US 12,338,473 B2
Fermentative production of oligosaccharides by total fermentation utilizing a mixed feedstock
Stefan Jennewein, Bad Honnef (DE); Dirk Wartenberg, Gau-Algesheim (DE); and Katja Parschat, Bonn (DE)
Assigned to Chr. Hansen A/S, Hoersholm (DK)
Filed by Chr. Hansen A/S, Hoersholm (DE)
Filed on Jun. 8, 2023, as Appl. No. 18/207,453.
Application 18/207,453 is a division of application No. 17/273,447, granted, now 11,713,475, previously published as PCT/EP2019/073363, filed on Sep. 2, 2019.
Claims priority of application No. 18192898 (EP), filed on Sep. 6, 2018.
Prior Publication US 2023/0399669 A1, Dec. 14, 2023
Int. Cl. C12P 19/12 (2006.01); C12N 1/20 (2006.01); C12N 9/10 (2006.01); C12N 9/12 (2006.01); C12N 9/16 (2006.01); C12N 9/90 (2006.01); C12P 19/04 (2006.01); C12P 19/18 (2006.01)
CPC C12P 19/12 (2013.01) [C12N 1/20 (2013.01); C12N 9/1051 (2013.01); C12N 9/1205 (2013.01); C12N 9/16 (2013.01); C12N 9/90 (2013.01); C12P 19/04 (2013.01); C12P 19/18 (2013.01); C12N 2500/34 (2013.01); C12Y 204/01038 (2013.01); C12Y 207/01004 (2013.01); C12Y 207/01011 (2013.01); C12Y 207/01056 (2013.01); C12Y 207/07009 (2013.01); C12Y 301/03011 (2013.01); C12Y 501/03002 (2013.01); C12Y 504/02002 (2013.01)] 16 Claims
 
1. A genetically engineered microbial cell for the production of lactose or an oligosaccharide of interest which comprises a galactose-β1,4-glucose moiety at its reducing end, wherein said microbial cell is an Escheria coli cell and comprises:
at least one glucose transporter for translocating glucose from the culture medium into the microbial cell's cytoplasm such that free glucose becomes available for intracellular biosynthesis of lactose, wherein the at least one glucose transporter is selected from the group consisting of glucose facilitated diffusion proteins and glucose translocating permeases;
an UDP-galactose biosynthesis pathway comprising the activities of at least a phosphoglucomutase, an UTP-glucose-1-phosphate-uridyltransferase and an UDP-glucose-4-epimerase activity;
at least one β-1,4-galactosyltransferase being able to galactosylate free glucose to intracellularly produce lactose, wherein the β-1,4-galactosyltransferase is encoded by a gene selected from Neisseria meningitidis lgtB, Aggregatibacter aphrophilus lex-1, Pasteurella multocida galTpm 1141; and
wherein the glucokinase gene(s) of the microbial cell have been deleted or functionally inactivated.