Patent Application
20200347366
The present invention relates to genetically modified microorganisms for producing human milk oligosaccharide (HMO) and to a method of producing HMO using said microorganisms. The invention further relates to a medium, in which the microorganism of the invention has been cultured, to purified HMOs produced by the production method of the invention, and to uses of said medium and said HMOs.
Human milk oligosaccharides (HMOs) are structurally diverse unconjugated glycans that are found in human breast milk. More than 200 HMOs have been identified to date, which are all built from only five monosaccharide building blocks, i.e. D-galactose (Gal), D-glucose (Glc), N-acetyl-D-glucosamine (GlcNAc), L-fucose (Fuc) and the sialic acid derivative N-acetyl-neuraminic acid, coupled to a lactose moiety (Petschacher and Nidetzky, J Biotechnol. 2016, 235:61-83). The reducing end can either be lactose or lactose extended by several disaccharide units of two structure types, which are lacto-N-biose (Gal-β1,3-GlcNAc, type I) or lactosamine (Gal-β1,4-GlcNAc, type II, LacNAc). Structures and classification of HMOs can be found e.g. in
HMOs may be extracted from human breast milk or cow milk. However, in both cases it is difficult to obtain large amounts and/or to reach a satisfactory purity.
HMOs may also be chemically synthesized, however, the technology is not robust and the cost for chemically synthesized HMOs is high.
On the other hand, HMO production in genetically modified microorganisms holds promise to provide stable and safe means of production at low cost. U.S. Pat. No. 7,521,212 describes a method for producing oligosaccharides by a genetically modified E. coli cell from the precursor lactose, involving the inactivation of the lacZ gene. EP 2 675 899, EP 2 877 574, EP 2 440 661 and EP 3 191 499 further describe different fucosyltransferases for producing 2-FL or 3-FL in genetically modified microorganisms. Huang et al., 2017 describe the biosynthesis of 2-FL or 3-FL in genetically modified Escherichia coli (E. coli) and multiple modular optimization strategies thereto (Huang et al., Metab Eng. 2017; (41):23-38). EP 2 440 661 describes E. coli expressing sugar exporters to increase export of 2-FL into the medium. 2-FL consists of lactose and fucose (Pereira & McDonald, Sci Rep. 2012; 84(1):637-655). The biosynthesis pathway of 2-(3-)-FL in Escherichia coli is shown schematically in
Despite recent advances, there is still a need for improved methods of biotechnological production of HMOs, and particularly 2-FL and/or 3-FL, in genetically modified microorganisms. Relying on the known 2-FL biosynthetic pathway in E. coli, the invention significantly improves the synthesis of HMOs and their release into the medium.
The present invention provides microorganisms for the improved production of HMOs and HMO production methods using the same.
In particular the present invention provides microorganisms comprising an inducible lysis system, which allows for the easy extraction of intracellular and extracellular HMOs at the same time without the need of additional processing steps to obtain the intracellular fraction. A preferred example of the inducible lysis system is an auto-inducible lysis system which is Mg2+-regulated, wherein the cells are lysed under Mg2+-depleted conditions, i.e. when the extracellular Mg2+ concentration is below a certain threshold value. Upon lysis of the cells, intracellular HMO may be released into the medium, in which the cells are cultured, leading to an increased HMO concentration in the medium. This may advantageously lead to increased yields of HMO as well as time and cost savings.
The invention also provides microorganisms comprising an exogenous gene encoding a lactose permease. The lactose permease is preferably a mutant of LacY, which is less prone to inhibition by intracellular lactose compared to wildtype LacY. This may advantageously lead to an increased yield of HMO.
The present invention further provides a method for producing HMOs using the microorganism of the invention.
Also provided is a medium, in which the microorganism of the invention has been cultured during the method of producing HMOs, and purified HMOs, obtained by the method for producing HMOs of the invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Terms like “comprises” or “comprising” are used such that the terms may optionally be replaced by the terms “consists of” and “consisting of”, respectively.
Terms like “include” or “including” are used such that the terms may optionally be replaced by the terms “consists of” and “consisting of”, respectively.
The term “genetically modified” refers to any modification which has been introduced into a cell with respect to the genetic material of said cell. This may include the introduction of new genetic material, deletion of genetic material, mutation of genetic material etc.
The term “inducible lysis system” means that cell lysis may be induced by external stimuli. “Auto-inducible” means that the external stimuli do not have to be actively applied by the skilled person carrying out the invention.
The terms “external stimulus” or “external stimuli” refers to a chemical or physical stimulus or stimuli which is/are imposed on a cell from the outside.
The term “Mg2+-regulated” means that something is regulated by the presence or absence of free magnesium (Mg2+) ions. The “exogenous free Mg2+ concentration” refers to the concentration of free Mg2+ ions acting on the cells from the outside, e.g. from within the medium surrounding the cells. “Mg2+-depleted conditions” refer to a low free Mg2+ concentration in the medium or a complete absence of free Mg2+ in the medium.
A “lysis gene” is a gene encoding a factor, e.g. a protein, which causes cell lysis when expressed in a cell.
The term “inducibly expressed” means that a gene is not constitutively expressed but, instead, expression may be induced by external stimuli.
The term “the lysis genes are controlled by the Mg2+-regulated promoter” means that the genes are expressed or not expressed when the promoter is active or inactive, respectively, due to the absence or presence of free Mg2+ ions.
