Microbial production of glycolate from acetate by metabolically engineered Escherichia coli.

Escherichia coli was metabolically engineered to synthesize glycolate using acetate as the carbon source. The native glyoxylate bypass pathway was reinforced by the overexpression of isocitrate lyase and isocitrate dehydrogenase kinase/phosphatase. Glyoxylate/hydroxypyruvate reductase was overexpressed to convert glyoxylate to glycolate. Meanwhile, side reactions were eliminated by inactivating genes encoding malate synthase, glyoxylate carboligase, and glycolate oxidase to prevent loss of glyoxylate and glycolate. The engineered E. coli produced 1.78 g/L glycolate from 3.23 g/L acetate after 48 h shake flask cultivation using minimal medium supplemented with 1 g/L yeast extract. When citrate synthase, phosphotransacetylase, and acetate kinase were co-overexpressed to strengthen the tricarboxylic acid cycle and acetate utilization, glycolate production titer was improved to 2.75 g/L with pH control in shake flasks. The results of this work offer an approach for producing glycolate using acetate as the carbon source.

Indentation with atomic force microscope, Saccharomyces cerevisiae cell gains elasticity under ethanol stress.

During bioethanol fermentation process, Saccharomyces cerevisiae cell membrane is the first target to be attacked by the accumulated ethanol. In such a prominent position, S. cerevisiae cell membrane could reversely provide protection through changing fluidity or elasticity secondary to remodeled membrane components or structure during the fermentation process. However, there is yet to be a direct observation of the real effect of the membrane compositional change. In this study, atomic force microscope-based strategy was performed to determine Young's modulus of S. cerevisiae to directly clarify ethanol stress-associated changes and roles of S. cerevisiae cell membrane fluidity and elasticity. Cell survival rate decreased while the cell swelling rate and membrane permeability increased as ethanol concentration increased from 0% to 20% v/v. Young's modulus decreased continuously from 3.76MPa to 1.53MPa while ethanol stress increased from 0% to 20% v/v, indicating that ethanol stress induced the S. cerevisiae membrane fluidity and elasticity changes. Combined with the fact that membrane composition varies under ethanol stress, to some extent, this could be considered as a forced defensive act to the ethanol stress by S. cerevisiae cells. On the other hand, the ethanol stress induced loosening of cell membrane also caused S. cerevisiae cell to proactively remodel membrane to make cell membrane more agreeable to the increase of environmental threat. Increased ethanol stress made S. cerevisiae cell membrane more fluidized and elastic, and eventually further facilitated yeast cell's survival.

Four homoisoflavonoids isolated from traditional Chinese medicine: "gan luo xin".

Two new homoisoflavonoids, ( ± )-5,7-dihydroxy-8-methyl-3-(2',4'-dihydroxybenzyl) chroman-4-one (1) and ( ± )-5,7-dihydroxy-6,8-dimethyl-3-(2',4'-dihydroxybenzyl) chroman-4-one (2), along with two known homoisoflavonoids, 5,7-dihydroxy-6-methyl-3-(2',4'-dihydroxybenzyl)chroman-4-one (3) and disporopsin (4), were isolated from the EtOAc extract of traditional Chinese medicine--"Gan Luo Xin." Their structures were determined on the basis of spectroscopic analysis (UV, IR, HR-ESI-MS, 1D NMR, and 2D NMR).

A novel cleaning process for industrial production of xylose in pilot scale from corncob by using screw-steam-explosive extruder.

Steam explosion is the most promising technology to replace conventional acid hydrolysis of lignocellulose for biomass pretreatment. In this paper, a new screw-steam-explosive extruder was designed and explored for xylose production and lignocellulose biorefinery at the pilot scale. We investigated the effect of different chemicals on xylose yield in the screw-steam-explosive extrusion process, and the xylose production process was optimized as followings: After pre-impregnation with sulfuric acid at 80 °C for 3 h, corncob was treated at 1.55 MPa with 9 mg sulfuric acid/g dry corncob (DC) for 5.5 min, followed by countercurrent extraction (3 recycles), decoloration (activated carbon dosage 0.07 g/g sugar, 75 °C for 40 min), and ion exchange (2 batches). Using this process, 3.575 kg of crystal xylose was produced from 22 kg corncob, almost 90 % of hemicellulose was released as monomeric sugar, and only a small amount of by-products was released (formic acid, acetic acid, fural, 5-hydroxymethylfurfural, and phenolic compounds were 0.17, 1.14, 0.53, 0.19, and 1.75 g/100 g DC, respectively). All results indicated that the screw-steam-explosive extrusion provides a more effective way to convert hemicellulose into xylose and could be an alternative method to traditional sulfuric acid hydrolysis process for lignocellulose biorefinery.

Microbial biotransformation of kurarinone by Cunninghamella echinulata AS 3.3400.

