High-level expression of glutaryl-7-aminocephalosporanic acid acylase from Pseudomonas diminuta NK703 in Escherichia coli by combined optimization strategies

In this work, a glutaryl-7-aminocephalosporanic acid acylase (GLA) coding gene was cloned from Pseudomonas diminuta NK703 which was screened from oilfield. The concerted effects of the expression system, inducing condition and culture medium on the expression of NK703 GLA in E. coli were firstly investigated. The best combination was the recombinant E. coli strain of pET-28a + GLA/BL21(DE3) with 2.0% (w/v) lactose inducing in YT medium at 25 °C. Then, by optimizing the components of culture medium, a synthetic medium with dextrin and a feeding medium with glycerol as the carbon sources were developed to further enhance the GLA yield and improve the GLA solubility. In the end, the NK703 GLA activity increased about 50-fold, reached 14,470 ± 465 U/L, and the GLA productivity and the proportion of soluble GLA to the total soluble protein attained 206.0 ± 9.033 U L⁻¹ h⁻¹ and 60.13%, respectively. Scaling up the GLA production in 3.7 L fermenter under the optimized conditions identified in shake flask, the GLA activity also reached 12,406 ± 521 U/L, which was the highest report at fermenter level.     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

Engineered Escherichia coli capable of co-utilization of cellobiose and xylose

Natural ability to ferment the major sugars (glucose and xylose) of plant biomass is an advantageous feature of Escherichia coli in biofuel production. However, excess glucose completely inhibits xylose utilization in E. coli and decreases yield and productivity of fermentation due to sequential utilization of xylose after glucose. As an approach to overcome this drawback, E. coli MG1655 was engineered for simultaneous glucose (in the form of cellobiose) and xylose utilization by a combination of genetic and evolutionary engineering strategies. The recombinant E. coli was capable of utilizing approximately 6 g/L of cellobiose and 2 g/L of xylose in approximately 36 h, whereas wild-type E. coli was unable to utilize xylose completely in the presence of 6 g/L of glucose even after 75 hours. The engineered strain also co-utilized cellobiose with mannose or galactose; however, it was unable to metabolize cellobiose in the presence of arabinose and glucose. Successful cellobiose and xylose co-fermentation is a vital step for simultaneous saccharification and co-fermentation process and a promising step towards consolidated bioprocessing.     

Engineering of an N-acetylneuraminic acid synthetic pathway in Escherichia coli

N-acetylneuraminic acid (NeuAc) has recently drawn much attention owing to its wide applications in many aspects. Besides extraction from natural materials, production of NeuAc was recently focused on enzymatic synthesis and whole-cell biocatalysis. In this study, we designed an artificial NeuAc biosynthetic pathway through intermediate N-acetylglucosamine 6-phosphate in Escherichia coli. In this pathway, N-acetylglucosamine 2-epimerase (slr1975) and glucosamine-6-phosphate acetyltransferase (GNA1) were heterologously introduced into E. coli from Synechocystis sp. PCC6803 and Saccharomyces cerevisiae EBY100, respectively. By derepressing the feedback inhibition of glucosamine-6-phosphate synthase, increasing the accumulation of N-acetylglucosamine and pyruvate, and blocking the catabolism of NeuAc, we were able to produce 1.62 g l^?1 NeuAc in recombinant E. coli directly from glucose. The NeuAc yield reached 7.85 g l^?1 in fed-batch fermentation. This process offered an efficient fermentative method to produce NeuAc in microorganisms using glucose as carbon source and can be optimized for further improvement.     

A simple bacterial transformation method using magnesium- and calcium-aminoclays

An efficient and user-friendly bacterial transformation method by simple spreading cells with aminoclays was demonstrated. Compared to the reported transformation approaches using DNA adsorption or wrapping onto (in)organic fibers, the spontaneously generated clay-coated DNA suprastructures by mixing DNA with aminoclay resulted in transformants in both Gram-negative (Escherichia coli) and Gram-positive cells (Streptococcus mutans). Notably, the wild type S. mutans showed comparable transformation efficiency to that of the E. coli host for recombinant DNA cloning. This is a potentially promising result because other trials such as heat-shock, electroporation, and treatment with sepiolite for introducing DNA into the wild type S. mutans failed. Under defined conditions, the transformation efficiency of E. coli XL1-Blue and S. mutans exhibited ~ 2 × 10⁵ and ~ 6 × 10³ CFU/μg of plasmid DNA using magnesium-aminoclay. In contrast, transformation efficiency was higher in S. mutans than that in E. coli XL1-Blue for calcium-aminoclay. It was also confirmed that each plasmid transformed into E. coli and S. mutans was stably maintained and that they expressed the inserted gene encoding the green fluorescent protein during prolonged growth of up to 80 generations.     