“Free Mg2+ ions” are those dissolved in e.g. the culture medium, which are not e.g. complexed by a chelating agent, such as EDTA. Thus, addition of e.g. a chelating agent may be used to reduce the concentration of free Mg2+ in the medium. If not stated otherwise, “Mg2+” refers to “free Mg2+ ions” herein.
The term “exogenous gene” refers to a gene which is introduced into a cell from the outside, e.g. by transformation. However, the term “exogenous gene” does not dictate the origin of said gene, e.g. an exogenous gene in E. coli may be derived from E. coli or from another organism. An exogenous gene may be episomal, for example present in an (expression) vector, such as a plasmid, or it may be integrated into a chromosome of the cell.
The term “heterologous gene” refers to a gene which is introduced into a cell from the outside and is derived from different organism than the cell into which it is introduced, e.g. a heterologous gene in E. coli is not derived from E. coli. A heterologous gene may be episomal, for example present in an (expression) vector, such as a plasmid, or it may be integrated into a chromosome of the cell.
A “heterologous promoter” when operably linked to a gene, refers to a promoter which is not naturally associated with the gene.
Exogenous or heterologous genetic material may be introduced into the microorganism by techniques commonly known in the art. For example, genetic material may be introduced into bacteria by transformation. Transformation may be conducted e.g. by heat-shock of chemically competent bacteria or by electroporation of electrocompetent bacteria (see e.g. Hanahan et al., Methods Enzymol. 1991; 204:63-113; Miller and Nickoloff, Methods Mol Biol. 1995; 47:105-13).
Exogenous or heterologous genetic material may be integrated into a chromosome of a cell by techniques commonly known in the art, such as the lambda Red system-mediated integration or CRISPR/Cas9 (see e.g. Juhas and Ajioka, Microb Cell Fact. 2016; 15(1):172; Reisch and Prather, Sci Rep. 2015; 5:15096). A gene which is “derived from” a particular organism, such as E. coli, may be the respective gene (directly obtained) from said organism (i.e. without modifying the sequence by mutations and/or codon-optimization). Alternatively, it may also include mutants obtained by introducing (e.g. 1 to 10, preferably 1 to 5, more preferably 1 to 3) mutations in the original sequence from said organism, and/or it may also include codon-optimized variants.
The term “codon-optimized for expression in the microorganism” means that the nucleic acid sequence of the open-reading frame (ORF) of a gene is optimized for translation into a protein in the organism into which the gene is introduced.
Where the microorganism of the invention is defined to comprise a gene encoding a particular protein, it is intended that the microorganism may also express the gene to produce the encoded protein.
The terms “overexpressed” or “overexpression” in the context of genes mean that the expression level of a gene in a microorganism, in which the gene is overexpressed, are higher than the expression level of the same gene in the same microorganism which does not overexpress said gene. Genes may be overexpressed by techniques commonly known in the art. For example, additional exogenous gene copies may be introduced into a cell, a cell may be treated with an exogenous transcriptional activator, or an exogenous gene encoding a transcriptional activator may be introduced into the cell. Introduction of additional gene copies is preferred in the present invention.
“Inactivation” of a gene means that the gene does not express any functional protein anymore. This includes almost complete or complete lack of expression or expression of dysfunctional proteins. An almost complete or complete lack of expression may be achieved for example via deletion of control sequences, such as the promoter, deletion of (parts of) the open reading frame (ORF) of the gene or deletion of the entire gene including promoter and ORF. A knockout of the gene is thus also encompassed by the term. Expression of dysfunctional proteins may be achieved for example by partial deletion, point mutations and/or insertions in/of the ORF, leading to expression e.g. of truncated and/or catalytically inactive proteins. Methods for introducing deletions, point mutations and/or insertions are well-known in the art. See for example, Reisch and Prather, 2015 (supra) or Zerbini et al. (Microb Cell Fact. 2017; 16(1):68), Jensen et al. (Sci Rep. 2015; 5:17874).
A particular nucleotide sequence, for example a sequence underlying a particular gene of interest or a promoter sequence etc., can either be amplified by polymerase chain reaction from the genomic sequences of an organism, e.g. E. coli, or it can be chemically synthesized by methods commonly known in the art.
GenBank Accession numbers refer to the unique identifiers used in GenBank, accessible e.g. via https://www.ncbi.nlm.nih.gov/genbank/. Gene identifiers (GI) refer to the unique numbers used to identify a particular gene, and may be queried e.g. at https://www.ncbi.nlm.nih.gov/gene/.
The present invention provides genetically modified microorganisms for the production of HMOs.
The HMOs to be produced in the invention are based on the disaccharides lactose or N-acetyllactosamine. HMOs based on lactose are preferred.
Preferred human milk oligosaccharides in the context of the invention include, but are not limited to, 2′-Fucoyllactose (2-FL), 3′-Fucosyllactose (3-FL), 3′-Sialyllactose (3-SL), 6′-Sialyllactose (6-SL), lacto-N-tetraose (LNT), lacto-N-neotetraose (LNnT), lacto-N-hexaose (LNH), iso-lacto-N-octaose, iso-lacto-N-neooctaose, para-lacto-N-octaose, lacto-N-fucopentaose I (LNFP I), lacto-N-fucopentaose II (LNFP II), lacto-N-fucopentaose III (LNFP III), lacto-N-fucopentaose V (LNFP V), LS-tetrasaccharide a (LST a), LS-zetrasaccharide b (LST b), LS-zetrasaooharide c (LST c) and disialyllacto-N-tetraose (DSLNT).
