WO2022243307A1 - Cell factories for lnt-ii production - Google Patents

Cell factories for lnt-ii production Download PDF

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WO2022243307A1
WO2022243307A1 PCT/EP2022/063310 EP2022063310W WO2022243307A1 WO 2022243307 A1 WO2022243307 A1 WO 2022243307A1 EP 2022063310 W EP2022063310 W EP 2022063310W WO 2022243307 A1 WO2022243307 A1 WO 2022243307A1
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seq
pglpf
70utr
pmglb
cell
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Manos PAPADAKIS
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Dsm Ip Assets B.V.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01149—N-Acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase (2.4.1.149)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This disclosure relates to a method of producing human milk oligosaccharides (HMOs) with high titres, wherein the HMO is LNT-II.
  • HMOs human milk oligosaccharides
  • LNT-II human milk oligosaccharides
  • Several genetic engineering approaches have been applied to change the abundance of the HMO produced by cells that express a heterologous b-1 ,3-N-acetyl- glucosaminyltransferase.
  • the strain engineering strategies to achieve this goal depend on the b-1 ,3-N- acetyl-glucosaminyltransferase introduced in the host.
  • the disclosure highlights several strain engineering strategies to enable the formation of LNT-II at high titres in the E. coli DH1 K12 host. Specifically, these strategies deal with different aspects for the design of an efficient LNT-II production system, which include the introduction and optimization of the pathway enabling LNT-II biosynthesis, the modification and/
  • Human milk represents a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements.
  • the by far most predominant fraction is represented by carbohydrates, which can be further divided into lactose and more complex oligosaccharides (Human Milk Oligosaccharides, HMO).
  • lactose is used as an energy source
  • the complex oligosaccharides are not metabolized by the infant.
  • the fraction of complex oligosaccharides accounts for up to 1/10 of the total carbohydrate fraction and consists of probably more than 150 different oligosaccharides.
  • the occurrence and concentration of these complex oligosaccharides are specific to humans and thus cannot be found in large quantities in the milk of other mammals, like for example domesticated dairy animals.
  • HMOs have become of great interest in the last decade, due to the discovery of their important functionality in human development. Besides their prebiotic properties, HMOs have been linked to additional positive effects, which expands their field of application.
  • the health benefits of HMOs have enabled their approval for use in foods, such as infant formulas and foods, and for consumer health products.
  • Fermentation based processes have traditionally been developed for individual HMOs such as 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto- N-neotetraose, 3'-sialyllactose and 6'-sialyllactose.
  • Fermentation based processes typically utilize genetically engineered bacterial strains, such as recombinant Escherichia coli (E.
  • CN 111979168 addresses the production of lactoyl-N-triose II (LNT-II) by use of a genetically engineered E. coli host, wherein genetic manipulations to the N-acetylglucosamine (GlcNAc) glycolysis pathway resulted in an increased yield of LNT-II
  • This disclosure relates to multiple genetic engineering approaches applied to enhance the formation of the human milk oligosaccharide (HMO) lacto-N-triose II (LNT-II).
  • HMO human milk oligosaccharide
  • LNT-II lacto-N-triose II
  • LNT-II lacto-N-triose II
  • HD0466 GenBank ID: WP_010944479.1
  • expression of a single copy enhanced the production of LNT-II over cells expressing the b-1 ,3-N- acetyl-glucosaminyltransferase LgtA (GenBank ID: WP_033911473.1).
  • combination of the b-1 ,3-N-acetyl-glucosaminyltransferase HD0466 with a second b-1 ,3-N- acetyl-glucosaminyltransferase LgtA or PmnagT was capable of further enhancing the LNT-II titer.
  • combining expression of the b-1 ,3-N-acetyl-glucosaminyltransferases HD0466 and LgtA with overexpression of the E. coli native lactose permease also enhanced the LNT-II titer.
  • strain engineering strategies to achieve this goal comprise the manipulation of the following genetic traits of the HMO producer cell:
  • HMOs primarily LNT-II
  • the present disclosure relates to a method for the production of LNT-II, the method comprising the steps of: a. providing a genetically engineered cell capable of producing an HMO, wherein said cell comprises i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i); and b. culturing the cell according to (a) in a suitable cell culture medium; and c. harvesting the HMO(s) produced in step (b).
  • a. providing a genetically engineered cell capable of producing an HMO wherein said cell comprises i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD
  • the capability of the cell to take up lactose is advantageous, when synthesizing LNT- II from lactose, it is therefore preferred that said cell is able to import lactose into the cell.
  • the present disclosure relates to a genetically engineered cell comprising a) a nucleic acid sequence according to SEQ ID NO: 4 or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 4, encoding a heterologous b-1 ,3-N-acetyl- glucosaminyl-transferase, and b) a native or heterologous regulatory element for controlling the expression of a).
  • the disclosure relates to a nucleic acid construct comprising a nucleic acid sequence encoding i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i).
  • the disclosure relates to use of a genetically engineered cell, or a nucleic acid construct according to the present disclosure to produce LNT-II.
  • HMOs primarily LNT-II
  • present disclosure first of all pinpoints and compares different b-1 ,3-N-acetyl- glucosaminyl transferases that can efficiently convert lactose to LNT-II and indicates how their expression can be balanced for reaching optimal LNT-II production.
  • the E. coli DH1 K12 host can be engineered to form LNT-II at high levels by introducing the b-1 ,3-N-acetyl-glucosaminyl transferase HD0466 from Haemophilus ducreyi (SEQ ID NO: 1 , or functional homologues thereof), which can be further combined with the expression of the b-1 ,3-N-acetyl-glucosaminyl transferase LgtA from Neisseria meningitidis (SEQ ID NO: 2, , or functional homologues thereof) or PmnagT from Pasteurella multocida (SEQ ID NO: 28, , or functional homologues thereof), and the over-expression of the native E. coli lacY gene to reach even higher LNT-II levels.
  • the enhanced amounts of LNT-II gives a more sustainable manufacturing process; valuable HMOs are not discarded during the purification process and the conversion from carbon source to HMO product in fermentation is thus done at a higher overall yield.
  • Example 1 it is demonstrated how different GlcNAc transferases(GlcNAcTs) can be beneficially expressed at varied genomic copy numbers in the genetic background of E. coli K12 cells for high-level LNT-II production.
  • the Example reveals the HD0466 enzyme as a novel enzyme for the in vivo production of LNT-II.
  • the method for the production of LNT-II comprises the steps of: a.
  • a genetically engineered yeast or bacterial cell capable of producing an HMO wherein said cell comprises i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i); and b. culturing the cell according to (a) in a suitable cell culture medium; and c. harvesting the HMO(s) produced in step (b).
  • a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an
  • said cell is able to import lactose into the cell.
  • the present disclosure shows marked gains in LNT-II titers when different b-1 ,3-N-acetyl- glucosaminyl transferases are simultaneously expressed in the same production cell compared to LNT-II producers expressing a single transferase.
  • the most marked increase in LNT-II titers could be achieved by combined expression of the HD0466 and LgtA enzymes.
  • the b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof is expressed together with a second b-1 ,3-N-acetyl- glucosaminyl transferase.
  • Example 2 it is demonstrated that the pairwise expression of two different GlcNAcTs can be a more efficient approach in converting E. coli K12 cells to an efficient LNT-II cell factory than merely expressing a single GlcNAcT.
  • the method for the production of LNT-II comprises the steps of: a. providing a genetically engineered yeast or bacterial cell capable of producing an HMO, wherein said cell comprises i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i); and iii) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 [LgtA], or a functional homologue thereof having an amino acid sequence which is at
  • said cell is able to import lactose into the cell
  • the method comprises the steps of: a. providing a genetically engineered bacterial or yeast cell capable of producing an HMO, wherein said cell comprises: i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i); and iii) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 28 [PmnagT], or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85
  • said cell is able to import lactose into the cell.
  • the import of the lactose is preferably balanced with the level of b-1 ,3-N-acetyl-glucosaminyl-transferase activity.
  • the lactose is imported into the cell via the cells native lactose importer, such as lactose permease.
  • no additional lactose permease activity is added to the cell.
  • the expression level of b-1 ,3-N-acetyl-glucosaminyl-transferases is controlled such that it is higher than the expression level of a lactose permease.
  • a rational genetic engineering program includes the following major focus areas: a) the introduction one or more highly active b-1 ,3-N-acetyl-glucosaminyl transferase for the conversion of the externally added lactose to LNT- II and b) the enhancement of lactose import into the cells.
  • the genetic manipulation of genes involved in these cellular procedures could theoretically provide marked product yield gains.
  • the over-expression of the lacY gene in the cells expressing more than one b-1 ,3-N-acetyl- glucosaminyl transferases, namely HD0466 and LgtA can be advantageous for high-level LNT-II production, as seen in Example 3, the over-expression of the gene encoding the native lactose permease LacY can be beneficial for example when specific GlcNAcTs, namely HD0466 and LgtA, are coexpressed in the same strain.
  • the method comprises the steps of: a. providing a genetically engineered bacterial or yeast cell capable of producing an HMO, wherein said cell comprises i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i); and iii) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 [LgtA], or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85
  • the present disclosure demonstrates the superior activity of another enzyme, namely HD0466 from Haemophilus ducreyi, towards LNT-II synthesis. Moreover, it is here shown that the combinations of different enzymes of the same type can be a highly beneficial approach for achieving high LNT-II titers.
  • the genetically engineered cells comprise all the required enzymes to facilitate the production LNT-II.
  • One of these enzymes may for example be i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 %, such as at least 99 % or such as at least 99.9 % identical to SEQ ID NO: 1 .
  • the above enzyme can be exchanged or supplemented by others with similar functionality.
  • SEQ ID NO: 1 which can be supplemented with SEQ ID NO: 2 and/or SEQ ID NO: 28.
  • SEQ ID NO: 2 or SEQ ID NO: 28 When supplementing with SEQ ID NO: 2 or SEQ ID NO: 28, the level of produced LNT-II during culturing becomes increases, as shown in Example 2.
  • a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase is any protein which comprises the ability of transferring the N-acetyl-glucosamine of UDP-N-acetyl-glucosamine to lactose.
  • the b-1 ,3-N-acetyl- glucosaminyl-transferase used herein does not originate in the species of the genetically engineered cell i.e. the gene encoding the b-1 ,3-galactosyltransferase is of heterologous origin.
  • the examples below use the heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase HD0466, LgtA and/or PmnagT.
  • the HD0466 gene is as shown in SEQ ID NO: 4, or a functional homologue thereof having a nucleotide sequence that is at least 70 %, such as at least 75 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 4.
  • the IgtA gene is as shown in SEQ ID NO: 5 or is a functional homologue thereof having a nucleotide sequence that is at least 70 %, such as at least 75 %, such as at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 5.
  • the PmnagT gene is as shown in SEQ ID NO: 29, or a functional homologue thereof having a nucleotide sequence that is at least 70 %, such as at least 75 %, such as at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 29.
  • heterologous b-1 ,3-N-acetyl-glucosaminyltransferases of the present disclosure that may, upon expression, be used to produce LNT-II and/or HMOs using LNT-II as a precursor molecule, are shown in the below matrix.
  • the heterologous b-1 ,3-N-acetyl- glucosaminyltransferases and the HMO LNT-II can be generated using the protein of the amino acid sequence SEQ ID NO: 1 [HD0466] in combination enzymes are shown in the below matrix.
  • HD0466 can provide high LNT-II titers when expressed from one or two genomic copies ( Figure 1a).
  • the descending order of activity of the three selected GlcNAcTs on lactose, as it is indirectly revealed by the observed final LNT-II titers is as follows: HD0466 > PmnagT > LgtA.
  • the LNT-II titers reached by the strains MP5 and/or MP6, which express HD0466 from a different copy number, can be up to 40% or 15% higher than for strains expressing LgtA (strain MP1) or PmnagT (strain MP3), respectively.
  • lactose is used as the substrate for the synthesis of LNT-II.
  • a genetically engineered cell of the present invention should be capable of importing lactose into the cell. While lactose is naturally imported into some microorganisms, other microorganisms lack the ability to do so. To enable lactose import, such microorganisms would need to be genetically engineered to take up lactose.
  • the genetically engineered cell of the present invention is able to import lactose into the cell.
  • One way to enable lactose import into a cell of the present invention is by expression of a lactose permease.
  • the overexpression of an endogenous lactose import pathway such as but not limited to an endogenous lactose permease protein, and/or incorporation of a heterologous lactose import pathway, such as but not limited to a heterologous lactose permease, may be used to enhance the lactose import of said microorganism and thereby enhance LNT-II production.
  • the genetically engineered cell of the present invention overexpresses an endogenous lactose permease protein and/or expresses a heterologous lactose permease.
  • Lactose permease is a membrane protein which is a member of the major facilitator superfamily and can be classified as a symporter, which uses the proton gradient towards the cell to transport b-galactosides such as lactose in the same direction into the cell.
  • lactose is the molecule being decorated to produce any HMO of interest and bioconversions happen in the cell interior.
  • lactose permease e.g., the native lacY copy under the control of a promoter.
  • the lactose permease protein is as shown in SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 3.
  • the lactose permease gene is a nucleotide sequence as shown in SEQ ID NO: 6 or is a functional homologue thereof having a nucleotide sequence that is at least 70 %, such as at least 75 %, such as at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 6.
  • Example 3 the over-expression of the lacY gene coding lactose permease is used as a genetic tool to obtain an enhanced level of LNT-II produced by the genetically engineered cell of the present disclosure. As is shown in Example 3 only the combined expression of a specific pair of b-1 ,3-N- acetyl-glucosaminyltransferases and lacY over-expression results in an enhanced LNT-II production.
  • the genetically engineered cells disclosed herein may comprise a regulatory element for increasing the expression of the native lactose permease protein, such as but not limited to Ribosome Binding Sites (RBSs).
  • RBSs Ribosome Binding Sites
  • the RBSs may for example be the Shine-Dalgarno (SD) sequence. Mutations in the Shine- Dalgarno sequence can reduce or increase translation in prokaryotes. This change is due to a reduced or increased mRNA-ribosome pairing efficiency, as evidenced by the fact that compensatory mutations in the 3'-terminal 16S rRNA sequence can restore translation.
  • the regulatory element for increasing the expression of the native lactose permease protein could also be a promoter.
  • the genetically engineered cells disclosed herein may also comprise a heterologous episomal element for increasing the expression of the native lactose permease protein. This could for example be a plasmid-borne lacY gene.
  • the increased expression of the lactose permease may be achieved by direct integration of a copy of the lacY gene in the genome.
  • Examples 2 and 3 provide enough data to conclude that the combined expression of HD0466 and LgtA results in higher LNT-II titers regardless of intracellular lactose levels compared to when only one of these GlcNAcTs is expressed by the cell at the same copy number.
  • this trend is unique for this GlcNAcT pair and it is not observed for any other GlcNAcT pair that can be formed from HD0466, LgtA and PmnagT.
  • the increased expression of the lactose permease may be achieved by deleting a repressor of the lactose operon.
  • An example of such being the lad gene - UniProtKB - P03023 (LACI_ECOLI).
  • the amino acid sequence of the proteins of the present disclosure as shown in SEQ ID NO: 1-3 and 28, is at least 80% identical to SEQ ID NO: 1-3 and 28, such as at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85 % identical, at least 86 % identical, at least 87 % identical, at least 88 % identical, at least 89 % identical, at least 90 % identical, at least 91 % identical, at least 92 % identical, at least 93 % identical, at least 94 % identical, at least 95 % identical at least 96 % identical, at least 97 % identical, at least 98 % identical, or at least 99 % identical.
  • controlling the expression relates to gene expression where the transcription of a gene into mRNA and its subsequent translation into protein is controlled. Gene expression is primarily controlled at the level of transcription, largely as a result of binding of proteins to specific sites on DNA, such as but not limited to regulatory elements. As described above, engineering strategy to control expression can be applied in multiple ways
  • a variety of molecular mechanisms ensures that genes are expressed at the appropriate level and under conditions of relevance to the applied production process.
  • the regulation of transcription can be summarized into the following routes of influence; genetic (direct interaction of a control factor with the gene of interest), modulation and/or interaction of a control factor within the transcriptional machinery and epigenetic (non-sequence changes in DNA structure that influence transcription).
  • Over-expression of a gene may be achieved directly by transcriptional activators that bind to key gene regulatory sequences to promote transcription or enhancers that constitute sequence elements positively affecting transcription.
  • direct over-expression of a gene can be achieved by simply increasing its copy number in the genome, or replacing its native promoter with a promoter of higher strength or even modifying the sequence controlling the binding of the corresponding mRNA to the ribosomes, i.e. the Shine-Dalgarno sequence being present upstream of the gene’s coding sequence.
  • over-expression of a gene may also be achieved indirectly through the partial or full inactivation of transcriptional repressors that normally bind key regulatory sequences around the coding sequence of the gene of interest and thereby inhibit its transcription.
  • over-expression may for example refer to the native gene lacY and includes 1) the replacement of the native promoter of the any E. coli gene by another, stronger promoter, 2) the modification of the native Shine-Dalgarno sequence of these genes by a stronger sequence with the goal of promoting ribosomal binding, 3) the deletion of the gene encoding a direct repressor or the enhancement of the expression of a gene encoding a direct activator of the native promoter of the E.
  • coli gene(s) of interest 4) the increase in the copy number of the gene(s) of interest, where the gene(s) are expressed from a genomic locus other than the native locus and the expression is driven by the native or a synthetic promoter (e.g., PglpF), and 5) the episomal expression of the gene(s) of interest from a low (5-10 copies per cell) to a high-copy number plasmid (300-500 copies per cell).
  • a synthetic promoter e.g., PglpF
  • the over-expression of the b-1 ,3-N-acetyl-glucosaminyl- transferase proteins and/or the lactose permease of the present disclosure is provided by increasing the copy number of the genes coding said protein(s), and/or by choosing an appropriate element for or adding an extra genomic copy for the genes encoding the b-1 ,3-N-acetyl-glucosaminyl-transferase proteins and/or the genes encoding the lactose permease, and/or conferring a non-functional (or absent) gene product that normally binds to and repress the expression of any of the the b-1 ,3-N-acetyl- glucosaminyl-transferase proteins and/or the lactose permease of the present disclosure.
  • Copy number variation is a type of structural variation: specifically, it is a type of duplication or multiplication of a considerable number of base pairs. Specifically, in the context of the present invention the copy number refers to the number of sequences (genes) encoding the desired protein. The sequences may be located several places in the genome of the genetically modified cell, but they can also be inserted in one place operated by a single promoter.
  • expression is controlled by increasing the copy number of the gene encoding the desired protein.
  • the present disclosure relates to a method, wherein the overexpression of the b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease is provided by increasing the copy number of any of the genes coding for said protein(s) and/or by choosing an appropriate regulatory element.
  • the genetically engineered cell according to the methods described herein may comprise regulatory elements enabling the controlled overexpression of endogenous or heterologous and/or synthetic nucleic acid sequences.
  • the regulatory element for controlling and increasing the expression of the heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease protein(s) in the method(s) described above is a promoter.
  • regulatory element comprises promoter sequences, signal sequence, and/or arrays of transcription factor binding sites, which sequences affect transcription and/or translation of a nucleic acid sequence operably linked to the regulatory element.
  • RNA binding proteins are another class of post-transcriptional regulatory elements and are further classified as sequence elements or structural elements. Specific sequence motifs that may serve as regulatory elements are also associated with mRNA modifications.
  • DNA regulatory elements are involved in the regulation of gene expression and rely on the biochemical interactions involving DNA, the cellular proteins that make up chromatin, gene activators and repressors, and transcription factors.
  • transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, binding sites for gene regulators and enhancer sequences.
  • Promoters and enhancers are the primary genomic regulatory components of gene expression.
  • Promoters are DNA regions within 1-2 kilobases (kb) of a gene’s transcription start site (TSS); they contain short regulatory elements (DNA motifs) necessary to assemble RNA polymerase transcriptional machinery. However, transcription is often minimal without the contribution of DNA regulatory elements located more distal to the TSS. Such regions, often termed enhancers, are position-independent DNA regulatory elements that interact with site-specific transcription factors to establish cell type identity and regulate gene expression. Enhancers may act independently of their sequence context and at distances of several to many hundreds of kb from their target genes through a process known as looping. Because of these features, it is difficult to identify suitable enhancers and link them to their target genes based on DNA sequence alone.
  • control sequences are necessary to express a given gene or group of genes (an operon).
  • regulatory elements may or may not be post-translational regulators or it may or may not be translational regulators.
  • the regulatory element comprises one or more elements capable of enhancing the expression, of the one or more nucleic acid sequence(s) according to the present disclosure.
  • controlling the expression of nucleic acid sequences and/or genes encoding one or more glycosyltransferases and/or a lactose permease protein may be a promoter sequence.
  • promoter sequences may be used to drive transcription of different genes of interest integrated into the genome of the host cell or into episomal DNA.
  • nucleic acid sequences originating from the genome of the genetically engineered cell according to the method of the disclosure.
  • a nucleic acid sequence may be considered native if it originates from the E. coli K12 strain, and is not of heterologous origin and not a recombined nucleic acid sequence, with respect to the genetically engineered cell.
  • an amino acid sequence may be considered endogenous if it originates from the host cell and is thus not of heterologous origin, with respect to the genetically engineered cell.
  • a regulatory element may be endogenous or heterologous, and/or recombinant and/or synthetic nucleic acid sequences.
  • heterologous regulatory element is to be understood as a regulatory element that is not endogenously found in that genomic locus or heterologous to the original genetically engineered cell described herein.
  • a promoter sequence of the E. coli glpFKX operon, such as PglpF, in front of the native lactose permease is considered to be a heterologous regulatory element.
  • the heterologous regulatory element may also be a recombinant regulatory element, wherein two or more non-operably linked native regulatory element(s) are recombined into a heterologous and/or synthetic regulatory element.
  • the heterologous regulatory element may be introduced into the genetically engineered cell using methods known to the person skilled in the art.
  • a promoter which is regulating the transcription of a gene it is not naturally regulating is also considered to be heterologous, even if it is derived from another genomic locus in the same strain.
  • the regulatory element or elements regulating the expression of the genes and/or nucleic acid sequence(s), may comprise one or more promoter sequence(s), wherein the promoter sequence, is operably linked to the nucleic acid sequence of the gene of interest in that sense regulating the expression of the nucleic acid sequence of the gene of interest.
  • the heterologous regulatory element is a promoter sequence.
  • a promoter may comprise native, heterologous and/or synthetic nucleic acid sequences, and may be a recombinant nucleic acid sequence, recombining two or more nucleic acid sequences or same or different origin as described above, thereby generating a homologous, heterologous or synthetic nucleic promoter sequence, and/or a homologous, heterologous or synthetic nucleic regulatory element.
  • the regulatory element of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell comprises more than one native or heterologous promoter sequence.
  • the regulatory element of the genetically engineered cell comprises a single promoter sequence.
  • the regulatory element of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell comprises two or more regulatory elements with identical promoter sequences.
  • regulatory element of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell comprises two or more regulatory elements with nonidentical promoter sequences.
  • the regulatory architectures i.e., gene-by-gene distributions of transcription-factor-binding sites and identities of the transcription factors that bind those sites can be used multiple different growth conditions and there are more than 100 genes from across the E. coli genome, which acts as regulatory elements.
  • any promoter sequence enabling transcription and/or regulation of the level of transcription, of one or more heterologous or native nucleic acid sequences that encode one or more proteins as described herein may be suitable.
  • the promoter may be of heterologous origin, native to the genetically modified cell or it may be a recombinant promoter, combining heterologous and/or native elements.
  • One way to increase the production of a product may be to regulate the production of the desired enzyme activity used to produce the product or precursor/substrate import, such as the glycosyltransferases or a lactose permease.
  • Increasing the promoter strength driving the expression of the desired enzyme may be one way of doing this.
  • the strength of a promoter can be assed using a lacZ enzyme assay where b-galactosidase activity is assayed as described previously (see e.g. Miller J.H. Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972).
  • the cells are diluted in Z-buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform.
  • the LacZ assay is performed at 30°C. Samples are preheated, the assay initiated by addition of 200 pi ortho-nitro-phenyl-p-galactosidase (4 mg/ml) and stopped by addition of 500 pi of 1 M Na 2 CC>3 when the sample had turned slightly yellow. The release of ortho-nitrophenol is subsequently determined as the change in optical density at 420 nm. The specific activities are reported in Miller Units (MU) [A420/(min*ml*A600)].
  • a regulatory element with an activity above 10,000 MU is considered strong and a regulatory element with an activity below 3,000 MU is considered weak, what is in between has intermediate strength.
  • An example of a strong regulatory element is the PglpF promoter with an activity of approximately 14.000 MU and an example of a weak promoter is Plac which when induced with IPTG has an activity of approximately 2300 MU.
  • a promoter with the desired strength, e.g., middle or low strength.
  • Table 4 lists a series of wildtype and recombinant promoters according to their strength relative to the PglpF promoter.
  • the regulatory element is selected from the group consisting of PBAD, Pxyl, PsacB, PxylA, PrpR, PnitA, PT7, Ptac, PL, PR, PnisA, Pb, Pscr, Pscr_SD1, Pscr_SD7, PgatY_70UTR, PglpF, PglpF_SD1, PglpF_SD10, PglpF_SD2, PglpF_SD3, PglpF_SD4, PglpF_SD5, PglpF_SD6, PglpF_SD7, PglpF_SD8, PglpF_SD9, PglpF_B28, Plac_16UTR, Plac, PmglB_70UTR and PmglB_ 70UTR_SD4.
  • the regulatory element is a promoter selected from the group consisting of PglpF (SEQ ID NO: 11), PglpT(SEQ ID NO: 38), Plac (SEQ ID NO: 25, PmgIB (SEQ ID NO: 26, PglpA (SEQ ID NO: 37), and variants thereof. Specifically, the variants disclosed in table 4 are preferred.
  • the regulatory element is a promoter with high or middle strength, such as a promoter sequence selected from the group consisting of PmglB_70UTR_SD8, PmglB_70UTR_SD10, PmglB_54UTR, Plac_70UTR, PmglB_70UTR_SD9, PmglB_70UTR_SD4, PmglB_70UTR_SD5, PglpF_SD4, PmglB_70UTR_SD7, PmglB_70UTR, PglpA_70UTR, PglpT_70UTR, pgatY_70UTR, PglpF, PglpF_SD10, PglpF_SD5, PglpF_SD8, PglpF_B28, PglpF_B29, PmglB_16UTR, PglpF_SD9, PglpF_SD7, Pgl
  • the promoter is a strong promoter selected from the group consisting of PmglB_70UTR_SD8, PmglB_70UTR_SD10, PmglB_54UTR, Plac_70UTR, PmglB_70UTR_SD9, PmglB_70UTR_SD4, PmglB_70UTR_SD5, PglpF_SD4, PmglB_70UTR_SD7, PmglB_70UTR, PglpA_70UTR, PglpT_70UTR, pgatY_70UTR, PglpF, PglpF_SD10, PglpF_SD5, PglpF_SD8, andPmglB_16UTR.
  • This may in particular be advantageous for the expression the heterologous b-1 ,3-N- acetyl-glucosaminyl-transferase.
  • the promoter is selected from the group consisting of promoters with middle strength, such as PglpF_SD9, PglpF_SD7, PglpF_SD6 and PglpA_16UTR.
  • the promoter is selected from the group consisting of promoters with low strength, such as Plac_wt. PglpF_SD3 and PglpF_SD1. This may in particular be advantageous for the expression the lactose permease.
  • the regulatory element is PglpF or Plac, or a variant of PglpF or Plac.
  • the promoter sequence comprised in the regulatory element for the regulation of the expression of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell encompasses the glpFKX operon promoter sequence, PglpF.
  • the promoter sequence comprised in the regulatory element for the regulation of the expression of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell encompasses the lac operon promoter sequence, P lac.
  • the regulatory element for the regulation of the expression of a recombinant gene included in the construct of the disclosure is the mgIBAC ; galactose/methyl-galactoside ABC transporter periplasmic binding protein promoter PmgIB or variants thereof such as but not limited to Pmg/B_70UTR, or Pmg/B_70UTR_SD4.
  • the regulatory element for the regulation of the expression of a recombinant gene included in the construct of the disclosure is the gatYZABCD tagatose-1 ,6-bisP aldolase promoter PgatY or variants thereof.
  • the heterologous regulatory element is Pscr or variants thereof such as but not limited to SEQ ID NO: 7.
  • Pscr_SD1 Pscr_SD1
  • the heterologous regulatory element is Pscr_SD1 or variants thereof such as but not limited to SEQ ID NO: 8.
  • the heterologous regulatory element is Pscr_SD7 or variants thereof such as but not limited to SEQ ID NO: 9.
  • the heterologous regulatory element is PgatY_70UTR or variants thereof such as but not limited to SEQ ID NO: 10.
  • the heterologous regulatory element is PgipF or variants thereof such as but not limited to SEQ ID NO: 11 .
  • a GlcNAcT is hereby defined as “highly expressed” when the host strain expresses it from at least two PglpF-driven genomic copies.
  • the heterologous regulatory element is PglpF_SD1 or variants thereof such as but not limited to SEQ ID NO: 12.
  • the heterologous regulatory element is PglpF_SD10 or variants thereof such as but not limited to SEQ ID NO: 13.
  • the heterologous regulatory element is PglpF_SD2 or variants thereof such as but not limited to SEQ ID NO: 14.
  • the heterologous regulatory element is PglpF_SD3 or variants thereof such as but not limited to SEQ ID NO: 15.
  • the heterologous regulatory element is PglpF_SD4 or variants thereof such as but not limited to SEQ ID NO: 16.
  • the heterologous regulatory element is PglpF_SD5 or variants thereof such as but not limited to SEQ ID NO: 17.
  • the heterologous regulatory element is PglpF_SD6 or variants thereof such as but not limited to SEQ ID NO: 18.
  • the heterologous regulatory element is PglpF_SD7 or variants thereof such as but not limited to SEQ ID NO: 19.
  • the heterologous regulatory element is PglpF_SD8 or variants thereof such as but not limited to SEQ ID NO: 20.
  • the heterologous regulatory element is PglpF_SD9 or variants thereof such as but not limited to SEQ ID NO: 21.
  • the heterologous regulatory element is PglpF_B28 or variants thereof such as but not limited to SEQ ID NO: 22.
  • the heterologous regulatory element is PglpF_B29 or variants thereof such as but not limited to SEQ ID NO: 23. Plac_16UTR
  • the heterologous regulatory element is Plac_16UTR or variants thereof such as but not limited to SEQ ID NO: 24.
  • the heterologous regulatory element is Plac or variants thereof such as but not limited to SEQ ID NO: 25.
  • the heterologous regulatory element is PmglB_70UTR or variants thereof such as but not limited to SEQ ID NO: 26.
  • the heterologous regulatory element is PmglB_70UTR_SD4 or variants thereof such as but not limited to SEQ ID NO: 27.
  • an episomal nucleic acid sequences may be a plasmid that can integrate into the chromosome of the genetically engineered cell, i.e. not all plasmids are episomal elements.
  • episomal nucleic acid sequences may be a plasmid that is not integrated into the chromosome.
  • the episomal element refers to plasmid DNA sequences that carry an expression cassette of interest, with the cassette consisting of a promoter sequence, the coding sequence of the gene of interest and a terminator sequence.
  • episomal nucleic acid sequences may be a plasmid with only a part of it being integrated into the chromosome.
  • the expression cassette resembles the one described above but it further comprises two DNA segments that are homologous to the DNA regions up- and downstream of the locus that the gene of interest is to be integrated.
  • the genetically engineered cell disclosed herein comprises a non-functional or absent gene product that normally binds to and represses the expression of the required enzymes to facilitate the production of a human milk oligosaccharide (HMO) that is LNT-II.
  • HMO human milk oligosaccharide
  • non-functional (or absent) gene product that normally binds to and represses the expression driven by the regulatory element in the present context relates to DNA binding sites upstream of the coding sequence of a gene of interest and specifically at the promoter region of said gene.
  • the cell may have a non-functional (or absent) gene product(s) that would normally bind to and repress the expression of any of the b-1 ,3-N-acetyl-glucosaminyl- transferase protein(s) and/or the lactose permease protein or regions upstream of the regulatory element for controlling the expression of any of the b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease protein.
  • the method according to the present disclosure comprise a cell further comprising non-functional (or absent) gene product that binds to and represses the expression of any of the b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) of the present disclosure, and wherein the b-1 ,3-N-acetyl-glucosaminyl-transferase protein is HD0466 and LgtA or PmnagT.
  • the method according to the present disclosure comprise a cell further comprising non-functional (or absent) gene product that binds to and represses the expression of any of the b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) of the present disclosure, and wherein the b-1 ,3-N-acetyl-glucosaminyl-transferase protein is HD0466.
  • the method according to the present disclosure comprise a cell further comprising non-functional (or absent) gene product that binds to and represses the expression of any of b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease protein, and wherein the b-1 ,3-N-acetyl-glucosaminyl-transferase protein is HD0466 and LgtA.
  • said gene product is the DNA-binding transcriptional repressor GlpR.
  • GlpR belongs to the DeoR family of transcriptional regulators and acts as the repressor of the glycerol-3- phosphate regulon, which is organized in different operons.
  • This regulator is part of the glpEGR operon, yet it can also be constitutively expressed as an independent ( glpR ) transcription unit.
  • the operons regulated are induced when Escherichia coli is grown in the presence of inductor, glycerol, or glycerol-3-phosphate (G3P), and the absence of glucose. In the absence of inductor, this repressor binds in tandem to inverted repeat sequences that consist of 20-nucleic acid-long DNA target sites.
  • non-functional or absent in relation to the glpR gene refers to the inactivation of the glpR gene by complete or partial deletion of the corresponding nucleic acid sequence from the bacterial genome genome (e.g. SEQ ID NO: 41 or variants thereof encoding glpR capable of downregulating glpF derived promoters).
  • the glpR gene encodes the DNA-binding transcriptional repressor GlpR. In this way promoter sequences of the Pg/pF family are more active in the genetically engineered cell, due to deletion of the repressor gene that would otherwise reduce the transcriptional activity associated with the PglpF promoters.
  • the glpR gene is deleted.
  • the deletion of the glpR gene could eliminate the GlpR-imposed repression of transcription from all PglpF promoters in the cell and in this manner enhance gene expression from all PglpF- based cassettes.
  • the genetically engineered cell disclosed herein comprises an over-expressed gene product that enhances the expression of the gene(s) encoding the enzyme(s) required to facilitate the production of a human milk oligosaccharide (HMO) that is LNT-II.
  • HMO human milk oligosaccharide
  • the cell of the present disclosure may comprise an overexpressed gene product that enhances the expression of the gene(s) encoding any of the b-1 ,3-N-acetyl- glucosaminyl-transferase protein(s) and/or the lactose permease protein.
  • the cell of the present disclosure may comprise an overexpressed gene product that enhances the expression of the gene(s) encoding any of the b-1 ,3-N-acetyl- glucosaminyl-transferase protein(s) of the present disclosure, and wherein the b-1 ,3-N-acetyl- glucosaminyl-transferase protein is HD0466 and LgtA or PmnagT.
  • the cell of the present disclosure may comprise an overexpressed gene product that enhances the expression of the gene(s) encoding any of the b-1 ,3-N-acetyl- glucosaminyl-transferase protein(s) of the present disclosure, and wherein the b-1 ,3-N-acetyl- glucosaminyl-transferase protein is HD0466.
  • the cell of the present disclosure may comprise an overexpressed gene product that enhances the expression of any of the b-1 ,3-N-acetyl-glucosaminyl- transferase protein(s) and/or the lactose permease protein, and wherein the b-1 ,3-N-acetyl-glucosaminyl- transferase protein is HD0466 and LgtA.
  • said gene product is the cAMP DNA-binding transcriptional dual regulator CRP.
  • CRP belongs to the CRP-FNR superfamily of transcription factors. CRP regulates the expression of several of the E. coli genes, many of which are involved in catabolism of secondary carbon sources.
  • CRP cyclic-AMP
  • cAMP cyclic-AMP
  • RNA polymerase RNA polymerase
  • over-expression of CRP may lead to an enhanced expression of a gene/nucleic acid sequence of interest.
  • CRP exerts its function on the PglpF promoters, where it contrary to the repressor GlpR, activates promoter sequences of the PglpF family. In this way, over-expression of CRP in the genetically engineered cell of the present disclosure, promotes expression of genes that are regulated by promoters of the PglpF family.
  • the crp gene is over-expressed.
  • nucleic acid construct may comprise at least i) a nucleic acid sequence according to SEQ ID NO: 4 or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 4, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; and ii) a native or heterologous regulatory element for controlling the expression of i).
  • the nucleic acid construct also comprises: iii) a nucleic acid sequence according to SEQ ID NO: 5 [IgtA], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 5, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase, and iv) a native or heterologous regulatory element for controlling the expression of iii).
  • the nucleic acid construct also comprises: v) a nucleic acid sequence according SEQ ID NO: 6 [LacY], or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 6, to encoding a lactose permease, and vi) a native or heterologous regulatory element for controlling the expression of v).
  • the nucleic acid construct also comprises: vii) a nucleic acid sequence according to SEQ ID NO: 29 [PmnagT], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 29, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; viii) a native or heterologous regulatory element for controlling the expression of iii).
  • the nucleic acid construct may further comprise one or more regulatory element for controlling the expression of i), iii), v), vii).
  • the regulatory element(s) may be a native or heterologous or episomal.
  • the nucleic acid construct may further comprise a heterologous regulatory or episomal element for increasing the expression of v) a lactose permease protein as shown in SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3.
  • the nucleic acid construct may further comprise a non-functional (or absent) gene product that normally binds to and represses the expression of the regulatory elements).
  • the nucleic acid construct can be a recombinant nucleic acid sequence.
  • recombinant nucleic acid sequence “recombinant gene/nucleic acid/DNA encoding” or “recombinant coding nucleic acid sequence” used interchangeably is meant an artificial nucleic acid sequence (i.e. produced in vitro using standard laboratory methods for making nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) which is transcribed into mRNA and translated into a protein when under the control of the appropriate control sequences, i.e. a promoter sequence.
  • the boundaries of the coding sequence are generally determined by a ribosome binding site located just upstream of the open reading frame at the 5’end of the mRNA, a transcriptional start codon (AUG, GUG or UUG), and a translational stop codon (UAA, UGA or UAG).
  • a coding sequence can include, but is not limited to, genomic DNA, cDNA, synthetic, and recombinant nucleic acid sequences.
  • the term "nucleic acid” includes RNA, DNA and cDNA molecules. It is understood that, as a result of the degeneracy of the genetic code, a multitude of nucleic acid sequences encoding a given protein may be produced.
  • the recombinant nucleic sequence may be a coding DNA sequence e.g., a gene, or non-coding DNA sequence e.g., a regulatory DNA, such as a promoter sequence. It may be a native promoter in front of a heterologous nucleic acid sequence, which makes the native promoter a recombinant promoter.
  • the disclosure relates to a nucleic acid construct comprising a coding nucleic sequence, i.e. recombinant DNA sequence of a gene of interest, e.g. a b-1 ,3-N-acetyl- glucosaminyl-transferase gene, and a non-coding regulatory DNA sequence, e.g. a promoter DNA sequence, e.g. a recombinant promoter sequence derived from the promoter sequence of lac operon or an glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked.