Efficient production of polylactic acid and its copolymers by metabolically engineered Escherichia coli

Polylactic acid (PLA) is one of the promising biodegradable polymers, which has been produced in a rather complicated two-step process by first producing lactic acid by fermentation followed by ring opening polymerization of lactide, a cyclic dimer of lactic acid. Recently, we reported the production of PLA and its copolymers by direct fermentation of metabolically engineered Escherichia coli equipped with the evolved propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase using glucose as a carbon source. When employing these initially constructed E. coli strains, however, it was necessary to use an inducer for the expression of the engineered genes and to feed succinate for proper cell growth. Here we report further metabolic engineering of E. coli strain to overcome these problems for more efficient production of PLA and its copolymers. This allowed efficient production of PLA and its copolymers without adding inducer and succinate. The finally constructed recombinant E. coli JLXF5 strain was able to produce P(3HB-co-39.6 mol% LA) having the molecular weight of 141,000 Da to 20 g l⁻¹ with a polymer content of 43 wt% in a chemically defined medium by the pH-stat fed-batch culture.     

Large-scale production of GDP-fucose and Lewis X by bacterial coupling

A large-scale production system of GDP-fucose (GDP-Fuc) and fucosylated oligosaccharides was established by the combination of recombinant Escherichia coli cells overexpressing GDP-Fuc biosynthetic genes and Corynebacterium ammoniagenes cells. E. coli cells overexpressed the genes for glucokinase, phosphomannomutase, mannose-1-phosphate guanylyltransferase, GDP-mannose (GDP-Man) dehydratase, and GDP-4-keto-6-deoxy-mannose (GKDM) epimerase/reductase as well as phosphoglucomutase and phosphofructokinase. C. ammoniagenes contributed to the formation of GTP from GMP. GDP-Fuc accumulated to 29 mM (18.4 g l⁻¹) after a 22-h reaction starting with GMP and mannose through introducing the two-step reaction to overcome the inhibition of GDP-Fuc on GDP-Man dehydratase activity. When E. coli cells overexpressing the α1,3-fucosyltransferase gene of Helicobacter pylori were put into the GDP-Fuc production system, Lewis X [Galβ1–4(Fucα1–3)GlcNAc] was produced at an amount of 40 mM (21 g l⁻¹) for 30 h from GMP, mannose, and N-acetyl lactosamine. The production system through bacterial coupling can be applied to the industrial manufacture of fucosylated oligosaccharides. Journal of Industrial Microbiology & Biotechnology (2000) 25, 213–217. Received 01 May 2000/ Accepted in revised form 20 July 2000     

Production of succinic acid from sucrose and sugarcane molasses by metabolically engineered Escherichia coli

Sucrose-utilizing genes (cscKB and cscA) from Escherichia coli KO11 were cloned and expressed in a metabolically engineered E. coli KJ122 to enhance succinate production from sucrose. KJ122 harboring a recombinant plasmid, pKJSUC, was screened for the efficient sucrose utilization by growth-based selection and adaptation. KJ122-pKJSUC-24T efficiently utilized sucrose in a low-cost medium to produce high succinate concentration with less accumulation of by-products. Succinate concentrations of 51 g/L (productivity equal to 1.05 g/L/h) were produced from sucrose in anaerobic bottles, and concentrations of 47 g/L were produced in 10 L bioreactor within 48 h. Antibiotics had no effect on the succinate production by KJ122-pKJSUC-24T. In addition, succinate concentrations of 62 g/L were produced from sugarcane molasses in anaerobic bottles, and concentrations of 56 g/L in 10 L bioreactor within 72 h. These results demonstrated that KJ122-pKJSUC-24T would be a potential strain for bio-based succinate production from sucrose and sugarcane molasses.     

