Effects of knockout of 2,3-butanediol synthesis key enzyme genes on 1,3-propandediol production in Klebsiella pneumoniae / 生物工程学报

2,3-butanediol (2,3-BD) is a major byproduct of 1,3-propandediol (1,3-PDO) fermentation by Klebsiella pneumoniae. To decrease the formation of 2,3-BD, the budC and budA gene, coding two key enzymes of 2,3-BD synthetic pathway in K. pneumoniae, were knocked out using Red recombination technology. The growth of the two mutants were suppressed in different level. The budC deficient strain fermentation results showed that 1,3-PDO concentration increased to 110% and 2,3-butanediol concentration dropped to 70% of the parent strain. However, the budA deficient strain did not produce 1,3-PDO and 2,3-BD, and the final titer of lactic acid, succinic acid, ethanol and acetic acid increased remarkably compared with the parent strain. Further analysis of budC deficient strain fermentation inferred that K. pneumoniae possessed the 2,3-BD cycle as a replenishment pathway. The consequence provided a new evidence for reforming low-byproduct K. pneumoniae.

Cloning and application of a novel hydroxylase in lovastatin conversion / 生物工程学报

Wuxistatin, a novel and potent statin, is converted from lovastatin by Amycolatopsis sp. CGMCC1149. In the bioconversion, lovastatin is firstly hydroxylated by a hydroxylase. To obtain the critical hydroxylase, a novel hydroxylase gene was isolated from Amycolatopsis sp. CGMCC1149 by Degenerate PCR and Self-Formed Adaptor PCR and expressed in Escherichia coli. BLAST sequence analysis revealed that the gene belonged to cytochrome P450 gene superfamily and could encode a 403-amino-acid protein with a molecular weight of 44.8 kDa. The secondary structure prediction result showed that this protein contained many typical functional regions of P450, such as oxygen binding site, ion-pair region and heme binding region. Meanwhile, a catalytic function verification system was constructed by NADH, ferredoxin and ferredoxin reductase which could catalyze lovastatin hydroxylation into the target product. These would be helpful for further studies in large-scale production of wuxistatin.

Construction and fermentation of a recombinant Candida glycerinogenes strain with high glycerol production / 生物工程学报

Candida glycerinogenes WL2002-5 (C.g) is an important industrial strain for glycerol production. To further improve glycerol production, we reconstructed a binary vector pCAM3300-zeocin-CgGPD1, introduced it to Agrobacterium tumefaciens LBA4404 by electroporation, and then transformed the T-DNA harboring the CgGPD1 to Candida glycerinogenes by Agrobacterium tumefaciens-mediated transformation (ATMT). After 96 h fermentation with glucose as the substrate, we screened a transformant named C.g-G8 with high glycerol production. Compared with the wild strain, the glucose consumption rate of C.g-G8 and the glycerol production were 12.97% and 18.06% higher, respectively. During the fermentation, the activity of glycerol-3-phosphate dehydrogenase of C.g-G8 was 27.55% higher than that of the wild strain. The recombinant Candida glycerinogenes with high glycerol production was successful constructed by ATMT method.

Preliminary Study of Bio-transformation of Phytosterol by HPLC-MS / 微生物学通报

The method for analysis and determination the cleavage of soybean sterol, in which the soybean sterol was degraded and the products androst-1,4-diene-,17-dione (ADD) and androst-4-ene-3,17-dion (AD) were developed by Liquid Chromatography-mass Spectrometry. The HPLC conditions adopted were: a All- tima ODS-2 column (250 mm?4.6 mm, 5 ?m), a mobile phase consisted of menthanol-water (70:30), a flow rate of 1.0 mL/min, a room column temperature. and the detective wavelength was 244 nm.The ZMD Micromass electrospray ionization (ESI)-mass spectrometer was employed. In such conditions the corre- sponding HPLC chromatogram and MS spectrum were obtained. The method has a linear ranger of 0.01 mg/mL ~ 0.09 mg/mL, R2 =0.9999, the recoveries of ADD and AD were 102.6% and 105.90%, the RSD of ADD and AD were 3.02%, 3.5% and 3.08%, 3.24%. This method showed high sensitivity, accuracyand easy to perform. It is suitable to analysis the process cleavage of soybean sterol as well as quality control of product.

