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Articles by Wen Liu
Total Records ( 5 ) for Wen Liu
  Yun Chen , Wei Deng , Jiequn Wu , Jiangchao Qian , Ju Chu , Yingping Zhuang , Siliang Zhang and Wen Liu
  Erythromycin A (Er-A) is the most potent and clinically important member in the Er family produced by Saccharopolyspora erythraea. Er-B and Er-C, which are biologically much less active and cause greater side effects than Er-A, serve as the intermediates for Er-A biosynthesis and impurities in fermentation processes of many industrial strains. In this study, systematical modulation of the amounts of tailoring enzymes EryK (a P450 hydroxylase) and EryG (an S-adenosylmethionine-dependent O-methyltransferase) was carried out by genetic engineering in S. erythraea, including alterations of gene copy number ratio and organization and integrating the locus on the chromosome by homologous recombination. Introduction of additional eryK and eryG genes into S. erythraea showed significant impacts on their transcription levels and enhanced the biotransformation process from Er-D to Er-A with gene dose effects. At the eryK/eryG copy number ratio of 3:2 as well as their resultant transcript ratio of around 2.5:1 to 3.0:1, Er-B and Er-C were nearly completely eliminated and accordingly converted to Er-A, and the Er titer was improved by around 25% in the recombinant strain ZL1004 (genotype PermK*-K-K-G + PermE*-K + PermA*-G) and ZL1007 (genotype PermK*-K-G-K + PermE*-K + PermA*-G). This study may contribute to the continuous efforts toward further evaluation of the Er-producing system, with the aims of improving Er-A purity and production at the fermentation stage and lowering the production costs and environmental concerns in industry.
  Lei Li , Wei Deng , Jie Song , Wei Ding , Qun- Fei Zhao , Chao Peng , Wei- Wen Song , Gong -Li Tang and Wen Liu
  Saframycin A (SFM-A), produced by Streptomyces lavendulae NRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.
  Jie Fang , Yiping Zhang , Lijuan Huang , Xinying Jia , Qi Zhang , Xu Zhang , Gongli Tang and Wen Liu
  Tetrocarcin A (TCA), produced by Micromonospora chalcea NRRL 11289, is a spirotetronate antibiotic with potent antitumor activity and versatile modes of action. In this study, the biosynthetic gene cluster of TCA was cloned and localized to a 108-kb contiguous DNA region. In silico sequence analysis revealed 36 putative genes that constitute this cluster (including 11 for unusual sugar biosynthesis, 13 for aglycone formation, and 4 for glycosylations) and allowed us to propose the biosynthetic pathway of TCA. The formation of D-tetronitrose, L-amicetose, and L-digitoxose may begin with D-glucose-1-phosphate, share early enzymatic steps, and branch into different pathways by competitive actions of specific enzymes. Tetronolide biosynthesis involves the incorporation of a 3-C unit with a polyketide intermediate to form the characteristic spirotetronate moiety and trans-decalin system. Further substitution of tetronolide with five deoxysugars (one being a deoxynitrosugar) was likely due to the activities of four glycosyltransferases. In vitro characterization of the first enzymatic step by utilization of 1,3-biphosphoglycerate as the substrate and in vivo cross-complementation of the bifunctional fused gene tcaD3 (with the functions of chlD3 and chlD4) to ΔchlD3 and ΔchlD4 in chlorothricin biosynthesis supported the highly conserved tetronate biosynthetic strategy in the spirotetronate family. Deletion of a large DNA fragment encoding polyketide synthases resulted in a non-TCA-producing strain, providing a clear background for the identification of novel analogs. These findings provide insights into spirotetronate biosynthesis and demonstrate that combinatorial-biosynthesis methods can be applied to the TCA biosynthetic machinery to generate structural diversity.
  Shao-Bin Gu , Bin Yang , Ying Wu , Shi-Chang Li , Wen Liu , Xiao-Fei Duan and Meng-Wei Li
  It is undeniable that environmental sonic vibration can affect our emotions and mood, but so far the study of physical stimuli provoked by audible wave on single cells has been rarely concerned. To investigate the response of E. coli to audible wave exposure, the growth status and alterations in antioxidant enzyme activity were studied in liquid culture. The data showed that the growth of E. coli was promoted in the treatments of different frequencies sound wave. The most significant effect on growth promotion appeared when sound wave was maintained at 100 dB and 5000 Hz. Simultaneously, sonic vibration evoked significantly increases the level of total protein content contents. And the changes of activities of Super Oxide Dismutase (SOD) and catalase (CAT) were observed obviously. The results suggested that the growth promotion effect of audible sound may be non-linear and shows obvious frequency and intensity peculiarities. Moreover, the increase in activity of antioxidant enzymes implied that a number of active oxygen species generated in bacterial cell under the exposure of audible sound. We speculate that the audible sound may cause a secondary oxidative stress. Further studies are needed to elucidate the mechanisms of active oxygen species generation induced by audible sound.
  Ute Galm , Liyan Wang , Evelyn Wendt-Pienkowski , Runying Yang , Wen Liu , Meifeng Tao , Jane M. Coughlin and Ben Shen
  Bleomycin (BLM), an important clinically used antitumor compound, and its analogs are challenging to prepare by chemical synthesis. Genetic engineering of the biosynthetic pathway in the producer strain would provide an efficient and convenient method of generating new derivatives of this complex molecule in vivo. However, the BLM producing Streptomyces verticillus ATCC15003 has been refractory to all means of introducing plasmid DNA into its cells for nearly two decades. Several years after cloning and identification of the bleomycin biosynthetic gene cluster, this study demonstrates, for the first time, genetic accessibility of this pharmaceutically relevant producer strain by intergeneric Escherichia coli-Streptomyces conjugation. Gene replacement and in-frame deletion mutants were created by λRED-mediated PCR targeting mutagenesis, and the secondary metabolite profile of the resultant mutants confirmed the identity of the BLM biosynthetic gene cluster and established its boundaries. Ultimately, the in-frame blmD deletion mutant strain S. verticillus SB5 resulted in the production of a bleomycin intermediate. The structure of this compound, decarbamoyl-BLM, was elucidated, and its DNA cleavage activity was compared with the parent compounds.
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