Plasmodium falciparum: enhanced soluble expression, purification and biochemical characterization of lactate dehydrogenase

Plasmodium lactate dehydrogenase (pLDH), owing to unique structural and kinetic properties, is a well known target for antimalarial compounds. To explore a new approach for high level soluble expression of Plasmodium falciparum lactate dehydrogenase (PfLDH) in E. coli, PfLDH encoding sequence was cloned into pQE-30 Xa vector. When transformed E. coli SG13009 cells were induced at 37 °C with 0.5 mM isopropyl β-d-thiogalactoside (IPTG) concentration, the protein was found to be exclusively associated with inclusion bodies. By reducing cell growth temperature to 15 °C and IPTG concentration to 0.25 mM, it was possible to get approximately 82% of expressed protein in soluble form. Recombinant PfLDH (rPfLDH) was purified to homogeneity yielding 18 mg of protein/litre culture. rPfLDH was found to be biologically active with specific activity of 453.8 μmol/min/mg. The enzyme exhibited characteristic reduced substrate inhibition and enhanced k_cat [(3.2 ± 0.02) × 10⁴] with 3-acetylpyridine adenine dinucleotide (APAD⁺). The procedure described in this study may provide a reliable and simple method for production of large quantities of soluble and biologically active PfLDH.     

Photonic characteristics and ex vivo imaging of Escherichia coli-Xen14 within the bovine reproductive tract

The objectives of this study were to (1) characterize the photonic properties of Escherichia coli-Xen14 and (2) conduct photonic imaging of E. coli-Xen14 within bovine reproductive tract segments (RTS) ex vivo (Bos indicus). E. coli-Xen14 was grown for 24 h in Luria Bertani medium (LB), with or without kanamycin (KAN). Every 24 h, for an 8-d interval, inoculums were imaged and photonic emissions (PE) collected. Inoculums were subcultured and plated daily to determine the colony forming units (CFU) and ratio of photon emitters to nonemitters. In the second objective, abattoir-derived bovine reproductive tracts (n = 9) were separated into posterior and anterior vagina, cervix, uterine body, and uterine horns. Two concentrations (3.2 × 10⁸ and 3.2 × 10⁶ CFU/200 μL for relative [High] and [Low], respectively) of E. coli-Xen14 were placed in translucent tubes for detection of PE through RTS. The CFU did not differ (P = 0.31) over time with or without KAN presence; they remained stable with 99.93% and 99.98% photon emitters, respectively. However, PE were lower (P < 0.0001) in cultures containing KAN than in those containing no KAN (629.8 ± 117.7 vs. 3012.0 ± 423.5 relative lights units per second [RLU/sec], respectively). On average, the percentage of PE between RTS, for both concentrations, was higher (P < 0.05) in the uterine body. In summary, E. coli-Xen14 remained stable with respect to the proportions of photon emitters with or without KAN (used to selectively culture E. coli-Xen14). However, KAN presence suppressed photonic activity. The ability to detect PE through various segments of the reproductive tract demonstrated the feasibility of monitoring the presence of E. coli-Xen14 in the bovine reproductive tract ex vivo.     

Production of ferulic acid from lignocellulolytic agricultural biomass by Thermobifida fusca thermostable esterase produced in Yarrowia lipolytica transformant

A gene (axe) encoding the AXE thermostable esterase in Thermobifida fusca NTU22 was cloned into a Yarrowia lipolytica P01g host strain. Recombinant expression resulted in extracellular esterase production at levels as high as 70.94 U/ml in Hinton flask culture broth, approximately 140 times higher than observed in a Pichia pastoris expression system. After 72 h of fermentation by the Y. lipolytica transformant in the fed-batch fermentor, the fermentation broth accumulated 41.11 U/ml esterase activity. Rice bran, wheat bran, bagasse and corncob were used as hydrolysis substrates for the esterase, with corncob giving the best ferulic acid yield. The corncob was incubated with T. fusca xylanase (Tfx) for 12 h and then with the AXE esterase for an additional 12 h. Ferulic acid accumulated to 396 μM in the culture broth, a higher concentration than with esterase alone or with Tfx and esterase together for 24 h.     

Production of succinate and polyhydroxyalkanoate from substrate mixture by metabolically engineered Escherichia coli

The strategic design of this study aimed at producing succinate and polyhydroxyalkanoate (PHA) from substrate mixture of glycerol/glucose and fatty acid in Escherichia coli. To accomplish this, an E. coli KNSP1 strain derived from E. coli LR1110 was constructed by deletions of ptsG, sdhA and pta genes and overexpression of phaC1 from Pseudomonas aeruginosa. Cultivation of E. coli KNSP1 showed that this strain was able to produce 21.07 g/L succinate and 0.54 g/L PHA (5.62 wt.% of cell dry weight) from glycerol and fatty acid mixture. The generated PHA composed of 58.7 mol% 3-hydroxyoctanoate (3HO) and 41.3 mol% 3-hydroxydecanoate (3HD). This strain would be useful for complete utilization of byproducts glycerol and fatty acid of biodiesel production process.     

