title: Towards area-based in vitro metabolic engineering: assembly of Pfs enzyme onto patterned microfabricated chips.
abstract: We report an approach for spatially selective assembly of an enzyme onto selected patterns of microfabricated chips. Our approach is based on electrodeposition of the aminopolysaccharide chitosan onto selected electrode patterns and covalent conjugation of a target enzyme to chitosan upon biochemical activation of a genetically fused "pro-tag." We report assembly of S-adenosylhomocysteine nucleosidase (Pfs) fused with a C-terminal pentatyrosine pro-tag. Pfs is a member of the bacterial autoinducer-2 biosynthesis pathway, catalyzing the irreversible cleavage of S-adenosylhomocysteine. The assembled Pfs retains its catalytic activity and structure, as demonstrated by retained antibody recognition. Assembly is controlled by the electrode area, resulting in reproducible rates of catalytic conversion for a given area, and thus allowing for area-based manipulation of catalysis and small molecule biosynthesis. Our approach enables optimization of small molecule biosynthesis in 1-step as well as multistep enzymatic reactions, including entire metabolic pathways, and we envision a wide variety of potential applications.
authors.affiliation: Dept of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, USA.
authors.forename: Angela T
authors.initials: AT
authors.lastname: Lewandowski
authors.forename: William E
authors.initials: WE
authors.lastname: Bentley
authors.forename: Hyunmin
authors.initials: H
authors.lastname: Yi
authors.forename: Gary W
authors.initials: GW
authors.lastname: Rubloff
authors.forename: Gregory F
authors.initials: GF
authors.lastname: Payne
authors.forename: Reza
authors.initials: R
authors.lastname: Ghodssi
mesh_terms: D055162:Biocatalysis; D019149:Bioreactors; D048271:Chitosan / Q000737:chemistry; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D044085:Microfluidics / Q000379:methods*; D009699:N-Glycosyl Hydrolases / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors
chemical_list: D048271:Chitosan; D012435:S-Adenosylhomocysteine; D009699:N-Glycosyl Hydrolases; C019606:adenosylhomocysteine nucleosidase