Simple method for animal-free lactose synthesis

Abstract

A simple method for animal-free lactose synthesis. The present invention relates to a method for producing lactose, more particularly without separation from mammalian milk. Further, the present invention provides a method for purifying the lactose produced.

Description

[0001] The present invention relates to a method for producing lactose, more particularly without isolation from mammalian milk. Further, the present invention provides a method for purifying the produced lactose.

[0002] Lactose, as a disaccharide, has a wide variety of applications in the food and pharmaceutical industry (Hebbink GA, Dickhoff BHJ. Chapter 5 - Application of lactose in the pharmaceutical industry. In: Paques M, Lindner CBT-L, editors. Academic Press, 2019. p. 175-229. Available from https: / / www.sciencedirect.com / science / article / pii / B9780128117200000052; Hettinga KA. Chapter 6 - Lactose in the dairy production chain. In: Paques M, Lindner CBT-L, editors. Academic Press, 2019. p. 231-66. Available from https: / / www.sciencedirect.com / science / article / pii / B9780128117200000064). Currently, it is isolated from mammalian milk, mainly cow milk. Due to the environmental impact of this production process, there is a need for an alternative synthetic method that can compete with the well-established extraction from milk at low production costs.

[0003] Mammals synthesize lactose by a lactose synthase or a β-1,4 galactosyltransferase which transfers galactose to glucose with UDP-galactose as donor substrate and glucose as acceptor substrate. This principle has been adapted in microorganisms where the mammalian biosynthesis pathway was copied (Mao Z, Shin H-D, Chen RR. Engineering the E. coli UDP-glucose synthesis pathway for oligosaccharide synthesis. Biotechnol Prog [Internet]. Jan [cited 2016 Mar 13], 22(2):369-74. Available from, http: / / www.ncbi.nlm.nih.gov / pubmed / 16599548) and previously applied in vitro for the synthesis of lactose with enzymes by adding the expensive UDP-galactose as donor (Lau K, Thon V, Yu H, Ding L, Chen Y, Muthana MM, et al. Highly efficient chemoenzymatic synthesis of [small beta]1-4-linked galactosides with promiscuous bacterial [small beta]1-4-galactosyltransferases. Chem Commun [Internet]. 2010, 46(33):6066-8. Available from, http: / / dx.doi.org / 10.1039 / C0CC01381A).

[0004] One challenge encountered with these methods is the energy requirement for lactose synthesis. UDP-galactose synthesis on UDP-glucose requires UTP which has to be either added to the reaction mixture or synthesized by the microbial host. After lactose synthesis UDP is released which is either lost as substrate or can be recycled to UTP with the help of ATP, another expensive biochemical intermediate. Further, galactose needs to be added or synthesized which currently still originates from lactose and no alternative synthesis has been identified.

[0005] From an energy and cost perspective alone, even if it is more environmentally friendly, the prior art cannot compete with the extraction production of lactose. SUMMARY

[0007] It is an object of the present invention to provide a method that solves the problem of energy demand and production costs for lactose synthesis. The present invention provides a new method for producing lactose, wherein we solve the problem of stoichiometric addition of nucleotides and galactose.

[0008] According to the invention, this and other objects are achieved by providing a method for producing lactose, characterized in that the method comprises the use of i) a synthase, and ii) UDP, for forming UDP-glucose.

[0009] The method can further comprise the use of UDP-glucose-4-epimerase for UDP-glucose, which leads to the synthesis of UDP-galactose, which is then used by a galactosyltransferase for forming lactose. More particularly, a β1,4-galactosyltransferase is used for forming lactose, wherein glucose is used as acceptor. In a preferred method, the glucose is also produced from a carbon source, which is preferably selected from sucrose or trehalose, by adding a glucose isomerase, which converts fructose to glucose. In an alternative preferred method, the glucose is fed together with sucrose. This method allows the complete conversion of sucrose, trehalose or a combination of sucrose and glucose to lactose without the addition of stoichiometric amounts of nucleotides.

[0010] Key intermediates in the process from sucrose or trehalose to lactose include UDP-glucose, UDP-galactose and glucose. Key intermediates in the process from a combination feed of sucrose and glucose to lactose include UDP-glucose and UDP-galactose.

[0011] The present invention further provides an efficient method for purifying the produced lactose.

[0012] Further benefits of the teachings of the present invention will become clear to the skilled person upon reading the present invention.

[0013] Definitions

[0014] The words used in this specification to describe the invention and its various embodiments are to be understood not only in the sense of their commonly defined meanings, but to include by special definition in this specification structure, material or acts beyond the scope of the commonly defined meanings. Thus if an element can be understood in the context of this specification as including more than one meaning, then embodiments should be understood not to be limited to the normally defined meanings, but rather understood to include all compatible meanings.

[0015] The various aspects and embodiments of the application disclosed herein should not be construed as only being applicable in the specifically described order and context in the specification, but include any order and any combination thereof. Each embodiment identified herein can be combined together unless otherwise stated. All publications, patents and patent applications mentioned in the specification are hereby incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Unless otherwise indicated, all words used in the singular shall be interpreted to include the plural, and vice versa. 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 application belongs. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. Generally, enzymatic reactions and purification steps are performed according to the manufacturer's specifications. The specific techniques used to

[0016] In this specification, embodiments of the application have been disclosed and, although specific terms are employed, they are used in a descriptive sense only and should not be construed as limiting the application, the scope of which is set forth in the following claims. It is to be understood that the illustrative embodiments are presented by way of example only and that the application should not be limited by the illustrative embodiments. It will be apparent to those skilled in the art that changes, other embodiments, improvements, details and uses can be made with respect to the disclosure herein without departing from the scope of the disclosure, which is limited only by the claims, as interpreted according to the patent statute, including the doctrine of equivalents, as interpreted in the patent law. In the following claims, the reference characters used to designate steps are provided for convenience of description only and are not intended to imply any particular order to the steps, unless otherwise specifically stated.

[0017] Throughout the application, the feature "synthetic" is used interchangeably with the feature "produced" unless explicitly stated otherwise. Throughout the application, the expression "capable of" is preferably replaced by the active voice of the verb in question and vice versa. For example, the expression "capable of expressing" is preferably replaced by "expressing" and vice versa, i.e. "expressing" is preferably replaced by "capable of expressing". Throughout this document and in its claims, the verbs "comprise", "have" and "contain" and their conjugates are used in their non-limiting sense to mean that the items following the word comprise, have or contain, but do not exclude items not specifically mentioned. The verb "consist essentially of means, in the present context, that the solution or composition defined herein can contain, in addition to those particularly identified, further components which do not alter the unique characteristics of the invention. The further compounds can be unavoidable by-products, for example generated during the production of the lactose of the invention, and compounds which are introduced into the process stream from which the lactose is recovered but cannot be removed therefrom. With respect to the spray-dried powder, the term "consist essentially of includes a spray-dried powder which comprises at least 80%-wt, at least 85%-wt, at least 90%-wt, at least 93%-wt, at least 95%-wt or at least 98%-wt lactose with respect to the dry matter of the spray-dried powder. With respect to the spray-dried powder, the process stream and the solution comprising lactose, the term "consist essentially of is used likewise. Throughout this document and in its claims, the verbs "comprise", "have" and "contain" and their conjugates can preferably be replaced by "consist of" (and its conjugates) or "consist essentially of" (and its conjugates) and vice versa, unless specifically stated otherwise. Furthermore, the reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is only one of the elements. Thus, the indefinite article "a" or "an" often means "at least one". Throughout the document and in the claims, the articles "a" and "an" are preferably replaced by "at least one", more preferably by "at least two", even more preferably by "at least three", even more preferably by "at least four", even more preferably by "at least five", even more preferably by "at least six", most preferably by "at least two". When used in association with a numerical value (e.g. "about 10") or with a range (e.g. "about x to about y"), the words "about" or "approximately" preferably mean that the value or range is interpreted as accurately as the method used to measure it. If no error limits are specified, the expression "about" or "approximately" is interpreted as having the same rounding as the given value when used in association with a numerical value.Throughout this document and its claims, the expression "from x to y" (where x and y represent numerical values) means a range of values, wherein x is the lower limit of the range and y is the upper limit of the range. In this context, x and y are also included in the range.

[0018] Throughout the application, unless explicitly stated otherwise, the term "beta 1,4-galactosyltransferase" is understood to mean an enzyme capable of transferring galactose from UDP-galactose to glucose to form lactose. Such beta-1,4-galactosyltransferase is preferably a lactose synthase. An example of such a galactosyltransferase is the N-acetylglucosamine beta-1,4-galactosyltransferase GalT from Neisseria meningitidis (Uniprot ID Q51116, sequence version 2023_02 of May 3, 2023). Neisseria meningitidis Throughout the application, unless explicitly stated otherwise, the terms "UDP-glucose-4-epimerase" and "UDP-galactose-4-epimerase" are used interchangeably.

[0019] The term "bioconversion" is understood to mean the conversion of a product by resting cells or by permeabilized cells.

[0020] Throughout the application, unless explicitly stated otherwise, the terms "UDP-glucose-4-epimerase" and "UDP-galactose-4-epimerase" are used interchangeably.

[0021] The terms "glucose isomerase", "xylose isomerase" and "fructose isomerase" are used in the art to refer to an enzyme that converts fructose into glucose and vice versa.

[0022] Protein or polypeptide sequence information and functional information can be provided by extensive resources on protein sequences and annotation data such as the Universal Protein Resource (UniProt) (www.uniprot.org) (Nucleic Acids Res. 2021, 49(D1), D480-D489). UniProt comprises an expertly and richly curated protein database called UniProt Knowledgebase (UniProtKB), along with UniProt Reference Clusters (UniRef) and UniProt Archive (UniParc). The UniProt Identifier (UniProt ID) is unique for each protein present in the database. Throughout the application, the sequence of a polypeptide is denoted by the UniProt ID. Unless otherwise stated, the UniProt ID of a protein described corresponds to its sequence version 01 present in the UniProt database (www.uniprot.org) version release 2023_02 of May 3, 2023.

[0023] It will be understood by the skilled person that for each database used in the present text, the content of each database is fixed at each release and will not change. When the content of a particular database is changed, the particular database accepts a new release version with a new release date. All release versions on each database with their respective release dates and the particular content annotated at these particular release dates are available and known to the skilled person. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The production of lactose using sucrose as a starting material or carbon source is shown.

[0026] Figure 2 The production of lactose using trehalose as a starting material or carbon source is shown.

[0027] Figure 3 The production of lactose using sucrose and glucose as a starting material or carbon source is shown. DETAILED DESCRIPTION

[0029] In a first aspect, the present application provides a method for producing lactose, wherein the method comprises using i) a synthase, and ii) uridine diphosphate (UDP) for forming UDP-glucose.

[0030] UDP is preferably added in catalytic amounts, more preferably the UDP is provided in a concentration of 0.1 mM to 10 mM. Alternatively, UDP is preferably added in stoichiometric amounts.

[0031] Preferably, the method according to the present application further comprises using a UDP-glucose-4-epimerase to convert the UDP-glucose into UDP-galactose.

[0032] Alternatively or preferably, the UDP-galactose is a galactose donor for a b1,4-galactosyltransferase to form lactose.

[0033] Alternatively or preferably, the method can further comprise using a b1,4-galactosyltransferase to convert glucose and UDP-galactose to form lactose and UDP. Preferably, the b1,4-galactosyltransferase uses glucose as acceptor and UDP-galactose as donor.

[0034] Preferably, the UDP released by the b1,4-galactosyltransferase is recycled to be used by the synthase for forming UDP-glucose. Thus, the generated UDP can in turn be used in the reaction catalyzed by the sucrose synthase.

[0035] In a preferred embodiment of the present application, the synthase used also forms glucose or fructose. Preferably, in the method wherein the synthase also forms fructose, the fructose is converted into glucose.

[0036] In a preferred embodiment of the present application, the synthase is a sucrose synthase or a trehalose synthase.

[0037] Preferably, the sucrose synthase is selected from the GT4 glycosyltransferase subfamily, or from the KEGG enzyme class EC 2.4.1.13. Alternatively, the sucrose synthase is selected from the list of: sucrose synthase from Glycine max (Uniprot ID P13708, sequence version 2023_02 of May 3, 2023), sucrose synthase from Arabidopsis thaliana (Uniprot ID P49040, sequence version 2023_02 of May 3, 2023), sucrose synthase from Anabaena sp. PCC 7120 (Uniprot ID Q9ZEV2, sequence version 2023_02 of May 3, 2023), sucrose synthase from Acidithiobacillus ferrooxidans (Uniprot ID Q9ZEV2, sequence version 2023_02 of May 3, 2023). Glycine max Arabidopsis thaliana Anabaena Acidithiobacillus caldus ​​​the sucrose synthase from Anabaena sp. PCC 7119 (ATCC Accession No. 29151) (Uniprot ID Q9ZEV2, sequence version 2023_02 from May 3, 2023), the sucrose synthase from Acidithiobacillus ferrooxidans ATCC 51756 (Uniprot ID A0A059ZV61, sequence version 2023_02 from May 3, 2023), or any protein sequence comprising 80% or more sequence identity to any of the sucrose synthases from Glycine max (Uniprot ID P13708, sequence version 2023_02 from May 3, 2023), Arabidopsis thaliana (Uniprot ID P49040, sequence version 2023_02 from May 3, 2023), Anabaena sp. PCC 7119 (ATCC Accession No. 29151) (Uniprot ID Q9ZEV2, sequence version 2023_02 from May 3, 2023), Acidithiobacillus ferrooxidans ATCC 51756 (Uniprot ID A0A059ZV61, sequence version 2023_02 from May 3, 2023) and having sucrose synthase activity.