The trisaccharides 2-FL and/or 3-FL are preferably produced in the present invention, and 2-FL is particularly preferred. The structure of 2-FL and 3-FL is shown in (Pereira & McDonald, Sci Rep. 2012; 84(1):637-655).
The invention is described in more detail concerning the manipulation of genes and pathways within the enterobacterium Escherichia coli K-12 (E. coli) with regard to the production of 2-FL and/or 3-FL. However, the invention is similarly applicable to production of other HMOs in E. coli or microorganisms other than E. coli. Microbiological production of HMOs other than 2-FL and/or 3-FL has been described previously. For example, Priem et al. showed that lactose can be glycosylated to arrive at the trisaccharide GlcNAc-β1,3-Gal-β1,4-Glc, using expression of the β1,3-N-acetylglucosaminyltransferase LgtA (e.g. GI: 904226) from Neisseria meningitides, which can be further extended to lacto-N-neotetraose (LNnT) by the β1,4-galactosyltransferase LgtB (e.g. GI: 904227), also from N. meningitides (Priem et al., Glycobiology. 2002; 12(4):235-40). Subsequent intracellular fucosylation of LNnT, catalyzed by H. pylori β1,3-fucosyltransferase FutA or FutB, can provide a HMO mixture with lacto-N-neodifucohexaose II (LNnDFHII) as the main product (Dumon et al., Biotechnol Prog. 2004; 20(2):412-9). Different production methods are further discussed by Han et al. (Han et al., 2012, Biotechnol Adv. 2012; 30(6):1268-78).
The microorganism to be used in the present invention is not particularly limited. It is preferably a microorganism, which either has the natural ability of producing an HMO or is able to generate precursor molecules for production of an HMO intracellularly or to import such precursors from the extracellular space. Examples of the microorganism include a bacterium or a yeast. Examples of a bacterium include Escherichia coli, 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 modified using the methods of this invention, 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 jensenfi, and Lactococcus lactis. Streptococcus thermophiles and Proprionibacterium freudenreichii are also suitable bacterial species for the present invention. Also included as part of this invention are strains, modified 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). Examples of a yeast include Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris. The microorganism is preferably a bacterium, and E. coli is particularly preferred. The E. coli strain to be used is not particularly limited.
The release of HMOs from bacterial cells is an essential process for industrial production. About 50% of 2-FL yield remains in the cells while the remainder is discharged into the medium (Huang et al., 2017). Heat-induced lysis at 100° C. is required to gain access to the total 2-FL amount. To improve the recovery process, an autolysis system is established in the microorganism of the invention. The microorganism of the invention may thus comprise an inducible lysis system. The type of lysis system is not particularly limited as long as it can induce lysis due to an external stimulus. For example, the lysis genes of bacteriophage lambda (S, R and Rz) may be under control of the Mg2+-repressible PmgtA-UTR promoter (Tamekou Lacmata et al., Bioengineered. 2017; 26:1-6)). When free Mg2+ is consumed (e.g. at 10 μM or less), synthesis of the lysis genes is induced, facilitating the release of 2-FL into the medium by degradation of the cell wall component peptidoglycan. There are several alternatives available for the establishment of an autolysis system, which are based on an Mg2+-regulated promoter as described by Zhang et al. (J Microbiol Methods. 2009; 79(2):199-204), a nickel-inducible system as described by Liu and Curtiss, (Proc Natl Acad Sci USA. 2009; 106(51):21550-4), a thermosensitive lambda repressor (Juhas et al., PLoS One. 2016; 11(10):e0165778), a nisin-inducible promoter (Visweswaran et al., Appl Microbiol Biotechnol. 2017 February; 101(3):1099-1110), an arabinose-inducible promoter (Masuda et al., FEMS Microbiol Lett. 2016; 363(6)), an arabinose-inducible promoter, wherein MgSO4 counteracts the system (Li et al., Curr Microbiol. 2016; 72(4):390-6) or lysis induction by addition of solvents (Hajnal et al., Appl Microbiol Biotechnol. 2016; 100(21):9103-9110). These systems are also summarized in Table 1.
E. coli
E. coli
E. coli
Lactococcus
E. coli
E. coli
The Mg2+-regulatable system has the advantage that cell lysis can be promoted by the addition of e.g. Ethylenediaminetetraacetic acid (EDTA), a chelating agent that sequesters metal ions (such as Ca2+ or Mg2+). Moreover, Mg2+ as a divalent metal ion is required for α-1,2- and α-1,3-fucosyltransferase activities (Zhang et al., Glycobiology. 2010; 20(9):1077-88; Zhao et al., Chem Commun (Camb). 2016; 52(20):3899-902). Thus, in the presence of Mg2+, 2′-Fucosyllactose synthesis may be improved due to enhanced enzyme activity, while in its absence cell lysis is induced to facilitate product recovery via cell wall disruption. Accordingly, the Mg2+-regulated system is preferably used in the invention and preferably, the Mg2+-regulated system is Mg2+-repressible.
For the inducible lysis system a Mg2+-regulated promoter may be used. The Mg2+-regulated promoter is preferably regulated by the exogenous free Mg2+ concentration. It is preferably active under Mg2+-depleted conditions. Mg2+-depleted conditions are conditions wherein the growth medium comprises 500 μM or less, 100 μM or less, 50 μM or less, 10 μM or less, 1 μM or less or 0 μM of free Mg2+. Preferably, the Mg2+-depleted conditions are 10 μM or less of free Mg2+. The Mg2+-regulated promoter is preferably the PmgtB promoter from the mgtB gene (e.g. GI: 1250998) of Salmonella typhimurium, the PpagC promoter from the pagC gene (e.g. 1248241) of S. tyhphimurium or the PmgtA promoter from the mgtA gene (e.g. GI: 948778) of E. coli.
