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Synthesis of S-adenosyl-L-methionine in Escherichia coli
Xiao-Nan Wei,Minjie Cao,Jian Li,Huan Li,Yi Song,Cuihong Du 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.6
S-adenosyl-L-methionine (SAM) is an importantphysiological metabolite in vivo and may be useful inmedicines. SAM is produced from L-methionine and ATPcatalyzed by S-adenosyl-L-methionine synthetase (SAMS)in vivo. In this study, the gene encoding SAMS was clonedand a genetically engineered Escherichia coli (E. coli)BL21(pET-28a-SAMS) was constructed. The recombinantSAMS with a molecular mass of approximately 46 kDawas expressed by inducing the engineered E. coli usingisopropyl-β-D-1-thiogalactopyranoside (IPTG) as an inducer. To produce SAM using a low-cost, nontoxic and highperformanceexpression system, lactose was used as asubstitute for IPTG to induce BL21(pET-28a-SAMS). Byoptimizing the expression conditions, the concentration ofSAM produced by the engineered E. coli was 48 mg/L in theculture medium supernatant. To increase the concentrationof SAM produced, a coupled system was constructedconsisting of E. coli BL21(pET-28a-SAMS) and Saccharomycescerevisiae (S. cerevisiae) JM-310. In this coupled system,ATP generated from S. cerevisiae was provided to E. colifor producing a higher concentration of SAM. The SAMconcentration in the coupled system reached 1.7 g/L. SAMwas purified by a weak acid cationic exchange resin D113,and a simple and economical purification procedure forSAM isolation was achieved. SAM was confirmed byHigh Performance Liquid Chromatography-tandem MassSpectrometry analysis. Our study provides a feasible andconvenient approach to produce SAM.
Drought stress-induced changes of microRNAs in diploid and autotetraploid Paulownia tomentosa
Guoqiang Fan,Xibing Cao,Lin Cao,Minjie Deng,Zhenli Zhao,Suyan Niu,Zhe Wang,Yuanlong Wang 한국유전학회 2017 Genes & Genomics Vol.39 No.1
Drought stress adversely affects plant productivity. Growth and timber production of Paulownia trees are limited under drought stress. Changes in gene expression patterns and miRNA in different ploidy of Paulownia tomentosa have been investigated. However, the responses of P. tomentosa to drought stress at the microRNA (miRNA) level have not been reported so far. To identify miRNA candidates and their target genes involved in the drought stress response in diploid and tetraploid P. tomentosa, four small RNA and four degradome libraries from diploid and autotetraploid P. tomentosa under normal and drought stress conditions were constructed and sequenced. A total of 41 conserved and 90 novel miRNAs were identified. Among these miRNAs, 67 (26 conserved and 41 novel) and 53 (six conserved and 47 novel) were significantly differentially expressed in response to drought stress in diploid and autotetraploid P. tomentosa, respectively. Degradome analysis identified 356 candidate miRNA target genes that encoded proteins with functions that included plant defense, transcriptional regulation, and hormone metabolism. In particular, miR4 and miR156 were identified only in autotetraploid P. tomentosa under drought stress. These results will help us build a foundation for future studies of the biological functions of miRNAmediated gene regulation in P. tomentosa.