Directed evolution of phloroglucinol synthase PhlD with increased stability for phloroglucinol production

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• 作者：Guodong Rao (1)
  Jung-Kul Lee (2)
  Huimin Zhao (1) (3)
  
• 关键词：Type III polyketide synthase ; Protein engineering ; Phloroglucinol ; Thermostability ; Continuous product removal
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2013
• 出版时间：July 2013
• 年：2013
• 卷：97
• 期：13
• 页码：5861-5867
• 全文大小：198KB
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• 作者单位：Guodong Rao (1)
  Jung-Kul Lee (2)
  Huimin Zhao (1) (3)
  
  1. Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL, 61801, USA
  2. Department of Chemical Engineering, Konkuk University, Seoul, 143-701, Korea
  3. Departments of Chemical and Biomolecular Engineering, Biochemistry, and Bioengineering, Center for Biophysics and Computational Biology, Institute for Genomic Biology, 600 South Mathews Avenue, Urbana, IL, 61801, USA
  
• ISSN：1432-0614

文摘

Phloroglucinol synthase PhlD is a type III polyketide synthase capable of directly converting three molecules of malonyl-CoA to an industrially important chemical—phloroglucinol (1, 3, 5-trihydroxylbenzene). Although this enzymatic process provides an attractive biosynthetic route to phloroglucinol, the low productivity of PhlD limits its further practical application. Here we used protein engineering coupled with in situ product removal to improve the productivity of phoroglucinol biosynthesis in recombinant Escherichia coli. Specifically, directed evolution was used to obtain a series of thermostable PhlD mutants with the best one showing over 24-fold longer half-life of thermal inactivation than the wild-type enzyme at 37?°C. When introduced into a malonyl-CoA overproducing E. coli strain, one of the mutants showed 30?% improvement in phloroglucinol productivity compared to the wild-type enzyme in a shake-flask study and the final phloroglucinol concentration reached 2.35?g/L with 25?% of theoretical yield. A continuous product extraction strategy was designed to remove the toxic phloroglucinol product from the cell media, which further increased the titer of phloroglucinol to 3.65?g/L, which is the highest phloroglucinol titer ever reported to date.