The PmgtB promoter from the S. typhimurium mgtB gene is as described in e.g. Zhang et al., 2009 (supra) and is obtainable e.g. by polymerase chain reaction (PCR) using the S. typhimurium genome as template and using the primers 5′-GTATACTCCGGAGCAAACGCCTGAACTCCC-3′ (SEQ ID NO: 1) and 5′-TCTAGAGGAAGAATAAGTACGTGCTATATTTAG-3′ (SEQ ID NO: 2). The PmgtA promoter from E. coli is as described in e.g. Tamekou Lacmata et al., 2017 (supra) and is obtainable e.g. by polymerase chain reaction (PCR) using the E. coli genome as template and using the primers 5′-CGAGCTCCTTTCGTTATTCAGCACCCG-3′ (SEQ ID NO: 3) and 5′-CGAGCTCGCGATATAATACCTGCTGGC-3′ (SEQ ID NO: 4).
The Mg2+-regulated promoter may be combined with additional Mg2+-regulated elements, such as the 5′UTR of the mgtA gene of E. coli. The 5′UTR of the mgtA gene from E. coli is as described in e.g. Tamekou Lacmata et al., 2017 (supra) and the PmgtA promoter together with the mgtA 5′UTR is obtainable e.g. by polymerase chain reaction (PCR) using the E. coli genome as template and using the primers 5′-CGAGCTCCTTTCGTTATTCAGCACCCG-3′ (SEQ ID NO: 3) and 5′-CGAGCTCAAGGAGTCCCTCCGCACTGT-3′ (SEQ ID NO: 5).
The microorganism comprising an inducible lysis system may further comprise one or more lysis gene(s). The lysis gene is not particularly limited as long as it can lyse a cell when it is expressed therein. For example, the lysis gene may be from a bacteriophage, such as from λ bacteriophage, MS2 bacteriopage, ϕX174 bacteriophage or RLT bacteriophage. Examples of lysis genes include ydfD from E. coli as described by Masuda et al., 2016 (supra), sicG from Streptococcus dysgalactiase subsp. equisimilis, vanX from E. coli, SRRz from Salmonella phage P22, yncE from S. enterica serovar Paratyphi A, lysis genes from Staphylococcus aureus phage P68.
Preferred examples of lysis gene are the SRRz gene from λ bacteriophage (e.g. as found in the sequence of GenBank Accession number: NC_001416) or the Sam7 mutant thereof, carrying a G to A mutation at position 161 in the S gene, the MS2 phage lysis gene encoding the E protein or the ϕX174 bacteriophage lysis gene encoding the L protein. A SRRz gene is as described in e.g. Zhang et al., 2009 (supra) and is obtainable e.g. by PCR using λ bacteriophage genome as template and primers 5′-CCGCATATGCCAGAAAAACATGACCTG-3′ (SEQ ID NO: 6) and 5′-TTAGTCGACGCAACTCTATCTGCACTGCTC-3′ (SEQ ID NO: 7). MS2 bacteriopage or ϕX174 bacteriophage lysis genes are as described in e.g. Juhas et al., 2016 (supra).
The microorganism may also comprise two or more lysis genes, either of the same type or different. Optionally, a combination of two or more different lysis genes may be used, since a different mechanism of action may lead to more efficient lysis. For example, the microorganism may comprise a lysis gene from A and MS2 bacteriophages, from λ and ϕX174 bacteriophages, from MS2 and ϕX174 bacteriophages or from λ, MS2 and ϕX174 bacteriophages. However, the invention is not limited to these combination and other lysis genes as described may be used alone or in combination. Preferably, the two or more lysis genes are located in tandem and are controlled by the same promoter and the same optional additional regulatory elements in a polycistronic manner.
The one or more lysis gene(s) may be inducibly expressed under Mg2+-depleted conditions and is/are preferably controlled by the Mg2+-regulated promoter with or without an additional regulatory element. If the 5′UTR of the mgtA gene is used as additional regulatory element, the expression cassette of the lysis gene(s) preferably comprise(s) the Mg2+-regulated promoter followed by the 5′UTR of the mgtA gene, followed by the ORF (or ORFs) of the lysis gene(s).
The expression cassette which may be used for inducible lysis as described above may be present in the microorganism as an episomal vector, such as an expression vector, e.g. a plasmid, or integrated into a host chromosome.
Furthermore, as the precursor for HMOs is lactose, it is desirable for the intracellular production of HMOs to achieve high intracellular levels of lactose. Therefore, the microorganism of the invention may be unable to cleave lactose into glucose and galactose. This may be achieved by inactivating the lacZ gene encoding the β-galactosidase LacZ responsible for lactose cleavage, or inactivating the entire lac operon, which encodes LacZ as well as the lactose repressor LaI, the transacetylase LacA and the lactose permease LacY. Inactivation may be achieved by inhibitors of expression, by introducing inactivating mutations or by deletion. Inactivation of the whole lac operon, comprising the genes lacI, lacZ, lacY and lacA, may increase the intracellular lactose availability by preventing i) the degradation of lactose into glucose and galactose and ii) by preventing the formation of allo- or acetyl-lactose. The latter additionally simplifies the purification process by eliminating two contaminating sugar molecules.
