title: Negative feedback regulation of fatty acid production based on a malonyl-CoA sensor-actuator.
abstract: Engineering metabolic biosynthetic pathways has enabled the microbial production of many useful chemicals. However, pathway productivities and yields are often limited by metabolic imbalances. Synthetic regulatory circuits have been shown to be able to balance engineered pathways, improving titers and productivities. Here we developed a negative feedback regulatory circuit based on a malonyl-CoA-based sensor-actuator. Malonyl-CoA is biosynthesized from acetyl-CoA by the acetyl-CoA carboxylase, which is the rate-limiting step for fatty acid biosynthesis. Overexpression of acetyl-CoA carboxylase improves fatty acid production, but slows down cell growth. We have devised a malonyl-CoA sensor-actuator that controls gene expression levels based on intracellular malonyl-CoA concentrations. This sensor-actuator is used to construct a negative feedback circuit to regulate the expression of acetyl-CoA carboxylase. The negative feedback circuit is able to up-regulate acetyl-CoA carboxylase expression when the malonyl-CoA concentration is low and down-regulate acetyl-CoA carboxylase expression when excess amounts of malonyl-CoA have accumulated. We show that the regulatory circuit effectively alleviates the toxicity associated with acetyl-CoA carboxylase overexpression. When used to regulate the fatty acid pathway, the feedback circuit increases fatty acid titer and productivity by 34% and 33%, respectively.
authors.affiliation: Department of Energy, Environmental and Chemical Engineering, Washington University , 1 Brookings Drive, St. Louis, Missouri 63130, United States.
authors.forename: Di
authors.initials: D
authors.lastname: Liu
authors.forename: Yi
authors.initials: Y
authors.lastname: Xiao
authors.forename: Bradley S
authors.initials: BS
authors.lastname: Evans
authors.forename: Fuzhong
authors.initials: F
authors.lastname: Zhang
mesh_terms: D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000378:metabolism; D005227:Fatty Acids / Q000032:analysis / Q000096:biosynthesis*; D005818:Genetic Engineering; D008316:Malonyl Coenzyme A / Q000032:analysis / Q000378:metabolism*; D013058:Mass Spectrometry; D060847:Metabolic Engineering; D011401:Promoter Regions, Genetic
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D008316:Malonyl Coenzyme A; D000103:Acetyl-CoA Carboxylase
keywords: dynamic regulation; malonyl-CoA; metabolic control circuits; negative feedback; synthetic biology