Engineering of artificial plant cytochrome P450 enzymes for synthesis of isoflavones by Escherichia coli.
Engineered microbes are becoming increasingly important as recombinant production platforms. However, the nonfunctionality of membrane-bound cytochrome P450 enzymes precludes the use of industrially relevant prokaryotes such as Escherichia coli for high-level in vivo synthesis of many functional plant-derived compounds. We describe the design of a series of artificial isoflavone synthases that allowed the robust production of plant estrogen pharmaceuticals by E. coli. Through this methodology, a plant P450 construct was assembled to mimic the architecture of a self-sufficient bacterial P450 and contained tailor-made membrane recognition signals. The specific in vivo production catalyzed by one identified chimera was up to 20-fold higher than that achieved by the native enzyme expressed in a eukaryotic host and up to 10-fold higher than production by plants. This novel biological device is a strategy for the utilization of laboratory bacteria to robustly manufacture high-value plant P450 products.
Leonard
Effendi
E
Department of Chemical and Biological Engineering, The State University of New York, Buffalo, NY 14260, USA.
Koffas
Mattheos A G
MA
D000595:Amino Acid Sequence; D001483:Base Sequence; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D007529:Isoflavones / Q000096:biosynthesis* / Q000737:chemistry; D008957:Models, Genetic; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D007529:Isoflavones; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C048489:isoflavone synthase