An isolated cellulolytic Escherichia coli from bovine rumen produces ethanol and hydrogen from corn straw.

BACKGROUND: Lignocellulosic biomass is the most abundant resource on earth. Lignocellulose is mainly composed of cellulose, hemicelluloses, and lignin. The special construction of three kinds of constituents led to the prevention of effective degradation. The goal of this work was to investigate the great potentials of bovine rumen for novel cellulolytic bacterial isolation, which may be used for chemicals and biofuel production from lignocellulose. RESULTS: A cellulolytic strain, ZH-4, was isolated from Inner Mongolia bovine rumen. This strain was identified as Escherichia coli by morphological, physiological, and biochemical characteristics and 16S rDNA gene sequencing. The extracellular enzyme activity analysis showed that this strain produces extracellular cellulases with an exoglucanase activity of 9.13 IU, an endoglucanase activity of 5.31 IU, and a ß-glucosidase activity of 7.27 IU at the pH 6.8. This strain was found to produce 0.36 g/L ethanol and 4.71 mL/g hydrogen from corn straw with cellulose degradation ratio of 14.30% and hemicellulose degradation ratio of 11.39%. CONCLUSIONS: It is the first time that a cellulolytic E. coli was isolated and characterized form the bovine rumen. This provided a great opportunity for researchers to investigate the evolution mechanisms of the microorganisms in the rumen and provided great chance to produce biofuels and chemicals directly from engineered E. coli using consolidated bioprocess.

[Synthetic biology and rearrangements of microbial genetic material].

As an emerging discipline, synthetic biology has shown great scientific values and application prospects. Although there have been many reviews of various aspects on synthetic biology over the last years, this article, for the first time, attempted to discuss the relationship and difference between microbial genetics and synthetic biology. We summarized the recent development of synthetic biology in rearranging microbial genetic materials, including synthesis, design and reduction of genetic materials, standardization of genetic parts and modularization of genetic circuits. The relationship between synthetic biology and microbial genetic engineering was also discussed in the paper.

Pathway engineering results the altered polyhydroxyalkanoates composition in recombinant Escherichia coli.

To efficiently produce short-chain-length-medium-chain-length polyhydroxyalkanoates copolymer from substrate mixture containing sugars and/or fatty acids, fadA gene mutant was constructed in Escherichia coli DH5α phosphotransferase system (PTS) disrupted strain. Plasmids pCJY02, pBHR68 and pBHR71 were separately introduced into E. coli DH5α (ΔptsG, ΔFadA) by transformation, then the recombinants were cultivated in the medium containing glucose and/or decanoate as carbon resource, respectively. When cultivated in the medium containing decanoate, only pCJY02-harboring recombinant was able to accumulate SCL-MCL PHAs consisting of 3HB, 3HHx, 3HO and 3HD with mol ratios: 43.2:12.8:10.3:33.6. The copolymer content was 1.90wt% with 2.69gL(-1) cell dry weight. When cultivated in the medium containing both decanoate and glucose, the recombinant was found to utilize the mixture of glucose and fatty acids and accumulate SCL-MCL PHAs copolymer consisting of 3HB, 3HHx, 3HO and 3HD with mol ratios: 83.4:4.0:5.6:7.0. About 4.90gL(-1) cell dry weight was harvested and total PHAs content was 7.3wt% of CDW. This result indicated that the low-substrate-specificity PHA synthase PhaC2(Ps) endued hosts with the capability of synthesizing PHA copolymers, and the monomer composition of the synthesized PHA could be modulated by controlling the addition of carbon sources and by modifying metabolic pathways in the hosts.

Isolation and characterization of temperature and alkaline stable bioflocculant from Agrobacterium sp. M-503.