[0038] Preferably, the trehalose synthase is selected from the GT4 glycosyltransferase subfamily, or from the KEGG enzyme class EC 2.4.1.245. Alternatively, the trehalose synthase is selected from the list of: Thermococcus bergensis the trehalose synthase from Pyrococcus furiosus (strain T7324) (Uniprot ID Q7LYW5, sequence version 2023_02 from May 3, 2023), and the trehalose synthase from Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (Uniprot ID O58762, sequence version 2023_02 from May 3, 2023), or any protein sequence comprising 80% or more sequence identity to any of the trehalose synthases from Pyrococcus furiosus (strain T7324) (Uniprot ID Q7LYW5, sequence version 2023_02 from May 3, 2023) or from Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (Uniprot ID O58762, sequence version 2023_02 from May 3, 2023) and having trehalose synthase activity. Pyrococcus horikoshii

[0039] ​Preferably, the UDP-glucose-4-epimerase is selected from enzyme class EC 5.1.3.2, or from the list of the following: GalE from Escherichia coli K-12 MG1655 (Uniprot ID P09147, sequence version 2023_02, May 3, 2023), from Bifidobacterium longum subsp. longum (… Bifidobacterium longum subsp. longum GalE (Uniprot ID E8MF10, sequence version 2023_02, May 3, 2023) is from a species of the genus *Thermophyton* (*Thermophyton*). Pyrobaculum GalE (Uniprot ID A0A0K1E1Q5, sequence version 2023_02, May 3, 2023) of sp. WP30, is from Campylobacter jejuni subsp. ( Campylobacter jejuni subsp. jejuni GalE of serotype O:2 (Uniprot ID Q0P9C3, sequence version 2023_02 from May 3, 2023), from *Rhizobium sinense* (Osmanthus f. sinensis). Sinorhizobium meliloti GalE from strain 1021 (UniProt ID P26503, sequence version 2023_02, May 3, 2023), or containing GalE from *Escherichia coli* K-12 MG1655 (Uniprot ID P09147, sequence version 2023_02, May 3, 2023), GalE from *Bifidobacterium longum* subsp. *longum* (Uniprot ID E8MF10, sequence version 2023_02, May 3, 2023), GalE from *Thermoplasia* species WP30 (Uniprot ID A0A0K1E1Q5, sequence version 2023_02, May 3, 2023), or GalE from *Campylobacter jejuni* subsp. *jejuni* serotype O:2 (Uniprot ID P09147 ... Any protein sequence that has 80% or more sequence identity with either IDQ0P9C3 (sequence version 2023_02, May 3, 2023) or GalE (UniProt ID P26503, sequence version 2023_02, May 3, 2023) from *Rhizobium sinense* (strain 1021) and has UDP-glucose-4-epimerase activity.

[0040] Preferably, the β1,4-galactosyltransferase is lactose synthase, or is selected from enzyme class EC 2.4.1.87, GalT from Neisseria meningitidis (Uniprot ID Q51116, sequence version 2023_02, May 3, 2023), or Pasteurella multocida (…). Pasteurella multocida) the GalT of M1404 (UniProt ID D0EAD4 sequence version 2023_02 of May 3, 2023), or any protein having a sequence comprising 80% or more sequence identity with said GalT from Neisseria meningitidis (Uniprot ID Q51116, sequence version 2023_02 of May 3, 2023) or with said GalT from Pasteuria multiseptata (Uniprot ID Q9XQK8, sequence version 2023_02 of May 3, 2023) or with said GalT from M1404 (UniProt ID D0EAD4 sequence version 2023_02 of May 3, 2023). Pasteurella multocida ) any protein having a sequence of 80% or more sequence identity with the GalT of M1404 (UniProt ID D0EAD4 sequence version 2023_02 of May 3, 2023).

[0041] Preferably, said glucose isomerase is selected from the list of: the XylA from Streptomyces murinus (Uniprot ID P37031, sequence version 2023_02 of May 3, 2023), Streptomyces murinus the XylA from Arthrobacter sp. (strain NRRL B3728) (Uniprot ID P12070, sequence version 2023_02 of May 3, 2023), the XylA from Thermoanaerobacter ethanolicus (Uniprot ID D2DK62, sequence version 2023_02 of May 3, 2023), the XylA from Escherichia coli (Uniprot ID P00944, sequence version 2023_02 of May 3, 2023), Arthrobacter Thermoanaerobacter ethanolicus Escherichia coli the XylA from Escherichia coli K-12 MG1655 (Uniprot ID P00944, sequence version 2023_02 of May 3, 2023), or any protein having a sequence comprising 80% or more sequence identity with any one of said XylA from Streptomyces murinus (Uniprot ID P37031, sequence version 2023_02 of May 3, 2023), from Arthrobacter sp. (strain NRRL B3728) (Uniprot ID P12070, sequence version 2023_02 of May 3, 2023), from Thermoanaerobacter ethanolicus (Uniprot ID D2DK62, sequence version 2023_02 of May 3, 2023), from Escherichia coli K-12 MG1655 (Uniprot ID P00944, sequence version 2023_02 of May 3, 2023) and having a glucose isomerase activity.

[0042] In a preferred embodiment of the application, said method is a fermentation process, a biotransformation or an enzymatic process. The fermentation process used in the present application can be a microbial fermentation process or a cell culture fermentation process.​​

[0043] The cells used in such fermentation or biotransformation processes are selected from a list consisting of microorganisms, plant cells, animal cells, insect cells, or protozoan cells, more preferably microorganisms, more preferably bacteria or yeast, even more preferably bacteria, and even more preferably cells belonging to the genus Escherichia ( ). Escherichia ) or Bacillus spp. ( Bacillus The bacteria are more preferably Escherichia coli, more preferably Escherichia coli K-12 strain, and most preferably Escherichia coli MG1655.

[0044] Throughout the application and claims, unless otherwise specified, the microorganism is preferably bacteria, yeast or fungus, more preferably bacteria or yeast, and most preferably bacteria.

[0045] Throughout the application and claims, unless otherwise specified, the bacteria preferably belong to the phylum Proteobacteria, Firmicutes, Cyanobacteria, or Deinococcus-Thermus. The bacteria belonging to Proteobacteria preferably belong to the family Enterobacteriaceae, and more preferably to the species *Escherichia coli*. Escherichia coli The bacteria preferably refer to any strain belonging to the species *Escherichia coli*, such as, but not limited to, *Escherichia coli* B, *Escherichia coli* C, *Escherichia coli* W, *Escherichia coli* K12, and *Escherichia coli* Nissle. More particularly, the bacteria refer to cultured *Escherichia coli* strains – named *Escherichia coli* K12 strains – which are well adapted to laboratory environments and, unlike wild-type strains, have lost their ability to proliferate in the gut. Well-known examples of *Escherichia coli* K12 strains are K12 wild-type, W3110, MG1655, M182, MC1000, MC1060, MC1061, MC4100, JM101, NZN111, and AA200. Therefore, the present invention preferably specifically relates to *Escherichia coli* K12 strains, more preferably *Escherichia coli* MG1655 strains. The bacteria belonging to the phylum *Cladotomyces* preferably belong to the genus *Bacillus*, preferably from the genus *Bacillus* (…). Bacillus Bacillus species, such as Bacillus subtilis ( Bacillus subtilis ) or Bacillus amyloliquefaciens ( B. amyloliquefaciens The bacteria belonging to the phylum Actinobacteria preferably belong to the family Corynebacteriaceae, which includes the member Corynebacterium glutamicum (…). Corynebacteriumglutamicum ) or non-fermenting Corynebacterium ( C. afermentans ), or belong to the family Streptomycetaceae, which has the member Streptomyces griseus ( Streptomyces griseus ) or Streptomyces freundii ( S. fradiae ).

[0046] Throughout the application and claims, unless otherwise specified, the yeast cells preferably belong to the phylum Ascomycota, Basidiomycota, Deuteromycota, or Zygomycetes. The yeast cells preferably belong to the genus *Saccharomyces*. Saccharomyces (Having members such as brewer's yeast) Saccharomyces cerevisiae ), saccharin yeast ( S. bayanus ), Blakely yeast ( S. boulardii ), Pichia pastoris ( Pichia (Has members such as Pichia pastoris) Pichia pastoris ), abnormal Pichia pastoris ( P. anomala ), Klufer Pichia pastoris ( P. kluyveri ), genus *Komagata* ( Komagataella ), Hansenula genus ( Hansunella Kluyveromyces ( ) Kluyveromyces (Has members such as Kluyveromyces lactis) Kluyveromyces lactis ), Max Kluyveromycin ( K. marxianus ), heat-resistant Kluyveromycin ( K. thermotolerans ), Yersinia ( Yarrowia (For example, Yersinia lipophila) Yarrowia lipolytica )), Pseudomonas ( Eremothecium ), Zygosaccharomyces ( Zygosaccharomyces ), Stamozygosacchari ( Starmerella (For example, bumblebee Stamozyme) Starmerella bombicola )) or Debali yeast ( Debaromyces The yeast cells are more preferably selected from Pichia pastoris, Yersinia lipolytica, Saccharomyces cerevisiae, and Kluyveromyces lactis.

[0047] Throughout the application and claims, unless otherwise specified, the fungus preferably belongs to the genus Rhizopus (Rhizopus). Rhizopus ), genus *Dendrocalamus* Dictyostelium ), Penicillium ( Penicillium Mucor ( ) Mucor ) or Aspergillus ( Aspergillus ).

[0048] Throughout the application and claims, unless otherwise specifically indicated, plant cells include cells of flowering plants and non-flowering plants, as well as algal cells, such as Chlamydomonas, Chlorella, etc. Preferably, the plant cells are tobacco, alfalfa, rice, cotton, oilseed rape, tomato, maize, corn or soybean cells.

[0049] Throughout the application and claims, unless otherwise specifically indicated, animal cells are preferably derived from a non-human mammal (e.g., a cow, a bison, a pig, a sheep, a mouse, a rat), a bird (e.g., a chicken, a duck, an ostrich, a turkey, a pheasant), a fish (e.g., a marlin, a salmon, a tuna, a sea bass, a trout, a catfish), an invertebrate (e.g., a lobster, a crab, a shrimp, a clam, an oyster, a mussel, a sea urchin), a reptile (e.g., a snake, an alligator, a turtle), an amphibian (e.g., a frog) or an insect (e.g., a fly, a nematode), or a genetically modified cell line derived from a human cell (excluding embryonic stem cells). Both human and non-human mammalian cells are preferably selected from the list consisting of epithelial cells, such as mammary epithelial cells, embryonic kidney cells (e.g., HEK293 or HEK 293T cells), fibroblasts, COS cells, Chinese hamster ovary (CHO) cells, murine myeloma cells, such as N20, SP2 / 0 or YB2 / 0 cells, NIH-3T3 cells, non-mammary adult stem cells or derivatives thereof, such as described in WO21067641.

[0050] Throughout the application and claims, unless otherwise specifically indicated, insect cells are preferably derived from Spodoptera frugiperda (Sf) Spodoptera frugiperda )(e.g., Sf9 or Sf21 cells), Bombyx mori (Bm) Bombyx mori ), Mamestra brassicae (Mb) Mamestra brassicae ), Trichoplusia ni (Tn) Trichoplusia ni )(e.g., BTI-TN-5B1-4 cells) or Drosophila melanogaster (Dm) Drosophila melanogaster )(e.g., Drosophila S2 cells).

[0051] Throughout the application and claims, unless otherwise specifically indicated, protozoan cells are preferably Leishmania tarentolae (Lt) Leishmania tarentolae ) cells.

[0052] In another preferred embodiment, the cells used in a biotechnological production process according to the application are single cells. It is further preferred that the cells used in a biotechnological production process according to the application are isolated cells.

[0053] In a more preferred embodiment of the application, when the method is an enzymatic process, at least one enzyme used in the enzymatic process is immobilized. Preferably, the enzyme is immobilized on a support, or by entrapment, adsorption or covalent binding. The enzyme is immobilized by well-known techniques, for example by entrapment of the enzyme (“entrapped immobilized enzyme”), wherein the enzyme is entrapped in, for example, collagen, gelatin, cellulose triacetate, polyacrylamide and / or i-carrageenan, by binding (“bound immobilized enzyme”), by adsorption (“adsorbed bound immobilized enzyme”), wherein the enzyme is adsorbed to polysaccharide derivatives, synthetic polymers and porous glass, by covalent binding (“covalently bound immobilized enzyme”), wherein the enzyme is bound to glutaraldehyde, diazobenzenes and hexamethylenediisocyanate (see, for example, Rafiq Khan M, Bulletin of the National Research Centre 45, 207 (2021)).

[0054] In an alternative or more preferred embodiment of the application, when the method is an enzymatic process as described herein, at least one reaction in the enzymatic process is performed between 50°C and 80°C, preferably between 55°C and 70°C.

[0055] In a more preferred embodiment of the application, when the method is a fermentation process or a biotransformation, the enzymatic conversion in the fermentation process or biotransformation is performed by at least one enzyme produced by a cell synthesizing the enzyme.