  • a coding nucleic sequence i.e. recombinant DNA sequence of a gene of interest, e.g. a b-1 ,3-N-acetyl- glucosaminyl-transferase gene
  • a non-coding regulatory DNA sequence e.g
  • operably linked refers to a functional relationship between two or more nucleic acid (e.g.,
  • DNA segments operably linked refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
  • a promoter sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
  • promoter sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are c/s-acting.
  • the nucleic acid construct of the disclosure may be a part of the vector DNA, in another embodiment the construct it is an expression cassette/cartridge that is integrated in the genome of a host cell.
  • nucleic acid construct means an artificially constructed segment of nucleic acid, in particular a DNA segment, which is intended to be 'transplanted' into a target cell, e.g. a bacterial cell, to modify expression of a gene of the genome or express a gene/coding DNA sequence which may be included in the construct.
  • nucleic acid construct of interest comprised in the construct (expression cassette) into the bacterial genome
  • introduction of the nucleic acid construct of interest comprised in the construct (expression cassette) into the bacterial genome can be achieved by conventional methods, e.g. by using linear cartridges that contain flanking sequences homologous to a specific site on the chromosome, as described for the attTn7-site (Waddell C.S. and Craig N.L., Genes Dev. (1988) Feb;2(2):137-49.); methods for genomic integration of nucleic acid sequences in which recombination is mediated by the Red recombinase function of the phage l or the RecE/RecT recombinase function of the Rac prophage (Murphy, J Bacteriol.
  • the present disclosure relates to a recombinant nucleic acid shown in SEQ ID NO: 4-6 or 29, or a functional homologue thereof having a sequence that is at least 70% identical to SEQ ID NO: 4-6 or 29, such as at least 71% identical, at least 72% identical, at least 73% identical, at least 74% identical, at least 75% identical, at least 76% identical, at least 77% identical, at least 78% identical, at least 79% identical, at least 80 % identical, at least 81 % identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85 % identical, at least 86 % identical, at least 87 % identical, at least 88 % identical, at least 89 % identical, at least 90 % identical, at least 91 % identical, at least 92 % identical, at least 93 % identical, at least 94 % identical, at least 95 % identical at least 96 % identical, at least 97 % identical, at least
  • sequence identity of [a certain] %” in the context of two or more nucleic acid or amino acid sequences means that the two or more sequences have nucleic acids or amino acid residues in common in the given percent, when compared and aligned for maximum correspondence over a comparison window or designated sequences of nucleic acids or amino acids (i.e. the sequences have at least 90 percent (%) identity).
  • Percent identity of nucleic acid or amino acid sequences can be measured using a BLAST 2.0 sequence comparison algorithm with default parameters, or by manual alignment and visual inspection (see e.g. http://www.ncbi.nlm.nih.gov/BLAST/).
  • BLAST 2.2.20+ is used to determine percent sequence identity for the nucleic acids and proteins of the disclosure.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Examples of commonly used sequence alignment algorithms are
  • the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277,), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss needle/).
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix.
  • the sequence identity between two nucleotide sequences is determined using the Needleman- Wunsch algorithm (Needleman and Wunsch, 1 970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), 10 preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • a functional homologue of a protein/nucleotide as described herein is a protein/nucleotide with alterations in the genetic code, which retain its original functionality.
  • a functional homologue may be obtained by mutagenesis.
  • the functional homologue should have a remaining functionality of at least 50%, such as 60%, 70%, 80 %, 90% or 100% compared to the functionality of the protein/nucleotide.
  • a functional homologue of any one of the disclosed amino acid or nucleotide sequences can also have a higher functionality.
  • a functional homologue of any one of SEQ ID NOs: 1-29 should ideally be able to participate in the HMO production, in terms of HMO yield, purity, reduction in biomass formation, viability of the genetically engineered cell, robustness of the genetically engineered cell according to the disclosure, or reduction in consumables.
  • the present disclosure also relates to a genetically engineered cell comprising a) a nucleic acid sequence according to SEQ ID NO: 4 or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 4, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; and b) a native or heterologous regulatory element for controlling the expression of a).
  • the cell is able to import lactose into the cell.
  • the genetically engineered cell also comprises: c) a nucleic acid sequence according to SEQ ID NO: 5 [LgtA], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 5, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; and d) a native or heterologous regulatory element for controlling the expression of c).
  • the genetically engineered cell also comprises: e) a nucleic acid sequence according to SEQ ID NO: 6 [LacY], or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 6 encoding a lactose permease, and f) a native or heterologous regulatory element for controlling the expression of e).
  • the genetically engineered cell also comprises: g) a nucleic acid sequence according to SEQ ID NO: 29 [PmnagT], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 29, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; and h) a native or heterologous regulatory element for controlling the expression of g).
  • a “genetically modified” or genetically engineered” cell is used interchangeably herein and is understood as a cell whose genetic material has been altered by human intervention using a genetic engineering technique, such a technique is for example but not limited to transformation or transfection e.g., with a heterologous polynucleotide sequence, Crisper/Cas editing and/or random mutagenesis.
  • a genetically modified cell and “a host cell” are used interchangeably.
  • the "genetically engineered cell” is preferably a host cell which has been transformed or transfected by an exogenous polynucleotide sequence.
  • the HMO produced by the genetically engineered cell is LNT-II.
  • the genetically engineered cell may be any cell useful for HMO production including mammalian cell lines.
  • the host cell is a unicellular microorganism of eucaryotic or prokaryotic origin.
  • Appropriate microbial cells that may function as a host cell include yeast cells, bacterial cells, archaebacterial cells, algae cells, and fungal cells.
  • the genetically engineered cell may be e.g., a bacterial or yeast cell.
  • the genetically engineered cell is a bacterial or yeast cell.
  • the genetically engineered cell is preferably a prokaryotic cell, such as a bacterial cell.
  • the bacterial host cells there are, in principle, no limitations; they may be eubacteria (grampositive or gram-negative) or archaebacteria, as long as they allow genetic manipulation for insertion of a gene of interest and can be cultivated on a manufacturing scale.
  • the host cell has the property to allow cultivation to high cell densities.
  • Non-limiting examples of bacterial host cells that are suitable for recombinant industrial production of an HMO(s) according to the disclosure could be Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris.
  • Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans.
  • bacteria of the genera Lactobacillus and Lactococcus may be engineered using the methods of this disclosure, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius,
  • Lactobacillus plantarum Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis.
  • Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the disclosure described herein.
  • strains engineered as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).
  • Enterococcus e.g., Enterococcus faecium and Enterococcus thermophiles
  • Bifidobacterium e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifido
  • Non-limiting examples of fungal host cells that are suitable for recombinant industrial production of a HMO product could be yeast cells, such as Komagataella phaffii, Kluyveromyces lactis, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae or filamentous fungi such as Aspargillus sp, Fusarium sp or Thricoderma sp, exemplary species are A. niger, A. nidulans, A. oryzae, F. solani, F. graminearum and T. reesei.
  • the genetically engineered cell is selected from the group consisting of Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.
  • the genetically engineered cell is S. cerevisiae or P pastoris.
  • the genetically engineered cell is Pichia pastoris.
  • the genetically engineered cell is selected from the group consisting of E. coli, C. glutamicum, L lactis, B. subtilis, S. lividans, P. pastoris, and S. cerevisiae.
  • the genetically engineered cell is selected from the group consisting of B. subtilis, S. cerevisiae and E. coli.
  • the genetically engineered cell is selected from the group consisting of E. coli, C. glutamicum, L. lactis, B. subtilis, S. lividans.
  • the genetically engineered cell is B. subtilis.
  • the genetically engineered cell is Corynebacterium glutamicum.
  • the genetically engineered cell is S. cerevisiae.
  • the genetically engineered cell is E. coli.
  • the disclosure relates to a genetically engineered cell, wherein the cell is derived from the E. coli K12 or DE3 strain.
  • culturing refers to the process by which cells are grown under controlled conditions, generally outside their natural environment, thus a method used to cultivate, propagate and grow a large number of cells.
  • a growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms, cells, or small plants.
  • the medium comprises an appropriate source of energy and may comprise compounds which regulate the cell cycle.
  • the culture medium may be semi-defined, i.e. containing complex media compounds (e.g. yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined, without any complex compounds. Exemplary suitable medias are provided in experimental examples.
  • the culturing media is minimal media.
  • the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose, glucose and fructose.
  • the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose and glucose.
  • the culturing media is supplemented with glycerol, sucrose and/or glucose.
  • the culturing media is supplemented with glycerol and/or glucose.
  • the culturing media is supplemented with sucrose and/or glucose.
  • the culturing media is supplemented with glycerol and/or sucrose.
  • slaughtering in the context relates to collecting the produced HMO(s) following the termination of fermentation.
  • it may include collecting the HMO(s) included in both the biomass (i.e. the host cells) and cultivation media, i.e. before/without separation of the fermentation broth from the biomass.
  • the produced HMOs may be collected separately from the biomass and fermentation broth, i.e. after/following the separation of biomass from cultivation media (i.e. fermentation broth).
  • the separation of cells from the medium can be carried out with any of the methods well known to the skilled person in the art, such as any suitable type of centrifugation or filtration.
  • the separation of cells from the medium can follow immediately after harvesting the fermentation broth or be carried out at a later stage after storing the fermentation broth at appropriate conditions.
  • Recovery of the produced HMO(s) from the remaining biomass (or total fermentation) include extraction thereof from the biomass (i.e. the production cells). After recovery from fermentation, HMO(s) are available for further processing and purification.
  • HMO Human milk oligosaccharide
  • oligosaccharide means a saccharide polymer containing a number of monosaccharide units.
  • preferred oligosaccharides are saccharide polymers consisting of three or four monosaccharide units, i.e. trisaccharides ortetrasaccharides.
  • Preferable oligosaccharides of the disclosure are human milk oligosaccharides (HMOs).
  • human milk oligosaccharide or "HMO” in the present context means a complex carbohydrate found in human breast milk.
  • the HMOs have a core structure comprising a lactose unit at the reducing end that can be elongated by one or more beta-N-acetyl-lactosaminyl and/or one or more beta-lacto-N- biosyl units, and this core structure can be substituted by an alpha-L-fucopyranosyl and/or an alpha-N- acetyl-neuraminyl (sialyl) moiety.
  • non-acidic (or neutral) HMOs are devoid of a sialyl residue, and the acidic HMOs have at least one sialyl residue in their structure.
  • the non-acidic (or neutral) HMOs can be fucosylated or non- fucosylated.
  • Examples of such neutral non-fucosylated HMOs include lacto-N-triose 2 (LNT-II) lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH).
  • LNT-II lacto-N-triose 2
  • LNT lacto-N-triose 2
  • LNnT lacto-N-neotetraose
  • LNnH lacto-N-neohexaose
  • pLNnH para-lacto-N-neohexaose
  • pLNH para-lacto-N-hexao
  • neutral fucosylated HMOs examples include 2'-fucosyllactose (2’-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3’-FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N- fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-
  • acidic HMOs examples include 3’-sialyllactose (3’-SL), 6’- sialyllactose (6’-SL), 3-fucosyl-3’-sialyllactose (FSL), 3’-0-sialyllacto-N-tetraose a (LST a), fucosyl-LST a (FLST a), 6’-0-sialyllacto-N-tetraose b (LST b), fucosyl-LST b (FLST b), 6’-0-sialyllacto-N-neotetraose (LST c), fucosyl-LST c (FLST c), 3’-0-sialyllacto-N-neotetraose (LST d), fucosyl-LST d (FLST d), sialyl- lacto-N-hexaose (SLNH), sialy
  • lactose is not regarded as an HMO species.
  • the disclosure also relates to any commercial use of the genetically engineered cell(s) or the nucleic acid construct(s) disclosed herein.
  • the genetically engineered cell(s) or the nucleic acid construct(s) comprise at least one heterologous protein(s), such as but not limited to i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1 ; and optionally ii) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 or 28 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to any one of SEQ ID NO: 2 or 28.
  • the genetically engineered cell(s) or the nucleic acid construct(s) may further comprise a lactose permease protein as shown in SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3.
  • the genetically engineered cell(s) or the nucleic acid construct(s) may comprise a native or heterologous regulatory element for controlling the expression of the b-1 ,3-N-acetyl-glucosaminyl-transferase(s).
  • the genetically engineered cell(s) or the nucleic acid construct(s) may also comprise a native or heterologous regulatory or episomal element for increasing the expression of the lactose permease.
  • the genetically engineered cell(s) or the nucleic acid construct(s) may comprise a non-functional (or absent) gene product that normally binds to and represses the expression of the b-1 ,3-N-acetyl- glucosaminyl-transferase and/or the lactose permease.
  • the genetically engineered cell or the nucleic acid construct is used in the manufacturing of one or more HMOs.
  • the genetically engineered cell and/or the nucleic acid construct according to the disclosure is used in the manufacturing of LNT-II.
  • the genetically engineered cells as described herein are cultivated according to the procedures known in the art in the presence of a suitable carbon and energy source, e.g. glucose, glycerol or sucrose, and a suitable acceptor, e.g. lactose or any HMO, and the produced HMO blend is harvested from the cultivation media and the microbial biomass formed during the cultivation process. Thereafter, the HMOs are purified according to the procedures known in the art, e.g. such as described in WO2015188834, WO2017182965 or WO2017152918, and the purified HMOs are used as nutraceuticals, pharmaceuticals, or for any other purpose, e.g. for research.
  • a suitable carbon and energy source e.g. glucose, glycerol or sucrose
  • a suitable acceptor e.g. lactose or any HMO
  • the produced HMO blend is harvested from the cultivation media and the microbial biomass formed during the cultivation process.
  • the HMOs are purified according
  • Manufacturing of HMOs is typically accomplished by performing cultivation in larger volumes.
  • the term “manufacturing” and “manufacturing scale” in the meaning of the disclosure defines a fermentation with a minimum volume of 5 L culture broth.
  • a “manufacturing scale” process is defined by being capable of processing large volumes of a preparation containing the product of interest and yielding amounts of the HMO product of interest that meet, e.g., in the case of a therapeutic compound or composition, the demands for clinical trials as well as for market supply.
  • a manufacturing scale method is characterized by the use of the technical system of a bioreactor (fermenter) which is equipped with devices for agitation, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.).
  • a bioreactor which is equipped with devices for agitation, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.).
  • process parameters pH, temperature, dissolved oxygen tension, back pressure, etc.
  • the culture medium may be semi-defined, i.e. containing complex media compounds (e.g. yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined, without any complex compounds.
  • complex media compounds e.g. yeast extract, soy peptone, casamino acids, etc.
  • sucrose is used as the carbon and energy source, a minimal medium might be preferable.
  • the strain engineering strategy of the present invention contributes to a sustainable manufacturing process for the high-level production of LNT-II, where the conversion of the provided carbon source to HMO product in fermentation is done at a high overall yield.
  • concentration of the detected HMOs (in g/L) in each sample was used to calculate the % quantitative differences in the LNT-II content of the strains tested, i.e., the % differences in the LNT-II concentrations formed by strains expressing LgtA, PmnagT or HD0466, or pairwise combinations of all three GlcNAcTs relative to LgtA- expressing cells.
  • manufactured product refers to the one or more HMOs intended as the one or more product HMO(s).
  • the various products are described above.
  • the methods disclosed herein provides both a decreased ratio of by-product to product and an increased overall yield of the product (and/or HMOs in total). This, less by-product formation in relation to product formation facilitates an elevated product production and increases efficiency of both the production and product recovery process, providing superior manufacturing procedure of HMOs.
  • the manufactured product may be a powder, a composition, a suspension, or a gel comprising one or more HMOs.
  • beneficial features for the construction of efficient LNT-II cell factories described above do not provide an additive effect in a single production strain that produces any of the three GlcNAcTs or combinations thereof.
  • the features described in the present disclosure can be exploited only in the way presented here to provide the desired positive effect on LNT-II titers.
  • two given modifications that are proven beneficial for different strain backgrounds should not be expected to enhance LNT-II formation when combined in any of these strain backgrounds.
  • 1 1gtA gene coding for b-1 ,3-N-acetyloglucosamine transferase
  • LacY gene encoding for the lactose permease protein
  • fermentation and culturing are used interchangeably.
  • Lacto-N-triose LNT-II, LNT II, LNT2 and LNT 2 are used interchangeably.
  • the following figures and examples are provided below to illustrate the present disclosure. They are intended to be illustrative and are not to be construed as limiting in any way.
  • LNT-II titers reached by cells expressing a single GlcNAcT or a pairwise combination of GlcNAcTs at the same genomic copy number.
  • LNT-II titers are shown for strains that bear two genomic copies of a single GlcNAcT (LgtA/strain MP2 or PmnagT/strain MP4 or HD0466/strain MP6) and for strains bearing one copy of each of two GlcNAcTs (LgtA & HD0466/strain MP7 or PmnagT & HD0466/strain MP8 or PmnagT & LgtA/strain MP9).
  • the reference level (given as 100%) is shown for strain MP2.
  • LNT-II titers reached by cells expressing different GlcNAcTs with and without the native MFS transporter LacY.
  • LNT-II titers for strains that bear two genomic Pg/pF-driven copies of gene(s) encoding GlcNAcT (s) and a single Pg/pF-driven copy of the lacY gene strains MP10, MP11 , MP12, MP13, MP14, MP15
  • the reference level (given as 100%) is shown for strains MP2, MP4, MP6, MP7, MP8, MP9.
  • LNT-II titers reached by cells bearing beneficial modifications as described in the present disclosure.
  • LNT-II titers for strains that bear a genomic Pg/pF-driven copy of the IgtA and HD0466 genes and of the lacY gene (strains MP13), shown relative to the final LNT-II titers of cells that only express a Pg/pF-driven copy of the IgtA and HD0466 genes (strain MP7).
  • the reference level (given as 100%) is shown for strain MP7.
  • SEQ ID NO: 6 [lacY gene] SEQ ID NO: 28 [PmnagT protein] SEQ ID NO: 29 [PmnagT gene] SEQ ID NO: 41 [GlpR gene]
  • Example 1 Heterologous enzymes appropriate for the construction of efficient LNT-II production systems
  • strains (genetically engineered cells) constructed in the present application were based on Escherichia coli K-12 DH1 with the genotype: F ⁇ , A gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. Additional modifications were made to the E.
  • lacZ deletion of 1.5 kbp
  • nanKETA deletion of 3.3 kbp
  • melA deletion of 0.9 kbp
  • mdoH deletion of 0.5 kbp
  • Plac promoter upstream of the gmd gene lacZ: deletion of 1.5 kbp
  • nanKETA deletion of 3.3 kbp
  • melA deletion of 0.9 kbp
  • mdoH deletion of 0.5 kbp
  • insertion of Plac promoter upstream of the gmd gene deletion of Plac promoter upstream of the gmd gene.
  • MDO platform strain
  • the strains can produce the trisaccharide HMO LNT-II.
  • Each of the six strains expresses a single beta-1 ,3-N-acetyloglucosamine transferase (GlcNAcT) selected from the group consisting of (a) LgtA from Neisseria meningitidis (GenBank ID: WP_033911473.1)(MP1 and MP2), (b) PmnagT from Pasteurella multocida (GenBank ID:
  • WP_014390683.1 (MP3 and MP4), or (c) HD0466 from Haemophilus ducreyi (GenBank ID:
  • WP_010944479.1) MP5 and MP6.
  • the copy number of the GlcNAcT in each strain pair is the only difference among the strains of the pair.
  • the strain MP6 bears two copies of the HD0466 gene while the strain MP5 bears just one Pg/pF-driven copy of this gene.
