Redesigning enzymes based on adaptive evolution for optimal function in synthetic metabolic pathways.
Nature has balanced most metabolic pathways such that no one enzyme in the pathway controls the flux through that pathway. However, unnatural or nonnative, constructed metabolic pathways may have limited product flux due to unfavorable in vivo properties of one or more enzymes in the pathway. One such example is the mevalonate-based isoprenoid biosynthetic pathway that we previously reconstructed in Escherichia coli. We have used a probable mechanism of adaptive evolution to engineer the in vivo properties of two enzymes (3-hydroxy-3-methylglutaryl-CoA reductase [tHMGR] and many terpene synthases) in this pathway and thereby eliminate or minimize the bottleneck created by these inefficient or nonfunctional enzymes. Here, we demonstrate how we significantly improved the productivity (by approximately 1000 fold) of this reconstructed biosynthetic pathway using this strategy. We anticipate that this strategy will find broad applicability in the functional construction (or reconstruction) of biological pathways in heterologous hosts.
Yoshikuni
Yasuo
Y
UCSF/UCB Joint Graduate Group in Bioengineering, University of California at Berkeley, Berkeley, CA 94720, USA.
Dietrich
Jeffrey A
JA
Nowroozi
Farnaz F
FF
Babbitt
Patricia C
PC
Keasling
Jay D
JD
D019883:Alkyl and Aryl Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry; D001483:Base Sequence; D005075:Biological Evolution; D017931:DNA Primers; D008401:Gas Chromatography-Mass Spectrometry; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction
D000596:Amino Acids; D017931:DNA Primers; D006903:Hydroxymethylglutaryl CoA Reductases; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase