title: Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation.
abstract: Arthrobacter simplex 156 is a microorganism that is used for steroid drug biotransformation of cortisone acetate (CA) to prednisone acetate (PA). The enzyme 3-ketosteroid-△(1)-dehydrogenase encoded by the ksdD gene plays an important role in the bioconversion process. To further improve the biotransformation efficiencies of the industrial strain, a genetic manipulation system for A. simplex 156 was developed. Additional copies of the ksdD gene under the control of the cat promoter (from pXMJ19) were transferred into the strain A. simplex 156 and integrated into the 16S rDNA sites, yielding a series of recombinant strains. One of these recombinant strains, designated A. simplex M158, exhibited superior properties for CA biotransformation. At the substrate concentration of 83.6 g/l, the highest PA production of the recombinant strain reached 66.7 g/l, which is approximately 32.9 % higher than that of wild-type strains, and the incubation time for CA to PA bioconversion was reduced by 20 h. Southern blotting analysis of the recombinant strain indicated two copies of deregulated ksdD genes were integrated into the 16S rDNA sites, which means two of five 16S rRNA operons were insertionally disrupted in the recombinant strain. However, the disruption of the two 16S rRNA operons did not affect the growth rate of the recombinant strain, which survived and thrived under desired conditions. In addition, the new strain was genetically stable for more than 100 generations without the use of antibiotics for selection. These superior characteristics make the new strain more suitable than the wild-type strain for PA production.
authors.affiliation: Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, Tianjin, 300457, People's Republic of China.
authors.forename: Huitu
authors.initials: H
authors.lastname: Zhang
authors.forename: Yao
authors.initials: Y
authors.lastname: Tian
authors.forename: Jianling
authors.initials: J
authors.lastname: Wang
authors.forename: Yonghai
authors.initials: Y
authors.lastname: Li
authors.forename: Haikuan
authors.initials: H
authors.lastname: Wang
authors.forename: Shuhong
authors.initials: S
authors.lastname: Mao
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Liu
authors.forename: Chunxia
authors.initials: C
authors.lastname: Wang
authors.forename: Songtao
authors.initials: S
authors.lastname: Bie
authors.forename: Fuping
authors.initials: F
authors.lastname: Lu
mesh_terms: D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D001711:Biotransformation; D003348:Cortisone / Q000031:analogs & derivatives* / Q000378:metabolism; D018628:Gene Dosage; D015870:Gene Expression; D042822:Genomic Instability; D060847:Metabolic Engineering; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011241:Prednisone / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010088:Oxidoreductases; C022656:3-oxosteroid delta(1) dehydrogenase; D003348:Cortisone; D011241:Prednisone