  • the strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities and subsequently transferred to a medium that allowed induction of gene expression and product formation. More specifically, during day 1 , fresh precultures were prepared using a basal minimal medium supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7,5) in order to start the main culture.
  • BMM basal minimal medium
  • the new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20 % glucose solution (50 ul per 100 ml_) and a bolus of 10 % lactose solution (5 ml per 100 ml).
  • 50 % sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was released at a rate suitable for C-limited growth.
  • the main cultures were incubated for 72 hours at 28 °C and 1000 rpm shaking.
  • the 96-well plates were boiled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC.
  • the initial centrifugation of microtiter plates was followed by the removal of 0.1 ml_ supernatant for direct analysis by HPLC.
  • pellet samples the cells were initially washed, then dissolved in deionized water and centrifuged. Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supernatant.
  • a novel GlcNAcT namely HD0466
  • HD0466 can provide high LNT-II titers when expressed from one or two genomic copies ( Figure 1a).
  • the descending order of activity of the three selected GlcNAcTs on lactose, as it is indirectly revealed by the observed final LNT-II titers is as follows: HD0466 > PmnagT > LgtA.
  • a GlcNAcT is hereby defined as “highly expressed” when the host strain expresses it from at least two Pg/pF-driven genomic copies.
  • Example 2 Combination of heterologous beta-1 ,3-N-acetyloglucosamine transferases is highly beneficial for LNT-II titer enhancement
  • Example 2 Based on the platform strain (“MDO”) described in Example 1 , the modifications summarised in table 2, were made to obtain the fully chromosomal strains MP2, MP4, MP6, MP7, MP8 and MP9. The strains can produce the trisaccharide HMO LNT-II.
  • Each of the six strains bears in total two Pg/pF-driven copies of a single or two beta-1 ,3-N-acetyloglucosamine transferases (GlcNAcTs) selected from the group consisting of (a) LgtA from Neisseria meningitidis (GenBank ID: WP_033911473.1), (b) PmnagT from Pasteurella multocida (GenBank ID: WP_014390683.1), or (c) HD0466 from Haemophilus ducreyi (GenBank ID: WP_010944479.1).
  • GlcNAcTs beta-1 ,3-N-acetyloglucosamine transferases
  • the only difference among the strains is the GlcNAcT(s) being expressed, while the total copy number of the chosen transferase(s) is the same for every strain.
  • the strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities and subsequently transferred to a medium that allowed induction of gene expression and product formation. More specifically, during day 1 , fresh precultures were prepared using a basal minimal medium supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7,5) in order to start the main culture.
  • BMM basal minimal medium
  • the new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20 % glucose solution (50 ul per 100 mL) and a bolus of 10 % lactose solution (5 ml per 100 ml).
  • 50 % sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was released at a rate suitable for C-limited growth.
  • the main cultures were incubated for 72 hours at 28 °C and 1000 rpm shaking. For the analysis of total broth, the 96-well plates were boiled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC.
  • the initial centrifugation of microtiter plates was followed by the removal of 0.1 mL supernatant for direct analysis by HPLC.
  • the cells were initially washed, then dissolved in deionized water and centrifuged. Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supernatant.
  • Strains were characterized in deep well assays and samples were collected from the total broth. All samples were analysed for HMO content by HPLC following the 72-hour protocol described above. The concentration of the detected HMOs (in g/L) in each sample was used to calculate the % quantitative differences in the LNT-II content of the strains tested, i.e., the % differences in the LNT-II concentrations formed by strains expressing LgtA, PmnagT or HD0466, or pairwise combinations of all three GlcNAcTs relative to LgtA-expressing cells (set to 100%).
  • strains bearing a copy of HD0466 and a copy of one of the two other GlcNAcTs show higher titers than strains bearing two identical copies of any of the three GlcNAcTs (strains MP2, MP4 or MP6) or the strain that co-expresses LgtA and PmnagT (strain MP9) at the same GlcNAcT copy number ( Figure 2).
  • Example 3 Genetic manipulation of the native MFS transporter LacY can provide superior LNT-II production systems depending on the beta-1 ,3-N-acetyloglucosamine transferase(s) being expressed
  • strains can produce the trisaccharide HMO LNT-II.
  • Each of these strains bears in total two Pg/pF-d riven copies of a single or two beta-1 ,3-N- acetyloglucosamine transferases (GlcNAcTs) selected from the group consisting of (a) LgtA from Neisseria meningitidis (GenBank ID: WP_033911473.1), (b) PmnagT from Paste urella multocida (GenBank ID: WP_014390683.1), or (c) HD0466 from Haemophilus ducreyi (GenBank ID:
  • all strains express one or two GlcNAcTs from a total of 2 genomic copies with the strains differing in the identity of the selected GlcNAcTs. Moreover, apart from the identity of GlcNAcTs, the strains can differ in regard to the expression of the E. coli K12 lactose permease LacY (GenBank ID: NP_414877.1) (Table 3). In the present Example, it is demonstrated that the over-expression of the gene encoding the native lactose permease LacY can be beneficial only when specific GlcNAcTs, namely HD0466 and LgtA, are co-expressed in the same strain (MP13).
  • Examples 2 and 3 provide enough data to conclude that the combined expression of HD0466 and LgtA results in higher LNT-II titers regardless of intracellular lactose levels compared to when only one of these GlcNAcTs is expressed by the cell at the same copy number. Importantly, this trend is unique for this GlcNAcT pair and it is not observed for any other GlcNAcT pair.
  • the strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities and subsequently transferred to a medium that allowed induction of gene expression and product formation. More specifically, during day 1 , fresh precultures were prepared using a basal minimal medium supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7,5) in order to start the main culture.
  • BMM basal minimal medium
  • the new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20 % glucose solution (50 ul per 100 ml_) and a bolus of 10 % lactose solution (5 ml per 100 ml).
  • 50 % sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was released at a rate suitable for C-limited growth.
  • the main cultures were incubated for 72 hours at 28 °C and 1000 rpm shaking.
  • the 96-well plates were boiled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC.
  • the initial centrifugation of microtiter plates was followed by the removal of 0.1 ml_ supernatant for direct analysis by HPLC.
  • pellet samples the cells were initially washed, then dissolved in deionized water and centrifuged. Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supernatant.
  • Strains were characterized in deep well assays and samples were collected from the total broth. All samples were analysed for HMO content by HPLC following the 72-hour protocol described above. The concentration of the detected HMOs (in g/L) in each sample was used to calculate the % quantitative differences in the LNT-II content of the strains tested, i.e., the % differences in the LNT-II concentrations formed by strains expressing one of the three GlcNAcTs, namely LgtA, PmnagT or HD0466, or pairwise combinations of all three GlcNAcTs relative to similar strains that also express the native transporter LacY.

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Abstract

This invention relates to multiple genetic engineering approaches applied to enhance the formation of the human milk oligosaccharide (HMO) lacto-N-triose II (LNT-II). Several aspects towards the construction of a beneficial LNT-II cell factory have been investigated, including the LNT-II biosynthesis pathway per se, along with the cellular lactose import mechanism was investigated as to promote the cellular LNT-II production.

Description

CELL FACTORIES FOR LNT-II PRODUCTION
FIELD
This disclosure relates to a method of producing human milk oligosaccharides (HMOs) with high titres, wherein the HMO is LNT-II. Several genetic engineering approaches have been applied to change the abundance of the HMO produced by cells that express a heterologous b-1 ,3-N-acetyl- glucosaminyltransferase. The strain engineering strategies to achieve this goal depend on the b-1 ,3-N- acetyl-glucosaminyltransferase introduced in the host. The disclosure highlights several strain engineering strategies to enable the formation of LNT-II at high titres in the E. coli DH1 K12 host. Specifically, these strategies deal with different aspects for the design of an efficient LNT-II production system, which include the introduction and optimization of the pathway enabling LNT-II biosynthesis, the modification and/or introduction of a lactose and combinatorial solutions thereof.
BACKGROUND
Human milk represents a complex mixture of carbohydrates, fats, proteins, vitamins, minerals and trace elements. The by far most predominant fraction is represented by carbohydrates, which can be further divided into lactose and more complex oligosaccharides (Human Milk Oligosaccharides, HMO). Whereas lactose is used as an energy source, the complex oligosaccharides are not metabolized by the infant. The fraction of complex oligosaccharides accounts for up to 1/10 of the total carbohydrate fraction and consists of probably more than 150 different oligosaccharides. The occurrence and concentration of these complex oligosaccharides are specific to humans and thus cannot be found in large quantities in the milk of other mammals, like for example domesticated dairy animals.
To date, the structures of at least 115 HMOs have been determined, and considerably more are probably present in human milk. HMOs have become of great interest in the last decade, due to the discovery of their important functionality in human development. Besides their prebiotic properties, HMOs have been linked to additional positive effects, which expands their field of application. The health benefits of HMOs have enabled their approval for use in foods, such as infant formulas and foods, and for consumer health products.
To bypass the drawbacks associated with the chemical synthesis of HMOs, several enzymatic methods and fermentative approaches have been developed. Fermentation based processes have traditionally been developed for individual HMOs such as 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, lacto- N-neotetraose, 3'-sialyllactose and 6'-sialyllactose. Fermentation based processes typically utilize genetically engineered bacterial strains, such as recombinant Escherichia coli (E. coli ) (see for example Bych et al 2019 Current Opinion in Biotechnology 56:103-137), Corynebacterium glutamicum or Bacillus subtilis (see for example Lu et al 2021 ACS Synth. Biol. 10:923-938) or in recombinant yeast cells, such as Saccharomyces cerevisiae and Yarrowia lipolytica (see for example Hollands et al 2019 Metabolic Engineering 52: 232-242). Biotechnological production, such as a fermentation process, of HMOs is a valuable, cost-efficient and large-scale approach to HMO manufacturing. It relies on genetically engineered bacteria constructed so as to express the glycosyltransferases needed for synthesis of the desired oligosaccharides and takes advantage of the bacteria’s innate pool of nucleotide sugars as HMO precursors. For example, CN 111979168 addresses the production of lactoyl-N-triose II (LNT-II) by use of a genetically engineered E. coli host, wherein genetic manipulations to the N-acetylglucosamine (GlcNAc) glycolysis pathway resulted in an increased yield of LNT-II
At present, knowledge as to how to engineer cells to produce high titers of LNT-II, and how to select the optimal glycosyltransferases and subsequent genetic modifications which enable LNT-II production is limited, since LNT-II in many regards is considered a precursor HMO for production of more complex oligosaccharides.
SUMMARY
This disclosure relates to multiple genetic engineering approaches applied to enhance the formation of the human milk oligosaccharide (HMO) lacto-N-triose II (LNT-II). Several aspects towards the construction of a beneficial LNT-II cell factory have been investigated, including the LNT-II biosynthesis pathway perse, but also a mechanism that enhances lactose import into the cell interior, altogether enhancing the LNT-II titers.
We have observed that most genetic manipulations affected the level of lacto-N-triose II (LNT-II), produced, while only specific genetic modifications enhanced the LNT-II titer. This is particularly true for cells expressing the b-1 ,3-N-acetyl-glucosaminyltransferase, HD0466 (GenBank ID: WP_010944479.1), where expression of a single copy enhanced the production of LNT-II over cells expressing the b-1 ,3-N- acetyl-glucosaminyltransferase LgtA (GenBank ID: WP_033911473.1).
Furthermore, combination of the b-1 ,3-N-acetyl-glucosaminyltransferase HD0466 with a second b-1 ,3-N- acetyl-glucosaminyltransferase LgtA or PmnagT (WP_014390683.1) was capable of further enhancing the LNT-II titer. Also, combining expression of the b-1 ,3-N-acetyl-glucosaminyltransferases HD0466 and LgtA with overexpression of the E. coli native lactose permease also enhanced the LNT-II titer.
This disclosure highlights ways of achieving high LNT-II titers related to strain engineering strategies. The strain engineering strategies to achieve this goal comprise the manipulation of the following genetic traits of the HMO producer cell:
1. Introduction of a specific b-1 ,3-N-acetyl-glucosaminyltransferase, namely HD0466; and
2. Introduction of a second b-1 ,3-N-acetyl-glucosaminyltransferase, namely LgtA and/or PmnagT; or
3. Over-expression the native gene lacY encoding the lactose permease LacY in cells expressing the b-1 ,3-N-acetyl-glucosaminyltransferases HD0466 and LgtA. As stated in point 3 above, an important rate determining step in the synthesis of LNT-II is the availability of lactose. It is therefore advantageous if the genetically engineered cell of the present invention is capable of taking up lactose, to enable synthesis of LNT-II from lactose as the starting point.
This disclosure enables the skilled person to produce HMOs, primarily LNT-II, in enhanced amounts.
The enhanced amounts of LNT-II, gives a more sustainable manufacturing process; valuable HMOs are not discarded during the purification process and the conversion from carbon source to HMO product in fermentation is thus done at a higher overall yield.
In its broadest aspect, the present disclosure relates to a method for the production of LNT-II, the method comprising the steps of: a. providing a genetically engineered cell capable of producing an HMO, wherein said cell comprises i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i); and b. culturing the cell according to (a) in a suitable cell culture medium; and c. harvesting the HMO(s) produced in step (b).
In the above aspect, the capability of the cell to take up lactose is advantageous, when synthesizing LNT- II from lactose, it is therefore preferred that said cell is able to import lactose into the cell.
In another aspect, the present disclosure relates to a genetically engineered cell comprising a) a nucleic acid sequence according to SEQ ID NO: 4 or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 4, encoding a heterologous b-1 ,3-N-acetyl- glucosaminyl-transferase, and b) a native or heterologous regulatory element for controlling the expression of a).
In a third aspect, the disclosure relates to a nucleic acid construct comprising a nucleic acid sequence encoding i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i). In a fourth aspect, the disclosure relates to use of a genetically engineered cell, or a nucleic acid construct according to the present disclosure to produce LNT-II.
DETAILED DESCRIPTION
This disclosure enables the skilled person to produce HMOs, primarily LNT-II, in enhanced amounts. In this manner, the present disclosure first of all pinpoints and compares different b-1 ,3-N-acetyl- glucosaminyl transferases that can efficiently convert lactose to LNT-II and indicates how their expression can be balanced for reaching optimal LNT-II production.
Secondly, it shows that LNT-II production systems that simultaneously express specific combinations of b-1 ,3-N-acetyl-glucosaminyl transferases are more productive than the ones expressing the same transferases separately at the same copy number.
Thirdly, it shows that the over-expression of the native E. coli gene lacY, encoding lactose permease, at levels that are higher than the normal cell physiological level can further enhance the synthesis of LNT-II in cells expressing more than one b-1 ,3-N-acetyl-glucosaminyl transferases - but not necessarily, when a single transferase is expressed at the same copy number.
According to the present disclosure, the E. coli DH1 K12 host can be engineered to form LNT-II at high levels by introducing the b-1 ,3-N-acetyl-glucosaminyl transferase HD0466 from Haemophilus ducreyi (SEQ ID NO: 1 , or functional homologues thereof), which can be further combined with the expression of the b-1 ,3-N-acetyl-glucosaminyl transferase LgtA from Neisseria meningitidis (SEQ ID NO: 2, , or functional homologues thereof) or PmnagT from Pasteurella multocida (SEQ ID NO: 28, , or functional homologues thereof), and the over-expression of the native E. coli lacY gene to reach even higher LNT-II levels.
Thus, the enhanced amounts of LNT-II, gives a more sustainable manufacturing process; valuable HMOs are not discarded during the purification process and the conversion from carbon source to HMO product in fermentation is thus done at a higher overall yield.
Exemplary methods HD0466
In Example 1 , it is demonstrated how different GlcNAc transferases(GlcNAcTs) can be beneficially expressed at varied genomic copy numbers in the genetic background of E. coli K12 cells for high-level LNT-II production. The Example reveals the HD0466 enzyme as a novel enzyme for the in vivo production of LNT-II. In one or more exemplary embodiments, the method for the production of LNT-II comprises the steps of: a. providing a genetically engineered yeast or bacterial cell capable of producing an HMO, wherein said cell comprises i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i); and b. culturing the cell according to (a) in a suitable cell culture medium; and c. harvesting the HMO(s) produced in step (b).
In preferred embodiments, said cell is able to import lactose into the cell.
Moreover, the present disclosure shows marked gains in LNT-II titers when different b-1 ,3-N-acetyl- glucosaminyl transferases are simultaneously expressed in the same production cell compared to LNT-II producers expressing a single transferase. According to the present disclosure, the most marked increase in LNT-II titers could be achieved by combined expression of the HD0466 and LgtA enzymes.
In a further embodiment the b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof is expressed together with a second b-1 ,3-N-acetyl- glucosaminyl transferase.
In Example 2, it is demonstrated that the pairwise expression of two different GlcNAcTs can be a more efficient approach in converting E. coli K12 cells to an efficient LNT-II cell factory than merely expressing a single GlcNAcT.
HD0466/lgtA
Thus, in one or more exemplary embodiments the method for the production of LNT-II comprises the steps of: a. providing a genetically engineered yeast or bacterial cell capable of producing an HMO, wherein said cell comprises i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i); and iii) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 [LgtA], or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 2; and iv) a native or heterologous regulatory element for controlling the expression of iii), b. culturing the cell according to (a) in a suitable cell culture medium; and c. harvesting the HMO(s) produced in step (b).
In preferred embodiments, said cell is able to import lactose into the cell
HD0466/PmnagT
In one or more exemplary embodiments the method comprises the steps of: a. providing a genetically engineered bacterial or yeast cell capable of producing an HMO, wherein said cell comprises: i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i); and iii) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 28 [PmnagT], or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 28; and iv) a native or heterologous regulatory element for controlling the expression of iii), and b. culturing the cell according to (a) in a suitable cell culture medium; and c. harvesting the HMO(s) produced in step (b).
In preferred embodiments, said cell is is able to import lactose into the cell. The import of the lactose is preferably balanced with the level of b-1 ,3-N-acetyl-glucosaminyl-transferase activity. In one embodiment the lactose is imported into the cell via the cells native lactose importer, such as lactose permease. In one embodiment no additional lactose permease activity is added to the cell. In one embodiment the expression level of b-1 ,3-N-acetyl-glucosaminyl-transferases is controlled such that it is higher than the expression level of a lactose permease.
HD0466/LgtA/LacY
Since our goal is to convert e.g., the E. coli host to a cell factory for LNT-II production, a rational genetic engineering program includes the following major focus areas: a) the introduction one or more highly active b-1 ,3-N-acetyl-glucosaminyl transferase for the conversion of the externally added lactose to LNT- II and b) the enhancement of lactose import into the cells. The genetic manipulation of genes involved in these cellular procedures could theoretically provide marked product yield gains. In addition, the over-expression of the lacY gene in the cells expressing more than one b-1 ,3-N-acetyl- glucosaminyl transferases, namely HD0466 and LgtA, can be advantageous for high-level LNT-II production, as seen in Example 3, the over-expression of the gene encoding the native lactose permease LacY can be beneficial for example when specific GlcNAcTs, namely HD0466 and LgtA, are coexpressed in the same strain.
In one or more exemplary embodiments the method comprises the steps of: a. providing a genetically engineered bacterial or yeast cell capable of producing an HMO, wherein said cell comprises i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 1 ; and ii) a native or heterologous regulatory element for controlling the expression of i); and iii) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 [LgtA], or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 2; and iv) a native or heterologous regulatory element for controlling the expression of iii), v) a lactose permease protein as shown in SEQ ID NO: 3 [LacY], or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 3, and vi) a native or heterologous regulatory element for controlling the expression of v) and b. culturing the cell according to (a) in a suitable cell culture medium; and c. harvesting the HMO(s) produced in step (b).