A bacterium isolated from activated sludge of propylene epoxide wastewater was identified as Agrobacterium sp. M-503. It was confirmed to produce bioflocculant with excellent flocculation activity. The yield of the bioflocculant reached 14.9 g/l in batch cultivation with a carbon source conversion of 74.5%. This bioflocculant was temperature and alkaline stable, retaining almost all flocculation activity after being treated at 121°C for 20 minutes or at pH 12.0. It consisted of neutral sugar, uronic acid, aminosugar and protein in weight ratios of 85.0:9.9:2.1:3.0. The active polysaccharide fraction of the bioflocculant was purified to homogeneity by ethanol precipitation, DEAE ion-exchange and gel chromatography. Analysis of the purified polysaccharide showed that it consisted of glucose residues and had a molecular weight of 8.1 × 104 Da. Its low molecular weight endowed it with excellent solubility and favorable flocculation activity, especially for small particulates.

PEGylation markedly enhances the in vivo potency of recombinant human non-glycosylated erythropoietin: a comparison with glycosylated erythropoietin.

Recombinant human non-glycosylated erythropoietin (rh-ngEpo) expressed in E. coli was attached to polyethylene glycol (PEG) chains with different sizes and structures. The pharmacokinetic properties and in vivo potency of the PEGylated protein were investigated and comparisons were drawn between the conjugates and glycosylated recombinant Epo (rhEpo). The rh-ngEpo was modified with linear PEG-aldehyde (PEG-ALD, 20 kDa, 30 kDa, and 40 kDa) and a branched N-hydroxysuccinimide activated PEG (PEG(2)-NHS, 40 kDa). The monoPEGylated proteins were isolated by ion-exchange chromatography. The purified monoPEGylated conjugates suffered 6.5-86.1% loss of in vitro bioactivity compared to the unmodified rh-ngEpo. In addition, PEGylation remarkably increased the resistance of rh-ngEpo against plasma degradation. Pharmacokinetic studies showed that the plasma half-life of rh-ngEpo was increased 9.7-17.4 times by PEGylation, with the two 40k-PEG-rh-ngEpos-treated groups exhibiting better pharmacokinetic performances than rhEpo. Moreover, all the conjugates resulted in markedly enhanced Ret% (the percentage of reticulocyte count in red blood cells) compared with rh-ngEpo after subcutaneous injection. The two 40k-PEG conjugates demonstrated comparable in vivo efficacies compared with rhEpo. Overall, this research provides opportunities for the development of more cost-effective erythropoiesis-stimulating protein drugs.

[Construction of the Man8 GlcNAc2 glycosylation Saccharomyces cerevisiae mutant strain].

In Saccharomyces cerevisiae, protein glycosylation passed two different N-linked modification pathways after the export of predominantly Man8 GlcNAc2-containing glycoproteins from ER to the Golgi. The core oligosaccharide undergoes maturation in the Golgi resulting in a Man8-13 GlcNAc2 structure. Alternatively, core structures may be hypermannosylated with up to 200 mannose residues composing of a backbone of alpha1,6-mannosyl residues with branched alpha1, 2- and alpha1,3-mannosyl side chains. Mnn1p and Och1p play an important role in this process. The null disruption of MNN1, OCH1 was replaced by the S. cerevisiae URA3, HIS3, respectively. To characterize the N-glycosylation in the mnn1 och1 mutant, mannoproteins were obtained by hot citrate buffer extraction after the mnn1 och1 cells were crumbled. The extracted mannoprotein was precipitated by ethanol, and further purified by concanavalin A-sepharose 4B. The N-oligomannose saccharides were released from mannoprotein by PNGase F digestion, and then peptides and detergents were removed by passage through ion exchange columns. For desalting, glycans were applied to porous graphitic-carbon cartridge. 2-aminopyridine pyridylaminated sugars were profiled and purified by size fractionation HPLC with Shim-pack cle-NH2 column, and result showed dominantly a single peak. MALDI TOF/MS analysis ofthis peak revealed that its molecular weight was 1796.5Da, which corresponds to the calculated mass of Man8 GlcNAc2-PA. These results indicated that disruptions of MNN1 and OCH1 eliminated the hypermannosylation of the N-linked glycans, and glycoproteins were glycosylated with a single core type glycan, Man8 GlcNAc2, in the mnn1 och1 mutant.

