title: Down Regulation of ackA-pta Pathway in Escherichia coli BL21 (DE3): A Step Toward Optimized Recombinant Protein Expression System.
abstract: BACKGROUND
  One of the most important problems in production of recombinant protein is to attain over-expression of the target gene and high cell density. In such conditions, the secondary metabolites of bacteria become toxic for the medium and cause cells to die. One of these aforementioned metabolites is acetate, which enormously accumulated in the medium, so that both cell and protein yields are affected.
  
  
  OBJECTIVE
  To overcome this problem, several strategies applied. In this research we used antisense RNA strategy, where the transcription of phosphotransacetylase (PTA) and acetate kinase (ACK), two acetate pathway key enzymes, could be controlled, which led to reduced acetate production.
  
  
  METHODS
  In order to achieve this, recombinant plasmid harboring antisense sequences targeting both of pta and ackA was assembled, after transfecting to the cells, its effects on the cell growth and acetate accumulation in the minimal media was assessed and compared with the control, the plasmid without antisense cassette, in presence and absence of IPTG in Escherichia coli BL21 (DE3).
  
  
  RESULTS
  It was observed that the mentioned strategy partially affect the growth and amount of excreted acetate in comparison with the control. In addition it was found that high down-regulation of the acetate production pathway reduces the growth rate of E. coli BL21 (DE3).
  
  
  CONCLUSIONS
  The study principally proved the importance of this strategy in acetate excretion control.
authors.affiliation: Biochemistry Department, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IR Iran.
authors.forename: Nahid
authors.initials: N
authors.lastname: Bakhtiari
authors.affiliation: Biochemistry Department, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, IR Iran.
authors.forename: Manouchehr
authors.initials: M
authors.lastname: Mirshahi
authors.affiliation: Department of Bioscience and Biotechnology, Malek Ashtar University of Technology, Tehran, IR Iran.
authors.forename: Valiollah
authors.initials: V
authors.lastname: Babaeipour
authors.affiliation: Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, IR Iran.
authors.forename: Nader
authors.initials: N
authors.lastname: Maghsoudi
authors.affiliation: Food and Drug Organization, Ministry of Health of Iran, Tehran, IR Iran.
authors.forename: Abbas
authors.initials: A
authors.lastname: Tahzibi
keywords: Acetate; Antisense RNA; Down-Regulation; Escherichia coli