In this paper, microbial transformation of kurarinone (1) by Cunninghamella echinulata AS 3.3400 was investigated and four transformed products were isolated and identified as 6″-hydroxykurarinone (2), 4″,5″,8″-trihydroxynorkurarinone (3), norkurarinone (4), and kurarinone-7-O-ß-glucoside (5), respectively. Among them, 3 and 5 are new compounds, and the rare glycosylation in microbial transformation was observed. In addition, the cytotoxicities of transformed products (2-5) were also investigated.

Microbial transformation of Norkurarinone by Cunninghamella blakesleana AS 3.970.

In this paper, microbial transformation of norkurarinone (1) by Cunninghamella blakesleana AS 3.970 was investigated and seven transformed products were isolated and characterized as kurarinone (2), 4″,5″-dihydroxykurarinone (3), 6″-hydroxyl-2'-methoxyl-norkurarinone 7-O-ß-d-glucoside (4), 6″-hydroxyl-norkurarinone 4'-O-ß-d-glucoside (5), 4″,5″-dihydroxynorkurarinone (6), 7-methoxyl-norkurarinone (7), and 7-methoxyl-4″,5″-dihydroxynorkurarinone (8), respectively. Among them, 3-5 are new compounds, and the glycosylation reaction in microbial transformation process was reported rarely. In addition, the cytotoxicities of transformed products (1-8) were also investigated.

Molecular cloning and functional expression of two key carotene synthetic genes derived from Blakeslea trispora into E. coli for increased ß-carotene production.

Blakeslea trispora is used commercially to produce ß-carotene. Isopentenyl pyrophosphate isomerase (IPI) and geranylgeranyl pyrophosphate synthase (GGPS) are key enzymes in the biosynthesis of carotenoids. The cDNAs of genes ipi and carG were cloned from the fungus and expressed in Escherichia coli. Greater GGPS activity was needed in the engineered E. coli when IPP activity was increased. The introduction of GGPS and IPI increased the ß-carotene content in E. coli from 0.5 to 0.95 mg/g dry wt.

Improved lycopene production by Blakeslea trispora with isopentenyl compounds and metabolic precursors.

The highest lycopene production in mated cultures of Blakeslea trispora was 578 mg/l by adding 42 mg geraniol/l to the medium after 48 h of growth. The control gave 317 mg/l. Adding isopentenyl alcohol at 40 mg/l, mevalonic acid at 17.5 mg/l or dimethyl allyl alcohol at 150 mg, each after 36 h growth, gave lycopene yields 62, 45 and 47%, respectively, higher than the control.

Enhanced production of coenzyme Q10 by overexpressing HMG-CoA reductase and induction with arachidonic acid in Schizosaccharomyces pombe.

Coenzyme Q10 (CoQ10) is a vitamin-like substance which plays a crucial role in the respiratory chain ranging from bacteria to humans and in the radical scavenging in human body. In this study, the full-length hmgR gene (encoding 3-hydroxy-3-methyl-glutaryl-CoA reductase, HMG-CoA reductase) was cloned and overexpressed in Schizosaccharomyces pombe. Using the pREPG yeast depressed under the thiamine as the control, CoQ10 contents increased up to 2.68 and 3.09 times when recombinant cells were incubated without and with arachidonic acid, respectively. It demonstrated that arachidonic acid could upregulate the activity of HMG-CoA reductase and that hmgR gene played a significant role in CoQ10 biosynthesis. So, it has an importance to be utilized for fermentation.

Preparation and characterization of poly glycidyl methacrylete-zirconium dioxide-beta-cyclodextrin composite matrix for separation of isoflavones through expanded bed adsorption.

The specially prepared adsorbent is most important in realizing the expanded bed adsorption (EBA) process. In the present work, a novel poly glycidyl methacrylete-zirconium dioxide-beta-cyclodextrin (PGMA-ZrO(2)-beta-CD) composite matrix for EBA has been first prepared. Wet density, water content and pore properties of the composite beads have been investigated, which shows good expansion and stability in EBA. The application of custom-made adsorbent has been investigated to recover isoflavones from soy molasses. The recovery is up to 90% and the purity of isoflavones obtained is 75.4%. Compared with the traditional purification processes, EBA has the advantage of high efficiency and integrality, which leads to large reduction in operation time and cost.

Microbial transformation of dehydroandrographolide by Cunninghamella elegans.

The biotransformation of dehydroandrographolide (1) by Cunninghamella elegans was performed and four transformed products were obtained. Based on their extensive spectral data, the structures of these metabolites were identified as 3-oxo-dehydroandrographolide (2), 3-oxo-2beta-hydroxy-dehydroandrographolide (3), 3-oxo-8beta,17alpha-epoxydehydroandrographolide (4), 3,19-dihydroxy-7,11,13-ent-labdatrien-15,16-olide (5), respectively. Among them, products 3-5 are new compounds.

Effects of an ergosterol synthesis inhibitor on gene transcription of terpenoid biosynthesis in Blakeslea trispora.