A lactose permease is responsible for uptake of lactose into the cell. Many bacteria have the inherent ability of transporting lactose from the culture medium into the cell, by using a transport protein that is either a homolog of the E. coli lactose permease (e.g., as found in Bacillus licheniformis), or a member of the PTS sugar transport family (e.g., as found in Lactobacillus casei and Lactobacillus rhamnosus). Accordingly, introducing an exogenous lactose permease gene, such as E. coli LacY, may increase the intracellular lactose levels. In the case of bacteria which do not have an inherent ability of transporting extracellular lactose into the cell (including those having the entire lac operon inactivated), this ability may be conferred or restored by introduction of an exogenous lactose permease gene, such as E. coli lacY. The microorganism of the invention may thus comprise an exogenous gene encoding a lactose permease. It is preferred that the lactose permease is E. coli LacY (e.g. GI: 949083). It is particularly preferred that the E. coli LacY has A198V and/or S209I mutations (Wilson et al., Biochim Biophys Acta. 1990; 1029(1):113-6). Both mutations are predicted to prevent the interaction between LacY and its inhibitor protein Enzyme-IlaGlc (Hogema et al., Mol Microbiol. 1999; 31(6):1825-33). As a result, the autoregulation mechanism may be suppressed at high intracellular lactose concentrations. Thus, the yield of HMOs may be enhanced by increasing the lactose uptake. Moreover, as lactose is a natural inducer of the T7 promoter expression system (see below), the expression e.g. of the enzymes required for 2-FL synthesis may be further improved by increased intracellular lactose availability.
Preferably, the microorganism of the invention is for the production of 2-FL and/or 3-FL.
Exemplary genetic modifications for high production of 2-FL in E. coli is shown in Table 2.
E. coli)
E. coli)
2-FL or 3-FL can be generated by coupling the activated sugar GDP-L-fucose to lactose. As described above, E. coli is capable of importing the precursor molecule lactose into the cells via the lactose permease LacY (a lactose/H+ symporter). Furthermore, E. coli is capable of importing glucose into the cell via the phosphotransferase system. Glucose may serve as energy source to promote growth and as a precursor for the production of GDP-L-fucose, which is a natural intermediate of colanic acid biosynthesis, via a de novo pathway (see
The fucose moiety of GDP-L-fucose can be transferred to the acceptor lactose in the cell via a fucosyltransferase (EC 2.4.1.x), thereby generating 2-FL or 3-FL (see
Several fucosyltransferases have been identified in bacteria such as Bacteroides fragilis, B. vulgatus, Campylobacter jejuni, E. coli, Helicobacter bilis, H. hepaticus, H. mustelae, H. pylori and Thermosynechococcus elongatus. Because many organisms have differential codon usages, the presence of codons with limited availability of cognate tRNA anticodons can lower heterologous gene expression due to ribosome stalling. To guarantee a high protein expression in the microorganism of the invention, the codon usage of the α-1,2- and α-1,3-fucosyltransferases encoding genes (e.g. futC and futA) may be optimized for the microorganism of the invention. Accordingly, the gene encoding a fucosyltransferase may be codon-optimized for expression in the microorganism. Codon-optimization may result in increased intracellular fucosyltransferase protein levels which may improve the yield of 2-FL and/or 3-FL.
Preferred examples of α-1,2-fucosyltransferase for use in the invention are FutC from Helicobacter pylori (e.g. NCBI accession no. KY499613), FutL from H. mustelae (e.g. NCBI accession no. WP_013023529.1), FutF from H. bilis (e.g. NCBI accession no. WP_020995676.1), WcfB from Bacillus fragilis NCTC 9343 (e.g. NCBI accession no. WP_005817145.1), WbsJ from E. coli O128 (e.g. NCBI accession no. AAO37698.1), WbwK from E. coli O86 (e.g. NCBI accession no. AAO37719.1), WbgL from E. coli O126 (e.g. NCBI accession no. ADN43847.1), WbiQ from E. coli O127 (e.g. NCBI accession no. CAS09719.1), FutG from Campylobacter jejuni (e.g. NCBI accession no. WP_002861859.1), FutN from Bacteroides vulgatus ATCC 8482 (e.g. NCBI accession no. ALK85429.1), WcfB and WcfW from Bacteroides fragilis NCTC9343 (e.g. NCBI accession no. WP_005817145.1 and WP_005813010.1).
Preferred examples of α-1,3-fucosyltransferase for use in the invention are FutA from Helicobacter pylori (e.g. NCBI accession no. AAD07447.1), FutB from H. pylori 26695 (e.g. NCBI accession no. AAD07710.1), FutD from H. trogontum (e.g. NCBI accession no. WP_052089242.1), FutE from H. bilis (e.g. NCBI accession no. WP_034580283.1), FutH from H. typhlonius (e.g. NCBI accession no. WP_052082154.1), FutJ (Hh0072) and FutK (Hh1776) from H. hepaticus (e.g. NCBI accession no. WP_011114915.1 and AAP78373.1), FutM from B. fragilis (e.g. NCBI accession No. CAH09151.1.1).
