US 12,031,170 B2
System for improved production titers in fermentations
Peter Rugbjerg, Copenhagen (DK); Kira Sarup-Lytzen, Frederiksberg (DK); and Morten Sommer, Virum (DK)
Assigned to DANMARKS TEKNISKE UNIVERSITET, Lyngby (DK)
Filed by DANMARKS TEKNISKE UNIVERSITET, Lyngby (DK)
Filed on May 13, 2022, as Appl. No. 17/744,300.
Application 17/744,300 is a continuation of application No. 15/763,257, granted, now 11,352,650, previously published as PCT/EP2016/073132, filed on Sep. 23, 2016.
Claims priority of application No. 15187150 (EP), filed on Sep. 28, 2015.
Prior Publication US 2022/0348971 A1, Nov. 3, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 7/42 (2006.01); C12N 1/21 (2006.01); C12N 9/16 (2006.01); C12N 15/10 (2006.01); C12N 15/67 (2006.01); C12N 15/72 (2006.01); C12P 1/02 (2006.01); C12P 1/04 (2006.01); C12P 5/02 (2006.01); C12P 7/40 (2006.01); C12P 7/64 (2022.01); C12P 21/02 (2006.01)
CPC C12P 7/42 (2013.01) [C12N 9/16 (2013.01); C12N 15/1086 (2013.01); C12N 15/67 (2013.01); C12N 15/72 (2013.01); C12P 1/02 (2013.01); C12P 1/04 (2013.01); C12P 5/026 (2013.01); C12P 7/40 (2013.01); C12P 7/64 (2013.01); C12P 21/02 (2013.01)] 13 Claims
 
1. A method for producing a biosynthetic metabolite comprising the steps of:
a) providing a pre-seed microbial cell culture of genetically modified microbial cells wherein each cell comprises:
i. a first nucleic acid molecule wherein said first nucleic acid molecule is transcribed and/or translated to yield a biosensor and said biosensor binds to said biosynthetic metabolite to form a complex; and
ii. a second nucleic acid molecule comprising a coding sequence encoding an essential protein wherein expression alone of said coding sequence is essential for cell growth and/or survival in a nutrient rich culture medium, wherein:
(1) said cell is not viable in a nutrient rich culture medium when said coding sequence encoding said essential protein is knocked-out;
(2) said essential protein encoded by said second nucleic acid molecule is expressed when said second nucleic acid molecule is induced by said complex when said biosensor and said biosynthetic metabolite form said complex; and
(3) growth of said cell is arrested by an absence of complex formation;
(4) arrest of growth does not depend on whether an externally supplied compound is present;
b) introducing the pre-seed microbial cell culture into a nutrient rich cultivation medium comprising a substrate for production of said biosynthetic metabolite,
c) cultivating the cells in said nutrient rich culture medium, wherein the cells of the cell culture produce said biosynthetic metabolite and undergo at least 40, 45, 50, 60, 70 or 150 generations of cell multiplication, and,
d) recovering the biosynthetic metabolite produced by said culture, wherein the productive life-time of said culture, is prolonged over at least 40, 45, 50, 60, 70 or 150 generations of cell multiplication by preventing proliferation of non-producing spontaneous mutants arising during said generations of cell multiplication.