Directed evolution of phloroglucinol synthase PhlD with increased stability for phloroglucinol production
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APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Directed evolution of phloroglucinol synthase PhlD with increased stability for phloroglucinol production Guodong Rao & Jung-Kul Lee & Huimin Zhao
Received: 2 September 2012 / Revised: 21 December 2012 / Accepted: 12 January 2013 / Published online: 29 January 2013 # Springer-Verlag Berlin Heidelberg 2013
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 Electronic supplementary material The online version of this article (doi:10.1007/s00253-013-4713-4) contains supplementary material, which is available to authorized users. G. Rao : H. Zhao Department of Chemistry, University of Illinois at UrbanaChampaign, 600 South Mathews Avenue, Urbana, IL 61801, USA J.-K. Lee Department of Chemical Engineering, Konkuk University, Seoul 143-701, Korea H. Zhao (*) 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 e-mail: [email protected]
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. Keywords Type III polyketide synthase . Protein engineering . Phloroglucinol . Thermostability . Continuous product removal
Introduction Phloroglucinol synthase PhlD is a type III polyketide synthase which can condense three molecules of malonyl-CoA into phloroglucinol (Fig. 1; Achkar et al. 2005). Phloroglucinol is a useful chemical in copying and textile dying processes and also a versatile precursor for the production of pharmaceuticals, food additives and cosmetics (Mitchell et al. 2002). However, the low productivity of PhlD impeded its application as a biosynthetic tool for large-scale synthesis of phloroglucinol. In this work, we report a successful example of improving the productivity of phloroglucinol biosynthesis by using directed evolution, a powerful tool for i
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