Directed evolution of phloroglucinol synthase PhlD with increased stability for phloroglucinol production.
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.
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Guodong
G
Rao
Jung-Kul
JK
Lee
Huimin
H
Zhao
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D008316:Malonyl Coenzyme A / Q000378:metabolism; D010696:Phloroglucinol / Q000378:metabolism*; D015202:Protein Engineering; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; C096183:PhlD protein, Pseudomonas; D011994:Recombinant Proteins; D008316:Malonyl Coenzyme A; D010696:Phloroglucinol