[0056] In an alternative more preferred embodiment of the application, when the method is a fermentation process, the lactose is produced by a cell genetically engineered for producing all enzymes needed for lactose production.

[0057] In a preferred embodiment of the application, the fermentation process or biotransformation process is performed at a temperature of about 25°C to about 50°C, preferably about 25°C to about 40°C.

[0058] In a preferred embodiment of the application, the carbon source used for conversion to lactose by the synthase is selected from the list of sucrose, trehalose, a combination of sucrose and glucose, and even starch.

[0059] In a preferred embodiment of the application, the method uses a synthase which is sucrose synthase and the method starts the conversion using sucrose. The sucrose is converted to UDP-glucose and fructose with the sucrose synthase. The formed UDP-glucose is converted to UDP-galactose. The fructose is converted to glucose and then the UDP-galactose and the glucose are converted to lactose. In An exemplary embodiment thereof can be found in Figure 1 An exemplary embodiment thereof can be found in

[0060] In an alternative preferred embodiment of the application, the method uses a synthase which is trehalose synthase and the method starts the conversion using trehalose. The trehalose is converted to UDP-glucose and glucose with the trehalose synthase. The formed UDP-glucose is converted to UDP-galactose and then the UDP-galactose together with glucose is converted to lactose. In An exemplary embodiment thereof can be found in Figure 2 An exemplary embodiment thereof can be found in

[0061] In another alternative preferred embodiment of the application, the method uses a synthase which is sucrose synthase and the method starts the conversion using sucrose and glucose. The sucrose is converted to UDP-glucose and fructose with the sucrose synthase. The formed UDP-glucose is converted to UDP-galactose. The fructose does not need to be converted to glucose because glucose is also present as carbon source. Then, the UDP-galactose and the glucose are converted to lactose. In An exemplary embodiment thereof can be found in Figure 3 An exemplary embodiment thereof can be found in

[0062] In a further preferred embodiment of the application, the method is the enzymatic production of lactose and uses i) sucrose, ii) trehalose or iii) sucrose and glucose as starting material or carbon source. Then, lactose is produced by using i) sucrose, ii) trehalose or iii) sucrose and glucose as starting material or carbon source and its reaction is catalyzed by a combination of enzymes. In a preferred embodiment, all enzymatic reactions are performed in a single tank. In another preferred embodiment, some enzymatic reactions are performed separately from others in the method.

[0063] According to a preferred embodiment of the application, the method described herein further comprises the step of separating the lactose.

[0064] According to a preferred embodiment of the application, the method described herein further comprises the step of purifying the lactose.

[0065] According to a preferred embodiment of the present application, the process described herein comprises a step of drying or crystallizing said lactose, preferably said drying step comprises any one or more of the following: spray drying, lyophilization, evaporation, precipitation, spray freeze drying, freeze spray drying, band drying, belt drying, vacuum band drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying and agitated thin film drying.

[0066] In a second aspect, the present application provides a process for purifying lactose synthetized biotechnologically, wherein said lactose is purified by at least one of the following purification steps: microfiltration, centrifugation, ultrafiltration, nanofiltration, ion exchange, simulated moving bed (SMB), color removal, removal of carbon source, removal of sucrose, glucose or trehalose by addition of yeast, removal of trehalose by addition of trehalase, removal of sucrose by addition of invertase; and preferably further comprising concentration, drying and / or crystallization as described herein. Preferably, said ion exchange step is a cation and / or anion exchange step.

[0067] According to a preferred embodiment of the present application, the lactose purity before purification is < 70%, < 60%, < 50%, < 40%, < 30%, < 20%, < 10% based on total solids, and / or the purity at the end of the process including purification is higher than > 80%, preferably > 85%, more preferably > 90%, even more preferably > 95%, even more preferably > 97%, even more preferably > 98%, most preferably > 99% based on dry solids.

[0068] In a further preferred embodiment, the purification of lactose synthetized enzymatically comprises a step of microfiltration or ultrafiltration to recover used enzymes.

[0069] In an alternative further preferred embodiment, the purification of lactose synthetized biotechnologically comprises a step of centrifugation, microfiltration and / or ultrafiltration to remove biomass.

[0070] In a further preferred embodiment, the cells obtained from the fermentation or biotransformation after purification of the fermentation or biotransformation medium are reused in a further fermentation or biotransformation process.

[0071] In a further preferred embodiment of the present application, the process described herein can comprise: i) a step of nanofiltration to remove / reduce monosaccharides and / or salts; and / or ii) a step of ion exchange to remove charged materials.

[0072] According to the present application described herein, the purified lactose preferably has an ash content of less than 1 %, more preferably less than 0.5 % by total solids. According to a further preferred embodiment, the purified lactose has one or more of the following:

[0073] a) a lead content of less than 0.1 mg / kg solids, preferably less than 0.02 mg / kg solids;

[0074] b) an arsenic content of less than 0.2 mg / kg solids, preferably less than 0.02 mg / kg solids;

[0075] c) a cadmium content of less than 0.1 mg / kg solids, preferably less than 0.01 mg / kg solids; or

[0076] d) a mercury content of less than 0.5 mg / kg solids, preferably less than 0.1 mg / kg solids.

[0077] In another preferred embodiment according to the present application, the purified lactose has at least one or more of the following: i) a protein content of less than 100 mg / kg dry solids, ii) a DNA content of less than 10 ng / g dry solids, and iii) an endotoxin content of less than 10000 EU / g dry solids.

[0078] All other embodiments which are obtained by a person skilled in the art without inventive effort on the basis of the description, the embodiments and the examples in the present application fall within the scope of protection of the present application.

[0079] In summary, the process for producing lactose of the present application is completely different from known processes for producing lactose, and the end product lactose can be used in industry, e.g. in food.

[0080] Further advantages can be taken from the specific embodiments and examples. It goes without saying that the above-mentioned features and the features still to be explained below can be used not only in the respectively indicated combinations, but also in other combinations or alone, without departing from the scope of the present application.

[0081] The present application relates to the following particular embodiments:

[0082] 1. A method for producing lactose, characterized in that the method comprises the use of i) a synthase, and ii) UDP, for forming UDP-glucose.

[0083] 2. The method according to embodiment 1, wherein UDP is provided in catalytic amounts, preferably UDP is provided in a concentration of 0.1 mM to 10 mM.

[0084] 3. The method according to embodiment 1, further comprising using a UDP-glucose-4-epimerase to convert the UDP-glucose to UDP-galactose.

[0085] 4. The method according to any one of embodiments 1 or 2, wherein the UDP-galactose is the galactose donor for a b1,4-galactosyltransferase to form lactose.

[0086] 5. The method according to any one of embodiments 1 to 3, further comprising using a b1,4-galactosyltransferase to convert glucose and UDP-galactose to form lactose and UDP.

[0087] 6. The method according to any one of embodiments 1 to 5, wherein UDP released by the b1,4-galactosyltransferase is recycled to be used by the synthase to form UDP-glucose.

[0088] 7. The method according to any one of embodiments 1 to 6, wherein the synthase further forms glucose or fructose.

[0089] 8. The method according to any one of embodiments 1 to 7, wherein the synthase is a sucrose synthase or a trehalose synthase.

[0090] 9. The method according to any one of embodiments 1 to 8, wherein the b1,4-galactosyltransferase uses glucose as acceptor and UDP-galactose as donor.

[0091] 10. The method according to any one of embodiments 8 to 9, wherein the fructose is converted to glucose.

[0092] 11. The method according to any one of embodiments 1 to 10, wherein the method is a microbial fermentation process, a biotransformation or an enzymatic process.

[0093] 12. The method according to any one of embodiments 1 to 11, wherein the method is an enzymatic process, and wherein at least one of the enzymes is immobilized, preferably the enzymes are immobilized on a support, or by entrapment, adsorption or covalent binding.

[0094] 13. The method according to any one of embodiments 1 to 12, wherein the method is an enzymatic process, and wherein the reaction is performed between 50 °C and 80 °C, preferably between 55 °C and 70 °C.

[0095] 14. The method according to any one of embodiments 1 to 11, wherein the method is a microbial fermentation process or a bioconversion, characterized in that the enzymatic conversion is carried out by at least one enzyme produced by a cell synthesizing the enzyme.

[0096] 15. The method according to any one of embodiments 1 to 11, wherein the method is a microbial fermentation process, characterized in that the lactose is produced by a cell genetically engineered for producing all enzymes needed for lactose production.

[0097] 16. The method according to any one of the preceding embodiments, wherein the carbon source for conversion by the synthase is selected from the list of sucrose, trehalose, a combination of sucrose and glucose.

[0098] 17. The method according to any one of embodiments 11 to 13 for the enzymatic production of lactose using i) sucrose, ii) trehalose or iii) sucrose and glucose as starting material, characterized in that lactose is produced by using i) sucrose, ii) trehalose or iii) sucrose and glucose as starting material and its reaction is catalyzed by a combination of enzymes.

[0099] 18. The method according to any one of embodiments 1 to 17, wherein the synthase is a trehalose synthase and wherein i) trehalose is converted to UDP-glucose and glucose with the trehalose synthase and ii) the UDP-glucose is converted to UDP-galactose and iii) glucose and UDP-galactose are converted to lactose.

[0100] 19. The method according to any one of embodiments 1 to 17, wherein the synthase is a sucrose synthase and wherein i) sucrose is converted to UDP-glucose and fructose with the sucrose synthase, ii) the UDP-glucose is converted to UDP-galactose and iii) fructose is converted to glucose and iv) the UDP-galactose from step ii) and the glucose from step iii) are converted to lactose.

[0101] 20. The method according to any one of embodiments 1 to 17, wherein the synthase is a sucrose synthase and the carbon source is sucrose with glucose, wherein i) sucrose is converted to UDP-glucose and fructose with the sucrose synthase, ii) the UDP-glucose is converted to UDP-galactose and iii) the UDP-galactose from step ii) and the glucose provided with sucrose are converted to lactose.

[0102] 21. The method according to any one of embodiments 4, 5 or 9, wherein the beta 1,4- galactosyltransferase is a lactose synthase.

[0103] 22. The method according to any one of the preceding embodiments, wherein the method further comprises a step of separating the lactose.

[0104] 23. The method according to any one of the preceding embodiments, wherein the method further comprises a step of purifying the lactose.

[0105] 24. A method for purifying lactose synthetized enzymatically or biotechnologically, wherein the lactose is purified by a purification step selected from the group consisting of a microfiltration step, a centrifugation step, an ultrafiltration step, a nanofiltration step, an ion exchange step, preferably a cation and / or anion exchange step, a simulated moving bed step, a color removal step, a step of removing sucrose, glucose or trehalose by adding yeast, a step of removing trehalose by adding trehalase, a step of removing sucrose by adding invertase, and preferably further comprising a concentration step, a spray drying step and / or a crystallization step.

[0106] 25. The method according to any one of the preceding embodiments, wherein the method comprises a step of drying or crystallizing the lactose, preferably the drying step comprises any one or more of the group consisting of spray drying, freeze drying, evaporation, precipitation, spray freeze drying, freeze spray drying, belt drying, belt drying, vacuum belt drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying and agitated thin film drying.

[0107] 26. The method according to any one of embodiments 23 to 25, wherein the lactose purity before purification is < 70%, < 60%, < 50%, < 40%, < 30%, < 20%, < 10% based on total dry solids, and / or the purity at the end of the method including purification is higher than > 80%, preferably > 85%, more preferably > 90%, even more preferably > 95%, even more preferably > 97%, even more preferably > 98%, most preferably > 99% based on total dry solids.

[0108] 27. The method according to any one of embodiments 23 to 26, wherein the purification of the lactose synthetized enzymatically comprises a step of microfiltration or ultrafiltration to recover used enzymes.

[0109] 28. The method according to any one of embodiments 23 to 27, wherein the purification of the biotechnologically synthesized lactose comprises a step of centrifugation, microfiltration and / or ultrafiltration to remove biomass.

[0110] 29. The method according to any one of embodiments 23 to 28, comprising i) a step of nanofiltration to remove / reduce monosaccharides and / or salts; and / or ii) a step of ion exchange to remove charged materials.

[0111] 30. The method according to any one of embodiments 23 to 29, wherein the purified lactose has an ash content of below 1% based on total dry solids, preferably below 0.5% based on total dry solids, preferably has one or more of:

[0112] a) a lead content of below 0.1 mg / kg dry solids, preferably below 0.02 mg / kg dry solids;

[0113] b) an arsenic content of below 0.2 mg / kg dry solids, preferably below 0.02 mg / kg dry solids;

[0114] c) a cadmium content of below 0.1 mg / kg dry solids, preferably below 0.01 mg / kg dry solids; or

[0115] d) a mercury content of below 0.5 mg / kg dry solids, preferably below 0.1 mg / kg dry solids.

[0116] 31. The method according to any one of embodiments 23 to 30, wherein the purified lactose has at least one or more of: i) a protein content of below 100 mg / kg dry solids, ii) a DNA content of below 10 ng / g dry solids, and iii) an endotoxin content of below 10000 EU / g dry solids.

