Qingyang Xu

Modification of tryptophan transport system and its impact on production of L-tryptophan in Escherichia coli.

The production of L-tryptophan through chemical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of transport system for aromatic amino acids in L-tryptophan producing strains has not been fully explored. In this study, the fact was revealed that L-tryptophan production and cell growth were affected by the modification of transport systems based on YddG functioning as aromatic amino acid excretion and AroP functioning as general aromatic amino acid permease. Through comparing glucose conversion rates of recombinant strains such as Escherichia coli TRTH ΔaroP, E. coli TRTH-Y, and E. coli TRTH ΔaroP-Y, the moderate modification of transport system resulted in the metabolic flux redistribution of L-tryptophan biosynthesis pathway. In the fed-batch fermentation by E. coli TRTH and E. coli TRTH-Y in 30-liter fermentor, the final production of L-tryptophan fermented by E. coli TRTH-Y was 36.3 g/L, which was 12.6% higher than fermentation by E. coli TRTH.

Low-molecular-mass purine nucleoside phosphorylase: characterization and application in enzymatic synthesis of nucleoside antiviral drugs

Purine nucleoside phosphorylase (PNP) that catalyzes the reversible phosphorolysis of various purine nucleosides is widely distributed in prokaryotes and eukaryotes. Four pnp genes from Bacillussubtilis 168, Escherichia coli K-12 and Pseudoalteromonas sp. XM2107 were cloned by PCR and expressed in E. coli XL1-Blue. Recombinant PNPs (rPNPs) were purified by Ni2+-NTA chromatography. Compared with other rPNPs, PNP816 was a low-molecular-mass homotrimer, which exhibited 11-, 4- and 1.5-fold higher values in kcat/Km using inosine as the substrate at 37°C. The PNP816 or engineered strain XBlue (pQE-816) had a higher catalytic activity than other rPNPs or engineered strains during the enzymatic synthesis of ribavirin, which suggested that the low-molecular-mass homotrimer derived from microorganisms has higher catalytic activity for synthesis of nucleoside antiviral drugs.

Expression of the Escherichia Coli TdcB gene encoding threonine dehydratase in L-isoleucine-overproducing Corynebacterium Glutamicum Yilw

Threonine dehydratase (EC 4.3.1.19, TDH) catalyzing the degradation of Thr to α-ketobutyrate, is a rate-limiting enzyme in L-Ile pathway. The tdcB gene encoding TDH was obtained from Escherichia coli K12 by PCR and expressed at E. coli BL21 (DE3). Then the tdcB gene was inserted into the shuttle expression vector pXMJ19 and the recombinant plasmid was electroporated into the L-isoleucine-producing strain of Corynebacterium glutamicum YILW. Crude extracts of the microbial strain containing the plasmid pXMJ19tdcB retained 60% of the original TDH activity even in the presence of 300 mM L-Ile. The recombinant strain of bacteria showed 7.5% higher enzyme activity and 11.3% higher L-Ile production compared to the original strain.

Optimization of technical conditions of producing ribavirin byBacillus subtilis

In this study, the fed-batch fermentation technique was applied to improve the yield of ribavirin produced byBacillus subtilis AG208-1. Various fermentation substrates and conditions were investigated to identify the optimal concentration of carbon source, Tween-80 and fermentation temperature in the production of ribavirin. The optimal initial glucose concentration was determined to be 120 g/L based on the results of fermentations conducted in a baffled flask. Then, different concentrations of Tween-80 and feeding time were also investigated in this work, respectively. Our results showed that the production of ribavirin byBacillus subtilis was enhanced when Tween-80 concentration was 0.5%. Furthermore, variable temperature control strategy was predominant factor for ribavirin overproduction and the optimal strategy was: temperature of fermentation at 36 °C from 0 h to 24 h, with a gradual increase in temperature of 2 °C every 6 h. Under the optimal conditions, a final ribavirin concentration of 4.21 g/L was achieved after 60 h.

Effects of aroP gene disruption on L-tryptophan fermentation

The production of L-tryptophan through chemical synthesis, direct fermentation, bioconversion and enzymatic conversion has been reported. However, the role of the transport system for the aromatic amino acids in Ltryptophan producing strains has not been fully explored. In this study, the aroP gene of the L-tryptophan producing Escherichia coli TRTH strain was disrupted using Red recombination technology and an aroP mutant E. coli TRTH ΔaroP was constructed. Fed-batch fermentation of E. coli TRTH ΔaroP was carried out in 30-L fermentor to investigate the L-tryptophan production. Compared with E. coli TRTH, the aroP mutant was able to maintain a higher growth rate during the exponential phase of the fermentation and the L-tryptophan production increased by 13.3%.

Structure–activity relationship of a cold-adapted purine nucleoside phosphorylase by site-directed mutagenesis

Purine nucleoside phosphorylase can be expressed in Escherichia coli and the intact cells can be used as a catalyst for the biosynthesis of nucleosides. The purine nucleoside phosphorylases from E. coli (EcPNP) and Pseudoalteromonas sp. XM2107 (PsPNP) have been purified. In order to improve the catalytic efficiency, the model of three-dimensional structure of PsPNP was constructed, and then 9 active/binding-site mutants were constructed by one-step site-directed mutagenesis and characterized by steady-state kinetics. Double mutations exhibited the largest change of catalytic activity. The T90R:T156S mutant revealed 1000 fold enhancements in k(cat)/K(m) for inosine phosphorolysis. However, the T90A:T156A mutant revealed 500 fold reduction in catalytic activity when compared with wild-type one. These results in combination with the predicted locations of Thr90 and Thr156 side chains by homology modeling suggested that: (i) a complete hydrophobic pocket played an important role in the catalytic function of PsPNP; (ii) a potential transition state structure was present in hydrogen bond between the carboxyl groups of Thr90 in the phosphate binding site. Therefore, the application of site-directed mutagenesis will be benefit to further improve catalytic efficiency of PsPNP during the enzymatic synthesis of antivirus drug ribavirin.

Molecular Cloning, Expression and Enzymatic Characterization of Inosine Monophosphate Dehydrogenase from Bacillus amyloliquefaciens

Inosine monophosphate dehydrogenase(IMPDH, Ec1.1.1.205) is the rate-limiting enzyme for de nove guanosine monophosphate synthesis. The IMPDH encoding gene guaB has been clonded and sequenced from Bacillus amyloliquefaciens GR600, a overproduction-guanosine strain. A fragment contained the stuctrural gene guaB encoding IMPDH from GR600 was constructed into expression vector pET-His. The recombinant expression plamid was transformed into Escherichia coil strain BL21(DE3), induced by IPTG and expressed. The recombinat IMPDH was purified by Ni-NTA resins. The result of SDS-PAGE showed that molecular weight of the recombinat IMPDH was 54 kD. Enzyme activity assay showned that the optimum pH value and temperatrure of the recombinat IMPDH were 8.0 and 40 °C. The results have great significance in genetical modify of producing strain.

Construction of Ribavirin Engineering Bacteria

In this study, the gene encoding purine nucleoside phosphorylase (PNPase) from Bacillus subtilis W168 was identified, cloned and expressed in Bacillus subtilis AG208. The gene encodes a polypeptide of 233 amino acids with a calculated molecular weight of 25,018 Da. The enzyme activity of the recombinant protein (AGPNP) was analyzed by temperature and pH perturbation difference spectra. Results showed that the optimum of temperature was 65°C and the optimum pH was 7.5 and the PNPase activity of the recombinant bacteria was increased by 193.9%.