Construction of novel recombinant strain harboring glycerol dehydratase reactivating factor capable of producing 1,3-propanediol / 生物工程学报

The dhaB gene encoding glycerol dehydratase and dhaG dhaF gene encoding glycerol dehydratase reactivating factor from Citrobacterfreundii were amplified by PCR. The temperature control expression vector pHsh harboring yqhD, dhaB, dhaG and dhaF gene was transformed into E. coli JM109 to yield the recombinant strain E. coli JM109 (pHsh-dhaB-dhaG-dhaF-yqhD). The results from SDS-PAGE analysis show that the recombinant product was consistent with the molecular weight predicted from gene sequence. The fermentation result show that the yield of 1,3-propanediol was increased by 28% compared with E. coli JM109(pHsh-dhaB-yqhD).

Disruption of hom Gene Encoding for Homoserine Dehydrogenase of Corynebacterium glutamicum / 中国生物工程杂志

The hom gene encoding for homoserine dehydrogenase was amplified from the genomic DNA of Corynebacterium glutamicum ATCC 13032.After the kanamycin-resistant gene(Km)cassette from plasmid pET28a was inserted into the center of hom,the hom::Km cassette was then electroporated into the competent cell of C.glutamicum ATCC 13032.And kanamycin-resistant clones were obtained.PCR was performed to confirm whether the Km gene was integrated into the hom gene of these clones and the recombinant strains of hom-disrupted were screened out.Fermentation results showed that the lysine yield of the hom-disrupted strain C.g-hom::Km-8 reached 4.7 g/L,which was 6.7 times that of C.glutamicum ATCC 13032.

Expression,Purification and Enzymatic Characterization of Klebsiella sp.Glycerol Dehydrogenase in E.coli / 中国生物工程杂志

The dhaD gene encoding glycerol dehydrogenase(GDH) from Klebsiella sp.was amplified,and was inserted into expression vector pET-28a(+),the plasmid pET-28a-dhaD was constructed and was transformed into Escherichia coli BL21(DE3).SDS-PAGE showed that the gene dhaD was expressed successfully in recombinant E.coli BL21.Then GDH was purified by Ni-NTA affinity chromatography,the results showed a single band about 39kDa on SDS-PAGE gel,and the specified activity was about 156U/mg.The special activity of GDH is 4.6-fold higher than that of unpurified and the activity recovery is 67.4%.The optimum reaction pH was 11.0,and the GDH activity have little changed when incubated in the buffer of pH7.0～11.0.The optimum reactive temperature was 30℃,and the GDH was more stable on the temperature of 25℃～45℃.The Km value was 0.54mmol/L and Vmax was 0.49 ?mol/ml?min in the glycerol.

Genetic Engineering Reconstruction of Klebsiella pneumoniae Producing 1,3-propanediol by the Gene yqhD Encoding 1,3-propanediol Oxidoreductase Isoenzyme / 中国生物工程杂志

In the reductive branch, glycerol is first dehydrated to 3-hydroxypropionaldehyde that is then reduced to 1,3-PD under the consumption of reducing power (NADH). If over-expression of the gene dhaB encoding glycerol dehydrase is achieved,the reducing power will be scarce and 3-hydroxypropionaldehyde will be accumulated,which is disadvantage to produce 1,3-propanediol.The structure gene yqhD from E.coli encoding 1,3-propanediol oxidoreductase isoenzyme[under the consumption of reducing power (NADPH)]and the gene dhaB encoding glycerol dehydrase from Klebsiella pneumoniae was amplified using PCR method.The two gene were transferred into expression vector pEtac to construct a novel recombinant Klebsiella pneumoniae (pEtac-dhaB-tac-yqhD).Over-expression of yqhD and dhaB in Klebsiella pneumoniae was achieved with pEtac-dhaB-tac-yqhD.The fermentation result on aerobic conversion showed the increase of 20% of 1,3-propanediol yield by Klebsiella pneumoniae(pEtac-dhaB-tac-yqhD) was obtained compared with Klebsiella pneumoniae.The main by-products,acetic acid and butanediol decreased estrogen receptors 30% obviously.