[0117] Example

[0118] Example 1: Materials and methods

[0119] Analytical analysis

[0120] Standards, such as but not limited to sucrose, lactose, glucose, fructose, UDP-glucose, UDP-galactose were purchased from Sigma, Carbosynth (UK), Elicityl (France) and IsoSep (Sweden). Other compounds were analyzed with in-house prepared standards.

[0121] Carbohydrates were analyzed on a Waters Acquity H-Class UPLC with evaporative light scattering detector (ELSD) or refractive index (RI) detection. A volume of 0.7 μL of sample was injected onto a Waters Acquity UPLC BEH Amide column (2.1 x 100 mm; 130 Å; 1.7 μm) with an Acquity UPLC BEH Amide VanGuard column (130 Å, 2.1 x 5 mm). The column temperature was 50 °C. The mobile phase consisted of ¼ water and ¾ acetonitrile to which 0.2% triethylamine was added. The method was isocratic with a flow rate of 0.130 mL / min. The ELS detector had a drift tube temperature of 50 °C, and N2 gas pressure was 50 psi, gain was 200, and data transfer rate was 10 pps. The temperature of the RI detector was set at 35 °C.

[0122] Ash content

[0123] Ash content is a measure of the total amount of minerals present in a food or ingredient, such as an oligosaccharide, while mineral content is a measure of the amount of specific inorganic components (e.g., Ca, Na, K, Mg, phosphate, sulfate, and CI) present in a food. Determination of ash and mineral content of a food or oligosaccharide is important for many reasons. Nutrition labeling: The concentration and type of minerals present must often be specified on the label of a food or ingredient, such as an oligosaccharide. The quality of many foods depends on the concentration and type of minerals they contain, including their taste, appearance, texture, and stability. Microbiology stability: High mineral content is sometimes used to retard the growth of certain microorganisms. Nutrition: Some minerals are essential for a healthy diet (e.g., calcium, phosphorus, potassium, and sodium), while others can be toxic (e.g., lead, mercury, cadmium, and aluminum). Processing: Knowing the mineral content of a food / product during processing is often important because it affects the physicochemical properties of the food or ingredient, such as an oligosaccharide.

[0124] Ash is the inorganic residue remaining after water and organic matter are removed by heating in the presence of an oxidizing agent, which provides a measure of the total amount of minerals within a food. Analytical techniques used to provide information about total mineral content are based on the fact that the minerals (analytes) can be distinguished from all other components (matrix) within the food in some measurable way. The most widely used method is based on the fact that minerals are not destroyed by heat and they have a lower volatility than other food components. Three main types of analytical procedures for determining the ash content of foods are based on this principle: dry ashing, wet ashing, and low-temperature plasma ashing. The method chosen for a particular analysis depends on the reason for performing the analysis, the type of food or ingredient being analyzed, and the equipment available. Ashing can also be used as a first step in preparing a sample for the analysis of specific minerals (by atomic spectroscopy or various conventional methods described below).

[0125] For sample preparation, a sample whose composition is representative of the composition of the ingredient is selected to ensure that its composition does not change significantly prior to analysis. For example, dry oligosaccharide samples are typically hygroscopic, and the selected sample should be kept under dry conditions to avoid the absorption of moisture. Typically, 1-10 g of sample is used in the analysis of ash content. Solid ingredients are finely ground and then carefully mixed to facilitate the selection of a representative sample. Prior to performing ash analysis, samples with high moisture or solution content are usually dried to prevent spattering during ashing. Other possible problems include contamination of the sample by minerals in the mill, glassware, or crucible that come into contact with the sample during analysis. For the same reason, deionized water is used when preparing the sample, and deionized water is used in the blank sample.

[0126] The dry ashing procedure uses a muffle furnace capable of maintaining temperatures between 500 and 600 °C. Water and other volatile materials are vaporized, and organic matter is combusted in the presence of oxygen in air to CO2, H2O, and N2. Most minerals are converted to oxides, sulfates, phosphates, chlorides, or silicates. While most minerals have a fairly low volatility at these high temperatures, some are volatile and can be partially lost, such as iron, lead, and mercury, for which ICP-MS analysis of the product is more suitable for quantification.

[0127] The food sample is weighed before and after ashing to determine the concentration of ash present. Ash content can be expressed on a dry basis, calculated by dividing the mass of the ashed material, ingredient or food by the mass of the dry material, ingredient or food before ashing. Multiplying by 100, this gives the percentage of ash in the material, ingredient or food. In a similar manner, wet ash percentage can be determined for liquid products, using the mass of the liquid before and after ashing, instead of the mass of the dry material, ingredient or food.

[0128] Heavy metal determination

[0129] For each of the following elements, detection and quantification was performed using a robust, general inductively coupled plasma-mass spectrometry (ICP-MS) based method: arsenic (As), selenium (Se), cadmium (Cd), tin (Sn), lead (Pb), silver (Ag), palladium (Pd), platinum (Pt), mercury (Hg), molybdenum (Mo), sodium (Na), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn), manganese (Mn), phosphorus (P), selenium (Se).

[0130] Nitric acid (> 65%, Sigma-Aldrich) was used for microwave digestion and standard / sample preparation. All dilutions were performed using 18.2 MΩ-cm (Millipore, Bedford, MA, USA) deionized water (DIW). Approximately 0.2 g of each oligosaccharide, ingredient, sample was digested in 5 mL of HNO3, using the following microwave digestion (CEM, Mars 6) program schedule: 15 minutes ramping time and 15 minutes holding time (at 100 W and 50°C), followed by 15 minutes ramping time and 20 minutes holding time (at 1800 W and 210°C). The sample was allowed to cool for 30 minutes after digestion. 1. The fully digested sample was then diluted to 50 mL with DIW.

[0131] Analysis was performed using a standard Agilent 7800 ICP-MS, including the fourth-generation ORS cup system, which effectively controls polyatomic interferences using helium collision mode (He mode). The ORS uses He to control polyatomic interferences to reduce the propagation of all common matrix-based polyatomic interferences. Kinetic energy discrimination (KED) was used to separate smaller, faster analyte ions from larger, slower interfering ions. All elements, except Se, were measured in He mode at a flow rate of 5 mL / min. Se was measured in high-energy He (HEHe) mode using a cup gas flow rate of 10 mL / min. The 7800 ICP-MS was equipped with a standard sample introduction system consisting of a MicroMist glass concentric nebulizer, a quartz spray chamber, a quartz torch with a 2.5 mm inner diameter injector, and a nickel interface cone. ICP-MS operating conditions are: 1550 W RF power, 8 mm sampling depth, 1.16 l / min nebulizer gas, automatically adjusted lens tuning, 5 or 10 ml / min helium gas flow, 5 V KED.

[0132] Quantification of dry matter and moisture content

[0133] The dry matter content of oligosaccharides was determined using a Sartorius MA150 infrared moisture analyzer. 0.5 g of oligosaccharide was weighed on an analytical balance and dried in the infrared moisture analyzer until the sample weight stabilized. The dry matter content (in percentage) of the oligosaccharide, or a sample containing oligosaccharides, was given by dividing the mass of the dried sample by the mass of the sample before drying. Liquid samples were weighed in a similar manner, however, the amount of liquid weighed was adjusted to suit the expected amount of dry matter in the liquid so that the mass of the dry matter could be accurately measured on the analytical balance.

[0134] Moisture analyzers measure dry matter, but not water content. Karl Fischer titration is used to determine the amount of water present in powders, ingredients, or food products. The KF titration is performed using a Karl Fischer titrator from Mettler Toledo. The titration was performed using a Fischer DL31 titrator with a two-component technique, employing Hydra-Point Solvent G and Hydra-Point titrant (5 mg H2O / ml), both purchased from JT Baker (Deventer, Holland). The polarization current for endpoint determination using the dual-indicating electrode potentiometric titration was 20 μA, and the termination voltage was 100 mV. Endpoint criteria were drift stability (15 μg H2O / min) or the maximum titration time (10 min).

[0135] The moisture content (MC) of the sample is calculated by using the following equation:

[0136] MC = V_KF W_eq 100 / W_sample; where V_KF is the consumption of titrant in mL, W_eq is the titer of the titrant in mg H20 / mL, and W_sample is the weight of the sample in mg.

[0137] Protein quantification

[0138] For protein quantification, a method compatible with the reducing agent (e.g., a reducing sugar or oligosaccharide with a reducing end) is used. For this purpose, the Bradford assay (Thermo Scientific, Pierce) is used, with a linear range from 1 to 1500 pg / ml. The assay is calibrated with a standard curve of BSA. The protein content of the dried oligosaccharide product is quantified by dissolving a pre-weighed amount in 18.2 MΩ-cm (Millipore, Bedford, MA, USA) deionized water (DIW) up to an amount of 50% (m / v). The amount of protein is measured at 595 nm and converted to concentration using the BSA-based calibration curve.

[0139] DNA quantification

[0140] Production host-specific DNA residues are quantified by RT-qPCR for which specific primers for the host are designed, amplifying the residual DNA of the production host. The RT-qPCR is performed according to the standard operating protocol of the kit obtained from Sigma and is based on SYBR Green detection.

[0141] Total DNA is measured by threshold assay (Molecular Devices), based on an immunoassay that allows measuring as little as 2 pg of DNA in a sample in solution. Double-stranded DNA is measured by SpectraMax ® Quant TM AccuBlue TM Pico dsDNA Assay kit (Molecular Devices).

[0142] Endotoxin measurement

[0143] Endotoxins in liquids are measured by LAL test.

[0144] A. Escherichia coli

[0145] Culture medium, cultivation and cell lysis

[0146] Luria Broth (LB) medium consists of 1% tryptone (Difco, Erembodegem, Belgium), 0.5% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). The minimal medium used in cultivation experiments in 96-well plates or shake flasks contains 2.00 g / L NH4CI, 5.00 g / L (NH4)2SO4, 2.993 g / L KH2PO4, 7.315 g / L K2HPO4, 8.372 g / L MOPS, 0.5 g / L NaCI, 0.5 g / L MgSO4.7H2O, 30 g / L sucrose or 30 g / L glycerol, 1 ml / L vitamin solution, 100 μl / L molybdate solution and 1 mL / L selenium solution. The minimal medium is set to a pH of 7 with 1 M KOH. The vitamin solution consists of 3.6 g / L FeCI2.4H2O, 5.0 g / L CaCI2.2H2O, 1.3 g / L MnCI2.2H2O, 0.38 g / L CuCI2.2H2O, 0.5 g / L CoCI2.6H2O, 0.94 g / L ZnCI2, 0.0311 g / L H3BO4, 0.4 g / L Na2EDTA.2H2O and 1.01 g / L thiamine hydrochloride. The molybdate solution contains 0.967 g / L NaMoO4.2H2O. The selenium solution contains 42 g / L SeO2. The minimal medium used for fermentation contains 6.75 g / L NH4CI, 1.25 g / L (NH4)2SO4, 2.93 g / L KH2PO4 and 7.31 g / L KH2PO4, 0.5 g / L NaCI, 0.5 g / L MgSO4.7H2O, 30 g / L sucrose or 30 g / L glycerol, 1 mL / L vitamin solution, 100 μL / L molybdate solution and 1 mL / L selenium solution, with the same composition as described above. The complex medium is sterilized by autoclaving (121°C, 21 min) and the minimal medium is sterilized by filtration (0.22 μm Sartorius). When required, the medium is made selective by the addition of antibiotics: e.g. chloramphenicol (20 mg / L), carbenicillin (100 mg / L), spectinomycin (40 mg / L) and / or kanamycin (50 mg / L). When required, inducers are added.

[0147] For fermentation production, pre-cultures for 96-well microtiter plate experiments were started from frozen vials in 150 μΙ_ LB and incubated at 37°C on an orbital shaker at 800 rpm overnight. This culture was used as inoculum for 96-well square microtiter plates (with 400 μΙ_ minimal medium) by dilution 400x. Then, these final 96-well culture plates were incubated at 37°C on an orbital shaker at 800 rpm for 72 hours, or shorter, or longer. To measure sugar concentrations at the end of the cultivation experiment, whole culture broth samples (= average of intracellular and extracellular sugar concentrations) were taken from each well by boiling the culture broth at 60°C for 15 min before spinning down the cells.

[0148] Pre-cultures for bioreactor were started from whole 1 mL frozen vials, inoculated in 250 mL or 500 mL minimal medium in 1 L or 2.5 L shake flasks and incubated at 37°C on an orbital shaker at 200 rpm for 24 hours. Then, a 5 L bioreactor was inoculated (250 mL inoculum in 2 L batch medium); the process was controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). The cultivation conditions were set to 37°C and maximum agitation; the pressure gas flow rate depends on the strain and the bioreactor. The pH was controlled at 6.8 by using 0.5 M H2SO4 and 20% NH4OH. The off-gas was cooled. When the foam rose during the fermentation, a 10% silicone antifoam solution was added.

[0149] For heterologous expression of enzymes, pre-cultures for 96-well microtiter plate experiments were started from frozen vials in 175 μΙ_ LB and incubated at 37°C on an orbital shaker at 800 rpm overnight. This culture was used as inoculum for 96-well deep well microtiter plates (with 1 mL LB medium) by dilution 100x. Then, these final 96-well culture plates were incubated at 37°C on an orbital shaker at 800 rpm, induced if necessary, and further grown at 16-37°C for 5-24 hours. After expression, the DW plates were centrifuged at 4200 rpm for 30 min at 4°C, the supernatant was discarded, and the pellet was frozen at -80°C for at least one hour. The pellet was then resuspended in a buffer containing 1 mg / mL lysozyme, 10 U / mL DNase, and 1 mM protease inhibitor and incubated at 37°C for 30 min. Optionally, the cell lysate was clarified by centrifuging the plates (at 4200 rpm for 30 min at 4°C) and transferring the supernatant to a new plate.

