Fitness analysis between the L-sorbosone dehydrogenase modules and Ketogulonigenium vulgare chassis / 生物工程学报

Ketogulonigenium vulgare is an acid-producing strain in the process of two-step vitamin C fermentation. L-sorbosone dehydrogenase (SNDH) is one of the key enzymes during the biosynthesis of 2-keto-L-gulonic acid (2-KGA), the precursor of vitamin C. However, the catalytic mechanism of SNDH is unclear. According to the whole genome sequencing of K. vulgare, two genes encoding sorbosone dehydrogenases, one derived from the chromosome (named as sndhg) and one from plasmid (named as sndhp), were introduced into an industrial strain K. vulgare. The overexpression of gene sndhg had hardly effect on 2-KGA production, and the overexpression of gene sndhp produced an obvious byproduct in the fermentation broth. Combinational expression of sndhg/sndhp with pqqA (obtaining sndhg-pqqA and sndhp-pqqA modules) in K. vulgare resulted in the similar fermentation phenotype to two previous strains. After serial sub-cultivation of co-cultured Bacillus endophyticus with each engineered K. vulgare for 50 d, the conversion rate of 2-KGA increased by 15.4%, 179%, 0.65% and 125% compared with that of the parental K. vulgare with B. endophyticus. This study shows that adaptive evolution of microbial consortium is an effective strategy to increase the fitness between functional modules and chassis, thus quickly getting better strains for production of 2-KGA.

Effect of integration loci of genome on heterologous gene expression in Saccharomyces cerevisiae / 生物工程学报

Chromosomal integration of heterologous genes or pathways is preferred over the use of episomal plasmids for its inherently stability and thus more desirable in the industrial setting. However, the position of integration of heterologous genes in the genome influences the expression levels. In combination of high throughput transformation of the Yeast Knock-out Collection (YKO) and FACS analysis, the position effect on heterologous reporter gene gfp was identified across the whole genome in yeast. In total 428 high-expressed sites and 444 low-expressed sites were spotted, providing massive data to analyze patterns and reasons for region dependency of gene expression on the genome-wide scale.

Metabolomics analysis of taxadiene producing yeasts / 生物工程学报

In order to study the inherent difference among terpenes producing yeasts from the point of metabolomics, we selected taxadiene producing yeasts as the model system. The changes of cellular metabolites during fermentation log phase of artificial functional yeasts were determined using metabolomics methods. The results represented that compared to W303-1A as a blank control, the metabolites in glycolysis, tricarboxylic acid cycle (TCA) cycle and several amino acids were influenced. And due to the changes of metabolites, the growth of cells was inhibited to a certain extent. Among the metabolites identified, citric acid content in taxadiene producing yeasts changed the most, the decreasing amplitude reached 90% or more. Therefore, citric acid can be a marker metabolite for the future study of artificial functional yeasts. The metabolomics analysis of taxadiene producing yeasts can provide more information in further studies on optimization of terpenes production in heterologous chassis.

Match of functional module with chassis in 7-dehydrocholesterol synthesis / 生物工程学报

The key challenge to generate engineered cells by synthetic biology for producing 7-dehydrocholesterol (7-DHC) in a high titer is the match between functional module and chassis. Our study focused on solving this problem by combining different promoters and yeast chassis to increase 7-DHC production. To optimize the chassis in order to accumulate zymosterol, the substrate for 7-DHC synthesis, we overexpressed truncated HMG-CoA reductase (tHmglp) and squalene epoxidase (Erglp), both are key genes of yeast endogenous zymosterol biosynthetic pathway. In addition, we knocked out C-24 methyl transferase (Erg6p) and C-22 dehydrogenase (Erg5p) to inhibit the conversion of zymosterol to ergosterol. By introducing heterologous C-24 reductase under three promoters with different strengths, namely TDH3p, PGK1p and TDH1p, we constructed functional modules of diverse activities. Nine engineeredcells were generated based on the combination of these three modules and three chassis. The result shows that the engineered cell composed of functional module regulated by TDH3p and chassis SyBE_000956 had the highest 7-DHC production, indicating a better match than others. This study provides evidences for importance of match and empirical support for rational design of subsequent researches.

Preparation of galactosylated hyaluronic acid/chitosan scaffold for liver tissue engineering / 生物医学工程学杂志

The purpose of this research is to construct a kind of 3D-Scaffold with galactose-carrying polysaccharide for improving the function of hepatocytes in vitro. Galactose moieties were covalently coupled with hyaluronic acid through ethylenediamine. Galactosylated hyaluronic acid/chitosan scaffolds were prepared by lyophilization. The characteristics of the scaffolds such as morphology, hydrophilicity, and mechanical properties were investigated. The results indicated that the porosity and the pore size of the scaffolds made in -20 degrees C were useful used for culturing hepatocytes. And, the incorporating of hyaluronic acid in chitosan network improved the hydrophilicity and mechanical properties of the scaffolds. Rat primary hepatocytes growing in the scaffolds observed by phase-contrast microscope showed the multicellular spheroid morphologies. Therefore, galactosylated hyaluronic acid/chitosan scaffolds could be used as a promising scaffold for liver tissue engineering.

Studies on paclitaxel-loaded methoxy poly (ethylene glycol)/poly (L-lactic acid) diblock copolymer nanoparticles / 生物医学工程学杂志

Paclitaxel-loaded methoxy poly (ethylene glycol )-b-poly (L-lactic acid) diblock copolymer nanoparticles (PMT) were prepared by a self-emulsification/solvent evaporation method. The PMT morphology, size and its distribution, and drug release in vitro were investigated by DLS, UV, TEM and HPLC. The results indicate that PMT show a spherical morphology with inner core and outer shell. The diameter (nm) of PMT increases with the increase of the drug-loaded amount. The initial burst release is not observed, the drug releasing rate in vitro is lower, and the accumulated release increases with the increase of replacement amout of the pH7. 4 medium. This study develops a new formulation for paclitaxel and provides an experimental basis for the intravenous administration of paclitaxel.