Highly efficient induction of carotene biosynthesis of Blakeslea trispora by ketoconazole (KCZ), an inhibitor of ergosterol biosynthesis, was found previously. To get some insight into the regulatory mechanisms of KCZ controlling terpenoid (including carotene) biosynthesis, the transcript levels of gene hmgR, encoding HMGR, which initiates the biosynthesis of all terpenoids, and gene carRA, encoding lycopene cyclase and phytoene synthase in the carotene biosynthsis pathway, were investigated in B. trispora cells treated with KCZ. Upon KCZ treatment, up-regulation of hmgR and carRA genes, increased beta-carotene and ubiquinone contents, and decreased ergosterol content were all observed. The results suggest that the inhibition of ergosterol biosynthesis by KCZ triggered hmgR gene transcription, which might present a positive feedback regulation of gene hmgR in response to a depletion of ergosterol. Furthermore, KCZ could be used as a new agent to improve not only beta-carotene but also ubiquinone production, whose regulatory mechanisms controlling terpenoid biosynthesis differ from the agents reported previously.

[Cloning and secretion expression of hepcidin in Pichia pastoris].

Hepcidin is a liver-expressed, small cysteine rich peptide that acts as a regulator of systemic iron homeostasis. In this work, according to the partiality codon of Pichia pastoris, a DNA fragment containing the coding sequence of hepcidin was designed and synthesized, especially a Kex2 signal cleavage site was fused in 5' end of the antibacterial peptide genes. Then the modified hepcidin gene was inserted into the Pichia pastoris expression vector plasmid pPICZalpha-A. After electroporation of the resulting vector, pPICZalpha-A-Hepc, into the yeast host strain GS115, transformants with high copy inserts were selected by 1500 mg/L Zeocin selection. Under the control of the promoter AOX1 (alcohol oxidase 1), recombinant hepcidin secreted from P. pastoris had a molecular weight of 2.7kD. After optimization of the flask-shaking culture fermentation, the yield of hepcidin reached 100 mg/L in the clarified broth. Through antibacterial assay, the recombinant hepcidin displayed obvious antibacterial activity against Bacillus subtilis. But it could not distinctly inhibit the growth of E. coli BL21 (DE3).

[Properties and advance of hepcidin].

Hepcidin is a small cystein-rich cationic peptide produced mainly by the liver. It was initially isolated from human plasma and exhibited antimicrobial activity. Recently, several lines of evidence have suggested that hepcidin is a key regulator of iron metabolism at the whole body level and is relative to inflammation, infection, hypoxia and anemia. Hepcidin, is implicated in duodenal iron absorption and iron mobilization from reticuloendothelial macrophages. The major mechanism of hepcidin function seems to be the regulation of transmembrane iron transport. As both iron deficiency and iron excess are associated with cellular dysfunction, so hepcidin or hepcidin-related therapeutics could find a place in the treatment of various diseases such as hemochromatosis and anemia of chronic disease. To elucidate biological function of hepcidin further and use it for other research, it is necessary to produce enough hepcidin through DNA recombinant technique. As a highly successful system for the production of a variety of heterologous proteins, the methylotrophic Pichia pastoris system has the probability for a high level production of hepcidin. The subject of this paper is to summarize the regulation of hepcidin gene expression and the understanding of functions of hepcidin. At last, giving a prospect of production hepcidin by gene engineer.

A novel approach for improving the productivity of ubiquinone-10 producing strain by low-energy ion beam irradiation.

Agrobacterium tumefaciens ATCC4452 cells were irradiated by nitrogen ion beam, a new mutagen, with energy of 10 keV and fluence ranging from 2.6x10(14) ions/cm2 to 6.5x10(15) ions/cm2. A similar "saddle shape" survival curve due to ion beam irradiation appeared again in this study. Some mutants with high yield of ubiquinone-10 were induced by ion implantation. High mutation rate and wide mutation spectrum were also observed in the experiment. These results suggested that the mutagenic effect of such low-energy ion influx into bacterium cells could result from multiple processes involving direct collision of particles with cytoplasm, nucleolus, and cascade atomic and molecular reactions due to plentiful primary and secondary particles.

Effect of iontophoresis on skin permeation of defibrase.

AIM: To investigate the effect of iontophoresis on skin permeation of defibrase. METHODS: Iontophoresis was carried out in side-by-side chambers, excised rat skin membrane (RSM) or human epidermis membrane (HEM). The effects of electrode polarity, permeation medium pH and ionic strength were evaluated. RESULTS: Permeation of defibrase caused by anodal iontophoresis was more effective [the apparent permeability coefficient was (1.2 +/- 0.4) x 10(-4) cm x h(-1)] than that of cathodal iontophoresis [(4.3 +/- 1.4) x 10(-5) cm x h(-1)]. The amount of permeated defibrase caused by anodal iontophoresis in pH 7.4 medium was (25 +/- 5) x 10(-14) mol x cm(-2), which was higher than that of in pH 6. 4 permeation medium [(15 +/- 4) x 10(-14) mol x cm(-2)]. CONCLUSION: Iontophoresis could enhance skin permeation of defibrase. Electroosmotic flow effect played an important role.