New major facilitator superfamily (mfs) protein (fred) in hmo production

Abstract

The present invention relates to the field of recombinant production of biological molecules in genetically modified cells. More particularly, it relates to a method for recombinant production of human milk oligosaccharides (HMO) using a genetically modified cell expressing a protein of the major facilitator superfamily (MFS), the protein expressed being Fred.

1 . A genetically modified cell capable of producing one or more Human Milk Oligosaccharides (HMOs), wherein said genetically modified cell comprises a heterologous nucleic acid sequence encoding a major facilitator superfamily (MFS) polypeptide shown in SEQ ID NO: 1, or a functional homologue thereof which amino acid sequence is more than 95.4% identical to SEQ ID NO: 1. 
     
     
         2 . The genetically modified cell according to  claim 1 , wherein said functional homologue is at least 96% identical to SEQ ID NO: 1. 
     
     
         3 . The genetically modified cell according to  claim 1 , wherein the cell further comprises a nucleic acid sequence comprising a regulatory element for the regulation of the expression of the heterologous nucleic acid sequence. 
     
     
         4 . The genetically modified cell according to  claim 3 , wherein the regulatory element regulates the expression of the MFS polypeptide shown in SEQ ID NO: 1, or a functional homologue thereof which amino acid sequence is more than 95.4% identical to SEQ ID NO: 1. 
     
     
         5 . The genetically modified cell according to  claim 3  wherein the regulatory element is selected from the group consisting of PglpF, PglpF_SD4 and PglpF_SD7. 
     
     
         6 . The genetically modified cell according to  claim 1 , wherein the genetically modified cell is  Escherichia coli.    
     
     
         7 . The genetically modified cell according to  claim 1 , wherein the one or more HMOs are selected from the group consisting of 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL) and Lacto-N-tetraose (LNT); or a mixture thereof. 
     
     
         8 . A nucleic acid construct comprising a nucleic acid sequence encoding an MFS polypeptide according to SEQ ID NO: 1, or a functional homologue thereof, having more than 95.4% sequence identity to SEQ ID NO: 1, wherein the nucleic acid sequence encoding the MFS polypeptide has at least 70% sequence identity to SEQ ID NO: 2. 
     
     
         9 . The nucleic acid construct according to  claim 8 , wherein the construct further comprises a nucleic acid sequence comprising a regulatory element. 
     
     
         10 . The nucleic acid construct according to  claim 9 , wherein the regulatory element regulates the expression of the nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 2. 
     
     
         11 . The nucleic acid construct according to  claim 9 , wherein the regulatory element is selected from the group consisting of PglpF, PglpF_SD4 and PglpF_SD7. 
     
     
         12 . A method for the biosynthetic production of one or more Human Milk Oligosaccharides (HMOs), the method comprising the steps of:
 (i) providing a genetically modified cell according to  claim 1 ;   (ii) culturing the genetically modified cell according to (i) in a suitable cell culture medium to express said polypeptide capable of efflux sugar transportation and produce one or more Human Milk Oligosaccharides (HMOs) and;   (iii) harvesting one or more HMOs produced in step (ii).   
     
     
         13 .- 14 . (canceled) 
     
     
         15 . The method according to  claim 12 , wherein the one or more HMOs are selected from the group consisting of 2′-fucosyllactose (2′-FL), 3-fucosyllactose (3-FL), difucosyllactose (DFL) and Lacto-N-tetraose (LNT); or a mixture thereof. 
     
     
         16 . The method according to  claim 15 , wherein one or more HMOs comprises 2′-FL or 3-FL. 
     
     
         17 . The genetically modified cell according to  claim 1 , wherein the one or more HMOs are selected from the group consisting of LNT-2, LNT, and LNFP I; or a mixture thereof. 
     
     
         18 . The genetically modified cell according to  claim 17 , wherein the one or more HMOs comprises LNT. 
     
     
         19 . The method according to  claim 12 , wherein the one or more HMOs are selected from the group consisting of LNT-2, LNT, and LNFP I; or a mixture thereof. 
     
     
         20 . The method according to  claim 19 , wherein the one or more HMOs comprises LNT. 
     
     
         21 . The nucleic acid construct according to  claim 4 , wherein the regulatory element is selected from the group consisting of PglpF, PglpF_SD4 and PglpF_SD7. 
     
     
         22 . A method for the biosynthetic production of one or more Human Milk Oligosaccharides (HMOs), the method comprising the steps of:
 (iv) providing a genetically modified cell according to  claim 21 ;   (v) culturing the genetically modified cell according to (i) in a suitable cell culture medium to express said polypeptide capable of efflux sugar transportation and produce one or more Human Milk Oligosaccharides (HMOs) and;   (vi) harvesting the one or more HMOs produced in step (ii).