title: Phenotypic knockouts of selected metabolic pathways by targeting enzymes with camel-derived nanobodies (V(HH)s).
abstract: Surveying the dynamics of metabolic networks of Gram-negative bacteria often requires the conditional shutdown of enzymatic activities once the corresponding proteins have been produced. We show that given biochemical functions can be entirely suppressed in vivo with camel antibodies (VHHs, nanobodies) that target active sites of cognate enzymes expressed in the cytoplasm. As a proof of principle, we raised VHHs against 2,5-dihydroxypyridine dioxygenase (NicX) of Pseudomonas putida, involved in nicotinic acid metabolism. Once fused to a thioredoxin domain, the corresponding nanobodies inhibited the enzyme both in Escherichia coli and in P. putida cells, which then accumulated the metabolic substrate of NicX. VHHs were further engineered to track the antigen in vivo by C-terminal fusion to a fluorescent protein. Conditional expression of the resulting VHHs allows simultaneously to track and target proteins of interest and enables the design of transient phenotypes without mutating the genetic complement of the bacteria under study.
authors.affiliation: Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain; Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom.
authors.forename: José I
authors.initials: JI
authors.lastname: Jiménez
authors.affiliation: Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain.
authors.forename: Sofía
authors.initials: S
authors.lastname: Fraile
authors.affiliation: Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain.
authors.forename: Olga
authors.initials: O
authors.lastname: Zafra
authors.affiliation: Systems Biology Program, Centro Nacional de Biotecnología-CSIC, Campus de Cantoblanco, Madrid 28049, Spain. Electronic address: vdlorenzo@cnb.csic.es.
authors.forename: Víctor
authors.initials: V
authors.lastname: de Lorenzo
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D002162:Camelus; D049308:Dioxygenases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D061905:Single-Domain Antibodies / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D061905:Single-Domain Antibodies; D049308:Dioxygenases
keywords: Nanobodies; Networks; NicX; Nicotinic acid; Pseudomonas putida; Systems Biology; V(HH)