Research in Extraction of GPDH with Ultrasonic from Tobacco Leaf / 中国生物工程杂志

Nicotiana tabacum is an important and classical model plant which can respond to the change of environmental conditions by accumulating osmoprotectants, such as glycerol and proline which contribute to the re-establishment of homeostasis when exposed to various adverse environmental stresses, such as drought, salinity, high and low temperatures. The optimization of ultrasonic extraction (UE) conditions of glycerol-3-phosphate dehydrogenase (GPDH) of tobacco leaf have been built by orthogonal test. It showed that optimum of the powers, treatment times, slot times and leaf-to-solvent ratios of UE was 75w, 2h, 2s, and 1[DK]∶12 g/ml, respectively. Under these conditions, the activity of GPDH has been tested as 0.3937U/mg protein, which was higher than other extraction methods such as liquid nitrogen and grinding on ice bath. According to investigation, it is the first description of determination of content of GPDH with ultrasonic in tobacco. It could provide basis for the further research in the relation of content of glycerol and osmotic pressure in tobacco.

Determination of Dihydroxyacetone in Fermentation Broth by HPLC / 中国生物工程杂志

A method to determine dihydroxyacetone (DHA) in fermentation broth was developed by high performance liquid chromatography (HPLC). DHA was separated on a Alltima C18(5?m,250?4.6mm). The mobile phase was 0.5% methanol solution (pH adjusted to 3.0 with H3PO4), the flow-rate was 1.0 ml/min and the detective wavelength was 200 nm. The detection limits of DHA was 0.1 g/L～10.0 g/L. 6.2 g/L DHA in the fermentation broth was detected by HPLC, which was in agreement with the result by spectrophotometric method.The method was applicable for DHA determination in the fermentation process.

Stimulatory effects of some amino acids on glycerol production by Candida glycerinogenes / 生物工程学报

By using some intermediate metabolites in EMP pathway and TCA cycle as the control, the effects of amino acid supplements on glycerol production by Candida glycerinogenes in shake-flask fermentations with urea as nitrogen resource were investigated. The results showed that ten kinds of amino acids, including L-glutamic acid, L-glutamine, L-aspartic acid, L-asparagine, L-glycin, L-lysine, L-tyrosine, L-proline, L-histidine, and L-serine, had strong promotional effects on glycerol production; The optimal concentrations of these amino acids were 0.40, 0.45, 0.36, 0.35, 0.39, 0.36, 0.35, 0.45, 0.26, and 0.45 g/L, respectively. Accordingly the optimum contents of pyruvic acid, a-oxoglutarate, oxaloacetic acid, citrate, and succinate were 0.24, 0.42, 0.40, 0.37, and 0.38 g/L, respectively. The advantageous opportunities of supplement were as follows: L-lysine at the beginning of fermentation; pyruvic acid and oxaloacetic acid at the fourteenth hour; L-glutamic acid, L-glutamine, L-histidine, L-proline, L-aspartic acid, L-tyrosine, L-glycin, alpha-oxoglutarate, and succinate at the thirtieth hour; and L-asparagine, L-serine, and citrate at the forty-eighth hour. The addition of each stimulant at the optimal conditions could significantly promote glycerol production, with the glycerol yield on initial glucose and its increased speed exceeding 60% and 16%, respectively. The possible stimulatory mechanism due to the amino acid supplements is that the increased intermediate metabolites levels from amino acids degradation may have enhanced the flux through TCA cycle, which improve cell energetics. Meanwhile, the shift of carbon metabolism flux at the glyceraldhyde-3-phosphate node can result in the incremental flux through glycerol biosynthesis pathway.