[0150] Pre-cultures for shake flask experiments are started from frozen vials in 5 mL LB medium and incubated at 37°C on an orbital shaker at 200 rpm overnight. This culture is used as inoculum for shake flasks (with 50-250 mL LB medium) by diluting 50x. Then, these final cultures are incubated at 37°C on an orbital shaker at 200 rpm, induced if necessary, and further grown at 16-37°C for 5-24 hours, or longer, or shorter. After expression, cells are harvested by centrifugation at 4°C at 4200 rpm for 30 minutes. The supernatant is discarded and the pellet is frozen at -20°C for at least one hour. The pellet is resuspended and lysed by sonication. Optionally, the cell lysate is clarified by centrifugation (at 4°C at 4200 rpm for 30 minutes) and the supernatant is transferred to a new vessel.

[0151] Pre-cultures for bioreactor are started from whole 1 mL frozen vials of a strain, which are inoculated in 250 mL or 500 mL of LB medium in 1 L or 2.5 L shake flasks and incubated at 37°C on an orbital shaker at 200 rpm for 24 hours. Then, a 5 L bioreactor is inoculated (250 mL inoculum in 2 L batch medium); the process is controlled by MFCS control software (Sartorius Stedim Biotech, Melsungen, Germany). The cultivation conditions are set to 25-37°C and maximum agitation; the pressure gas flow rate depends on the strain and the bioreactor. The pH is controlled at 6.8 by using 0.5 M H2SO4 and 20% NH4OH. The off-gas is cooled. When the foam rises during the fermentation, a 10% silicone antifoam solution is added. The slow release of product is established by physically destroying the cells by means of sonication. Other commonly used methods known in the art are methods such as freeze-thaw and / or by mixed shear stress, homogenizer and / or French press.

[0152] Cells for whole-cell bioconversion are obtained by centrifugation of the culture (produced as described above) at 4°C at 4200 rpm overnight for 30 minutes. The obtained pellet is then resuspended in 0.9% NaCl solution and transferred to a 50 mL tube and centrifuged at 4°C at 4200 rpm for 30 minutes. The supernatant is removed and 1000 mg of each pellet is taken and resuspended in 2 mL of 0.9% NaCl (cell concentration of 0.5 g / mL). Optionally, the cells can be permeabilized (e.g., xylene).

[0153] Strains and mutations

[0154] Escherichia coli K12 MG1655 [λ] - , F - [rph-1] was obtained from Coli Genetic Stock Center (US) in March 2007, CGSC strain #: 7740, and Escherichia coli NiCo21 (DE3) was obtained from New England Biolabs in November 2017. Gene disruption, gene introduction, and gene replacement were performed using techniques published by Datsenko and Wanner (PNAS 97(2000), 6640-6645). All constitutive promoter, UTR, and terminator sequences were derived from libraries described by Cambray et al. (Nucleic Acids Res. 2013, 41(9), 5139-5148), Dunn et al. (Nucleic Acids Res. 1980, 8, 2119-2132), Edens et al. (Nucleic Acids Res. 1975, 2, 1811-1820), Kim and Lee (FEBS Letters 1997, 407, 353-356), and Mutalik et al. (Nat. Methods 2013, No.10, 354-360). Genes were synthesized and ordered from Twist Bioscience (twistbioscience.com) or IDT (eu.idtdna.com), and the vendor's tools were used to tune codon usage. All strains were stored in frozen vials at -80°C (overnight LB culture, which was mixed with 70% glycerol in a 1:1 ratio).

[0155] In cases where lactose is produced via whole-cell biotransformation, cell extracts, enzymatic synthesis using (partially purified) enzymes, or fermentation, *E. coli* K12 MG1655 or NiCo21 (DE3) uses an *E. coli* gene encoding β-galactosidase (e.g., ...). lacZ Modification is achieved by knocking out a specific enzyme. In the case of lactose enzymatic synthesis using purified enzymes, lacZ Knockout may not be necessary. For fermentation production, the strain is further modified with a suitable sugar input (e.g., cscB for sucrose). The strain may also be modified by, for example, knocking out proteases (e.g., lon, OmpT), nucleotide sugar degrading enzymes (e.g., ushA), or glucose-consuming enzymes (e.g., glk). Optionally, the strain may also be modified by overexpressing an export protein (e.g., setA) to secrete lactose from the cell.

[0156] B. Saccharomyces cerevisiae

[0157] Culture media, cultivation and cell lysis

[0158] Strains were grown on synthetic complete component defined yeast medium (SD CSM) with complete supplement mixture or CSM drop-out (SD CSM-Ura, SD CSM-Trp, SD CSM-His) containing 6.7 g / L Yeast Nitrogen Base without amino acids (YNB w / o AA, Difco), 20 g / L agar (Difco) (solid cultures), 22 g / L sucrose monohydrate or glucose or fructose and 0.79 g / L CSM or 0.77 g / L CSM-Ura, 0.77 g / L CSM-Trp or 0.77 g / L CSM-His (MP Biomedicals). Typically, yeast strains were initially grown on SD CSM plates to obtain single colonies. These plates were grown at 30°C for 2-3 days. Starting from single colonies, pre-cultures were grown at 30°C overnight in 5 mL with shaking at 200 rpm. Subsequently, 125 mL shake flask experiments were inoculated with 2% of this pre-culture in 25 mL medium. These shake flasks were incubated at 30°C with orbital shaking at 200 rpm. Product release was established by physical cell disruption by means of sonication. Other commonly used methods known in the art are methods such as freeze-thaw and / or by mixed shear stress, homogenizer and / or French press.

[0159] Strains, plasmids and mutations

[0160] Yeast BY4742 created by Brachmann et al. (Yeast (1998) 14: 115-32), which is available at the Euroscarf culture collection. All mutant strains were created by either homologous recombination or plasmid transformation, using the method of Gietz (Yeast 11 :355-360, 1995). Synthetic constitutive promoters were used to express genes, as described by, e.g., Blazeck (Biotechnology and Bioengineering, Vol. 109, No. 11, 2012), Redden and Alper (Nat. Commun. 2015, 6, 7810), Liu et al. (Microb. Cell Fact. 2020, 19, 38), Xu et al. (Microb. Cell Fact. 2021, 20, 148), and Lee et al. (ACS Synth. Biol. 2015, 4(9), 975-986).

[0161] C. Bacillus subtilis

[0162] Culture media, cultivation and cell lysis

[0163] Two media were used to grow B. subtilis: complex media such as rich Luria Broth (LB) and minimal media for shake flask cultures. LB medium consists of 1% tryptone (Difco), 0.5% yeast extract (Difco), and 0.5% sodium chloride (VWR). Luria Broth agar (LBA) plates consist of LB medium with the addition of 12 g / L agar (Difco). Minimal medium contains 2.00 g / L (NH4)2SO4, 7.5 g / L KH2PO4, 17.5 g / L K2HPO4, 1.25 g / L sodium citrate, 0.25 g / L MgSO4.7H2O, 0.05 g / L tryptophan, 10 up to 30 g / L glucose (or another carbon source, including but not limited to fructose, maltose, sucrose, glycerol, and maltotriose), 10 mL / L trace element mixture, and 10 mL / L ferric citrate solution. The medium was set to a pH of 7 with 1 M KOH. The trace element mixture consists of 0.735 g / L CaCl2.2H2O, 0.1 g / L MnCl2.2H2O, 0.033 g / L CuCl2.2H2O, 0.06 g / L CoCl2.6H2O, 0.17 g / L ZnCl2, 0.0311 g / L H3BO4, 0.4 g / L Na2EDTA.2H2O, and 0.06 g / L Na2MoO4. The ferric citrate solution contains 0.135 g / L FeCl3.6H2O, 1 g / L sodium citrate (Hoch 1973 PMC1212887). Complex media (e.g., LB) were sterilized by autoclaving (121 °C, 21 min), and minimal media were sterilized by filtration (0.22 μm Sartorius). When needed, the media were made selective by the addition of antibiotics. B. subtilis strains were initially grown on LB agar to obtain single colonies. These plates were grown overnight at 37 °C. Starting from single colonies, pre-cultures were grown overnight at 37 °C in 5 mL with shaking at 200 rpm. Subsequently, 125 mL shake flask experiments were inoculated with 2% of this pre-culture in 25 mL medium. The shake flasks were incubated at 37 °C for 72 h, or shorter, or longer, with orbital shaking at 200 rpm. At the end of the cultivation experiments, samples were taken to measure the supernatant concentration (extracellular sugar concentration, after spinning down the cells for 5 min), or by boiling the culture broth at 90 °C for 15 min or at 60 °C for 60 min before spinning down the cells (= whole broth concentration, i.e., intracellular and extracellular sugar concentration). The product was and the slow release was established by physically destroying the cells by means of sonication.Other commonly used methods known in the art are methods such as freeze-thaw and / or by shear stress of mixing, homogenizer and / or French press.

[0164] Strains, plasmids and mutations

[0165] Bacillus subtilis 168 was used, which is available at the Bacillus Genetic Stock Center (Ohio, USA). The plasmids for gene deletion via Cre / lox were constructed as described by Yan et al. (Appl & Environm. Microbial., September 2008, pp. 5556-5562). Gene disruption was performed by homologous recombination with linear DNA and transformation via electroporation as described by Xue et al. (J. microb. Meth. 34 (1999) 183-191). The method of gene knockout is described by Liu et al. (Metab. Engine. 24 (2014) 61-69). The integration vectors described by Popp et al. (Sci. Rep., 2017, 7, 15158) were used as expression vectors and can further be used for genomic integration if needed. Suitable promoters for expression can be derived from the partial repository (iGem): sequence id: BBa_K143012, BBa_K823000, BBa_K823002 or BBa_K823003. Cloning can be performed by using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

[0166] D. Corynebacterium glutamicum

[0167] Culture media, cultivation and cell lysis

[0168] Two different media were used, i.e. complex media such as rich tryptone yeast extract (TY) medium and minimal medium for shake flasks (MMsf). The minimal medium used a 1000x stock trace elements mixture. The trace elements mixture consisted of 10 g / L CaCI2, 10 g / L FeS04.7H20, 10 g / L MnS04.H20, 1 g / L ZnS04.7H20, 0.2 g / L CuS04, 0.02 g / L NiCI2.6H20, 0.2 g / L biotin (pH 7) and 0.03 g / L protocatechuic acid. The minimal medium for shake flasks (MMsf) experiments contained 20 g / L (NH4)2S04, 5 g / L urea, 1 g / L KH2P04, 1 g / L K2HPO4, 0.25 g / L MgS04.7H20, 42 g / L MOPS, from 10 up to 30 g / L glucose or another carbon source (including but not limited to fructose, maltose, sucrose, glycerol and maltotriose) (when specifically indicated in the examples) and 1 mL / L trace elements mixture. The TY medium consisted of 1.6% tryptone (Difco, Erembodegem, Belgium), 1% yeast extract (Difco) and 0.5% sodium chloride (VWR. Leuven, Belgium). TY agar (TYA) plates consisted of TY medium with the addition of 12 g / L agar (Difco, Erembodegem, Belgium). Complex media (e.g. TY) were sterilized by autoclaving (121°C, 21 min) and minimal media by filtration (0.22 μm Sartorius). When needed, the media were made selective by the addition of antibiotics.

[0169] Pre-cultures were started in 6 mL TY from frozen vials or from single colonies from TY plates and incubated at 37°C on a orbital shaker at 200 rpm overnight. Subsequently, 125 mL shake flask experiments were inoculated with 2% of this pre-culture in 25 mL MMsf medium. The shake flasks were incubated at 37°C for 72 hours, or shorter, or longer, with orbital shaking at 200 rpm. At the end of the cultivation experiments, samples were taken to measure the supernatant concentration (extracellular sugar concentration, after spinning down the cells for 5 minutes) or by cooking the culture broth at 90°C for 15 minutes or at 60°C for 60 minutes (= whole broth concentration, i.e. intracellular and extracellular sugar concentration) before spinning down the cells. Product and slow release was established by physically disrupting the cells by means of sonication. Other commonly used methods known in the art are methods such as freeze-thaw and / or by mixed shear stress, homogenizer and / or French press.

[0170] Strains and mutations

[0171] Corynebacterium glutamicum was used, which is available at the American Type Culture Collection (ATCC 13032). Integration plasmid vectors were made by using the Cre / loxP technology described by Suzuki et al. (Appl. Microbiol. Biotechnol., 2005 Apr, 67(2):225-33), and temperature sensitive shuttle vectors for gene deletion, mutation and insertion were constructed as described by Okibe et al. (Journal of Microbiological Methods 85, 2011, 155-163). Suitable promoters for (heterologous) gene expression can be derived from Yim et al. (Biotechnol. Bioeng., 2013 Nov, 110(11):2959-69). Cloning can be performed by using Gibson Assembly, Golden Gate assembly, Cliva assembly, LCR or restriction ligation.