In a preferred embodiment, the activity of a fucosyltransferase is increased by the presence of Mg2+. Fucosyltransferases may form insoluble intracellular inclusion bodies, which may reduce the availability of active fucosyltransferases in the cell. The presence of an exogenously introduced chaperone may advantageously influence the solubility of the fucosyltransferase and thereby improve production of 2-FL and/or 3-FL. The microorganism of the invention may thus further comprise an exogenous gene encoding a chaperone. In a preferred embodiment, the chaperone is human Hsp70 (e.g. GenBank Accession Number: BC112963). Apart from chaperon function, human Hsp70 may further be utilized to establish a self-inducible expression system (Briand et al., Sci Rep. 2016; 6:33037), which may be used to avoid the addition of the expression inducer IPTG in the culture medium. As IPTG is also a substrate for the lactose permease, omission of IPTG may increase lactose uptake and thereby further improve the production of HMOs, particularly 2-FL and/or 3-FL.
Apart from the above modifications, a number of other genes may be introduced into or inactivated in the cells in order to further optimize production of HMOs, particularly of 2-FL and/or 3-FL. RcsA is a positive transcriptional regulator of colanic acid biosynthesis in E. coli, which uses GDP-L-fucose as intermediate. An overexpression of rcsA (e.g. GenBank Accession Number M58003) increases the transcription of the genes involved in biosynthesis of the GDP-L-fucose intermediate (see
However, RcsA is under the control of the Lon- and ClpYQ proteases. Thus, the respective Ion and clpYQ-genes may be inactivated in order to prevent a down-regulation of the required GDP-L-fucose biosynthesis genes. Overexpression of rcsA in combination with a Ion/clpYQ inactivation may dramatically improve GDP-L-fucose and thus 2-FL production. Accordingly, in the microorganism of the invention a Lon protease family gene, preferably E. coli Lon protease (e.g. GenBank Accession Number L20572), and/or the clpYQ genes (NC_000913.3) may be inactivated.
Furthermore, the wcaJ gene (e.g. GenBank Accession Number (amino acid) BAA15900) codes for an UDP-glucose lipid carrier transferase predicted to initiate the colanic acid biosynthesis. Inactivation of the wcaJ gene may be performed in order to i) prevent the consumption of GDP-L-fucose by the competitive colanic acid pathway, ii) prevent high viscosity of the culture medium due to export of the exopolysaccharide colanic acid and iii) simplify the 2-FL purification process by eliminating another contaminating sugar molecule (colanic acid). Thus, in the microorganism of the invention the wcaJ gene, preferably the E. coli wcaJ gene, may be inactivated.
The co-factors NADPH and GTP also play an important role in GDP-L-fucose biosynthesis, whose availability may limit the synthesis of the activated sugar (GDP-L-fucose) and thereby also the total yield of HMOs, such as 2-FL and/or 3-FL. To increase the availability of co-factors for GDP-L-fucose synthesis, four genes may be overexpressed in the microorganism of the invention. Zwf is part of the pentose phosphate pathway, while PntA and PntB belong to the transhydrogenase system. Overexpression of zwf and pntAB may significantly contribute to NADPH-regeneration, thereby providing the required co-factor for the NADPH-dependent WcaG enzyme involved in GDP-L-fucose synthesis (
The second co-factor, GTP, is synthesized via two alternative routes, the de novo GTP biosynthesis pathway via the formation of phosphoribosyl pyrophosphate or the salvage pathway from exogenous delivered guanosine. In both cases, an adequate supply of GMP can increase the GTP level and thus improve the efficiency of GDP-L-fucose production. The overexpression of the gsk gene (guanosine inosine kinase, e.g. GI: 946584), involved in both metabolic pathways, in the microorganism of the invention may increase the GTP-availability required for ManC (
Primers for cloning of exemplary genes discussed above may be found e.g. in Table S2 of Huang et al., 2017 (supra).
Additional optimization may involve manipulation of thioredoxin activity. The trxA gene (KEGG Entry, E. coli K-12 MG1655: b3781) codes for the major physiological electron donor in E. coli. Overexpression of thioredoxin (TrxA) is known to improve heterologous protein expression by promoting the formation of disulphide bond formation in poorly soluble proteins. However, two other functions have also been linked to TrxA, a potential chaperone function and a function as a processivity factor of the T7-DNA polymerase (Weickert et al., Curr Opin Biotechnol. 1996; 7(5):494-9). Thus, overexpression of trxA may be performed to i) increase the solubility of enzymes needed for HMO synthesis and ii) positively influence the expression of the respective genes. Accordingly, the microorganism of the invention may overexpress the thioredoxin trxA gene, which is preferably derived from E. coli.
Furthermore, the trxB gene (KEGG Entry, E. coli BL21 (DE3): ECD_00892) codes for a NADPH-dependent thioredoxin reductase (TrxB) that regenerates TrxA from its oxidized form. Inactivation of trxB limits the reducing potential in the cytoplasm and allows the formation of disulfide bonds (Weickert et al., 1996, supra). Inactivation of trxB may be performed to i) improve the protein solubility and enzyme activity and ii) prevent the exhaustion of NADPH, the co-factor required for WcaG (
The present invention also provides a method for producing HMOs using the genetically modified microorganism of the invention.
By culturing the microorganism of the invention in a medium, HMOs may be produced.