Isolation of alkaloids and structure elucidation of lehmannine from / 中草药

To study the alkaloid constituents in the seed of Sophora alopecuroides L. and isolation of lehmannine by transhydrogenation of sophocarbine to matrine present in the mixture. Methods　Various processes of exchange, ion exchange and column chromatograph with very similar physical and chemical properties was successfully separated by transhydrogenation. Results　5 alkaloids were obtained and identified by elementary analysis, IR, MS, 1HNMR and 13CNMR as oxymatrine, oxysophoridine, matrine, sophoridine and lehmannine (12, 13-dehydromatrine). Conclusion　Lehmannine was isolated from the seed of S. alopecuroides for the first time.

Cell metabolism of Taxus chinensis var. mairei in suspension cultures treated with methyl jasmonate / 中草药

Object To study the physiological changes of Taxus chinensis var. mairei (Lemee et L?vl.) Cheng et L. K. Fu in the case of methyl jasmonate (MJ). Methods TTC assay, soluble protein measurement and enzyme analysis were used. Results It was observed that MJ inhibited Taxus cell growth in the view of primary metabolism. MJ induced phenylalanine ammonia lyase(PAL) activity while it inhibited polyphenoloxidase (PPO) activity. Extracellular phenolic content after addition of MJ increased to the maximum at the three days than that of the control group. Conclusion It was demonstrated that MJ induced the transition of Taxus cell from primary metabolism to secondary metabolism. This is favorable for secondary metabolism of Taxus cells. It is important to study the physiology of Taxus cells for revealing the mechanism of MJ.

Isolation of alkaloids and structure elucidation of lehmannine from seed of Sophora alopecuroides / 中草药

Object To study the alkaloid constituents in the seed of Sophora alopecuroides L and isolation of lehmannine by transhydrogenation of sophocarbine to matrine present in the mixture Methods Various processes of exchange, ion exchange and column chromatograph with very similar physical and chemical properties was successfully separated by transhydrogenation Results 5 alkaloids were obtained and identified by elementary analysis, IR, MS, 1HNMR and 13 CNMR as oxymatrine, oxysophoridine, matrine, sophoridine and lehmannine (12, 13 dehydromatrine) Conclusion Lehmannine was isolated from the seed of S alopecuroides for the first time

Biotechnology in modernization of Chinese materia medica / 中草药

Chinese materia medica (CMM) plays an important role both in the disease prevention and therapy system and also in natural drug screening. Biotechnology exhibits applicable prospects in modern research of CMM with progress of natural science and technologies. Amplification fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP), random amplification polymorphism of DNA (RAPD), and microsatellite DNA have been used to discriminate and breed herbal varieties. Genetic transformation and techniques of tissue and cell culture are explored to protect herbal resouces and to produce active components or parts on a commercial scale. High throughput technologies of proteome and biochip are expected to probe molecular targets and routes of CMM with the changes of proteome and gene expression. The results can be helpful to novel drug development and secondary exploitation, so as to promote the modernization process of CMM.

Application of proteomics to modernization research of Chinese materia medica / 中草药

To summarize the application of proteomics to modernization research of Chinese materia medica (CMM) and offer the reference for modernization of CMM and new medicine preparation exploitation. Based on the references and our project group studies in proteomic of drug biosynthesis pathway and metabolization;the main content,technology strategies and important application of the proteomics were introduced significantly;and the further development of protemics was analyzed. The proteomics will become one of absolutely necessary tackles in the modernization research of CMM.

Effects of fed-batch carbohydrate, nitrogen, phosphorus on growth of Taxus chinensis var. mairei cell during suspension culture / 中草药

Object To solve the problem that Taxus chinensis var. mairei (Lemee et L?vl.) Cheng et L. K.Fu grows slowly. Methods The consumption of phosphorus during the cell suspension culture and the effect of fed-batch carbohydrate, nitrogen and phosphorus on cell growth and living-cell activity were assayed. Results The carbohydrate was exhausted in the middle phase of suspension culture, dificiency of carbohydrate led to inhibition of cell growth in the late culture. Conclusion Compared with the control group, the cell growth rate and the cell density in the fed-batch carbohydrate group were increased significantly, and the cell growth rate was up to 83%.

Study on Immobilization Plant Cell Culture and Release of Intracellular Metabolites / 中草药

Membrane immobilization of Catharathus roseus (L. )G. Don cell culture was stuudied. Results showed that the production capacity can attain a higher level when the cell layer was 7~8mm thick. Stainless-steel screen(80?m)was suitable for immobllization of C. roses cell. Pressure pulse can enhance the release of intracelluler metabolites. A method to determine allowable cell layer thickness was developed. The calculated value agreed well with experimental results.

MUTAGENICITY OF STREPTOMYCES REGENSIS PRODUCING A NOVEL ANTIBIOTICS AGPM / 微生物学通报

of Streptomyces regensis was isolated from soil to produce a novel antibiotic AGPM of a strong biological activity of antitumor The strain was irradiated by UV after treatment with LiCl to give a AGPM yield of 1 87?10 2 mg mL 1 , 2 2 times higher than that of the original strain The optimum UV irradiation time was 30～60 s and the best LiCl concentration was 0 05～0 09 mol/L The fermentation of AGPM was conducted in a 30 L stirred tank, the maximum yield of AGPM using the mutants reached 1 85?10 2 mg mL 1 , while that using the original strain was only 0 85?10 2 mg mL 1