title: [Expression of glycerol dehydrogenase gene in Escherichia coli by codon optimization].
abstract: OBJECTIVE
  To improve expression level of glycerol dehydrogenase gene gldA in Escherichia coli by means of codon optimization.
  
  
  METHODS
  For immediately downstream region of initiation codon in gldA, we designed optimized sequence by choosing higher AT-content synonymous, in order that this region's AT-content was increased without changing the corresponding amino acids. Then we had wild gene gldA-WT site-directed mutagenesis depending on mega-primers PCR, so that physically optimized gene gldA-4 was acquired. We cloned gldA-4 into pET-32a(+) to construct expression plasmid pET-gldA4, which was transformed into Escherichia coli BL21 (DE3) for gaining engineering bacteria E. coli-4, by contrast engineering bacteria involved gldA-WT named E. coli-WT. After E. coli-4 and E. coli-WT were fermented in shake flasks,we measured enzyme activities of expression products with glycerol as substrate.
  
  
  RESULTS
  Four gldA-4's bases in the second, fifth and sixth codon were different with gldA-WT, so AT-content of the optimized gene was up to 80.0% higher than the wild gene's 53.3%. Furthermore, enzyme activity of E. coli-4's crude extract was 191.3 U/mL more three times than E. coli-WT's 48.3 U/mL.
  
  
  CONCLUSIONS
  This optimization scheme was quick and easy, but indeed increased dehydrogenase's activity. It possible becomes a universal method to improve heterogenous expression level of target genes.
authors.affiliation: Key Laboratory for Industrial Biotechnology of Fujian Higher Education (Huaqiao University), Xiamen 361021, China. tang3433@sina.com
authors.forename: Longpan
authors.initials: L
authors.lastname: Tang
authors.forename: Jincong
authors.initials: J
authors.lastname: Yu
authors.forename: Danfeng
authors.initials: D
authors.lastname: Dai
authors.forename: Baishan
authors.initials: B
authors.lastname: Fang
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003062:Codon; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005990:Glycerol / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D015202:Protein Engineering; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D013401:Sugar Alcohol Dehydrogenases; C020672:glycerol dehydrogenase; D005990:Glycerol