The LNT-II enzymes
The present disclosure demonstrates the superior activity of another enzyme, namely HD0466 from Haemophilus ducreyi, towards LNT-II synthesis. Moreover, it is here shown that the combinations of different enzymes of the same type can be a highly beneficial approach for achieving high LNT-II titers.
For the production of LNT-II, the genetically engineered cells comprise all the required enzymes to facilitate the production LNT-II. One of these enzymes may for example be i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 %, such as at least 99 % or such as at least 99.9 % identical to SEQ ID NO: 1 . The above enzyme can be exchanged or supplemented by others with similar functionality. Especially, SEQ ID NO: 1 , which can be supplemented with SEQ ID NO: 2 and/or SEQ ID NO: 28. When supplementing with SEQ ID NO: 2 or SEQ ID NO: 28, the level of produced LNT-II during culturing becomes increases, as shown in Example 2.
Heterologous b- 1, 3-N-acetyl-glucosaminyl-transferase
A heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase is any protein which comprises the ability of transferring the N-acetyl-glucosamine of UDP-N-acetyl-glucosamine to lactose. The b-1 ,3-N-acetyl- glucosaminyl-transferase used herein does not originate in the species of the genetically engineered cell i.e. the gene encoding the b-1 ,3-galactosyltransferase is of heterologous origin. The examples below use the heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase HD0466, LgtA and/or PmnagT.
HD0466 genes
In one or more exemplary embodiments, the HD0466 gene is as shown in SEQ ID NO: 4, or a functional homologue thereof having a nucleotide sequence that is at least 70 %, such as at least 75 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 4.
IgtA genes
In one or more exemplary embodiments, the IgtA gene is as shown in SEQ ID NO: 5 or is a functional homologue thereof having a nucleotide sequence that is at least 70 %, such as at least 75 %, such as at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 5.
PmnagT genes
In one or more exemplary embodiments, the PmnagT gene is as shown in SEQ ID NO: 29, or a functional homologue thereof having a nucleotide sequence that is at least 70 %, such as at least 75 %, such as at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 29.
In one or more exemplary embodiments, the heterologous b-1 ,3-N-acetyl-glucosaminyltransferases of the present disclosure that may, upon expression, be used to produce LNT-II and/or HMOs using LNT-II as a precursor molecule, are shown in the below matrix.
Figure imgf000010_0001
In one or more exemplary preferred embodiments, the heterologous b-1 ,3-N-acetyl- glucosaminyltransferases and the HMO LNT-II can be generated using the protein of the amino acid sequence SEQ ID NO: 1 [HD0466] in combination enzymes are shown in the below matrix.
Figure imgf000010_0002
Lactose permease
HD0466, can provide high LNT-II titers when expressed from one or two genomic copies (Figure 1a). The descending order of activity of the three selected GlcNAcTs on lactose, as it is indirectly revealed by the observed final LNT-II titers is as follows: HD0466 > PmnagT > LgtA. The LNT-II titers reached by the strains MP5 and/or MP6, which express HD0466 from a different copy number, can be up to 40% or 15% higher than for strains expressing LgtA (strain MP1) or PmnagT (strain MP3), respectively.
In the present invention, lactose is used as the substrate for the synthesis of LNT-II. Thus, a genetically engineered cell of the present invention should be capable of importing lactose into the cell. While lactose is naturally imported into some microorganisms, other microorganisms lack the ability to do so. To enable lactose import, such microorganisms would need to be genetically engineered to take up lactose. Thus, in embodiments of the present invention, the genetically engineered cell of the present invention, is able to import lactose into the cell. One way to enable lactose import into a cell of the present invention is by expression of a lactose permease. In microorganisms comprising a lactose import pathway, the overexpression of an endogenous lactose import pathway, such as but not limited to an endogenous lactose permease protein, and/or incorporation of a heterologous lactose import pathway, such as but not limited to a heterologous lactose permease, may be used to enhance the lactose import of said microorganism and thereby enhance LNT-II production. Thus, in embodiments of the present invention, the genetically engineered cell of the present invention overexpresses an endogenous lactose permease protein and/or expresses a heterologous lactose permease.
Lactose permease is a membrane protein which is a member of the major facilitator superfamily and can be classified as a symporter, which uses the proton gradient towards the cell to transport b-galactosides such as lactose in the same direction into the cell. In HMO production, lactose is the molecule being decorated to produce any HMO of interest and bioconversions happen in the cell interior. Thus, there is a desire to be able to import lactose into the cell, which can mainly be done by a certain activity of lactose permease, e.g., the native lacY copy under the control of a promoter.
In one or more exemplary embodiments, the lactose permease protein is as shown in SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence which is at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 3.
In one or more exemplary embodiments, the lactose permease gene is a nucleotide sequence as shown in SEQ ID NO: 6 or is a functional homologue thereof having a nucleotide sequence that is at least 70 %, such as at least 75 %, such as at least 80 %, such as at least 85 %, such as at least 90 %, such as at least 95 % or such as at least 99 % identical to SEQ ID NO: 6.
Lactose permease over-expression
As shown in Example 3, the over-expression of the lacY gene coding lactose permease is used as a genetic tool to obtain an enhanced level of LNT-II produced by the genetically engineered cell of the present disclosure. As is shown in Example 3 only the combined expression of a specific pair of b-1 ,3-N- acetyl-glucosaminyltransferases and lacY over-expression results in an enhanced LNT-II production.
The genetically engineered cells disclosed herein may comprise a regulatory element for increasing the expression of the native lactose permease protein, such as but not limited to Ribosome Binding Sites (RBSs). The RBSs may for example be the Shine-Dalgarno (SD) sequence. Mutations in the Shine- Dalgarno sequence can reduce or increase translation in prokaryotes. This change is due to a reduced or increased mRNA-ribosome pairing efficiency, as evidenced by the fact that compensatory mutations in the 3'-terminal 16S rRNA sequence can restore translation. The regulatory element for increasing the expression of the native lactose permease protein could also be a promoter.
The genetically engineered cells disclosed herein may also comprise a heterologous episomal element for increasing the expression of the native lactose permease protein. This could for example be a plasmid-borne lacY gene.
The increased expression of the lactose permease may be achieved by direct integration of a copy of the lacY gene in the genome. In this manner, Examples 2 and 3 provide enough data to conclude that the combined expression of HD0466 and LgtA results in higher LNT-II titers regardless of intracellular lactose levels compared to when only one of these GlcNAcTs is expressed by the cell at the same copy number. Importantly, this trend is unique for this GlcNAcT pair and it is not observed for any other GlcNAcT pair that can be formed from HD0466, LgtA and PmnagT.
The increased expression of the lactose permease may be achieved by deleting a repressor of the lactose operon. An example of such being the lad gene - UniProtKB - P03023 (LACI_ECOLI).
As shown in the examples, an additional genomic copy of the lacY gene which encodes the lactose permease under control of the PglpF promoter, in a HD0466 expressing cell resulted in a decrease in produced LNT-II. Contrary, the over-expression of the /acYgene by an additional genomic PglpF-driven copy in HD0466- and LgtA- expressing cells resulted in an increase in produced LNT-II, as is shown in Example 3.
Amino acid identity
In one or more exemplary embodiments, the amino acid sequence of the proteins of the present disclosure as shown in SEQ ID NO: 1-3 and 28, is at least 80% identical to SEQ ID NO: 1-3 and 28, such as at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85 % identical, at least 86 % identical, at least 87 % identical, at least 88 % identical, at least 89 % identical, at least 90 % identical, at least 91 % identical, at least 92 % identical, at least 93 % identical, at least 94 % identical, at least 95 % identical at least 96 % identical, at least 97 % identical, at least 98 % identical, or at least 99 % identical.
Controlling the expression
In the present context the term “controlling the expression” relates to gene expression where the transcription of a gene into mRNA and its subsequent translation into protein is controlled. Gene expression is primarily controlled at the level of transcription, largely as a result of binding of proteins to specific sites on DNA, such as but not limited to regulatory elements. As described above, engineering strategy to control expression can be applied in multiple ways
1) the copy number of the gene of interest,
2) controlling the expression of any copy of these genes at the transcriptional or the translational level using heterologous or native regulatory elements,
3) the deletion of regulators that repress the expression of key genes in the HMO production process,
4) the over-expression of regulators that activate and/or enhance the expression of key genes in the HMO production process.
Over-expression
A variety of molecular mechanisms ensures that genes are expressed at the appropriate level and under conditions of relevance to the applied production process. For instance, the regulation of transcription can be summarized into the following routes of influence; genetic (direct interaction of a control factor with the gene of interest), modulation and/or interaction of a control factor within the transcriptional machinery and epigenetic (non-sequence changes in DNA structure that influence transcription).
It is known that a reduction in gene expression below a critical threshold for any gene will result in a mutant phenotype, since such a defect essentially mimics either a partial or complete loss of function of the target gene, whereas increased expression of a native gene can be both beneficial or disruptive to a cell or organism.
Over-expression of a gene may be achieved directly by transcriptional activators that bind to key gene regulatory sequences to promote transcription or enhancers that constitute sequence elements positively affecting transcription. Similarly, direct over-expression of a gene can be achieved by simply increasing its copy number in the genome, or replacing its native promoter with a promoter of higher strength or even modifying the sequence controlling the binding of the corresponding mRNA to the ribosomes, i.e. the Shine-Dalgarno sequence being present upstream of the gene’s coding sequence.
Moreover, over-expression of a gene may also be achieved indirectly through the partial or full inactivation of transcriptional repressors that normally bind key regulatory sequences around the coding sequence of the gene of interest and thereby inhibit its transcription.
The term “over-expression” that is used here may for example refer to the native gene lacY and includes 1) the replacement of the native promoter of the any E. coli gene by another, stronger promoter, 2) the modification of the native Shine-Dalgarno sequence of these genes by a stronger sequence with the goal of promoting ribosomal binding, 3) the deletion of the gene encoding a direct repressor or the enhancement of the expression of a gene encoding a direct activator of the native promoter of the E. coli gene(s) of interest, 4) the increase in the copy number of the gene(s) of interest, where the gene(s) are expressed from a genomic locus other than the native locus and the expression is driven by the native or a synthetic promoter (e.g., PglpF), and 5) the episomal expression of the gene(s) of interest from a low (5-10 copies per cell) to a high-copy number plasmid (300-500 copies per cell).
Thus, in one or more exemplary embodiments, the over-expression of the b-1 ,3-N-acetyl-glucosaminyl- transferase proteins and/or the lactose permease of the present disclosure is provided by increasing the copy number of the genes coding said protein(s), and/or by choosing an appropriate element for or adding an extra genomic copy for the genes encoding the b-1 ,3-N-acetyl-glucosaminyl-transferase proteins and/or the genes encoding the lactose permease, and/or conferring a non-functional (or absent) gene product that normally binds to and repress the expression of any of the the b-1 ,3-N-acetyl- glucosaminyl-transferase proteins and/or the lactose permease of the present disclosure.
Increasing the copy number
As shown in Table 1 below, the only difference among the strains is the beta-1 ,3-N-acetyloglucosamine transferase being expressed or the copy number of the chosen transferase.
Copy number variation is a type of structural variation: specifically, it is a type of duplication or multiplication of a considerable number of base pairs. Specifically, in the context of the present invention the copy number refers to the number of sequences (genes) encoding the desired protein. The sequences may be located several places in the genome of the genetically modified cell, but they can also be inserted in one place operated by a single promoter.
In one or more exemplary embodiments, expression is controlled by increasing the copy number of the gene encoding the desired protein.
Thus, in one or more exemplary embodiments, the present disclosure relates to a method, wherein the overexpression of the b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease is provided by increasing the copy number of any of the genes coding for said protein(s) and/or by choosing an appropriate regulatory element.
Regulatory element
The genetically engineered cell according to the methods described herein may comprise regulatory elements enabling the controlled overexpression of endogenous or heterologous and/or synthetic nucleic acid sequences. In one or more exemplary embodiments, the regulatory element for controlling and increasing the expression of the heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease protein(s) in the method(s) described above is a promoter.
The term “regulatory element", comprises promoter sequences, signal sequence, and/or arrays of transcription factor binding sites, which sequences affect transcription and/or translation of a nucleic acid sequence operably linked to the regulatory element.
Regulatory elements are found at transcriptional and post-transcriptional levels and further enable molecular networks at those levels. For example, at the post-transcriptional level, the biochemical signals controlling mRNA stability, translation and subcellular localization are processed by regulatory elements. RNA binding proteins are another class of post-transcriptional regulatory elements and are further classified as sequence elements or structural elements. Specific sequence motifs that may serve as regulatory elements are also associated with mRNA modifications. A variety of DNA regulatory elements are involved in the regulation of gene expression and rely on the biochemical interactions involving DNA, the cellular proteins that make up chromatin, gene activators and repressors, and transcription factors.
In general, the transcriptional and translational regulatory sequences include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, binding sites for gene regulators and enhancer sequences.
Promoters and enhancers are the primary genomic regulatory components of gene expression.
Promoters are DNA regions within 1-2 kilobases (kb) of a gene’s transcription start site (TSS); they contain short regulatory elements (DNA motifs) necessary to assemble RNA polymerase transcriptional machinery. However, transcription is often minimal without the contribution of DNA regulatory elements located more distal to the TSS. Such regions, often termed enhancers, are position-independent DNA regulatory elements that interact with site-specific transcription factors to establish cell type identity and regulate gene expression. Enhancers may act independently of their sequence context and at distances of several to many hundreds of kb from their target genes through a process known as looping. Because of these features, it is difficult to identify suitable enhancers and link them to their target genes based on DNA sequence alone.
The promoter, together with other transcriptional and translational regulatory nucleic acid sequences (also termed "control sequences") is necessary to express a given gene or group of genes (an operon).
Identification of suitable promoter sequences that promote the expression of the specific gene of interest is a tedious task, which in many cases requires laborious efforts. In relation to the present disclosure regulatory elements may or may not be post-translational regulators or it may or may not be translational regulators. Thus, in one embodiment of the disclosure the regulatory element comprises one or more elements capable of enhancing the expression, of the one or more nucleic acid sequence(s) according to the present disclosure.
In that regard the regulatory element, controlling the expression of nucleic acid sequences and/or genes encoding one or more glycosyltransferases and/or a lactose permease protein may be a promoter sequence.
In carrying out the methods as disclosed herein, different or identical promoter sequences may be used to drive transcription of different genes of interest integrated into the genome of the host cell or into episomal DNA.
Native
In relation to the present disclosure, the term “native” and “endogenous” are used interchangeably and refers to nucleic acid sequences originating from the genome of the genetically engineered cell according to the method of the disclosure. In that regard a nucleic acid sequence may be considered native if it originates from the E. coli K12 strain, and is not of heterologous origin and not a recombined nucleic acid sequence, with respect to the genetically engineered cell. Also, an amino acid sequence may be considered endogenous if it originates from the host cell and is thus not of heterologous origin, with respect to the genetically engineered cell.
Heterologous regulatory element
A regulatory element may be endogenous or heterologous, and/or recombinant and/or synthetic nucleic acid sequences. In the present context, the term “heterologous regulatory element” is to be understood as a regulatory element that is not endogenously found in that genomic locus or heterologous to the original genetically engineered cell described herein. As an example, a promoter sequence of the E. coli glpFKX operon, such as PglpF, in front of the native lactose permease is considered to be a heterologous regulatory element. The heterologous regulatory element may also be a recombinant regulatory element, wherein two or more non-operably linked native regulatory element(s) are recombined into a heterologous and/or synthetic regulatory element. The heterologous regulatory element, may be introduced into the genetically engineered cell using methods known to the person skilled in the art.
In the context of the present invention a promoter which is regulating the transcription of a gene it is not naturally regulating is also considered to be heterologous, even if it is derived from another genomic locus in the same strain. Promoter sequences
The regulatory element or elements regulating the expression of the genes and/or nucleic acid sequence(s), may comprise one or more promoter sequence(s), wherein the promoter sequence, is operably linked to the nucleic acid sequence of the gene of interest in that sense regulating the expression of the nucleic acid sequence of the gene of interest.
In one or more exemplary embodiments, the heterologous regulatory element is a promoter sequence.
In general, a promoter may comprise native, heterologous and/or synthetic nucleic acid sequences, and may be a recombinant nucleic acid sequence, recombining two or more nucleic acid sequences or same or different origin as described above, thereby generating a homologous, heterologous or synthetic nucleic promoter sequence, and/or a homologous, heterologous or synthetic nucleic regulatory element.
In one or more exemplary embodiments, the regulatory element of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell comprises more than one native or heterologous promoter sequence.
In one or more exemplary embodiments, the regulatory element of the genetically engineered cell comprises a single promoter sequence.
In one or more exemplary embodiments, the regulatory element of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell comprises two or more regulatory elements with identical promoter sequences.
In one or more exemplary embodiments, regulatory element of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell comprises two or more regulatory elements with nonidentical promoter sequences.
The regulatory architectures i.e., gene-by-gene distributions of transcription-factor-binding sites and identities of the transcription factors that bind those sites can be used multiple different growth conditions and there are more than 100 genes from across the E. coli genome, which acts as regulatory elements. Thus, any promoter sequence enabling transcription and/or regulation of the level of transcription, of one or more heterologous or native nucleic acid sequences that encode one or more proteins as described herein may be suitable.
The promoter may be of heterologous origin, native to the genetically modified cell or it may be a recombinant promoter, combining heterologous and/or native elements. One way to increase the production of a product may be to regulate the production of the desired enzyme activity used to produce the product or precursor/substrate import, such as the glycosyltransferases or a lactose permease. Increasing the promoter strength driving the expression of the desired enzyme may be one way of doing this. The strength of a promoter can be assed using a lacZ enzyme assay where b-galactosidase activity is assayed as described previously (see e.g. Miller J.H. Experiments in molecular genetics, Cold spring Harbor Laboratory Press, NY, 1972). Briefly the cells are diluted in Z-buffer and permeabilized with sodium dodecyl sulfate (0.1%) and chloroform. The LacZ assay is performed at 30°C. Samples are preheated, the assay initiated by addition of 200 pi ortho-nitro-phenyl-p-galactosidase (4 mg/ml) and stopped by addition of 500 pi of 1 M Na2CC>3 when the sample had turned slightly yellow. The release of ortho-nitrophenol is subsequently determined as the change in optical density at 420 nm. The specific activities are reported in Miller Units (MU) [A420/(min*ml*A600)]. A regulatory element with an activity above 10,000 MU is considered strong and a regulatory element with an activity below 3,000 MU is considered weak, what is in between has intermediate strength. An example of a strong regulatory element is the PglpF promoter with an activity of approximately 14.000 MU and an example of a weak promoter is Plac which when induced with IPTG has an activity of approximately 2300 MU.
Alternatively, if there is a need for balancing the expression level of one or more proteins to optimize the production it may be beneficial to use a promoter with the desired strength, e.g., middle or low strength. Table 4 below lists a series of wildtype and recombinant promoters according to their strength relative to the PglpF promoter.
Table 4 - Promoter sequences according to strength
Figure imgf000018_0001
Figure imgf000019_0001
na = not accessed
*The promoter activity is assessed in the LacZ assay described below with the PglpF promoter run as positive reference in the same assay. To compare across assays the activity is calculated relative to the PglpF promoter, a range indicates results from multiple assays
In one or more exemplary embodiments, the regulatory element is selected from the group consisting of PBAD, Pxyl, PsacB, PxylA, PrpR, PnitA, PT7, Ptac, PL, PR, PnisA, Pb, Pscr, Pscr_SD1, Pscr_SD7, PgatY_70UTR, PglpF, PglpF_SD1, PglpF_SD10, PglpF_SD2, PglpF_SD3, PglpF_SD4, PglpF_SD5, PglpF_SD6, PglpF_SD7, PglpF_SD8, PglpF_SD9, PglpF_B28, Plac_16UTR, Plac, PmglB_70UTR and PmglB_ 70UTR_SD4.
