Recombinant cellular iysate system for producing a product of interest
AssigneesThe Board of Trustees of the Leland Stanford Junior University, GreenLight Biosciences, Inc.
InventorJames R. Swartz
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Abstract
The embodiments described herein pertain to cells, and methods for preparing cells, that can be used as biocatalysts by altering enzymes that compete for a substrate or product of a pathway of interest such that the targeted enzyme is sensitive to a site-specific protease, which protease is expressed but relocated in the cell to a site where it is not in contact with the targeted enzyme in the intact cell. Upon cell lysis, the protease contacts the target enzyme, which is then inactivated by protease cleavage.
Claims18
A bacterial cell lysate obtained from bacterial cells genetically modified to comprise:
- nucleic acids that encode
- (a) enzymes of a biosynthetic pathway of interest that catalyze production of a product,
- (b) a recombinant targeted enzyme that competes for substrates or cofactors with an enzyme in the biosynthetic pathway of interest, decreases the overall yield and/or the rate of production of the product, and includes a site-specific protease recognition sequence, and
- (c) a recombinant site-specific protease that cleaves the site-specific protease recognition sequence of the targeted enzyme and is genetically modified to include a periplasmic targeting sequence,
- wherein the bacterial cell lysate comprises a substrate and supports the production of the product of interest from said substrate.
Dependencies are inferred from the claim text.