[0172] E. Optical density

[0173] Cell density of the cultures was monitored regularly by measuring the optical density at 600 nm (Implen Nanophotometer NP80, Westburg, Belgium, or with Spark 10M microplate reader, Tecan, Switzerland). The maximum growth rate (mumax) was calculated based on the observed optical density at 600 nm using the R package grofit.

[0174] F. Growth rate / speed measurements

[0175] The maximum growth rate (μMax) was calculated based on the observed optical density at 600 nm using the R package grofit.

[0176] G. Heterologous and homologous expression

[0177] Genes that needed to be expressed, whether they were from a plasmid or from the genome, were synthesized synthetically by one of the following companies: IDT or Twist Bioscience. The proteins described in the present disclosure are summarized in Table 1. Unless otherwise noted, the UniProt ID of the described protein corresponds to its sequence version 01 as present in UniProt database release 2023_02 dated May 3, 2023. Expression can be further pushed by optimizing codon usage to that of the expression host. The supplier’s tools were used to optimize the genes.

[0178] Table 1. Overview of genes used to describe the invention and their UniProt ID.

[0179]

[0180] H. Analysis

[0181] For determination of carbohydrates, a HPAEC-PAD system (Dionex) was used in which a Dionex CarboPac PA1 Guard (2 x 50 mm) and a Dionex CarboPac PA1 Separation (2 x 250 mm) were used with a gradient of sodium acetate (1 M) (A), sodium hydroxide (200 mM) (B), water (C) and sodium acetate (25 mM) (D) in which the gradient was 5% B, 88% C and 7% D from 0-10 min, B changed from 5% to 17% and C changed from 88% to 76% from 10-15 min, B changed from 17% to 93% and C changed from 76% to 0% from 15-25 min, A changed from 0% to 20% and B changed from 93% to 73% from 25-28.1 min, the percentages of A, B, C and D were kept from 28.1 min to 32 min, A changed from 20% to 0%, B changed from 73% to 0%, C changed from 0% to 88% from 32 to 32.1 min, the percentages of A, B, C and D were kept from 32.1 to 50 min.

[0182] For the determination of nucleotide sugars, the same system was used, although with a different gradient: sodium hydroxide (1 mM) (A) and sodium acetate (1 M) in sodium hydroxide (1 mM) (B), where the gradient changes A from 80% to 45% and B from 20% to 55% in 0-10 min, 45% A and 55% B in 10-25 min, A from 45% to 20% and B from 55% to 80% in 25-35 min, A from 20% to 0% and B from 80% to 100% in 35-40 min, 0% A and 100% B in 40-50 min, A from 0% to 80% and B from 100% to 20% in 50-50.001 min, 80% A and 20% B in 50.001-60 min.

[0183] I. Enzyme purification

[0184] The clarified lysate containing the enzyme with N-terminal histidine tag in 50 mM Tris pH 7.0, 250 mM NaCl and 10 mM imidazole is applied to an AKTA Pure Protein Purification system equipped with a Ni-NTA column. Bound protein is washed with five column volumes of wash buffer (50 mM Tris, pH 7.0, 250 mM NaCl, 20-50 mM imidazole) and eluted with elution buffer (50 mM Tris, pH 7.0, 250 mM NaCl, 250 mM imidazole) and the protein of interest is collected. Buffer exchange is then performed on Amicon centrifugal filters with appropriate MW cut-off. Other commonly used purification methods known in the art are methods such as salting out, size exclusion chromatography, affinity chromatography, ion exchange chromatography and / or any combination of the methods mentioned before.

[0185] J. SDS-PAGE and Western blotting

[0186] Three microliters of Laemmli 4x buffer containing 2-mercaptoethanol (1 : 10) was added to 9 microliters of protein sample (5 mg / mL final protein concentration for cell extracts; or 0.05-0.5 mg / mL final protein concentration for purified proteins) and heated at 95°C for 5 minutes. Protein samples were loaded with protein ladder and run at 120-220 V. After electrophoresis, gels were either stained (1 hour in Coomassie Brilliant Blue R-250) or gels were transferred to blotting membranes for Western blotting. Membranes with transferred proteins were blocked with 5% milk powder and 0.2% Tween 20 for at least 30 minutes prior to application of primary antibody (anti-His). After 2 hours of incubation, the membranes were washed and secondary antibody with HRP was applied for one hour. Finally, the membranes were washed again and his-tagged proteins were visualized by chemiluminescence.

[0187] Example 2: Expression of active sucrose synthase

[0188] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with inducible expression plasmids containing genes encoding sucrose synthases (SuSy) from Glycine max (UniProt ID P13708), Arabidopsis thaliana (UniProt ID P49040), Anabaena sp. (UniProt ID Q9ZEV2), or Acidithiobacillus ferrooxidans (UniProt ID A0A059ZV61). Expression of enzymes was carried out in shake flasks as described in Example 1 and cells were harvested and lysed. Western blot analysis of clarified cell lysates was carried out as described in Example 1 and confirmed expression of the target enzymes. Activity was verified by incubating 100 mM sucrose, 10 mM UDP, and 50% (v / v) SuSy cell extract in 50 mM MOPS buffer (pH 7.0) at 37°C for 24 hours and analyzing the formation of UDP-glucose and fructose (as described in Example 1).

[0189] Example 3: Expression of active UDP-glucose-4-epimerase

[0190] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an inducible expression plasmid comprising a gene encoding a UDP-glucose-4-epimerase from E. coli (UniProt ID P09147), Bifidobacterium longum (UniProt ID 8MF10), Caloramator subterraneus (UniProt ID A0A0K1E1Q5), or Campylobacter jejuni (UniProt ID Q0P9C3). Expression of the enzyme was carried out in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysate was carried out as described in Example 1, and it confirmed the expression of the target enzyme. Activity was verified by incubating 10 mM UDP-glucose and 50% (v / v) GalE cell extract in 50 mM MOPS buffer (pH 6.5) at 37°C for 24 hours and analyzing the formation of UDP-galactose (as described in Example 1).

[0191] Example 4: Expression of active β1,4-galactosyltransferase

[0192] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an inducible expression plasmid comprising a gene encoding a β1,4-galactosyltransferase (GalT) from Neisseria meningitidis with UniProt ID Q51116. Expression of the enzyme was carried out in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysate was carried out as described in Example 1, and it confirmed the expression of the target enzyme. Activity was verified by incubating 10 mM UDP-galactose, 10 mM glucose, 10 mM MgCl2, and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0) at 37°C for 24 hours and analyzing the formation of lactose (as described in Example 1).

[0193] Example 5: Expression of active glucose isomerase

[0194] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an inducible expression plasmid comprising a gene encoding a trehalose synthase (treT) from Pyrococcus furiosus (UniProt ID Q7LYW5) or Horikoshia tokdonicus (with UniProt ID 058762). Expression of the enzyme was carried out in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysate was carried out as described in Example 1, and it confirmed the expression of the target enzyme. Activity was verified by incubating 100 mM trehalose, 10 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 6.5) at 37°C for 24 hours and analyzing the formation of glucose (as described in Example 1).

[0195] Example 6: Expression of active trehalose synthase

[0196] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an inducible expression plasmid comprising a gene encoding a trehalose synthase (treT) from Pyrococcus furiosus (UniProt ID Q7LYW5) or Horikoshia tokdonicus (with UniProt ID 058762). Expression of the enzyme was carried out in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis of the clarified cell lysate was carried out as described in Example 1, and it confirmed the expression of the target enzyme. Activity was verified by incubating 100 mM trehalose, 10 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 6.5) at 37°C for 24 hours and analyzing the formation of glucose (as described in Example 1).

[0197] Example 7: Activity of sucrose synthase at elevated temperatures

[0198] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an inducible expression plasmid comprising a gene encoding sucrose synthase (SuSy) from Acidithiobacillus ferroxidans with UniProt ID A0A059ZV61. Expression of the enzyme was carried out in shake flasks as described in Example 1 and cells were harvested and lysed. Activity was verified by incubating 100 mM sucrose, 10 mM UDP and 50% (v / v) SuSy cell extract in 50 mM MOPS buffer (pH 7.0) at 60 °C for 1 h and analyzing the formation of UDP-glucose and fructose (as described in Example 1).

[0199] Example 8: Activity of UDP-glucose-4-epimerase at elevated temperatures

[0200] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an inducible expression plasmid comprising a gene encoding UDP-glucose-4-epimerase from Caldicellulosiruptor sp. WP30 with UniProt ID A0A0K1E1Q5. Expression of the enzyme was carried out in shake flasks as described in Example 1 and cells were harvested and lysed. Activity was verified by incubating 10 mM UDP-glucose and 50% (v / v) GalE cell extract in 50 mM MOPS buffer (pH 6.5) at 60 °C for 1 h and analyzing the formation of UDP-galactose (as described in Example 1).

[0201] Example 9: Activity of glucose isomerase at elevated temperatures

[0202] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an inducible expression plasmid comprising a gene encoding glucose isomerase from Thermoanaerobacter ethanolicus with UniProt ID D2DK62. Expression of the enzyme was carried out in shake flasks as described in Example 1 and cells were harvested and lysed. Activity was verified by incubating 100 mM fructose, 10 mM MgCl2 and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 6.5) at 60 °C for 1 h and analyzing the formation of glucose (as described in Example 1).

[0203] Example 10: Activity of trehalose synthase at elevated temperatures

[0204] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an inducible expression plasmid comprising a gene encoding trehalose synthase (treT) from Pyrococcus furiosus (UniProt ID Q7LYW5) or Pyrococcus horikoshii (with UniProt ID 058762). Expression of the enzyme was carried out in shake flasks as described in Example 1 and cells were harvested and lysed. Activity was verified by incubating 10 mM trehalose, 10 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 6.5) at 60°C for 1 hour and analyzing the formation of glucose (as described in Example 1).

[0205] Example 11: Co-expression of sucrose synthase, UDP-glucose-4-epimerase, and β1,4-galactosyltransferase

[0206] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an expression plasmid comprising a constitutive transcriptional unit for sucrose synthase (SuSy) from Acidithiobacillus ferroxidans with UniProt ID A0A059ZV61, a constitutive transcriptional unit for UDP-glucose-4-epimerase (GalE) from Caloramator species WP30 with UniProt ID A0A0K1E1Q5, and a constitutive transcriptional unit for β1,4-galactosyltransferase (GalT) from Neisseria meningitidis with UniProt ID Q51116. Expression of the enzymes was carried out in shake flasks as described in Example 1 and cells were harvested and lysed. Western blot analysis was performed on the clarified cell lysate as described in Example 1 and it confirmed the expression of the target enzymes.

[0207] Example 12: Co-expression of sucrose synthase, UDP-glucose-4-epimerase, β1,4-galactosyltransferase, and glucose isomerase

[0208] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an expression plasmid comprising a constitutive transcriptional unit for sucrose synthase (SuSy) from Acidithiobacillus ferroxidans with UniProt ID A0A059ZV61, a constitutive transcriptional unit for UDP-glucose-4-epimerase (GalE) from Caloramator celer with UniProt ID A0A0K1E1Q5, a constitutive transcriptional unit for β1,4-galactosyltransferase (GalT) from Neisseria meningitidis with UniProt ID Q51116, and a constitutive transcriptional unit for glucose isomerase (XylA) from Ethanoligenens harbinense with UniProt ID D2DK62. Expression of the enzymes was carried out in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis was performed on the clarified cell lysate as described in Example 1, and it confirmed the expression of the target enzymes.

[0209] Example 13: Co-expression of trehalose synthase, UDP-glucose-4-epimerase, and β1,4-galactosyltransferase

[0210] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an expression plasmid comprising a constitutive transcriptional unit for trehalose synthase (treT) from Pyrococcus furiosus with UniProt ID Q7LYW5, a constitutive transcriptional unit for UDP-glucose-4-epimerase (GalE) from Caloramator sp. WP30 with UniProt ID A0A0K1E1Q5, and a constitutive transcriptional unit for β1,4-galactosyltransferase (GalT) from Neisseria meningitidis with UniProt ID Q51116. Expression of the enzymes was carried out in shake flasks as described in Example 1, and cells were harvested and lysed. Western blot analysis was performed on the clarified cell lysate as described in Example 1, and it confirmed the expression of the target enzymes.

[0211] Example 14: In vitro synthesis of lactose via cell extracts of individually expressed enzymes

[0212] Cell lysates from strains expressing sucrose synthase (described in Example 2), UDP-glucose 4-epimerase (described in Example 3), and βl,4-galactosyltransferase (described in Example 4) were mixed with substrate in a single-vessel multi-enzyme reaction containing 100 mM sucrose, 2 mM UDP, 10 mM MgCl2, and three times 16.6% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0), and incubated at 37°C for 24 hours. Formation of lactulose was analyzed as described in Example 1.

[0213] Example 15: In vitro synthesis of lactose via cell extracts of individually expressed enzymes

[0214] Cell lysates from strains expressing sucrose synthase (described in Example 2), UDP-glucose 4-epimerase (described in Example 3), βl,4-galactosyltransferase (described in Example 4), and glucose isomerase (described in Example 5) were mixed with substrate in a single-vessel multi-enzyme reaction containing 100 mM sucrose, 2 mM UDP, 10 mM MgCl2, and four times 12.5% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0), and incubated at 37°C for 24 hours. Formation of lactose was analyzed as described in Example 1.