Construction of novel recombinant Escherichia coli capable of producing 1,3-propanediol / 生物工程学报

The 1,3-propanediol oxidoreductase isoenzyme encoding gene (yqhD) from E. coli was amplified by PCR. yqhD was inserted in pEtac to yield the recombinant expression vector pEtac-yqhD. Over-expression of yqhD in E. coli JM109 was achieved with pEtac-yqhD. SDS-PAGE analysis showed an over-expressed recombinant product at about 43 kD, consistent with the molecular weight predicted from gene sequence. Compared with E. coli JM109 (pEtac), the 1,3-propanediol oxidoreductase isoenzyme activity of the recombinant E. coli (pEtac-yqhD) reached 120 u/mg protein under the induction of 1.0 mmol/L IPTG at 37 degrees C for 4 hours; at similar conditions, enzyme activity of E. coli JM109 (pEtac) was only 0.5 u/mg protein. The recombinant E. coli JM109 (pUCtac-dhaB, pEtac-yqhD) was constructed. After induction with 1.0 mmol/L IPTG, the recombinant strain could transform 50 g/L glycerol to 38 g/L 1,3-propanediol under aerobic conditions. This work demonstrated firstly that the 1,3-propanediol oxidoreductase isoenzyme could show high activity under aerobic conditions.

Glyoxylate cycle is required for the overproduction of glutamate but is not essential for Corynebacterium glutamicum growth on glucose / 生物工程学报

The glyoxylate cycle was hypothesed to be indispensable for glutamate overproduction in coryneform bacteria, for it was thought to fulfill anaplerotic functions and to supply energy during the growth phase. During glutamate overproduction phase, however, it has been noted that the high level of the cycle is detrimental to the glutamate production. In order to clarify the relationship between the glutamate production and the glyoxylate cycle, a chromosomal aceA-disrupted mutant of wild-type C. glutamicum ATCC 13032 was constructed. The isocitrate lyase (ICL) activity of the parental strain was 0.011 u/mg of protein and reached 1.980 u/mg of protein after acetate induction; the mutant strain WTdeltaA, however, had no detectable ICL activity and was no longer able to grow on minimal medium with acetate as the sole carbon source. Compared with the wild-type C. glutamicum WT, the mutant strain WTdeltaA, exhibited the same growth rate with glucose as the sole carbon source, indicating glyoxylate cycle is not required for its growth on glucose. On the contrary, the glutamate production in WTdeltaA was severely impaired and more residual glucose was found in the fermentation broth at the end of fermentation with the mutant strain than with the wild-type strain. Further investigations into the relationship between the glutamate production and the glyoxylate cycle are under the way, which may help to elucidate the mechanism of glutamate overproduction.

Establishment of a HepG2 cell line stably expressing human cytochrome P450 1A2 and its metabolic activity / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To establish a HepG2 cell line stably expressing the human cytochrome P450 1A2 and to study its metabolic activity.</p><p><b>METHODS</b>The human wild-type CYP1A2 cDNA was subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP1A2 to HepG2 cells. The expression of CYP1A2 mRNA was validated by RT PCR. The metabolic activation of HepG2 CYP1A2 cells on aflatoxin B1 (AFB1) was assayed by cytotoxicity test.</p><p><b>RESULT</b>The HepG2-CYP1A2 cells expressed CYP1A2 mRNA and could increase the cytotoxicity to AFB1 in comparison with that of wild type HepG2 cells.</p><p><b>CONCLUSION</b>The established HepG2-CYP1A2 can express the mRNA and has the metabolic activity to AFB1. The cell line may be useful for testing the toxicity and metabolism of xenobiotics, which might possibly be activated or metabolized by CYP1A2.</p>

Study on fermentation condition of alkaline protease gene engineering strain and the purification and characterization of recombinant enzyme / 生物工程学报