[The product specificity evolution of cyclodextrin glucanotransferase: problems and challenges].

Cyclodextrin glucanotransferase, the essential enzyme for the production of cyclodextrins, has become the focus of scientific research nowadays. Although many related enzyme properties are well known, the crucial factors in product specificity determination remain to be answered. Here, the recent research progresses of cyclodextrin glucanotransferase, especially those about the evolution of product specificity, were reviewed, and the scientific problems were discussed.

[Progress of oligosaccharides biosynthesis in recombinant Escherichia coli].

As more bioactivities of oligosaccharides have been elucidated, researches on biosynthesis of oligosaccharides have drawn more concerns in Glycobiology. A lot of enzymatic methods for the synthesis of oligosacchrides have been developed employing recombinant E. coli expressed glycosyltranferase or synthase of nucleotide-sugar. This review focuses on the recent progress in the production of oligosaccharides using bacteria especially by genetically engineered bacteria. The key point concering the oligosaccharides biosynthesis in recombinant E. coli, such as enzyme expression, NDP-sugar provision and biosynthesis pathway, was discussed.

Immobilization of UDP-galactose 4-epimerase from Escherichia coli on the yeast cell surface.

UDP-galactose 4-epimerase (EC 5.1.3.2, Gal E) from Escherichia coli catalyzes the reversible reaction between UDP-galactose and UDP-glucose. In this study, the Gal E gene from E. coli, coding UDP-galactose 4-epimerase, was cloned into pYD1 plasmid and then transformed into Saccharomyces cerevisiae EBY100 for expression of Gal E on the cell surface. Enzyme activity analyses with EBY100 cells showed that the enzyme displayed on the yeast cell surface was very active in the conversion between UDP-Glc and UDP-Gal. It took about 3 min to reach equilibrium from UDP-galactose to UDP-glucose.

Delivery of nitric oxide released from beta-Gal-NONOate activation by beta-galactosidase and its activity against Escherichia coli.

Beta-galactosyl-pyrrolidinyl diazeniumdiolates (beta-Gal-NONOate) is a new site-specific nitric oxide (NO)-releasing compound, which releases NO once activated by beta-galactosidase. In the present experiment, we used beta-Gal-NONOate as a bactericidal reagent to investigate its effectiveness of NO releasing. Through the evaluation of intracellular NO level and the comparison of survival of E. coli transformed with lacZ gene but treated with beta-Gal-NONOate and NONOate, respectively, it's evident that beta-Gal-NONOate had a higher bactericidal activity than conventional NONOate. While either beta-Gal-NONOate- or NONOate-treated E. coli without transferred lacZ gene showed low bactericidal activity. The results revealed that beta-Gal-NONOate was a potentially efficient NO donor, which took on a novel and promising approach to deliver NO into cells.

[High level expression of PNGase F in Escherichia coli and its bioactivities].

In order to obtain active recombinant PNGase F in Escherichia coli, a prokaryotic expression vector pET28a/PNGase F was constructed. Amplification of PNGase F was obtained using PCR technique employing suitable primers designed according to the PNGase F gene sequence from Flavobacterium nmeningosepticum. The expression of PNGase F gene in LB medium or M9 medium led to the formation of inclusion body and soluble protein, respectively. The refolding of the denatured inclusion body was successful by gradual dilution. Further purification of the refolded protein and soluble crude extract from M9 medium with Ni2+ -NTA argarose resulted a 90% purified PNGase F. The purified protein catalyzed the complete and intact cleavage of N-linked oligosaccharides from various glycoproteins. The efficiency of this cleavage was affected by the substrate status in the reaction system. In summary, we have developed an enzyme production system where PNGase F was over-expressed in recombinant E. coli. This system can provide more than 15 mg/L purified active PNGase F. This purified active PNGase F can be used as tools in analyzing the oligosaccharide structure.