[0215] Example 16: In vitro synthesis of lactose via cell extracts of individually expressed enzymes

[0216] Cell lysates from strains expressing trehalose synthase (described in Example 6), UDP-glucose 4-epimerase (described in Example 3), and βl,4-galactosyltransferase (described in Example 4) were mixed with substrate in a single-vessel multi-enzyme reaction containing 100 mM trehalose, 2 mM UDP, 10 mM MgCl2, and three times 16.6% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0), and incubated at 37°C for 24 hours. Formation of lactose was analyzed as described in Example 1.

[0217] Example 17: In vitro synthesis of lactose via cell extracts of co-expressed enzymes

[0218] Cell lysate from a strain co-expressing the three enzymes described in Example 11 was mixed with substrate in a one-pot multi-enzyme reaction containing 100 mM sucrose, 100 mM glucose, 2 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 hours. Formation of lactose was analyzed as described in Example 1.

[0219] Example 18: In vitro synthesis of lactose via cell extract of co-expressed enzymes

[0220] Cell lysate from a strain co-expressing the four enzymes described in Example 12 was mixed with substrate in a one-pot multi-enzyme reaction containing 100 mM sucrose, 2 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 hours. Formation of lactose was analyzed as described in Example 1.

[0221] Example 19: In vitro synthesis of lactose via cell extract of co-expressed enzymes

[0222] Cell lysate from a strain co-expressing the three enzymes described in Example 13 was mixed with substrate in a one-pot multi-enzyme reaction containing 100 mM trehalose, 2 mM UDP, 10 mM MgCl2, and 50% (v / v) cell extract in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 hours. Formation of lactose was analyzed as described in Example 1.

[0223] Example 20: In vitro synthesis of lactose via purified enzymes

[0224] Cell lysate from a strain expressing sucrose synthase (described in Example 2), glucose isomerase (described in Example 3), β1,4-galactosyltransferase (described in Example 4), and glucose isomerase (described in Example 5) was purified as described in Example 1. The purified enzymes were then mixed in a one-pot multi-enzyme reaction containing 100 mM sucrose, 2 mM UDP, 10 mM MgCl2, and 0.1 mg / ml purified enzymes in 50 mM MOPS buffer (pH 7.0) and incubated at 37°C for 24 hours. Formation of lactose was analyzed as described in Example 1.

[0225] Example 21: In vivo synthesis of lactose

[0226] An E. coli K-12 MG1655 strain (described in Example 1) that was modified to grow on sucrose and was deficient in lactose catabolism was transformed with expression plasmids comprising a constitutive transcriptional unit for sucrose synthase (SuSy) from Glycine max (UniProt ID P13708), Arabidopsis thaliana (UniProt ID P49040), Anabaena sp. (UniProt ID Q9ZEV2), or Acidithiobacillus ferrooxidans (UniProt ID A0A059ZV61), a constitutive transcriptional unit for UDP-glucose-4-epimerase (GalE) from E. coli (UniProt ID P09147), Bifidobacterium longum (UniProt ID E8MF10), Caloramator sp. WP30 (UniProt ID A0A0K1E1Q5), or Campylobacter jejuni (UniProt ID Q0P9C3), a constitutive transcriptional unit for β1,4-galactosyltransferase (GalT) from Neisseria meningitidis with UniProt ID Q51116, and a constitutive transcriptional unit for glucose isomerase (XylA) from Streptomyces murinus (UniProt ID P37031), Arthrobacter sp. (UniProt ID P12070), or Thermoanaerobacter ethanolicus (UniProt ID D2DK62), as described in Example 6. The new strains were evaluated for lactose production in 96-well plates according to the culture conditions provided in Example 1, wherein the strains were cultured in minimal medium with 30 g / l sucrose. After 72 hours of incubation, the culture broth was harvested and analyzed for lactose formation as described in Example 1.

[0227] Example 22: Whole-cell biotransformation synthesis of lactose

[0228] The E. coli K-12 MG1655 strain described in Example 1 was transformed with an expression plasmid comprising a constitutively transcribed unit for sucrose synthase (SuSy) from Acidithiobacillus ferroxidans with UniProt ID A0A059ZV61, a constitutively transcribed unit for UDP-glucose-4-epimerase (GalE) from Caldicellulosiruptor owczarzaki WP30 with UniProt ID A0A0K1E1Q5, a constitutively transcribed unit for β1,4-galactosyltransferase (GalT) from Neisseria meningitidis with UniProt ID Q51116, and a constitutively transcribed unit for glucose isomerase (XylA) from Thermoanaerobacterium saccharolyticum with UniProt ID D2DK62. The strain was cultivated in shake flasks as described in Example 1, and cells were harvested and prepared for whole-cell bioconversion. Reaction vessels containing 100 mM sucrose, 50 mM UDP, 100 mM MOPS, 15 mM MgS04, and 50 mg / mL cells (pH 7) were incubated at 21 °C and 175 rpm for 48 hours. The formation of lactulose was analyzed as described in Example 1.

[0229] Example 23: Recovery of enzymes for synthesis

[0230] Enzymes can be recovered from solution by ultrafiltration, where the molecular cut-off of the ultrafiltration membrane is less than the molecular weight of the enzyme (i.e., between 80 and 440 kDa for sucrose synthase; between 30 and 80 kDa for UDP-glucose-4-epimerase; between 30 and 45 kDa for β1,4-galactosyltransferase; between 40 and 200 kDa for glucose isomerase; and between 45 and 200 kDa for trehalose synthase). The retained enzymes are recirculated to the reaction vessel of the column, while the filtrate with residual substrates, byproducts, and salts is further purified to form highly purified lactulose.

[0231] Typical ultrafiltration membranes are PES membranes (Synder), PAN membranes (Synder), PVDF membranes (Synder), SPES membranes, or ceramic membranes (Tami) with molecular weight cut-offs varying between 1 kDa and 100 kDa.

[0232] In a preferred production setup, the ultrafiltration recirculation of enzymes is performed in a continuous manner, making continuous lactulose synthesis possible.

[0233] Example 24: Removal of proteins, cells, other carbohydrates, and salts from lactulose solution

[0234] After synthesis of lactose as described in the previous examples, typical impurities are sucrose, glucose, fructose, galactose, UDP-glucose, UDP-galactose and buffer components (phosphate and cations such as magnesium).

[0235] These impurities need to be removed from the solution, however separating the sugars from each other in solution is not trivial. The sugars have similar properties and molecular weight and are therefore not easily separated.

[0236] Use of selective yeast to purify lactose

[0237] The solution containing lactose, sucrose, glucose, fructose, galactose, UDP-glucose, UDP-galactose and buffer components and enzymes for synthesis is inoculated with baker's yeast (e.g. Bruggeman yeast, Saccharomyces cerevisiae). The yeast converts sucrose, fructose, galactose, UDP-glucose, UDP-galactose into biomass and ethanol, but leaves lactose in the solution.

[0238] After this conversion, the yeast is centrifuged off, leaving lactose, buffer and metabolites (e.g. ethanol, acetate, glycerol) formed by the Saccharomyces cerevisiae and cell debris in the supernatant. The formation of these metabolites and debris poses a new challenge, which needs to be removed. One alternative method for removing the yeast is flocculation and decanting or microfiltration (ceramic, PES, PVDF membranes) with a pore size of 0.1 to 10 pm.

[0239] To this end, the cell debris is removed by ultrafiltration, resulting in a solution with less than 100 mg protein / kg dry solids, a DNA content of less than 10 ng / g dry solids and endotoxins of less than 10000 EU / g dry solids. The ultrafiltration is followed by a nanofiltration step, in which the buffer salts and yeast metabolites are separated from the lactose. For nanofiltration, a NFX membrane (Synder) with a molecular weight cut-off of 300 Da is used at 50°C, which has a >99% rejection for lactose. The membrane has a <40% salt rejection, which enables easy removal of the salts and yeast metabolites while diafiltrating with water. The lactose yield after this step is higher than 95%. During nanofiltration, the solution is further concentrated to a solution with >15% dry matter.

[0240] An alternative nanofiltration membrane is NFW (Synder, with a molecular weight cut-off of <600 Da). This membrane has a >98.5% lactose rejection, <20.0% salt rejection, which enables easy removal of the salts and yeast metabolites in a diafiltration step. The ash content after diafiltration is reduced to <5% on total solids.

[0241] Use of invertase to selectively hydrolyze sucrose ​

[0242] A solution comprising lactose, sucrose, glucose, fructose, galactose, UDP-glucose, UDP-galactose and buffer components and synthesis enzymes is treated with invertase (beta-fructofuranosidase, Novozymes, approximately 1 g / l, pH 5, 50°C). During this treatment, residual sucrose is converted to glucose and fructose, while lactose remains intact.

[0243] After sucrose conversion, the enzymes are removed by ultrafiltration, resulting in a solution with less than 100 mg protein / kg dry solids, less than 10 ng DNA / g dry solids and less than 10000 EU endotoxin / g dry solids. Ultrafiltration is followed by a nanofiltration step, in which the buffer salts and monosaccharides are separated from the lactose. For nanofiltration, a DL series membrane (Suez) with a molecular weight cut-off of 300 Da is used at 50°C, which has >99% rejection for lactose, but low rejection (5-10%) for monosaccharides. By means of diafiltration, the monosaccharides and salts are removed in this step. During nanofiltration, the solution is further concentrated to a solution with >15% dry matter. The ash content after diafiltration is reduced to <5% on total solids.

[0244] Example 25: Removal of proteins, cells, other carbohydrates and salts from a lactose solution

[0245] After synthesis of lactose as described in the previous examples, typical impurities are trehalose, glucose, galactose, UDP-glucose, UDP-galactose and buffer components (phosphate and cations such as magnesium).

[0246] These impurities need to be removed from the solution, however it is not trivial to separate the sugars from each other in the solution. The sugars have similar properties and molecular weights, and are therefore not easily separated.

[0247] Use of selective yeast to purify lactose

[0248] A solution comprising lactose, trehalose, glucose, galactose, UDP-glucose, UDP-galactose and buffer components and synthesis enzymes is inoculated with baker's yeast (e.g. Bruggeman yeast, Saccharomyces cerevisiae). The yeast converts trehalose, glucose, galactose, UDP-glucose, UDP-galactose to biomass and ethanol, but leaves lactose in the solution.

[0249] After this conversion, the yeast is centrifuged off, leaving the lactose, buffer and metabolites formed by S. cerevisiae (e.g. ethanol, acetate, glycerol) and cell debris in the supernatant. The formation of these metabolites and debris poses new challenges, which need to be removed. One alternative method for removing the yeast is flocculation and decanting or microfiltration (ceramic, PES, PVDF membranes) with a pore size of 0.1 to 10 pm.

[0250] To this end, the cell debris is removed by ultrafiltration, resulting in a solution with less than 100 mg protein / kg dry solids, a DNA content of less than 10 ng / g dry solids and endotoxins of less than 10,000 EU / g dry solids. The ultrafiltration is followed by a nanofiltration step, in which the buffer salts and yeast metabolites are separated from the lactose. For nanofiltration, NFX membranes (Synder) with a molecular weight cut-off of 300 Da are used at 50°C, which have a rejection of >99% for lactose. The membranes have a salt rejection of <40%, which enables easy removal of salts and yeast metabolites while diafiltration with water. The lactose yield after this step is higher than 95%. During nanofiltration, the solution is further concentrated to a solution with >15% dry matter.

[0251] An alternative nanofiltration membrane is NFW (Synder, with a molecular weight cut-off of <600 Da). This membrane has a lactose rejection of >98.5%, a salt rejection of <20.0%, which enables easy removal of salts and yeast metabolites in a diafiltration step. The ash content after diafiltration is reduced to <5% on total solids.

[0252] Use of trehalase to selectively hydrolyze trehalose

[0253] The solution comprising lactose, trehalose, glucose, fructose, galactose, UDP-glucose, UDP-galactose and buffer components as well as the synthetic enzymes is treated with trehalase (a,a-trehalase, Megazymes, approximately 100 mg / l, 40°C, pH 5.5). During this treatment, the residual trehalose is converted, while the lactose remains intact.

[0254] ​Following trehalose hydrolysis, the enzymes were removed by ultrafiltration, resulting in a solution with less than 100 mg protein / kg dry solids, less than 10 ng / g dry solids DNA, and less than 10,000 EU / g dry solids endotoxin. Ultrafiltration was followed by a nanofiltration step, in which buffer salts and monosaccharides were separated from lactose. For nanofiltration, a DL-series membrane (Suez) with a molecular weight cutoff of 300 Da was used at 50°C, exhibiting >99% repulsion for lactose but low repulsion (5-10%) for monosaccharides. Monosaccharides and salts were removed in this step by percolation. During nanofiltration, the solution was further concentrated to a solution with >15% dry matter. After percolation, the ash content (based on total solids) was reduced to <5% (based on total solids).

[0255] Example 26: Demineralization of lactose solution

[0256] Solutions from any of Examples 14 to 22 contained residual salts and nucleotide sugars after treatment. To completely remove the salts from the solutions, the solutions were treated with cation and anion exchange resins.

[0257] First, the solution is passed through a column containing a strong acid cation exchange resin (1 L Amberlite IR120) in proton form at 10°C, resulting in the exchange of all cations with protons in the liquid. The liquid resulting from the cation exchange step is then passed through a column containing a weak base anion exchange resin (1 L Amberlite IR400) in hydroxide form at 10°C, thereby exchanging the anions in the liquid for hydroxide ions. After both cation and anion exchange, the pH is set to between 6 and 7. Lactose recovery is 95% to 98%. Ash content, based on total solids, is reduced to <1% after ion exchange.

