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Methods for control of flux in metabolic pathways through protease manipulation

  • US 8,916,358 B2
  • Filed: 08/31/2011
  • Issued: 12/23/2014
  • Est. Priority Date: 08/31/2010
  • Status: Active Grant
First Claim
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1. A method of producing a product of a biosynthetic pathway of interest, the method comprising:

  • (i) growing genetically modified bacterial cells that comprise nucleic acids that encode;

    (a) enzymes of the biosynthetic pathway of interest that catalyze production of the product,(b) a 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 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;

    (ii) lysing at least a portion of the bacterial cells to produce a lysate, thereby bringing the site-specific protease into contact with the targeted enzyme to cleave and inactivate or reduce the activity of the targeted enzyme;

    (iii) adding substrate to the lysate; and

    (iv) incubating the lysate and substrate, thereby producing the product of the biosynthetic pathway of interest.

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