In a 5L fermentor the production conditions of alkaline protease gene engineering strain BA071 were investigated. The maximum activity of alkaline protease reached 24,480 u/mL in 40 hours of fermentation by combination of enhancing aeration and changing the agitation rate. The fast purification method of recombinant protease was conducted with FPLC (Fast Protein Liquid Chromatography). The crude enzyme, treated with ammonium sulfate fractionation and decolored with DEAE-A-50 and polyethylene glycol concentration, was purified with CM-Sephadex-C-50 and Sephadex-G-75. The purified enzyme appears homologous on SDS-PAGE. The purity of enzyme was increased 76.2 times. SDS-PAGE analysis showed that the molecular weights of expressed recombinant products were about 28 kD. The optimal reaction pH and temperature of recombinant enzyme were at pH11 and 60 degrees C, respectively. The recombinant enzyme exhibited high temperature tolerance and was stable at a wide range of pH. Ca2+, MG2+ can enhance the stability of the recombinant enzyme. While the protease activity of the enzyme was strongly inhibited by Hg2+, Ag+, PMFS [symbol: see text] DFP, and was not affected by SDS and Urea.

Researches on the polymorphism of cytochrome P450 2A6 / 中华医学遗传学杂志

Cytochrome P450 2A6(CYP2A6) is known as a major enzyme responsible for C-oxidation of nicotine and 7-hydroxylation of coumarin. The article reviews different alleles of CYP2A6 that have been discovered, their effect on CYP 2A6 activity and the relationship between genetic polymorphism of CYP2A6 and smoking behavior as well as susceptibility of lung and esophageal cancer in different individuals.

Mechanism of Corn Steep Liquor during Glycerol Fermentation by Candida glycerinogenes / 微生物学通报

Using chemically defined medium as the control, mechanism of corn steep liquor (CSL) in complex medium during glycerol production by Candida glycerinogenes was studied.The results showed that there were three key factors in CSL that had some great influences on glycerol fermentation of C.glycerinogenes, including phosphorus, nitrogen, and trace elements.The maximum glycerol yield of 53.44% was achieved at an optimal phosphorus concentration of 121.75mg/L, where the CSL concentration was 14g/L.Phosphorus in CSL could control the distribution of carbon metabolism flux between EMP pathway and HMP pathway.With the increase in CSL concentrations, superfluous phosphorus could restrain HMP pathway and activate EMP pathway, thus resulting in remarkable changes in various fermentation parameters of complex medium.Nitrogen in CSL could play a cooperative role in the regulative function of phosphorus.However, it was not a suitable nitrogen source for C.glycerinogenes.Trace elements in CSL could markedly improve the glucose consumption rate, accelerate the cell growth, and enhance the glycerol yield.

A Rapid Method for Preparation of Fungal Chromosome DNA / 微生物学通报

The article introduce a rapid method for preparation of fungal chromosome DNA In this method the quartz sand is used to break the fungal cell wall and the chromosome DNA is harvested rapidly in 1～2 h The method is applied successfully by the author to four kinds of fungi Neurospora crassa , Aspergillus oryzae , Morchella esculcnta , Saccharomyces cervisae All the chromosome DNA extracted has the fragment size lager than 20 kb and can be used directly for either digestion with restriction endoenzyme or PCR

THE TWO ISOENZYMES OF S.CEREVISIAE GLYCEROL 3-PHOSPHATE DEHYDROGENASE / 微生物学通报

NAD +-dependent cytosolic glycerol-3-phosphate dehydrogenase in Sacch aromyces cerevisiae is one of the key enzymes in metabolic pathway of glycerol . catalysing the reduction of dihydroxyacetone phosphate to glycerol-3-phosph ate.It has two isoenzymes.To study the differences between their structures, their expression of encoding genes and their functions may help increase the understan ding of the cell response mechanism to the hyperosmotic and anoxic conditions. In this paper the research on the two isoenzymes was reviewed.

STUDY ON RESISTANCE GENE KNOCK OUT FROM INTEGRATED ALKALINE PROTEASE GENE ENGINEERING STRAIN / 微生物学通报

The knock out vector pHK was constructed with E coli vector pET 28a and shuttle vector pHY300PL, by using denatured DNA and homologous recombination technique, the kanamycin resistance gene ( Kan r) from integrated alkaline protease gene engineering strain BP071 was knocked out successfully, and the 11 positive clones were obtained The yield of the best positive clone BP0715 was stable as same as BP071 The methods provided the good experience for the industrial microbiology research, and it was foundation for studying on the safety of genetically modified organisms