In one or more exemplary embodiments, the regulatory element is a promoter selected from the group consisting of PglpF (SEQ ID NO: 11), PglpT(SEQ ID NO: 38), Plac (SEQ ID NO: 25, PmgIB (SEQ ID NO: 26, PglpA (SEQ ID NO: 37), and variants thereof. Specifically, the variants disclosed in table 4 are preferred.
In one or more exemplary embodiments, the regulatory element is a promoter with high or middle strength, such as a promoter sequence selected from the group consisting of PmglB_70UTR_SD8, PmglB_70UTR_SD10, PmglB_54UTR, Plac_70UTR, PmglB_70UTR_SD9, PmglB_70UTR_SD4, PmglB_70UTR_SD5, PglpF_SD4, PmglB_70UTR_SD7, PmglB_70UTR, PglpA_70UTR, PglpT_70UTR, pgatY_70UTR, PglpF, PglpF_SD10, PglpF_SD5, PglpF_SD8, PglpF_B28, PglpF_B29, PmglB_16UTR, PglpF_SD9, PglpF_SD7, PglpF_SD6 and PglpA_16UTR. In on referred embodiment the promoter is a strong promoter selected from the group consisting of PmglB_70UTR_SD8, PmglB_70UTR_SD10, PmglB_54UTR, Plac_70UTR, PmglB_70UTR_SD9, PmglB_70UTR_SD4, PmglB_70UTR_SD5, PglpF_SD4, PmglB_70UTR_SD7, PmglB_70UTR, PglpA_70UTR, PglpT_70UTR, pgatY_70UTR, PglpF, PglpF_SD10, PglpF_SD5, PglpF_SD8, andPmglB_16UTR. This may in particular be advantageous for the expression the heterologous b-1 ,3-N- acetyl-glucosaminyl-transferase.
In another embodiment the promoter is selected from the group consisting of promoters with middle strength, such as PglpF_SD9, PglpF_SD7, PglpF_SD6 and PglpA_16UTR.
In another embodiment the promoter is selected from the group consisting of promoters with low strength, such as Plac_wt. PglpF_SD3 and PglpF_SD1. This may in particular be advantageous for the expression the lactose permease.
In preferred embodiments, the regulatory element is PglpF or Plac, or a variant of PglpF or Plac.
In a preferred exemplary embodiment, the promoter sequence comprised in the regulatory element for the regulation of the expression of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell, encompasses the glpFKX operon promoter sequence, PglpF.
In one or more exemplary embodiments, the promoter sequence comprised in the regulatory element for the regulation of the expression of the genes and/or heterologous nucleic acid sequences of the genetically engineered cell, encompasses the lac operon promoter sequence, P lac.
In one or more exemplary embodiments, the regulatory element for the regulation of the expression of a recombinant gene included in the construct of the disclosure is the mgIBAC ; galactose/methyl-galactoside ABC transporter periplasmic binding protein promoter PmgIB or variants thereof such as but not limited to Pmg/B_70UTR, or Pmg/B_70UTR_SD4.
In one or more exemplary embodiments, the regulatory element for the regulation of the expression of a recombinant gene included in the construct of the disclosure is the gatYZABCD tagatose-1 ,6-bisP aldolase promoter PgatY or variants thereof.
Pscr
In one or more exemplary embodiments, the heterologous regulatory element is Pscr or variants thereof such as but not limited to SEQ ID NO: 7. Pscr_SD1
In one or more exemplary embodiments, the heterologous regulatory element is Pscr_SD1 or variants thereof such as but not limited to SEQ ID NO: 8.
Pscr_SD7
In one or more exemplary embodiments, the heterologous regulatory element is Pscr_SD7 or variants thereof such as but not limited to SEQ ID NO: 9.
PgatY_70UTR
In one or more exemplary embodiments, the heterologous regulatory element is PgatY_70UTR or variants thereof such as but not limited to SEQ ID NO: 10.
PgipF
In one or more exemplary embodiments, the heterologous regulatory element is PgipF or variants thereof such as but not limited to SEQ ID NO: 11 .
It is also obvious from the data shown in Figures 1 a and 1 b that even a single genomic copy of the HD0466 gene suffice to reach the highest LNT-II titers when any of these GlcNAcTs is highly expressed in the cell. A GlcNAcT is hereby defined as “highly expressed” when the host strain expresses it from at least two PglpF-driven genomic copies.
PglpF_SD1
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD1 or variants thereof such as but not limited to SEQ ID NO: 12.
PglpF_SD10
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD10 or variants thereof such as but not limited to SEQ ID NO: 13.
PglpF_SD2
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD2 or variants thereof such as but not limited to SEQ ID NO: 14. PglpF_SD3
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD3 or variants thereof such as but not limited to SEQ ID NO: 15.
PglpF_SD4
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD4 or variants thereof such as but not limited to SEQ ID NO: 16.
PglpF_SD5
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD5 or variants thereof such as but not limited to SEQ ID NO: 17.
PglpF_SD6
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD6 or variants thereof such as but not limited to SEQ ID NO: 18.
PglpF_SD7
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD7 or variants thereof such as but not limited to SEQ ID NO: 19.
PglpF_SD8
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD8 or variants thereof such as but not limited to SEQ ID NO: 20.
PglpF_SD9
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_SD9 or variants thereof such as but not limited to SEQ ID NO: 21.
PglpF_B28
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_B28 or variants thereof such as but not limited to SEQ ID NO: 22.
PglpF_B29
In one or more exemplary embodiments, the heterologous regulatory element is PglpF_B29 or variants thereof such as but not limited to SEQ ID NO: 23. Plac_16UTR
In one or more exemplary embodiments, the heterologous regulatory element is Plac_16UTR or variants thereof such as but not limited to SEQ ID NO: 24.
Plac
In one or more exemplary embodiments, the heterologous regulatory element is Plac or variants thereof such as but not limited to SEQ ID NO: 25.
PmglB_70UTR
In one or more exemplary embodiments, the heterologous regulatory element is PmglB_70UTR or variants thereof such as but not limited to SEQ ID NO: 26.
PmglB_ 70UTR_SD4
In one or more exemplary embodiments, the heterologous regulatory element is PmglB_70UTR_SD4 or variants thereof such as but not limited to SEQ ID NO: 27.
Episomal element
The term “episomal element” refers to an extrachromosomal nucleic acid sequence, that can replicate autonomously or integrate into the genome of the genetically engineered cell. Thus, an episomal nucleic acid sequences may be a plasmid that can integrate into the chromosome of the genetically engineered cell, i.e. not all plasmids are episomal elements.
In one or more exemplary embodiments, episomal nucleic acid sequences may be a plasmid that is not integrated into the chromosome. In the present context, the episomal element refers to plasmid DNA sequences that carry an expression cassette of interest, with the cassette consisting of a promoter sequence, the coding sequence of the gene of interest and a terminator sequence.
In one or more exemplary embodiments, episomal nucleic acid sequences may be a plasmid with only a part of it being integrated into the chromosome. In the present context, the expression cassette resembles the one described above but it further comprises two DNA segments that are homologous to the DNA regions up- and downstream of the locus that the gene of interest is to be integrated. Repressors
In one or more exemplary embodiment(s), the genetically engineered cell disclosed herein comprises a non-functional or absent gene product that normally binds to and represses the expression of the required enzymes to facilitate the production of a human milk oligosaccharide (HMO) that is LNT-II.
The term a non-functional (or absent) gene product that normally binds to and represses the expression driven by the regulatory element in the present context relates to DNA binding sites upstream of the coding sequence of a gene of interest and specifically at the promoter region of said gene.
In one or more exemplary embodiments, the cell may have a non-functional (or absent) gene product(s) that would normally bind to and repress the expression of any of the b-1 ,3-N-acetyl-glucosaminyl- transferase protein(s) and/or the lactose permease protein or regions upstream of the regulatory element for controlling the expression of any of the b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease protein.
Moreover, the deletion of regulators that repress the expression of key steps in the biosynthesis of LNT-II can lead to increased levels of the mRNAs and eventually to higher total HMO titers.
In one or more exemplary preferred embodiments, the method according to the present disclosure comprise a cell further comprising non-functional (or absent) gene product that binds to and represses the expression of any of the b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) of the present disclosure, and wherein the b-1 ,3-N-acetyl-glucosaminyl-transferase protein is HD0466 and LgtA or PmnagT.
In one or more exemplary preferred embodiments, the method according to the present disclosure comprise a cell further comprising non-functional (or absent) gene product that binds to and represses the expression of any of the b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) of the present disclosure, and wherein the b-1 ,3-N-acetyl-glucosaminyl-transferase protein is HD0466.
In one or more exemplary preferred embodiments, the method according to the present disclosure comprise a cell further comprising non-functional (or absent) gene product that binds to and represses the expression of any of b-1 ,3-N-acetyl-glucosaminyl-transferase protein(s) and/or the lactose permease protein, and wherein the b-1 ,3-N-acetyl-glucosaminyl-transferase protein is HD0466 and LgtA.
In one or more exemplary embodiments, said gene product is the DNA-binding transcriptional repressor GlpR. GlpR
GlpR belongs to the DeoR family of transcriptional regulators and acts as the repressor of the glycerol-3- phosphate regulon, which is organized in different operons. This regulator is part of the glpEGR operon, yet it can also be constitutively expressed as an independent ( glpR ) transcription unit. In addition, the operons regulated are induced when Escherichia coli is grown in the presence of inductor, glycerol, or glycerol-3-phosphate (G3P), and the absence of glucose. In the absence of inductor, this repressor binds in tandem to inverted repeat sequences that consist of 20-nucleic acid-long DNA target sites.
The term “non-functional or absent” in relation to the glpR gene refers to the inactivation of the glpR gene by complete or partial deletion of the corresponding nucleic acid sequence from the bacterial genome genome (e.g. SEQ ID NO: 41 or variants thereof encoding glpR capable of downregulating glpF derived promoters). The glpR gene encodes the DNA-binding transcriptional repressor GlpR. In this way promoter sequences of the Pg/pF family are more active in the genetically engineered cell, due to deletion of the repressor gene that would otherwise reduce the transcriptional activity associated with the PglpF promoters.
In one or more exemplary embodiments, the glpR gene is deleted.
The deletion of the glpR gene could eliminate the GlpR-imposed repression of transcription from all PglpF promoters in the cell and in this manner enhance gene expression from all PglpF- based cassettes.
Activators
In one or more exemplary embodiment(s), the genetically engineered cell disclosed herein comprises an over-expressed gene product that enhances the expression of the gene(s) encoding the enzyme(s) required to facilitate the production of a human milk oligosaccharide (HMO) that is LNT-II.
In one or more exemplary embodiments, the cell of the present disclosure may comprise an overexpressed gene product that enhances the expression of the gene(s) encoding any of the b-1 ,3-N-acetyl- glucosaminyl-transferase protein(s) and/or the lactose permease protein.
In one or more exemplary embodiments, the cell of the present disclosure may comprise an overexpressed gene product that enhances the expression of the gene(s) encoding any of the b-1 ,3-N-acetyl- glucosaminyl-transferase protein(s) of the present disclosure, and wherein the b-1 ,3-N-acetyl- glucosaminyl-transferase protein is HD0466 and LgtA or PmnagT.
In one or more exemplary embodiments, the cell of the present disclosure may comprise an overexpressed gene product that enhances the expression of the gene(s) encoding any of the b-1 ,3-N-acetyl- glucosaminyl-transferase protein(s) of the present disclosure, and wherein the b-1 ,3-N-acetyl- glucosaminyl-transferase protein is HD0466.
In one or more exemplary embodiments, the cell of the present disclosure may comprise an overexpressed gene product that enhances the expression of any of the b-1 ,3-N-acetyl-glucosaminyl- transferase protein(s) and/or the lactose permease protein, and wherein the b-1 ,3-N-acetyl-glucosaminyl- transferase protein is HD0466 and LgtA.
CRP
In one or more exemplary embodiments, said gene product is the cAMP DNA-binding transcriptional dual regulator CRP.
CRP belongs to the CRP-FNR superfamily of transcription factors. CRP regulates the expression of several of the E. coli genes, many of which are involved in catabolism of secondary carbon sources.
Upon activation by cyclic-AMP, (cAMP) CRP binds directly to specific promoter sequences, the binding recruits the RNA polymerase through direct interaction, which in turn activates the transcription of the nucleic acid sequence following the promoter sequence leading to expression of the gene of interest. Thus, over-expression of CRP may lead to an enhanced expression of a gene/nucleic acid sequence of interest. Amongst other functions, CRP exerts its function on the PglpF promoters, where it contrary to the repressor GlpR, activates promoter sequences of the PglpF family. In this way, over-expression of CRP in the genetically engineered cell of the present disclosure, promotes expression of genes that are regulated by promoters of the PglpF family.
Thus, in one or more exemplary embodiments, the crp gene is over-expressed.
Genetic engineering of GlpR and/or CRP, as suggested in the present disclosure, in 2’-FL producing strains is beneficial for the overall production of 2’-FL by these strains.
Nucleic acid constructs
An aspect of the present disclosure is the provision of a nucleic acid construct. Thus, in one or more exemplary embodiments the nucleic acid construct may comprise at least i) a nucleic acid sequence according to SEQ ID NO: 4 or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 4, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; and ii) a native or heterologous regulatory element for controlling the expression of i).
In one or more exemplary embodiments the nucleic acid construct also comprises: iii) a nucleic acid sequence according to SEQ ID NO: 5 [IgtA], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 5, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase, and iv) a native or heterologous regulatory element for controlling the expression of iii).
In one or more exemplary embodiments the nucleic acid construct also comprises: v) a nucleic acid sequence according SEQ ID NO: 6 [LacY], or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 6, to encoding a lactose permease, and vi) a native or heterologous regulatory element for controlling the expression of v).
In one or more exemplary embodiments the nucleic acid construct also comprises: vii) a nucleic acid sequence according to SEQ ID NO: 29 [PmnagT], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 29, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; viii) a native or heterologous regulatory element for controlling the expression of iii).
The nucleic acid construct may further comprise one or more regulatory element for controlling the expression of i), iii), v), vii). The regulatory element(s) may be a native or heterologous or episomal.
The nucleic acid construct may further comprise a heterologous regulatory or episomal element for increasing the expression of v) a lactose permease protein as shown in SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3.
The nucleic acid construct may further comprise a non-functional (or absent) gene product that normally binds to and represses the expression of the regulatory elements).
Recombinant nucleic acid sequence
The nucleic acid construct can be a recombinant nucleic acid sequence. By the term “recombinant nucleic acid sequence”, “recombinant gene/nucleic acid/DNA encoding” or "recombinant coding nucleic acid sequence" used interchangeably is meant an artificial nucleic acid sequence (i.e. produced in vitro using standard laboratory methods for making nucleic acid sequences) that comprises a set of consecutive, non-overlapping triplets (codons) which is transcribed into mRNA and translated into a protein when under the control of the appropriate control sequences, i.e. a promoter sequence.
The boundaries of the coding sequence are generally determined by a ribosome binding site located just upstream of the open reading frame at the 5’end of the mRNA, a transcriptional start codon (AUG, GUG or UUG), and a translational stop codon (UAA, UGA or UAG). A coding sequence can include, but is not limited to, genomic DNA, cDNA, synthetic, and recombinant nucleic acid sequences. The term "nucleic acid" includes RNA, DNA and cDNA molecules. It is understood that, as a result of the degeneracy of the genetic code, a multitude of nucleic acid sequences encoding a given protein may be produced.
The recombinant nucleic sequence may be a coding DNA sequence e.g., a gene, or non-coding DNA sequence e.g., a regulatory DNA, such as a promoter sequence. It may be a native promoter in front of a heterologous nucleic acid sequence, which makes the native promoter a recombinant promoter.
Accordingly, in one exemplified embodiment the disclosure relates to a nucleic acid construct comprising a coding nucleic sequence, i.e. recombinant DNA sequence of a gene of interest, e.g. a b-1 ,3-N-acetyl- glucosaminyl-transferase gene, and a non-coding regulatory DNA sequence, e.g. a promoter DNA sequence, e.g. a recombinant promoter sequence derived from the promoter sequence of lac operon or an glp operon, or a promoter sequence derived from another genomic promoter DNA sequence, or a synthetic promoter sequence, wherein the coding and promoter sequences are operably linked.
Operably linked
The term “operably linked” refers to a functional relationship between two or more nucleic acid (e.g.,
DNA) segments operably linked refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
Generally, promoter sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are c/s-acting.
In one exemplified embodiment, the nucleic acid construct of the disclosure may be a part of the vector DNA, in another embodiment the construct it is an expression cassette/cartridge that is integrated in the genome of a host cell.
Nucleic acid construct
Accordingly, the term “nucleic acid construct” means an artificially constructed segment of nucleic acid, in particular a DNA segment, which is intended to be 'transplanted' into a target cell, e.g. a bacterial cell, to modify expression of a gene of the genome or express a gene/coding DNA sequence which may be included in the construct.
Integration of the nucleic acid construct of interest comprised in the construct (expression cassette) into the bacterial genome can be achieved by conventional methods, e.g. by using linear cartridges that contain flanking sequences homologous to a specific site on the chromosome, as described for the attTn7-site (Waddell C.S. and Craig N.L., Genes Dev. (1988) Feb;2(2):137-49.); methods for genomic integration of nucleic acid sequences in which recombination is mediated by the Red recombinase function of the phage l or the RecE/RecT recombinase function of the Rac prophage (Murphy, J Bacteriol. (1998);180(8):2063-7; Zhang et al., Nature Genetics (1998) 20: 123-128 Muyrers et al., EMBO Rep. (2000) 1(3): 239-243); methods based on Red/ET recombination (Wenzel et al., Chem Biol. (2005), 12(3):349-56.; Vetcher et al., Appl Environ Microbiol. (2005);71(4):1829-35); or positive clones, i.e., clones that carry the expression cassette, can be selected e.g. by means of a marker gene, or loss or gain of gene function.
Nucleic acid identity
In one or more exemplary embodiments, the present disclosure relates to a recombinant nucleic acid shown in SEQ ID NO: 4-6 or 29, or a functional homologue thereof having a sequence that is at least 70% identical to SEQ ID NO: 4-6 or 29, such as at least 71% identical, at least 72% identical, at least 73% identical, at least 74% identical, at least 75% identical, at least 76% identical, at least 77% identical, at least 78% identical, at least 79% identical, at least 80 % identical, at least 81 % identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85 % identical, at least 86 % identical, at least 87 % identical, at least 88 % identical, at least 89 % identical, at least 90 % identical, at least 91 % identical, at least 92 % identical, at least 93 % identical, at least 94 % identical, at least 95 % identical at least 96 % identical, at least 97 % identical, at least 98 % identical, or at least 99 % identical.
Sequence identity
The term “sequence identity of [a certain] %” in the context of two or more nucleic acid or amino acid sequences means that the two or more sequences have nucleic acids or amino acid residues in common in the given percent, when compared and aligned for maximum correspondence over a comparison window or designated sequences of nucleic acids or amino acids (i.e. the sequences have at least 90 percent (%) identity). Percent identity of nucleic acid or amino acid sequences can be measured using a BLAST 2.0 sequence comparison algorithm with default parameters, or by manual alignment and visual inspection (see e.g. http://www.ncbi.nlm.nih.gov/BLAST/). This definition also applies to the complement of a test sequence and to sequences that have deletions and/or additions, as well as those that have substitutions. An example of an algorithm that is suitable for determining percent identity, sequence similarity and for alignment is the BLAST 2.2.20+ algorithm, which is described in Altschul et al. Nucl. Acids Res. 25, 3389 (1997). BLAST 2.2.20+ is used to determine percent sequence identity for the nucleic acids and proteins of the disclosure. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). Examples of commonly used sequence alignment algorithms are
CLUSTAL Omega (http://www.ebi.ac.uk Tools/msa/clustalo/), EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss needle/),
MAFFT (http://mafft.cbrc.ip/aiiqnment/server/), or
MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/).
