A functional protein chip for pathway optimization and in vitro metabolic engineering.
Pathway optimization is difficult to achieve owing to complex, nonlinear, and largely unknown interactions of enzymes, regulators, and metabolites. We report a pathway reconstruction using RNA display-derived messenger RNA-enzyme fusion molecules. These chimeras are immobilized by hybridization of their messenger RNA end with homologous capture DNA spotted on a substrate surface. Enzymes thus immobilized retain activity proportional to the amount of capture DNA, allowing modulation of the relative activity of pathway enzymes. Entire pathways can thus be reconstructed and optimized in vitro from genomic information. We provide concept validation with the sequential reactions catalyzed by luciferase and nucleoside diphosphate kinase and further illustrate this method with the optimization of the five-step pathway for trehalose synthesis.
Jung
Gyoo Yeol
GY
Department of Chemical Engineering, Massachusetts Institute of Technology, Room 56-469, Cambridge, MA 02139, USA.
Stephanopoulos
Gregory
G
D002384:Catalysis; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D020869:Gene Expression Profiling; D005818:Genetic Engineering; D005947:Glucose / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D006593:Hexokinase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008660:Metabolism; D009693:Nucleic Acid Hybridization; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010733:Phosphoglucomutase / Q000235:genetics / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D040081:Protein Array Analysis; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D014199:Trehalose / Q000096:biosynthesis*; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000235:genetics / Q000378:metabolism
D004800:Enzymes, Immobilized; D012333:RNA, Messenger; D004247:DNA; D014199:Trehalose; D008156:Luciferases; D005964:Glucosyltransferases; C023180:trehalose-6-phosphate synthase; D006593:Hexokinase; D009701:Nucleoside-Diphosphate Kinase; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; C023178:trehalose-phosphatase; D010744:Phosphoric Monoester Hydrolases; D010733:Phosphoglucomutase; D005947:Glucose