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Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation

  • Applied genetics and molecular biotechnology
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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.

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References

  • Choi K-P, Murooka Y, Molnár I (1995a) Secretory overproduction of Arthrobacter simplex 3-ketosteroid △1-dehydrogenase by Streptomyces lividans with a multi-copy shuttle vector. Appl Microbiol Biotechnol 43(6):1044–1049

    Article  PubMed  CAS  Google Scholar 

  • Choi KP, Molnar I, Yamashita M, Murooka Y (1995b) Purification and characterization of the 3-ketosteroid-delta 1-dehydrogenase of Arthrobacter simplex produced in Streptomyces lividans. J Biochem 117(5):1043–1049

    PubMed  CAS  Google Scholar 

  • Croxatto HB (2000) Progestin implants. Steroids 65(10–11):681–685

    Article  PubMed  CAS  Google Scholar 

  • Dunny GM, Lee L, LeBlanc D (1991) Improved electroporation and cloning vector system for Gram-positive bacteria. Appl Environ Microbiol 57(4):1194–1201

    PubMed  CAS  Google Scholar 

  • Flett F, Mersinias V, Smith CP (1997) High efficiency intergeneric conjugal transfer of plasmid DNA from Escherichia coli to methyl DNA–restricting streptomycetes. FEMS Microbiol Lett 155(2):223–229

    Article  PubMed  CAS  Google Scholar 

  • He JY, Wang P, Yang YF, Xie SL (2011) Enhanced whole-cell biodehydrogenation of 11β-hydroxyl medroxyprogesterone in a biphasic system containing ionic liquid. Biotechnol Bioprocess 16(5):852–857

    Article  CAS  Google Scholar 

  • Hocknull M, Lilly M (1990) The use of free and immobilised Arthrobacter simplex in organic solvent/aqueous two-liquid-phase reactors. Appl Microbiol Biotechnol 33(2):148–153

    Article  PubMed  CAS  Google Scholar 

  • Holland AD, Rothfuss HM, Lidstrom ME (2006) Development of a defined medium supporting rapid growth for Deinococcus radiodurans and analysis of metabolic capacities. Appl Microbiol Biotechnol 72(5):1074–1082

    Article  PubMed  CAS  Google Scholar 

  • IMAI Y, SATO R (1967) Studies on the substrate interactions with P450 in drug hydroxylation by liver microsomes. J Biochem 62(2):239–249

    PubMed  CAS  Google Scholar 

  • Kaul R, Mattiasson B (1986) Extractive bioconversion in aqueous two-phase systems. Appl Microbiol Biotechnol 24(4):259–265

    Article  CAS  Google Scholar 

  • Jakoby M, Ngouoto-Nkili CE, Burkovski A (1999) Construction and application of new Corynebacterium glutamicum vectors. Biotechnol Tech 13(6):437–441

    Article  CAS  Google Scholar 

  • Liu W, Chow L, Lo C (1996) Strain improvement of A. simplex by protoplast fusion. J Ind Microbiol Biotechnol 16(4):257–260

    CAS  Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC T method. Methods 25(4):402–408

    Article  PubMed  CAS  Google Scholar 

  • Manosroi J, Saowakhon S, Manosroi A (2007) A novel one-step biotransformation of cortexolone-21-acetate to hydrocortisone acetate using Cunninghamella blakesleeana ATCC 8688a. Enzyme Microb Tech 41(3):322–325

    Article  CAS  Google Scholar 

  • Manosroi J, Sripalakit P, Manosroi A (2003) Biotransformation of chlormadinone acetate to delmadinone acetate by free and immobilized Arthrobacter simplex ATCC 6946 and Bacillus sphaericus ATCC 13805. Enzyme Microb Tech 33(2):320–325

    Article  CAS  Google Scholar 

  • Ohlson S, Larsson PO, Mosbach K (1979) Steroid transformation by living cells immobilized in calcium alginate. Appl Microbiol Biotechnol 7(2):103–110

    Article  CAS  Google Scholar 

  • Overhage J, Priefert H, Rabenhorst J, Steinbuchel A (1999) Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene. Appl Microbiol Biotechnol 52(6):820–828

    Article  PubMed  CAS  Google Scholar 

  • Prasad SB, Ramachandran K, Jayaraman G (2012) Transcription analysis of hyaluronan biosynthesis genes in Streptococcus zooepidemicus and metabolically engineered Lactococcus lactis. Appl Microbiol Biotechnol 94(6):1593–1607

    Article  PubMed  CAS  Google Scholar 

  • Saxena S (2009) Fungal biotransformation of cannabinoids: potential for new effective drugs. CURR Opin Drug Disc 12(2):305–312

    CAS  Google Scholar 

  • Spassov G, Pramatarova V, Wandrey C (1996) Conversion of corticosteroid hormones by immobilized cells of Flavobacterium dehydrogenans, Curvularia lunata and Arthrobacter simplex on ceramic carriers. Appl Microbiol Biotechnol 46(5):481–488

    Article  CAS  Google Scholar 

  • Wei M-Q, Rush CM, Norman JM, Hafner LM, Epping RJ, Timms P (1995) An improved method for the transformation of Lactobacillus strains using electroporation. J Microbiol Meth 21(1):97–109

    Article  Google Scholar 

  • Zhang L, Wang M, Shen Y, Ma Y, Luo J (2009) Improvement of steroid biotransformation with hydroxypropyl-β-cyclodextrin induced complexation. Appl Biochem Biotechnol 159(3):642–654

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by National High-tech Research and Development Program of China (863 Program) (no. 2011AA02A211) and Natural Science Foundation of China (no. 21206127).

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Authors and Affiliations

  1. Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Bioengineering, Tianjin University of Science & Technology, Tianjin, 300457, People’s Republic of China

    Huitu Zhang, Yao Tian, Jianling Wang, Haikuan Wang, Shuhong Mao, Xiaoguang Liu, Chunxia Wang, Songtao Bie & Fuping Lu

  2. School of Life Sciences and Technology, Henan cultivating Key laboratory of Heredity disease targeted Molecular drugs, Xinxiang Medical University, Xinxiang, 453003, People’s Republic of China

    Yonghai Li

  3. Industrial Microbiology Laboratory, College of Biotechnology, Tianjin University of Science & Technology, No. 29, 13 Main Street, Tianjin Economic and Technological Development Zone, Tianjin, 300457, People’s Republic of China

    Fuping Lu

Authors
  1. Huitu Zhang
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  2. Yao Tian
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  3. Jianling Wang
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  4. Yonghai Li
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  5. Haikuan Wang
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  6. Shuhong Mao
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  7. Xiaoguang Liu
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  8. Chunxia Wang
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  9. Songtao Bie
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  10. Fuping Lu
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Corresponding author

Correspondence to Fuping Lu.

Additional information

H. Zhang and Y. Tian contributed equally to this work

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Zhang, H., Tian, Y., Wang, J. et al. Construction of engineered Arthrobacter simplex with improved performance for cortisone acetate biotransformation. Appl Microbiol Biotechnol 97, 9503–9514 (2013). https://doi.org/10.1007/s00253-013-5172-7

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  • DOI: https://doi.org/10.1007/s00253-013-5172-7

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