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

Guodong Rao, Jung Kul Lee, Huimin Zhao

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)5861-5867
Number of pages7
JournalApplied Microbiology and Biotechnology
Volume97
Issue number13
DOIs
StatePublished - Jul 1 2013

Keywords

  • Continuous product removal
  • Phloroglucinol
  • Protein engineering
  • Thermostability
  • Type III polyketide synthase

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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