The culture medium is not particularly limited as long as it supports the growth of the microorganism. Examples of a medium for culturing the microorganism of the invention are LB medium (10 g/L tryptone; 5 g/L yeast extract; 10 g/L NaCl, pH 7.5), modified M9 minimal medium containing glucose (or glycerol) as carbon source (36 g/L glucose; 12.8 g/L Na2HPO4.7H2O, 3 g/L KH2PO4, 2 g/L NH4Cl, 0.5 g/L NaCl, 0.25 g/L MgSO4.7H2O, 14.7 mg/L CaCl2.2H2O, 10 mg/L thiamine, 2 g/L yeast extract, 0.1%(v/v) Triton-X 100, and 1 mL/L stock trace metal solution; stock trace metal solution: 25 g/L FeCl3.6H2O, 2 g/L CaCl2.2H2O, 2 g/L ZnCl2, 2 g/L Na2MoO4.2H2O, 1.9 g/L CuSO4.5H2O, and 0.5 g/L H3BO3, pH 7.2) (see Huang et al., 2017, supra), minimal medium containing 2.68 g/L (NH4)2SO4, 1 g/L (NH4)2—H-citrate, 26.42 g/L glycerol, 14.6 g/L K2HPO4, 0.241 g/L MgSO4, 2 g/L Na2SO4, 4 g/L NaH2PO4.H2O, 0.5 g/L NH4Cl, 10 mg/L thiamine and 3 ml/L trace element solution (0.5 g/L CaCl2.2 H2O, 16.7 g/L FeCl3.6 H2O, 20.1 g/L Na2-EDTA, 0.18 g/L ZnSO4.7 H2O, 0.1 g/L MnSO4.H2O, 0.16 g/L CuSO4.5 H2O and 0.18 g/L CoCl2.6 H2O) (Baumgärtner et al., Microb Cell Fact. 2013; 12:40), or minimal medium containing 20 g/L glycerol, 13.5 g/L KH2PO4, 4.0 g/L (NH4)2HPO4, 1.7 g/L citric acid, 1.4 g/L MgSO4.7H2O, 10 ml/L trace element solution (10 g/L Fe(III) citrate, 2.25 g/L ZnSO4.7H2O, 1.0 g/L CuSO4.5H2O, 0.35 g/L MnSO4.H2O, 0.23 g/L Na2B4O7.10H2O, 0.11 g/L (NH4)6Mo7O24, 2.0 g/L CaCl2.2H2O (Chin et al., J Biotechnol. 2016. pii: S0168-1656(16)31632-7).
The microorganism of the invention may be cultured at any temperature suitable for growth, e.g. at a temperature from 16° C. to 37° C., such as from 16° C. to 30° C., and preferably from 16° C. to 25° C.
In a preferred embodiment, the medium comprises lactose. The lactose concentration in the medium before the beginning of the culture may be 20 g/L or more, 12 g/L or more, 10 g/L or more, 8 g/L or more, between 8 and 20 g/L, between 10 and 20 g/L, or between 12 and 20 g/L. Preferably the lactose concentration in the medium before the beginning of the culture is between 10 and 20 g/L.
In another preferred embodiment, the medium comprises free Mg2+.
The free Mg2+ concentration (i.e. the concentration of free Mg2+) in the medium before beginning of the culture may be 80 mM or more, 70 mM or more, 60 mM or more, 50 mM or more, 40 mM or more, 30 mM or more, 20 mM or more, 10 mM or more, 5 mM or more, 1 mM or more, between 1 and 50 mM, between 5 and 50 mM, between 10 and 50 mM, between 20 and 50 mM, between 1 and 10 mM, between 5 and 10 mM, between 1 and 5 mM, or between around 50 and 60 μM. Preferably the free Mg2+ concentration in the medium before beginning of the culture is between 5 and 20 mM.
The free Mg2+ concentration at the end of the culture may be 500 μM or less, 100 μM or less, 50 μM or less, 10 μM or less, 1 μM or less, or 0 μM. Preferably, the free Mg2+ concentration at the end of the culture is 10 μM or less.
Preferably, the Mg2+ is added to the medium in the form of MgSO4.
At the end of the culture, a chelating agent, such as EDTA or EGTA, may optionally be added, preferably in sufficient amounts to sequester residual free Mg2+ ions and trigger lysis.
The method for producing HMOs of the invention is preferably conducted in a batch culture, or a fed-batch culture. The batch culture is a closed system culture in which cells are grown in a fixed volume of nutrient culture medium under specific environmental conditions without addition of further nutrients. In a fed-batch culture, nutrients are supplied to the bioreactor during cultivation. In both cases, the cells and products remain in the bioreactor until the end of the culture.
The culture volume in the method for producing HMOs of the invention is preferably 10 L or more, 100 L or more, 1000 L or more, 10000 L or more, 50000 L or more, between 10 L and 100 L, between 100 L and 1000 L, between 1000 L and 10000 L or between 10000 L or more than 50000 L. Preferably the culture volume is between 100 L and 1000 L.
The concentration of HMOs in the medium at the end of the culture is preferably at least 15 g/L, more preferably at least 20 g/L, even more preferably at least 30 g/L, and most preferably at least 50 g/L.
In the invention, a single HMO may be produced or two or more HMOs may be produced at the same time, preferably by a single genetically modified microorganism. For example, expression of a α-1,2-fucosyltransferase and a α-1,3-fucosyltransferase in the same cell may lead to the production of 2-FL, 3-FL and/or difucosylated structures such as lactodifucotetraose or lacto-N-difucohexaose. The HMO produced in the present invention is preferably 2-FL and/or 3-FL, and 2-FL is particularly preferred.
The HMO may be retrieved from the medium and/or from the microorganism itself. In order to retrieve the HMO from the microorganism, the microorganism has to be disrupted. Cell disruption by cell lysis may be conducted by chemical or physical means as commonly known in the art, or lysis may be induced by an inducible lysis system as described herein.
