and without bound sugar revealed ligand induced conformational changes. The intrinsic fluorescence quenching data indicate that DLL-II exhibits weak binding to Gal in the presence of (NH₄)₂SO₄ with a stoichiometry of one bound ligand per dimer. ITC data fitted using a two sets of sites binding model presented a similar picture. The K_a’s for Gal, Lac and GalNAc in the presence of (NH₄)₂SO₄ were 0.16 ± 0.002, 0.21 ± 0.004 and 8.45 ± 0.78 (×10^?3) M^?1 respectively. The Hill plot for the binding of these sugars to DLL-II was curvilinear with a tangent slope <1.0 indicating negative cooperativity. DLL-II thus exhibits half-of-the-site binding, an extreme form of negative cooperativity in which the second ligand does not bind at all. This is the first report of a legume lectin, exhibiting half-of-the-sites binding.     

Synthesis of polyhydroxyalkanoates from glucose that contain medium-chain-length monomers via the reversed fatty acid β-oxidation cycle in Escherichia coli

Polyhydroxyalkanoates that contain the medium-chain-length monomers (mcl-PHAs) have a wide range of applications owing to their superior physical and mechanical properties. A challenge to synthesize such mcl-PHAs from unrelated and renewable sources is exploiting the efficient metabolic pathways that lead to the formation of precursor (R)-3-hydroxyacyl-CoA. Here, by engineering the reversed fatty acid β-oxidation cycle, we were able to synthesize mcl-PHAs in Escherichia coli directly from glucose. After deletion of the major thioesterases, the engineered E. coli produced 6.62 wt% of cell dry weight mcl-PHA heteropolymers. Furthermore, when a low-substrate-specificity PHA synthase from Pseudomonas stutzeri 1317 was employed, recombinant E. coli synthesized 12.10 wt% of cell dry weight scl–mcl PHA copolymers, of which 21.18 mol% was 3-hydroxybutyrate and 78.82 mol% was medium-chain-length monomers. The reversed fatty acid β-oxidation cycle offered an efficient metabolic pathway for mcl-PHA biosynthesis in E. coli and can be further optimized.     

Studies on construction of a recombinant Eimeria tenella SO7 gene expressing Escherichia coli and its protective efficacy against homologous infection

Eimeria spp. are the causative agents of coccidiosis, a major disease affecting the poultry industry. A recombinant non-antibiotic Escherichia coli that expresses the Eimeria tenella SO7 gene was constructed and its protective efficacy against homologous infection in chickens was determined. The three-day-old chickens were orally immunized with the recombinant non-antibiotic SO7 gene expressing E. coli and boosted two weeks later. Four weeks after the second immunization, the chickens were challenged with 5 × 10⁴ homologous sporulated oocysts. The protective effects of the recombinant non-antibiotic E. coli were determined by measuring body weight change, mortality, histopathology, lesion scores, oocyst counts, the specific antibody response and the frequency of CD4⁺ and CD8⁺ lymphocytes in peripheral blood. The results showed that immunization with SO7 expressing E. coli resulted in significantly improved body weight gain, reduced lesion scores and oocyst shedding in immunized chickens compared to controls. Furthermore, administration of recombinant SO7 expressing E. coli leads to a significant increase in serum antibody, CD4⁺ and CD8⁺ T cells in peripheral blood of chickens. These results, therefore, suggest that the recombinant non-antibiotic E. coli that expresses the SO7 gene is able to effectively stimulate host protective immunity as evidenced by the induction of development of both humoral and cell-mediated immune responses against homologous challenge in chickens.