Carbohydrate binding specificities and crystal structure of the cholera toxin-like B-subunit from Citrobacter freundii

Enterotoxigenic Escherichia coli and Vibrio cholerae are well known causative agents of severe diarrheal diseases. Both pathogens produce AB₅ toxins, with one enzymatically active A-subunit and a pentamer of receptor-binding B-subunits. The primary receptor for both B-subunits is the GM1 ganglioside (Galβ3GalNAcβ4(NeuAcα3)Galβ4GlcβCer), but the B-subunits from porcine isolates of E. coli also bind neolacto-(Galβ4GlcNAcβ-)terminated glycoconjugates and the B-subunits from human isolates of E. coli (hLTB) have affinity for blood group A type 2-(GalNAcα3(Fucα2)Galβ4GlcNAcβ-)terminated glycoconjugates.     

Enhanced production of recombinant streptokinase in Escherichia coli using fed-batch culture

Fed-batch culture strategy is often used for increasing production of heterologous recombinant proteins in Escherichia coli. This study was initiated to investigate the effects of dissolved oxygen concentration (DOC), complex nitrogen sources and pH control agents on cell growth and intracellular expression of streptokinase (SK) in recombinant E. coli BL21(DE3). Increase in DOC set point from 30% to 50% did not affect SK expression in batch culture where as similar increase in fed-batch cultivation led to a significant improvement in SK expression (from 188 to 720 mg l⁻¹). This increase in SK could be correlated with increase in plasmid segregational stability. Supplementation of production medium with yeast extract and tryptone and replacement of liquid ammonia with NaOH as pH control agent further enhanced SK expression without affecting cell growth. Overall, SK concentration of 1120 mg l⁻¹ representing 14-fold increase in SK production on process scale-up from flask to bioreactor scale fed-batch culture is the highest reported concentration of SK to date.     

Conversion of squid pen by Pseudomonas aeruginosa K187 fermentation for the production of N-acetyl chitooligosaccharides and biofertilizers

Pseudomonas aeruginosa K187, a protease- and chitinase-producing bacterium, exhibited protease and chitinase activity after three and five days of incubation, respectively. The protease and chitinase were both produced by using 1% squid pen powder (SPP) (w/v) as sole carbon and nitrogen source. After fermentation, the deproteinization rate of the recovered squid pen gradually increased up to 68% on the fourth day. After five days of fermentation, the production of GlcNAc, (GlcNAc)₂, (GlcNAc)₃, (GlcNAc)₄ and (GlcNAc)₅ were 1.18 mg/mL, 0.76 mg/mL, 1.02 mg/mL, 0.93 mg/mL and 0.90 mg/mL, respectively. The culture supernatant of K187 also exhibited activity of enhancing vegetable growth. For Brassica chinensis Linn treated with the fifth day culture supernatant, the total weight and total length increased up to 529% and 148%, respectively, compared to the control group. With this method, the production of protease, chitinase, N-acetyl chitooligosaccharides and biofertilizers may be useful for biological applications.     

Optimized production of scygonadin in Pichia pastoris and analysis of its antimicrobial and antiviral activities

The crab antimicrobial peptide scygonadin is confirmed to have antimicrobial activity against bacteria and it is probably associated with the reproductive immunity in Scylla paramamosain. To obtain large quantity of scygonadin for further biological assays, a 306 bp cDNA sequence encoding the mature peptide of scygonadin was cloned into a secretion vector of pPIC9K, and a high-level of the recombinant scygonadin was achieved in Pichia pastoris. The optimal expression condition was determined as incubation with 0.5% methanol for 48 h at 28 °C under pH 6.0, and a total of 70 mg scygonadin was expressed in 1 L culture medium. The recombinant product was purified and 97% pure scygonadin was obtained using immobilized metal affinity chromatography with a yield of 46 mg/L. The recombinant scygonadin was confirmed using SDS-PAGE analysis and MS-fingerprinting. P. pastoris-derived scygonadin exhibited relatively higher antimicrobial activities against bacteria than Escherichia coli-derived scygonadin. The antimicrobial activity of the recombinant scygonadin against pathogenic Aeromonas hydrophila showed salt resistant and the killing kinetics of A. hydrophila was time dependent. Besides, the antiviral assay demonstrated that scygonadin could interfere with white spot syndrome virus (WSSV) replication in vitro-cultured crayfish haematopoietic (Hpt) cells. Taken together, this is the first report on the heterologous expression of scygonadin in P. pastoris, and P. pastoris is an effective expression system for producing large quantities of biological active scygonadin for both research and agricultural application.     