The method for producing HMOs of the invention may further comprise the step of purifying the HMO from the culture medium. Purifying in this context means that the HMO is present in pure form or essentially pure form after purification. After purification, the HMO, e.g., 2-FL and/or 3FL, preferably constitutes at least 90%, 95%, 98%, 99%, or 100% (w/w) of the purified product with respect to the dry weight. Purification may be done by techniques well known to the person skilled in the art. For example, HMO may be purified from the medium by methods commonly known to the person skilled in the art, e.g. by column chromatography using a charcoal step and elution with 35-50% ethanol, by an ethanol gradient or by size exclusion.
Purity can be assessed by any known method, such as thin layer chromatography or other electrophoretic or chromatographic techniques commonly known in the art.
Preferably, the HMO is purified from the culture medium after lysis has been induced via an inducible lysis system. Upon lysis of the cells, intracellular HMO is released into the medium leading to an increased HMO concentration in the medium. This may lead to increased yields of HMO as well as time and cost savings due to said increased yield and fewer processing steps. Accordingly, the HMO is preferably purified from the culture medium when the concentration of free Mg2+ in the medium is 10 μM or less and/or when the lysis can be directly visualized. Lysis can be directly visualized for example by a visual clearing of the culture and/or by measuring the OD600 absorbance values of the culture at 600 nm (optical density). OD600 values of the culture may be measured by a method commonly known to the person skilled in the art, e.g. using a spectrophotometer. A drop in OD600 values indicates that lysis of cells has taken place. Accordingly, in the invention, lysis can be directly visualized by a drop in OD600 values of the culture. The invention further provides a medium obtainable by the method for producing HMOs of the invention. Due to the production of HMOs by culturing the microorganism of the invention, said medium contains HMOs. The concentration of HMOs in the medium at the end of culturing is preferably at least 15 g/L, more preferably at least 20 g/L, even more preferably at least 30 g/L, and most preferably at least 50 g/L. The medium of the invention may further be dehydrated, for example by lyophilisation.
The invention also provides purified HMOs obtainable by the method of producing HMOs of the present invention.
The medium and the purified HMOs of the present invention may be used for the preparation of products for the consumption by humans, such as infant nutrition, and/or animals. The medium and the purified HMOs of the present invention may be used for the preparation of animal feed. The animal feed may be for companion animals (dogs, cats), livestock (bovine, equine, ovine, caprine, or porcine animals, as well as poultry) and/or fish. It is preferable that the animal feed is for cattle. Preferably the animal feed is a milk replacement product for calves.
The present invention will be illustrated by the following non-limiting examples.
The strains, plasmids and primers used are listed in Table 3 and 4. E. coli BL21 (DE3) is used as host strain for HMO production, E. coli TOP10 for plasmid construction and maintenance. E. coli is cultured in LB medium (10 gL−1 tryptone, 5 gL−1 yeast extract, 10 gL−1 NaCl) supplemented with ampicillin (100 μg ml−1), streptomycin (50 μg ml−1) or kanamycin (50 μg ml−1) when required. For gene deletions, the lambda Red recombinase/flippase system from Jensen et al. 2015 (Sci Rep 2015; 5:17874) may be used. Vector pSIJ8 is transformed into competent cells of E. coli BL21 (DE3). The lambda Red genes are induced in the presence of 15 mM L-arabinose. The kanamycin-resistance cassette (kanR) is amplified from vector pKD13 (Datsenko and Wanner, 2000), which is flanked by 50 bp long homologous target sequences and FRT sites, and transformed into L-arabinose induced competent cells. After deletion, removal of the antibiotic cassettes is achieved by induction of the flippase recombinase in the presences of 50 mM L-rhamnose. For curing of the temperature-sensitive helper plasmid (pSIJ8), cells are grown at 42° C. For gene expression, the vectors pETDuet-1, pCDFDuet-1 and pCOLADuet-1 may be used (Table 3,
E. coli K-12 MG1655
E. coli BL21 (DE3)
E. coli TOP10
GTGTAGGCTGGAGCTGCTTC
GATCCGTCGACCTGCAGTTC
GATCCGTCGACCTGCAGTTC
GTGTAGGCTGGAGCTGCTTC
GATCCGTCGACCTGCAGTTC
GTGTAGGCTGGAGCTGCTTC
GATCCGTCGACCTGCAGTTC
GTGTAGGCTGGAGCTGCTTC
GATCCGTCGACCTGCAGTTC
pET-manCB-gmd-wcaG
The genes manC-manB and gmd-wcaG are amplified from the genomic DNA of E. coli K-12 MG1655 using the primers listed in Table 4. First, the gmd-wcaG PCR product is digested with NdeI/KpnI and inserted into the likewise cut vector pETDuet-1 (
pCDF-futC-lacY*-hHSP70-trxA-SRRz
The human HSP70 gene with codons optimized for E. coli is synthesized and provided in a pUC57 vector (GeneScript). The hHSP70 gene is isolated from this vector via NdeI/BgIIII restriction and inserted into the likewise cut vector pCDFDuet-1 (
pCOLA-pntAB-rcsA-zwf-gsk
The rcsA gene is amplified from E. coli K12-MG1655, digested with NdeI/BgIII and inserted into the likewise cut vector pCOLADuet-1 (
The present invention provides means for improved production of human milk oligosaccharide (HMO). HMO produced by the microorganisms and production method of the present invention can be widely industrially applied, e.g. as functional ingredients in infant nutrition, medical nutrition, functional foods and animal feed.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17184232.1 | Aug 2017 | DE | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/070857 | 8/1/2018 | WO | 00 |