Preferably, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo/. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277,), preferably version 5.0.0 or later (available at https://www.ebi.ac.uk/Tools/psa/emboss needle/). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment).
Preferably, the sequence identity between two nucleotide sequences is determined using the Needleman- Wunsch algorithm (Needleman and Wunsch, 1 970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), 10 preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100)/(Length of Alignment — Total Number of Gaps in Alignment).
Functional homologue
A functional homologue of a protein/nucleotide as described herein is a protein/nucleotide with alterations in the genetic code, which retain its original functionality. A functional homologue may be obtained by mutagenesis. The functional homologue should have a remaining functionality of at least 50%, such as 60%, 70%, 80 %, 90% or 100% compared to the functionality of the protein/nucleotide.
A functional homologue of any one of the disclosed amino acid or nucleotide sequences can also have a higher functionality. A functional homologue of any one of SEQ ID NOs: 1-29, should ideally be able to participate in the HMO production, in terms of HMO yield, purity, reduction in biomass formation, viability of the genetically engineered cell, robustness of the genetically engineered cell according to the disclosure, or reduction in consumables. In particular functional homologous of the amino acid sequences of SEQ ID NO: 1 , 2, 3 or 28 or the nucleic acid sequences of SEQ ID NO: 4, 5, 6 or 29 should ideally be able to participate in the HMO production, in terms of HMO yield, purity, reduction in biomass formation, viability of the genetically engineered cell, robustness of the genetically engineered cell according to the disclosure, or reduction in consumables. Genetically engineered cell
The present disclosure also relates to a genetically engineered cell comprising a) a nucleic acid sequence according to SEQ ID NO: 4 or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 4, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; and b) a native or heterologous regulatory element for controlling the expression of a).
In additional embodiments, the cell is able to import lactose into the cell.
In one or more exemplary embodiments the genetically engineered cell also comprises: c) a nucleic acid sequence according to SEQ ID NO: 5 [LgtA], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 5, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; and d) a native or heterologous regulatory element for controlling the expression of c).
In one or more exemplary embodiments the genetically engineered cell also comprises: e) a nucleic acid sequence according to SEQ ID NO: 6 [LacY], or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 6 encoding a lactose permease, and f) a native or heterologous regulatory element for controlling the expression of e).
In one or more exemplary embodiments the genetically engineered cell also comprises: g) a nucleic acid sequence according to SEQ ID NO: 29 [PmnagT], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 29, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; and h) a native or heterologous regulatory element for controlling the expression of g).
A "genetically modified” or genetically engineered” cell is used interchangeably herein and is understood as a cell whose genetic material has been altered by human intervention using a genetic engineering technique, such a technique is for example but not limited to transformation or transfection e.g., with a heterologous polynucleotide sequence, Crisper/Cas editing and/or random mutagenesis. In the present context, the terms a” genetically modified cell” and “a host cell” are used interchangeably.
In the present invention the "genetically engineered cell” is preferably a host cell which has been transformed or transfected by an exogenous polynucleotide sequence.
In one or more exemplary embodiments, the HMO produced by the genetically engineered cell is LNT-II. The genetically engineered cell may be any cell useful for HMO production including mammalian cell lines. Preferably, the host cell is a unicellular microorganism of eucaryotic or prokaryotic origin. Appropriate microbial cells that may function as a host cell include yeast cells, bacterial cells, archaebacterial cells, algae cells, and fungal cells.
The genetically engineered cell (host cell) may be e.g., a bacterial or yeast cell. In embodiments the genetically engineered cell is a bacterial or yeast cell. In one preferred embodiment, the genetically engineered cell is preferably a prokaryotic cell, such as a bacterial cell.
Bacterial host cells
Regarding the bacterial host cells, there are, in principle, no limitations; they may be eubacteria (grampositive or gram-negative) or archaebacteria, as long as they allow genetic manipulation for insertion of a gene of interest and can be cultivated on a manufacturing scale. Preferably, the host cell has the property to allow cultivation to high cell densities. Non-limiting examples of bacterial host cells that are suitable for recombinant industrial production of an HMO(s) according to the disclosure could be Erwinia herbicola (Pantoea agglomerans), Citrobacter freundii, Pantoea citrea, Pectobacterium carotovorum, or Xanthomonas campestris. Bacteria of the genus Bacillus may also be used, including Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, and Bacillus circulans. Similarly, bacteria of the genera Lactobacillus and Lactococcus may be engineered using the methods of this disclosure, including but not limited to Lactobacillus acidophilus, Lactobacillus salivarius,
Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the disclosure described herein. Also included as part of this disclosure are strains, engineered as described here, from the genera Enterococcus (e.g., Enterococcus faecium and Enterococcus thermophiles), Bifidobacterium (e.g., Bifidobacterium longum, Bifidobacterium infantis, and Bifidobacterium bifidum), Sporolactobacillus spp., Micromomospora spp., Micrococcus spp., Rhodococcus spp., and Pseudomonas (e.g., Pseudomonas fluorescens and Pseudomonas aeruginosa).
Non-limiting examples of fungal host cells that are suitable for recombinant industrial production of a HMO product could be yeast cells, such as Komagataella phaffii, Kluyveromyces lactis, Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae or filamentous fungi such as Aspargillus sp, Fusarium sp or Thricoderma sp, exemplary species are A. niger, A. nidulans, A. oryzae, F. solani, F. graminearum and T. reesei. In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of Yarrowia lipolytica, Pichia pastoris, and Saccharomyces cerevisiae.
In one or more exemplary embodiments, the genetically engineered cell is S. cerevisiae or P pastoris.
In one or more exemplary embodiments, the genetically engineered cell is Pichia pastoris.
In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of E. coli, C. glutamicum, L lactis, B. subtilis, S. lividans, P. pastoris, and S. cerevisiae.
In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of B. subtilis, S. cerevisiae and E. coli.
In one or more exemplary embodiments, the genetically engineered cell is selected from the group consisting of E. coli, C. glutamicum, L. lactis, B. subtilis, S. lividans.
In one or more exemplary embodiments, the genetically engineered cell is B. subtilis.
In one or more exemplary embodiments, the genetically engineered cell is Corynebacterium glutamicum.
In one or more exemplary embodiments, the genetically engineered cell is S. cerevisiae.
In one or more exemplary embodiments, the genetically engineered cell is E. coli.
In one or more exemplary embodiments, the disclosure relates to a genetically engineered cell, wherein the cell is derived from the E. coli K12 or DE3 strain.
Culturing
In the present context, culturing refers to the process by which cells are grown under controlled conditions, generally outside their natural environment, thus a method used to cultivate, propagate and grow a large number of cells.
The terms culturing and fermentation are used interchangeably.
Cell culture medium
In the present context, a growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms, cells, or small plants. The medium comprises an appropriate source of energy and may comprise compounds which regulate the cell cycle. The culture medium may be semi-defined, i.e. containing complex media compounds (e.g. yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined, without any complex compounds. Exemplary suitable medias are provided in experimental examples.
In one or more exemplary embodiments, the culturing media is minimal media.
In one or more exemplary embodiments, the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose, glucose and fructose.
In one or more exemplary embodiments, the culturing media is supplemented with one or more energy and carbon sources selected form the group containing glycerol, sucrose and glucose.
In one or more exemplary embodiments, the culturing media is supplemented with glycerol, sucrose and/or glucose.
In one or more exemplary embodiments, the culturing media is supplemented with glycerol and/or glucose.
In one or more exemplary embodiments, the culturing media is supplemented with sucrose and/or glucose.
In one or more exemplary embodiments, the culturing media is supplemented with glycerol and/or sucrose.
Harvesting
The term “harvesting” in the context relates to collecting the produced HMO(s) following the termination of fermentation. In one or more exemplary embodiments it may include collecting the HMO(s) included in both the biomass (i.e. the host cells) and cultivation media, i.e. before/without separation of the fermentation broth from the biomass. In other embodiments, the produced HMOs may be collected separately from the biomass and fermentation broth, i.e. after/following the separation of biomass from cultivation media (i.e. fermentation broth).
The separation of cells from the medium can be carried out with any of the methods well known to the skilled person in the art, such as any suitable type of centrifugation or filtration. The separation of cells from the medium can follow immediately after harvesting the fermentation broth or be carried out at a later stage after storing the fermentation broth at appropriate conditions. Recovery of the produced HMO(s) from the remaining biomass (or total fermentation) include extraction thereof from the biomass (i.e. the production cells). After recovery from fermentation, HMO(s) are available for further processing and purification.
Human milk oligosaccharide (HMO)
In the context of the disclosure, the term “oligosaccharide” means a saccharide polymer containing a number of monosaccharide units. In some embodiments, preferred oligosaccharides are saccharide polymers consisting of three or four monosaccharide units, i.e. trisaccharides ortetrasaccharides. Preferable oligosaccharides of the disclosure are human milk oligosaccharides (HMOs).
The term “human milk oligosaccharide" or "HMO" in the present context means a complex carbohydrate found in human breast milk. The HMOs have a core structure comprising a lactose unit at the reducing end that can be elongated by one or more beta-N-acetyl-lactosaminyl and/or one or more beta-lacto-N- biosyl units, and this core structure can be substituted by an alpha-L-fucopyranosyl and/or an alpha-N- acetyl-neuraminyl (sialyl) moiety.
In this regard, the non-acidic (or neutral) HMOs are devoid of a sialyl residue, and the acidic HMOs have at least one sialyl residue in their structure. The non-acidic (or neutral) HMOs can be fucosylated or non- fucosylated. Examples of such neutral non-fucosylated HMOs include lacto-N-triose 2 (LNT-II) lacto-N- tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH), para-lacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH). Examples of neutral fucosylated HMOs include 2'-fucosyllactose (2’-FL), lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I), 3-fucosyllactose (3’-FL), difucosyllactose (DFL), lacto-N-fucopentaose II (LNFP-II), lacto-N- fucopentaose III (LNFP-III), lacto-N-difucohexaose III (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNH-II), lacto-N-fucopentaose V (LNFP-V), lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I), fucosyl-para-lacto-N-hexaose I (FpLNH-l), fucosyl-para-lacto-N-neohexaose II (F-pLNnH II) and fucosyl-lacto-N-neohexaose (FLNnH). Examples of acidic HMOs include 3’-sialyllactose (3’-SL), 6’- sialyllactose (6’-SL), 3-fucosyl-3’-sialyllactose (FSL), 3’-0-sialyllacto-N-tetraose a (LST a), fucosyl-LST a (FLST a), 6’-0-sialyllacto-N-tetraose b (LST b), fucosyl-LST b (FLST b), 6’-0-sialyllacto-N-neotetraose (LST c), fucosyl-LST c (FLST c), 3’-0-sialyllacto-N-neotetraose (LST d), fucosyl-LST d (FLST d), sialyl- lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I (SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II) and disialyl-lacto-N-tetraose (DSLNT).
In the context of the present disclosure lactose is not regarded as an HMO species.
Use of a genetically engineered cell
The disclosure also relates to any commercial use of the genetically engineered cell(s) or the nucleic acid construct(s) disclosed herein. The genetically engineered cell(s) or the nucleic acid construct(s) comprise at least one heterologous protein(s), such as but not limited to i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1 ; and optionally ii) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 or 28 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to any one of SEQ ID NO: 2 or 28.
The genetically engineered cell(s) or the nucleic acid construct(s) may further comprise a lactose permease protein as shown in SEQ ID NO: 3, or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3.
The genetically engineered cell(s) or the nucleic acid construct(s) may comprise a native or heterologous regulatory element for controlling the expression of the b-1 ,3-N-acetyl-glucosaminyl-transferase(s).
The genetically engineered cell(s) or the nucleic acid construct(s) may also comprise a native or heterologous regulatory or episomal element for increasing the expression of the lactose permease.
The genetically engineered cell(s) or the nucleic acid construct(s) may comprise a non-functional (or absent) gene product that normally binds to and represses the expression of the b-1 ,3-N-acetyl- glucosaminyl-transferase and/or the lactose permease.
In one or more exemplary embodiments, the genetically engineered cell or the nucleic acid construct is used in the manufacturing of one or more HMOs.
In another exemplified embodiment, the genetically engineered cell and/or the nucleic acid construct according to the disclosure, is used in the manufacturing of LNT-II.
Manufacturing ofHMOs
To produce one or more HMOs, the genetically engineered cells as described herein are cultivated according to the procedures known in the art in the presence of a suitable carbon and energy source, e.g. glucose, glycerol or sucrose, and a suitable acceptor, e.g. lactose or any HMO, and the produced HMO blend is harvested from the cultivation media and the microbial biomass formed during the cultivation process. Thereafter, the HMOs are purified according to the procedures known in the art, e.g. such as described in WO2015188834, WO2017182965 or WO2017152918, and the purified HMOs are used as nutraceuticals, pharmaceuticals, or for any other purpose, e.g. for research.
Manufacturing of HMOs is typically accomplished by performing cultivation in larger volumes. The term “manufacturing” and “manufacturing scale” in the meaning of the disclosure defines a fermentation with a minimum volume of 5 L culture broth. Usually, a “manufacturing scale” process is defined by being capable of processing large volumes of a preparation containing the product of interest and yielding amounts of the HMO product of interest that meet, e.g., in the case of a therapeutic compound or composition, the demands for clinical trials as well as for market supply. In addition to the large volume, a manufacturing scale method, as opposed to simple lab scale methods like shake flask cultivation, is characterized by the use of the technical system of a bioreactor (fermenter) which is equipped with devices for agitation, aeration, nutrient feeding, monitoring and control of process parameters (pH, temperature, dissolved oxygen tension, back pressure, etc.). To a large extent, the behavior of an expression system in a lab scale method, such as shake flasks, benchtop bioreactors or the deep well format described in the examples of the disclosure, does allow to predict the behavior of that system in the complex environment of a bioreactor.
With regard to the suitable cell medium used in the fermentation process, there are no limitations. The culture medium may be semi-defined, i.e. containing complex media compounds (e.g. yeast extract, soy peptone, casamino acids, etc.), or it may be chemically defined, without any complex compounds. Where sucrose is used as the carbon and energy source, a minimal medium might be preferable.
Manufactured product
The strain engineering strategy of the present invention contributes to a sustainable manufacturing process for the high-level production of LNT-II, where the conversion of the provided carbon source to HMO product in fermentation is done at a high overall yield. As shown in Example 2, the concentration of the detected HMOs (in g/L) in each sample was used to calculate the % quantitative differences in the LNT-II content of the strains tested, i.e., the % differences in the LNT-II concentrations formed by strains expressing LgtA, PmnagT or HD0466, or pairwise combinations of all three GlcNAcTs relative to LgtA- expressing cells.
The term “manufactured product” according to the use of the genetically engineered cell or the nucleic acid construct refer to the one or more HMOs intended as the one or more product HMO(s). The various products are described above.
Advantageously, the methods disclosed herein provides both a decreased ratio of by-product to product and an increased overall yield of the product (and/or HMOs in total). This, less by-product formation in relation to product formation facilitates an elevated product production and increases efficiency of both the production and product recovery process, providing superior manufacturing procedure of HMOs.
The manufactured product may be a powder, a composition, a suspension, or a gel comprising one or more HMOs.
Importantly, beneficial features for the construction of efficient LNT-II cell factories described above do not provide an additive effect in a single production strain that produces any of the three GlcNAcTs or combinations thereof. In other words, the features described in the present disclosure can be exploited only in the way presented here to provide the desired positive effect on LNT-II titers. Moreover, two given modifications that are proven beneficial for different strain backgrounds should not be expected to enhance LNT-II formation when combined in any of these strain backgrounds. Tables
Table 1. Genotypes of the strains MP1, MP2, MP3, MP4, MP5 and MP6
Figure imgf000038_0001
1 1gtA: gene coding for b- 1, 3-N-acetyloglucosamine transferase
2 PmnagT: gene coding for b-1 , 3-N-acetyloglucosamine transferase
3 HD0466: gene coding for b-1, 3-N-acetyloglucosamine transferase
Table 2. Genotypes of the strains MP2, MP4, MP6, MP7, MP8 and MP9
Figure imgf000038_0002
1 1gtA: gene coding for b-1 , 3-N-acetyloglucosamine transferase
2 PmnagT: gene coding for b-1, 3-N-acetyloglucosamine transferase
3 HD0466: gene coding for b-1, 3-N-acetyloglucosamine transferase
Table 3 - Genotypes of the strains MP2, MP10, MP4, MP11, MP6, MP12, MP7, MP13, MP8, MP14, MP9 and MP15.
Figure imgf000038_0003
Figure imgf000039_0001
1 1gtA: gene coding for b-1 ,3-N-acetyloglucosamine transferase
2 PmnagT: gene coding for b-1 ,3-N-acetyloglucosamine transferase
3 HD0466: gene coding for b-1 ,3-N-acetyloglucosamine transferase
4 LacY: gene encoding for the lactose permease protein
GENERAL
It should be understood that any feature and/or aspect discussed above in connections with the methods according to the disclosure apply by analogy to the engineered cell, the nucleic acid constructs and/or use described herein.
The terms fermentation and culturing are used interchangeably.
The terms Lacto-N-triose, LNT-II, LNT II, LNT2 and LNT 2, are used interchangeably. The following figures and examples are provided below to illustrate the present disclosure. They are intended to be illustrative and are not to be construed as limiting in any way.
BRIEF DESCRIPTION OF THE FIGURES Figure 1
Final LNT-II titers reached by cells expressing different GlcNAcTs at different genomic copy numbers (a) LNT-II titers for strains that bear a genomic copy of the PmnagT (strain MP3) or HD0466 (strains MP5 and MP6) genes, shown relative to the final LNT-II titers of /g/A-expressing cells (strain MP1). The reference level (given as 100%) is shown for strain MP1. (b) LNT-II titers for strains that bear two genomic copies of the PmnagT (strain MP4) or HD0466 (strain MP6) genes, shown relative to the final LNT-II titers of /g/A-expressing cells (strain MP2). The reference level (given as 100%) is shown for strain MP2.
Figure 2
Final LNT-II titers reached by cells expressing a single GlcNAcT or a pairwise combination of GlcNAcTs at the same genomic copy number. LNT-II titers are shown for strains that bear two genomic copies of a single GlcNAcT (LgtA/strain MP2 or PmnagT/strain MP4 or HD0466/strain MP6) and for strains bearing one copy of each of two GlcNAcTs (LgtA & HD0466/strain MP7 or PmnagT & HD0466/strain MP8 or PmnagT & LgtA/strain MP9). The reference level (given as 100%) is shown for strain MP2.
Figure 3
Final LNT-II titers reached by cells expressing different GlcNAcTs with and without the native MFS transporter LacY. LNT-II titers for strains that bear two genomic Pg/pF-driven copies of gene(s) encoding GlcNAcT (s) and a single Pg/pF-driven copy of the lacY gene (strains MP10, MP11 , MP12, MP13, MP14, MP15), shown relative to the corresponding final LNT-II titers of each GlcNAcT-expressing cells (strains MP2, MP4, MP6, MP7, MP8, MP9). The reference level (given as 100%) is shown for strains MP2, MP4, MP6, MP7, MP8, MP9.
Figure 4
Final LNT-II titers reached by cells bearing beneficial modifications as described in the present disclosure. LNT-II titers for strains that bear a genomic Pg/pF-driven copy of the IgtA and HD0466 genes and of the lacY gene (strains MP13), shown relative to the final LNT-II titers of cells that only express a Pg/pF-driven copy of the IgtA and HD0466 genes (strain MP7). The reference level (given as 100%) is shown for strain MP7.