The conformations of the O-specific polysaccharides of Shigella dysenteriae type 4 and Escherichia coli O159 studied with molecular mechanics (MM3) filtered systematic search

The branched O-antigens of Escherichia coli O159 and Shigella dysenteriae type 4 are structurally related and are known to show cross-reactivity with antibodies. In the present study, conformational analyses were performed on these two O-antigens using molecular mechanics MM3(96) with filtered systematic search. The results show very strong steric restrictions for the trisaccharide at the branch point of the E. coli O159 antigen, especially for the β-d-GlcNAc-(1 → 3)-β-d-GlcNAc linkage of the main chain. For the type 4 O-antigen the calculations show essentially a single conformation with respect to the α-d-GlcNAc-(1 → 3)-α-d-GlcNAc linkage of the main chain and three different favoured conformations for the fucose branch. Consecutive repeating units of the S. dysenteriae type 4 and E. coli O159 O-antigens form linear extended chains with significant flexibility between the branches. Comparative calculations carried out with the SWEET server indicate that our method of filtered systematic search is a superior method in the case of branched, constrained oligosaccharides. Based on the results of the MM3 calculations, we propose that the common epitope explaining the cross-reactivity comprises the fucose branch, the downstream GlcNAc and part of the uronic acid.     

New N-acyl-d-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate

N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-d-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-d-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117 ± 2 U mg⁻¹ at 37 °C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the K_m for the substrate than on the enzyme activity. At high substrate concentrations (≥200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess.     

Production and characterization of homopolymer poly(3-hydroxyvalerate) (PHV) accumulated by wild type and recombinant Aeromonas hydrophila strain 4AK4

Aeromonas hydrophila 4AK4 normally produces copolyesters (PHBHHx) consisting of 3-hydroxybutyrate (C4) and 3-hydroxyhexanoate (C6). Wild type and recombinant A. hydrophila 4AK4 (pSXW02) expressing vgb and fadD genes encoding Vitreoscilla haemoglobin and Escherichia coli acyl-CoA synthase respectively, were found able to produce homopolyester poly(3-hydroxyvalerate) (PHV) (C5) on undecanoic acid as a single carbon source. The recombinant grew to 5.59 g/L cell dry weight (CDW) containing 47.74 wt% PHV in shake flasks when growth was conducted in LB medium and PHV production in undecanoic acid. The cells grew to 47.12 g/L CDW containing 60.08 wt% PHV in a 6 L fermentor study. Physical characterization of PHV produced by recombinant A. hydrophila 4AK4 (pSXW02) in fermentor showed a weight average molecular weight (M_w) of 230,000 Da, a polydispersity of 3.52, a melting temperature of 103 °C and a glass transition temperature of −15.8 °C. The degradation temperature at 5% weight loss of the PHV was around 258 °C.     

Cloning expression and analysis of phytochelatin synthase (pcs) gene from Anabaena sp. PCC 7120 offering multiple stress tolerance in Escherichia coli

Phytochelatin synthase (PCS) is involved in the synthesis of phytochelatins (PCs), plays role in heavy metal detoxification. The present study describes for first time the functional expression and characterization of pcs gene of Anabaena sp. PCC 7120 in Escherichia coli in terms of offering protection against heat, salt, carbofuron (pesticide), cadmium, copper, and UV-B stress. The involvement of pcs gene in tolerance to above abiotic stresses was investigated by cloning of pcs gene in expression vector pGEX-5X-2 and its transformation in E. coli BL21 (DE3). The E. coli cells transformed with pGEX-5X-pcs showed better growth than control cells (pGEX-5X-2) under temperature (47 °C), NaCl (6% w/v), carbofuron (0.025 mg ml⁻¹), CdCl₂ (4 mM), CuCl₂ (1 mM), and UV-B (10 min) exposure. The enhanced expression of pcs gene revealed by RT-PCR analysis under above stresses at different time intervals further advocates its role in tolerance against above abiotic stresses.