title: Down-regulation of acetate pathway through antisense strategy in Escherichia coli: improved foreign protein production.
abstract: A problem with the use of Escherichia coli to produce foreign proteins is that although endogenously produced acetate is physiologically indispensable, it inhibits protein expression. Here we firstly employed an antisense RNA strategy as an elaborate metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, phosphotransacetylase (PTA) and acetate kinase (ACK). Three recombinant plasmids containing antisense genes targeting either or both of pta and ackA were constructed, and their effects on the acetate pathway and foreign protein productivity compared to control plasmid without any antisense genes were determined in E. coli BL21. Green fluorescent protein (GFP) was employed as a model foreign protein, and timing of antisense expression was controlled by using the intrinsic ackA promoter. We found that the antisense method partially reduced mRNA levels of target enzyme genes and, over time, lowered the concentration of acetate in culture media in all antisense-regulated strains. Notably, total production of GFP was enhanced 1.6- to 2.1-fold in antisense-regulated strains, even though the degree of acetate reduction was not significantly large. It was revealed that the acetate pathway has more critical roles in cellular physiology than expected in the previous reports. When the scale of culture was increased, enhancement of protein production became larger, demonstrating that this antisense strategy can be successfully applied to practical large-scale protein production processes.
authors.affiliation: Department of Chemical Engineering, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Korea.
authors.forename: Jaoon Y H
authors.initials: JY
authors.lastname: Kim
authors.forename: Hyung Joon
authors.initials: HJ
authors.lastname: Cha
mesh_terms: D000085:Acetates / Q000378:metabolism*; D001709:Biotechnology; D015153:Blotting, Western; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005947:Glucose / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D016372:RNA, Antisense / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic
chemical_list: D000085:Acetates; D008164:Luminescent Proteins; D011506:Proteins; D016372:RNA, Antisense; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D005947:Glucose