SEQUENCE ID’S
The current application contains a sequence listing in text format and electronical format which is hereby incorporated by reference as are the sequences listed in the priority application DK PA 202170252. Below is a summary of the sequences which are not present in the list of promoter sequences in table 4. SEQ ID NO: 1 [HD0466 protein]
SEQ ID NO: 2 [LgtA protein]
SEQ ID NO: 3 [LacY protein]
SEQ ID NO: 4 [HD0466 gene]
SEQ ID NO: 5 [IgtA gene]
SEQ ID NO: 6 [lacY gene] SEQ ID NO: 28 [PmnagT protein] SEQ ID NO: 29 [PmnagT gene] SEQ ID NO: 41 [GlpR gene]
EXAMPLES
Example 1 - Heterologous enzymes appropriate for the construction of efficient LNT-II production systems
Description of the genotype of strains tested in deep well assays
The strains (genetically engineered cells) constructed in the present application were based on Escherichia coli K-12 DH1 with the genotype: F~, A gyrA96, recA1, relA1, endA1, thi-1, hsdR17, supE44. Additional modifications were made to the E. coli K-12 DH1 strain to generate the platform strain “MDO” with the following modifications: lacZ: deletion of 1.5 kbp, lacA\ deletion of 0.5 kbp, nanKETA : deletion of 3.3 kbp, melA : deletion of 0.9 kbp, wcaJ\ deletion of 0.5 kbp, mdoH: deletion of 0.5 kbp, and insertion of Plac promoter upstream of the gmd gene.
Methods of inserting or deleting gene(s) of interest into the genome of E. coli are well known to the person skilled in the art. Insertion of genetic cassettes into the E. coli chromosome can be done using gene gorging (see e.g., Herring and Blattner2004 J. Bacteriol. 186: 2673-81 and Warming et al 2005 Nucleic Acids Res. 33(4): e36) with specific selection marker genes and screening methods.
Based on platform strain (“MDO”), the modifications summarized in table 1 , were made to obtain the fully chromosomal strains MP1 , MP2, MP3, MP4, MP5 and MP6 . The strains can produce the trisaccharide HMO LNT-II. Each of the six strains expresses a single beta-1 ,3-N-acetyloglucosamine transferase (GlcNAcT) selected from the group consisting of (a) LgtA from Neisseria meningitidis (GenBank ID: WP_033911473.1)(MP1 and MP2), (b) PmnagT from Pasteurella multocida (GenBank ID:
WP_014390683.1) (MP3 and MP4), or (c) HD0466 from Haemophilus ducreyi (GenBank ID:
WP_010944479.1) (MP5 and MP6). The only difference between the strain pairs, MP1-MP2, MP3-MP4 and MP5-MP6, is the GlcNAcT being expressed. The copy number of the GlcNAcT in each strain pair is the only difference among the strains of the pair. For example, the strain MP6 bears two copies of the HD0466 gene while the strain MP5 bears just one Pg/pF-driven copy of this gene.
In the present Example, it is demonstrated how different GlcNAcTs can be beneficially expressed at varied genomic copy numbers in the genetic background of E. coli K12 cells for high-level LNT-II production. The Example reveals the HD0466 enzyme as a novel enzyme for the in vivo production of LNT-II.
As shown in table 1 , the only difference among the strains is the beta-1 ,3-N-acetyloglucosamine transferase being expressed or the copy number of the chosen transferase. Description of the applied deep well assay protocol for strain characterization
The strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities and subsequently transferred to a medium that allowed induction of gene expression and product formation. More specifically, during day 1 , fresh precultures were prepared using a basal minimal medium supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7,5) in order to start the main culture. The new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20 % glucose solution (50 ul per 100 ml_) and a bolus of 10 % lactose solution (5 ml per 100 ml). Moreover, 50 % sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was released at a rate suitable for C-limited growth. The main cultures were incubated for 72 hours at 28 °C and 1000 rpm shaking.
For the analysis of total broth, the 96-well plates were boiled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC. For supernatant samples, the initial centrifugation of microtiter plates was followed by the removal of 0.1 ml_ supernatant for direct analysis by HPLC. For pellet samples, the cells were initially washed, then dissolved in deionized water and centrifuged. Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supernatant.
Results of the deep well assays
Strains were characterized in deep well assays and samples were collected from the total broth. All samples were analysed for HMO content by HPLC following the 72-hour protocol described above. The concentration of the detected HMOs (in g/L) in each sample was used to calculate the % quantitative differences in the LNT-II content of the strains tested, i.e., the % differences in the LNT-II concentrations of PmnagT- or HD0466-expressing cells relative to IgtA-ex pressing cells (set to 100%).
As revealed by the analysis of total samples in deep-well cultures, apart from the LgtA and PmnagT enzymes that have been previously applied for the production of various LNTII-core HMOs, a novel GlcNAcT, namely HD0466, can provide high LNT-II titers when expressed from one or two genomic copies (Figure 1a). The descending order of activity of the three selected GlcNAcTs on lactose, as it is indirectly revealed by the observed final LNT-II titers is as follows: HD0466 > PmnagT > LgtA. The LNT-II titers reached by the strains MP5 and/or MP6, which express HD0466 from a different copy number, can be up to 40% or 15% higher than for strains expressing LgtA (strain MP1) or PmnagT (strain MP3), respectively. It is also obvious from the data shown in Figures 1a and 1b that even a single genomic copy of the HD0466 gene suffice to reach the highest LNT-II titers when any of these GlcNAcTs is highly expressed in the cell. A GlcNAcT is hereby defined as “highly expressed” when the host strain expresses it from at least two Pg/pF-driven genomic copies.
Example 2 - Combination of heterologous beta-1 ,3-N-acetyloglucosamine transferases is highly beneficial for LNT-II titer enhancement
Description of the genotype of strains tested in deep well assays
Based on the platform strain (“MDO”) described in Example 1 , the modifications summarised in table 2, were made to obtain the fully chromosomal strains MP2, MP4, MP6, MP7, MP8 and MP9. The strains can produce the trisaccharide HMO LNT-II. Each of the six strains bears in total two Pg/pF-driven copies of a single or two beta-1 ,3-N-acetyloglucosamine transferases (GlcNAcTs) selected from the group consisting of (a) LgtA from Neisseria meningitidis (GenBank ID: WP_033911473.1), (b) PmnagT from Pasteurella multocida (GenBank ID: WP_014390683.1), or (c) HD0466 from Haemophilus ducreyi (GenBank ID: WP_010944479.1). Thus, all strains express two identical or two different GlcNAcTs from a total of 2 genomic copies with the only difference among the strains being the selected GlcNAcTs.
In the present Example, it is demonstrated that the pairwise expression of two different GlcNAcTs can be a more efficient approach in converting E. coli K12 cells to an efficient LNT-II cell factory than merely expressing two copies of a single GlcNAcT.
As shown in table 2, the only difference among the strains is the GlcNAcT(s) being expressed, while the total copy number of the chosen transferase(s) is the same for every strain.
Description of the applied deep well assay protocol for strain characterization
The strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities and subsequently transferred to a medium that allowed induction of gene expression and product formation. More specifically, during day 1 , fresh precultures were prepared using a basal minimal medium supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7,5) in order to start the main culture. The new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20 % glucose solution (50 ul per 100 mL) and a bolus of 10 % lactose solution (5 ml per 100 ml). Moreover, 50 % sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was released at a rate suitable for C-limited growth. The main cultures were incubated for 72 hours at 28 °C and 1000 rpm shaking. For the analysis of total broth, the 96-well plates were boiled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC. For supernatant samples, the initial centrifugation of microtiter plates was followed by the removal of 0.1 mL supernatant for direct analysis by HPLC. For pellet samples, the cells were initially washed, then dissolved in deionized water and centrifuged. Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supernatant.
Results of the deep well assays
Strains were characterized in deep well assays and samples were collected from the total broth. All samples were analysed for HMO content by HPLC following the 72-hour protocol described above. The concentration of the detected HMOs (in g/L) in each sample was used to calculate the % quantitative differences in the LNT-II content of the strains tested, i.e., the % differences in the LNT-II concentrations formed by strains expressing LgtA, PmnagT or HD0466, or pairwise combinations of all three GlcNAcTs relative to LgtA-expressing cells (set to 100%).
At physiological intracellular lactose concentrations (i.e., wild-type expression of the lacY gene coding the lactose permease), higher LNT-II titers can be achieved when HD0466 is co-expressed with either of the two other GlcNAcTs, namely PmnagT and LgtA (strains MP7 and MP8) rather than when HD0466 (strain MP6), PmnagT (strain MP4) or LgtA (strain MP2) alone are expressed at the same copy number (Figure 2). Specifically, strains bearing a copy of HD0466 and a copy of one of the two other GlcNAcTs (strains MP7 and MP8) show higher titers than strains bearing two identical copies of any of the three GlcNAcTs (strains MP2, MP4 or MP6) or the strain that co-expresses LgtA and PmnagT (strain MP9) at the same GlcNAcT copy number (Figure 2).
Example 3 - Genetic manipulation of the native MFS transporter LacY can provide superior LNT-II production systems depending on the beta-1 ,3-N-acetyloglucosamine transferase(s) being expressed
Description of the genotype genotype of strains tested in deep well assays
Based on platform strain (“MDO”) described in example 1 , the modifications summarised in table 3, were made to obtain a number of fully chromosomal strains. The strains can produce the trisaccharide HMO LNT-II. Each of these strains bears in total two Pg/pF-d riven copies of a single or two beta-1 ,3-N- acetyloglucosamine transferases (GlcNAcTs) selected from the group consisting of (a) LgtA from Neisseria meningitidis (GenBank ID: WP_033911473.1), (b) PmnagT from Paste urella multocida (GenBank ID: WP_014390683.1), or (c) HD0466 from Haemophilus ducreyi (GenBank ID:
WP_010944479.1). Thus, all strains express one or two GlcNAcTs from a total of 2 genomic copies with the strains differing in the identity of the selected GlcNAcTs. Moreover, apart from the identity of GlcNAcTs, the strains can differ in regard to the expression of the E. coli K12 lactose permease LacY (GenBank ID: NP_414877.1) (Table 3). In the present Example, it is demonstrated that the over-expression of the gene encoding the native lactose permease LacY can be beneficial only when specific GlcNAcTs, namely HD0466 and LgtA, are co-expressed in the same strain (MP13).
In this manner, Examples 2 and 3 provide enough data to conclude that the combined expression of HD0466 and LgtA results in higher LNT-II titers regardless of intracellular lactose levels compared to when only one of these GlcNAcTs is expressed by the cell at the same copy number. Importantly, this trend is unique for this GlcNAcT pair and it is not observed for any other GlcNAcT pair.
Description of the applied deep well assay protocol for strain characterization
The strains disclosed in the present example were screened in 96 deep well plates using a 4-day protocol. During the first 24 hours, precultures were grown to high densities and subsequently transferred to a medium that allowed induction of gene expression and product formation. More specifically, during day 1 , fresh precultures were prepared using a basal minimal medium supplemented with magnesium sulphate, thiamine and glucose. The precultures were incubated for 24 hours at 34 °C and 1000 rpm shaking and then further transferred to a new basal minimal medium (BMM, pH 7,5) in order to start the main culture. The new BMM was supplemented with magnesium sulphate, thiamine, a bolus of 20 % glucose solution (50 ul per 100 ml_) and a bolus of 10 % lactose solution (5 ml per 100 ml). Moreover, 50 % sucrose solution was provided as carbon source, accompanied by the addition of sucrose hydrolase (invertase), so that glucose was released at a rate suitable for C-limited growth. The main cultures were incubated for 72 hours at 28 °C and 1000 rpm shaking.
For the analysis of total broth, the 96-well plates were boiled at 100°C, subsequently centrifuged, and finally the supernatants were analysed by HPLC. For supernatant samples, the initial centrifugation of microtiter plates was followed by the removal of 0.1 ml_ supernatant for direct analysis by HPLC. For pellet samples, the cells were initially washed, then dissolved in deionized water and centrifuged. Following centrifugation, the pellets were analysed for HMO content in the cell interior after resuspension, boiling, centrifugation and analysis of the final supernatant.
Results of the deep well assays
Strains were characterized in deep well assays and samples were collected from the total broth. All samples were analysed for HMO content by HPLC following the 72-hour protocol described above. The concentration of the detected HMOs (in g/L) in each sample was used to calculate the % quantitative differences in the LNT-II content of the strains tested, i.e., the % differences in the LNT-II concentrations formed by strains expressing one of the three GlcNAcTs, namely LgtA, PmnagT or HD0466, or pairwise combinations of all three GlcNAcTs relative to similar strains that also express the native transporter LacY. As revealed by 72-hour experiments in 96-well plates and as shown in Figure 3, higher LNT-II levels (approximately 10%) can be reached when HD0466 is co-expressed with LgtA at saturating levels of intracellular lactose (i.e. when the lacY gene is over-expressed) (strain MP13) than at physiological lacY levels (strain MP7). This effect is even more obvious when the strains MP7 and MP13 have been tested in 24-well assays, with the strain MP13 reaching up to 30% higher LNT-II titers than the strain MP7 (Figure 4). Notably, the beneficial effect of the combined expression of HD0466 and LgtA and lacY overexpression is solely restricted to this pair of GlcNAcTs (Figure 3).

Claims

1. A method for the production of LNT-II, the method comprising the steps of: a) providing a genetically engineered bacterial or yeast cell capable of producing an HMO, wherein said cell comprises i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein [HD0466] as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1 ; and ii) a regulatory element for controlling the expression of i); and b) culturing the cell according to (a) in a suitable cell culture medium; and c) harvesting the HMO(s) produced in step (b).
2. The method according to claim 1 , wherein the cell is able to import lactose into the cell.
3. The method according to claim 1 or 2, wherein the cell further comprises, iii) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 2 [IgtA], or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 2; and iv) a regulatory element for controlling the expression of iii).
4. The method according to claim 3, wherein the cell further comprises v) a lactose permease protein as shown in SEQ ID NO: 3 [LacY], or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 3, and vi) a native or heterologous regulatory element for controlling the expression of v).
5. The method according to 4 , wherein the gene encoding the lactose permease protein is overexpressed.
6. The method according to any of the preceding claims, wherein the regulatory element for controlling and increasing the expression of i), iii) and/or v) is a heterologous or recombinant promoter.
7. The method according to claim 6, wherein the promoter is selected from a promoter sequence with a nucleic acid sequence as identified in Table 4, preferably a promoter sequence selected from the group consisting of SEQ ID NO: 11 (PglpF) or SEQ ID NO: 25 (Plac) or SEQ ID NO: 26 (PmglB_UTR70) or SEQ ID NO: 37 (PglpA_70UTR) or SEQ ID NO: 38 (PglpT_70UTR) or variants of these.
8. The method according to claim 6 or 7, wherein the promoter sequence is selected from the group consisting of PmglB_70UTR_SD8, PmglB_70UTR_SD10, PmglB_54UTR, Plac_70UTR, PmglB_70UTR_SD9, PmglB_70UTR_SD4, PmglB_70UTR_SD5, PglpF_SD4, PmglB_70UTR_SD7, PmglB_70UTR, PglpA_70UTR, PglpT_70UTR, pgatY_70UTR, PglpF, PglpF_SD10, PglpF_SD5, PglpF_SD8, PglpF_B28, PglpF_B29, PmglB_16UTR, PglpF_SD9, PglpF_SD7, PglpF_SD6 and PglpA_16UTR.
9. A genetically engineered bacterial or yeast cell comprising: a) a nucleic acid sequence according to SEQ ID NO: 4, or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 4, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase, and b) a regulatory element for controlling the expression of a).
10. The cell according to claim 9, wherein the cell is able to import lactose into the cell.
11. The cell according to claim 9 or 10, wherein the cell further comprises c) a nucleic acid sequence according to as shown in SEQ ID NO: 5 [IgtA], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 5, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; and d) a regulatory element for controlling the expression of c).
12. The cell according to claim 11 , further comprising e) a nucleic acid sequence according to as shown in SEQ ID NO: 6 [LacY], or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 6, encoding a lactose permease, and f) a native or heterologous regulatory element for controlling the expression of e).
13. The genetically engineered cell according to claim 12, wherein the lactose permease protein of e) is over-expressed.
14. The genetically engineered cell according to any of claims 9-13, wherein the regulatory element for controlling the expression is a promoter.
15. The genetically engineered cell according to claim 14, wherein the promoter is selected from the group consisting of with a nucleic acid sequence as identified in Table 4, preferably a promoter sequence selected from the group consisting of SEQ ID NO: 11 (PglpF) or SEQ ID NO: 25 (Plac) or SEQ ID NO: 26 (PmglB_UTR70) or SEQ ID NO: 37 (PglpA_70UTR) or SEQ ID NO: 38 (PglpT_70UTR) or variants of these.
16. The genetically engineered cell according to any of claims 14 or15, wherein the promoter sequence is selected from the group consisting of PmglB_70UTR_SD8, PmglB_70UTR_SD10, PmglB_54UTR, Plac_70UTR, PmglB_70UTR_SD9, PmglB_70UTR_SD4, PmglB_70UTR_SD5, PglpF_SD4, PmglB_70UTR_SD7, PmglB_70UTR, PglpA_70UTR, PglpT_70UTR, pgatY_70UTR, PglpF, PglpF_SD10, PglpF_SD5, PglpF_SD8, PglpF_B28, PglpF_B29, PmglB_16UTR, PglpF_SD9, PglpF_SD7, PglpF_SD6 and PglpA_16UTR.
17. The genetically engineered cell according to any of claims 9-16, wherein the glpR gene of the genetically engineered cell, encoding the DNA-binding transcriptional repressor GlpR, is deleted.
18. The genetically engineered cell according to any of claims 9-17, wherein the cell is Escherichia coli.
19. Use of a nucleic acid construct comprising a nucleic acid sequence encoding i) a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase protein as shown in SEQ ID NO: 1 or a functional homologue thereof having an amino acid sequence which is at least 80 % identical to SEQ ID NO: 1 ; and ii) a heterologous or recombinant regulatory element for controlling the expression of i), wherein the regulatory element is selected from the group consisting of PmglB_70UTR_SD8, PmglB_70UTR_SD10, PmglB_54UTR, Plac_70UTR, PmglB_70UTR_SD9, PmglB_70UTR_SD4, PmglB_70UTR_SD5, PglpF_SD4, PmglB_70UTR_SD7, PmglB_70UTR, PglpA_70UTR, PglpT_70UTR, pgatY_70UTR, PglpF, PglpF_SD10, PglpF_SD5, PglpF_SD8, PglpF_B28, PglpF_B29, PmglB_16UTR, PglpF_SD9, PglpF_SD7, PglpF_SD6 and PglpA_16UTR, in the production of LNT-II.
20. The use of a nucleic acid construct according to claim 19, further comprising iii) a nucleic acid sequence according to SEQ ID NO: 5 [IgtA], or a functional homologue thereof having a nucleic acid sequence which is at least 70% identical to SEQ ID NO: 5, encoding a heterologous b-1 ,3-N-acetyl-glucosaminyl-transferase; and iv) a regulatory element for controlling the expression of iii).
21. The use of a nucleic acid construct according to claim 20, further comprising v) a nucleic acid sequence according to SEQ ID NO: 6 [LacY], or a functional homologue thereof having a nucleic acid sequence which is at least 70 % identical to SEQ ID NO: 6, encoding a lactose permease, and vi) a native or heterologous regulatory element for controlling the expression of v).
22. Use of a genetically engineered cell according to any of claims 9-18, for the production of LNT-II.
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