[0258] The alternative cation and anion exchange resins are Amberlite IR100, Amberlite IR120, Amberlite FPC22, Dowex 50WX, Finex CS16GC, Finex CS13GC, Finex CS12GC, Finex CS11GC, Lewatit S, Diaion SK, Diaion UBK, Amberjet 1000, Amberjet 1200, Amberjet 4200, Amberjet 4600, Amberlite IR400, Amberlite IR410, Amberlite IR458, Diaion SA, Diaion UBA120, Lewatit MonoPlus M, and Lewatit S7468.

[0259] Example 27: Color removal

[0260] To achieve decolorization, several samples throughout the process were subjected to activated carbon treatment with Norit SX PLUS activated carbon (0.5% m / v). Color removal was measured at 420 nm with a spectrophotometer. In all samples, the color intensity at 420 nm was reduced by a factor of 50 to 100. The activated carbon was filtered off by a plate filter or a chamber filter press, preferably at elevated temperature.

[0261] Example 28: Concentration

[0262] Nanofiltration was performed with a NF-2540 membrane (DOW) with a cut-off of 200 Da to concentrate the deionized solution after ion exchange, nanofiltration, or the original solution after ultrafiltration up to 25 Brix. During the filtration process, a transmembrane pressure in the range of 20-25 bar and a process temperature of 45°C were used. The solution was continuously recirculated over the membrane for concentration, resulting in a dry matter content of the concentrate up to 25% Brix.

[0263] An alternative concentration method is falling film evaporation or wiped film evaporation, where the lactose concentration before drying or crystallization can be increased up to 80% at elevated temperature. Typical temperatures are 50-100°C.

[0264] Example 29: Spray drying of lactose synthesized enzymatically

[0265] The lactose containing solutions from examples 14 to 28 were spray dried with a pilot spray drying equipment. The equipment had an evaporation capacity of 2 kg / h.

[0266] For spray drying, the liquid was heated to a temperature of 50 to 100°C to reduce the viscosity. The pH of the liquid was set to a pH of 4 to 6. More preferably, the pH was set to 4 to 5 and the temperature was kept between 50 and 70°C.

[0267] Depending on whether the solution was concentrated (example 14) or treated with nanofiltration (example 11), the lactose concentration in the feed was 5% to 80% Brix. These concentrations were obtained by rotary evaporation or wiped film evaporation (example 14). The concentrated liquid was fed to the spray dryer at a rate of 50 to 90%. The higher the percentage of Brix, the faster the feed rate.

[0268] The inlet temperature used varied between 120°C and 280°C. The outlet temperature varied between 100°C and 180°C. The atomizer wheel rotational speed was set between 10,000 and 28,000 rpm. In one particular test, the inlet temperature was set at 184°C, the outlet temperature was set at 110°C, and the atomizer rate was set at 21,500 rpm.

[0269] The spray-dried, ultrafiltered, nanofiltered, and activated carbon treated powder obtained had a white to off-white color and a pH of 4 to 6 after dissolution in water at a concentration of 10%. The purity of the lactose was more than 80% oligosaccharides by dry solids. The spray-dried oligosaccharide mixture had a water content of about 3 to 10%, a protein content of less than 100 mg / kg dry solids, a DNA content of less than 10 ng / g dry solids, and an endotoxin of less than 10,000 EU / g dry solids. GMO DNA was not detectable in the powder. The powder treated with ion exchange resin had an ash content of less than 1% (by total dry solids), a lead content of less than 0.1 mg / kg dry solids, arsenic of less than 0.2 mg / kg dry solids, cadmium of less than 0.1 mg / kg dry solids, and mercury of less than 0.5 mg / kg dry solids.

[0270] Example 30: Crystallization of lactose

[0271] The crystallization process was performed in a stirred jacketed crystallizer. The concentrated lactose solution from Example 28 had a temperature above room temperature and was cooled to 20°C to 25°C to maximize the lactose crystal yield over a period of 12 to 48 hours. As the solution was cooled, lactose crystals crystallized due to the increase in supersaturation. The yield of crystallized lactose in one cycle was about 80%. The mother liquor can be re-concentrated by passing it again through a nanofiltration step to remove the concentrated impurities or by re-concentrating to approximately 50% dry matter content.

[0272] Example 31: Separation of lactose from disaccharides by SMB

[0273] For the SMB chromatography, a closed loop SMB system equipped with 12 glass columns of the described size can be used. Each glass column has a strong cation ion exchanger resin, a typical resin used is Dowex Monosphere in Ca2+form. The SMB can be run at ambient temperature, but is preferably run above 50°C. The flow rates are adjusted over the different zones in the SMB system, with a constant feed rate to the SMB. As eluent, typically water is used, but food grade ethanol can be used for better separation, up to about 10% (v / v).

[0274] Example 32: In vitro synthesis of lactose with purified enzymes

[0275] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with inducible expression plasmids comprising genes encoding sucrose synthase from Glycine max with UniProt ID P13708, UDP-glucose-4-epimerase from Sinorhizobium meliloti with UniProt ID P26503, β-1,4-galactosyltransferase from Neisseria meningitidis with UniProt ID Q51116, or β-1,4-galactosyltransferase from Pasteurella multocida with UniProt ID D0EAD4. Expression of the individual enzymes was performed in shake flasks. Cells were harvested and lysed, and the enzymes were purified by immobilized metal affinity chromatography (IMAC) as described in Example 1. Purified enzymes (1 mg / mL SuSy, 2 mg / mL GalE, and 1.5 mg / mL GalT) were mixed in a one-pot multi-enzyme reaction containing 100 mM sucrose, 25 mM glucose, 10 mM UDP, and 10 mM MgCl2in 50 mM HEPES buffer (pH 7.0), and incubated at 37 °C for 24 h. Formation of lactose was analyzed after 24 h as described in Example 1. Both combinations of enzymes approached the maximum amount of lactose that could be produced under the specific conditions (Table 2).

[0276] Table 2. Lactose production with different combinations of purified sucrose synthase, UDP-glucose-4-epimerase, and β-1,4-galactosyltransferase (production normalized to the theoretical maximum amount of lactose that could be produced under the specific conditions).

[0277]

[0278] Example 33: In vitro synthesis of lactose with purified enzymes

[0279] The E. coli NiCo21 (DE3) strain described in Example 1 was transformed with an inducible expression plasmid comprising a gene encoding sucrose synthase (SuSy) from Glycine max with UniProt ID P13708, UDP-glucose-4-epimerase (GalE) from Sinorhizobium meliloti with UniProt ID P26503, glucose isomerase (XylA) from Thermoanaerobacter ethanolicus with UniProt ID D2DK62, or β-1,4-galactosyltransferase (GalT) from Neisseria meningitidis with UniProt ID Q51116. Expression of the individual enzymes was performed in shake flasks. Cells were harvested and lysed, and the enzymes were purified by immobilized metal affinity chromatography (IMAC) as described in Example 1. Purified enzymes (2 mg / mL SuSy, 2 mg / mL GalE, 2 mg / mL XylA, and 0.4 mg / mL GalT) were mixed in a single-vessel multi-enzyme reaction containing 30 mM sucrose, 10 mM UDP, and 10 mM MgCl2(with or without 1 mM CoCl2) in 50 mM HEPES buffer (pH 7.0) and incubated at 30 °C for 24 h. Formation of lactose was analyzed as described in Example 1, and lactose was detected, demonstrating that the concerted action of the four enzymes can produce lactose from sucrose and catalytic amounts of UDP only (Table 3). Furthermore, the addition of Co 2+ enhanced production.

[0280] Table 3. Lactose production with purified sucrose synthase, UDP-glucose-4-epimerase, glucose isomerase, and β-1,4-galactosyltransferase in the presence and absence of Co 2+ conditions (standardized to the production in the absence of Co 2+ conditions).

[0281]

[0282] Example 34: In vitro synthesis of lactose via cell extracts of co-expressed enzymes

[0283] The E. coli K-12 MG1655 strain described in Example 1, which naturally expresses UDP-glucose-4-epimerase (GalE) with UniProt ID P09147, was transformed with expression plasmids comprising a combination of constitutive transcriptional units for sucrose synthase (SuSy) and constitutive transcriptional units for β-1,4-galactosyltransferase (GalT). The SuSy was either from Acidithiobacillus ferroxidans (with UniProt ID A0A059ZV61) or from Glycine max (with UniProt ID P13708) in combination with GalT from Neisseria meningitidis with UniProt ID Q51116 or GalT from Pasteurella multocida with UniProt ID D0EAD4. For the GalT enzymes, different transcriptional units (TUs) were evaluated, which differ in their promoters, 5'-untranslated regions, and / or terminator sequences, as described in Example 1. Co-expression of the three enzymes (SuSy, GalE, and GalT) in a single strain was performed in 24-deep well plates, and cells were harvested and lysed as described in Example 1. Cell lysates from each strain co-expressing the three enzymes were mixed with substrate in a single-tank multi-enzyme reaction comprising 100 mM sucrose, 25 mM glucose, 10 mM UDP, 10 mM MgCl2, and 80% (v / v) cell extract in 50 mM HEPES buffer (pH 7.0) and incubated at 30 °C for 24 h. Formation of lactose was analyzed as described in Example 1. Various combinations of the three co-expressed enzymes resulted in the production of lactose (Table 4).

[0284] Table 4. Lactose production (normalized to production of the Glycine max SuSy / Pasteurella multocida GalT (TU variant 2) combination) with cell extracts of different combinations of co-expressed sucrose synthase, β-1,4-galactosyltransferase, and UDP-glucose-4-epimerase.

[0285]

[0286] Example 35: In vitro synthesis of lactose via cell extracts of co-expressed enzymes

[0287] The E. coli K-12 MG1655 strain described in Example 1 naturally expressing UDP-glucose-4-epimerase (GalE) with UniProt ID P09147 was transformed with expression plasmids comprising a combination of a constitutive transcription unit on sucrose synthase from Glycine max (SuSy) with UniProt ID P13708, a constitutive transcription unit on glucose isomerase from Ethanoligenens harbinense (XylA) with UniProt ID D2DK62, and a constitutive transcription unit on β-1,4-galactosyltransferase (GalT) from Neisseria meningitidis with UniProt ID Q51116 or from Pasteurella multocida with UniProt ID D0EAD4. For the GalT enzymes, different transcription units (TUs) were evaluated which differ in their promoters, 5'-untranslated regions, and / or terminator sequences, as described in Example 1. Co-expression of the four enzymes (SuSy, GalE, XylA, and GalT) in a single strain was performed in 24-deep well plates and cells were harvested and lysed as described in Example 1. Cell lysates from each strain co-expressing the four enzymes were mixed with substrate in a single-tank multi-enzyme reaction comprising 100 mM sucrose, 10 mM UDP, 10 mM MgCl2, and 80% (v / v) cell extract in 50 mM HEPES buffer (pH 7.0) and incubated at 30°C for 24 hours. Formation of lactose was analyzed as described in Example 1. Various combinations of the four co-expressed enzymes resulted in the production of lactose (Table 5).

[0288] Table 5. Lactose production (normalized to production of Ethanoligenens harbinense XylA (TU variant 1) / Pasteurella multocida GalT (TU variant 1) combination) with cell extracts of different combinations of sucrose synthase, β-1,4-galactosyltransferase, glucose isomerase, and UDP-glucose-4-epimerase co-expressed in a single strain.

[0289]

[0290] Example 36: Whole-cell biotransformation synthesis of lactose

[0291] The E. coli K-12 MG1655 strain described in Example 1, which naturally expresses UDP-glucose-4-epimerase (GalE) with UniProt ID P09147, was transformed with expression plasmids comprising a combination of constitutive transcriptional units for sucrose synthase (SuSy) and for β-1,4-galactosyltransferase (GalT). The SuSy was either from Acidithiobacillus ferroxidans (with UniProt ID A0A059ZV61) or from Glycine max (with UniProt ID P13708) in combination with GalT from Neisseria meningitidis with UniProt ID Q51116 or from Pasteurella multocida with UniProt ID D0EAD4. For the SuSy and GalT enzymes from Glycine max, different transcriptional units (TUs) were evaluated, which differ in their promoter, 5'-untranslated region, and / or terminator sequences, as described in Example 1. Co-expression of enzymes was performed in 24-deep well plates, and cells were harvested and permeabilized by one freeze-thaw cycle, as described in Example 1. Whole-cell biotransformation was performed by incubation of 10 mg / mL cells in 25 mM phosphate buffer (pH 7), 100 mM sucrose, 25 mM glucose, 10 mM UDP, and 5 mM MgCl2, and incubated at 30 °C for 48 h. Formation of lactose was analyzed as described in Example 1. Various combinations of these three co-expressed enzymes resulted in the production of lactose in the whole-cell biotransformation setup (Table 6).

[0292] Table 6. Lactose production in whole-cell biotransformation with strains expressing different combinations of sucrose synthase, β-1,4-galactosyltransferase, and UDP-glucose-4-epimerase (normalized to the production of the Glycine max SuSy (TU variant 2) / Pasteurella multocida GalT (TU variant 1) combination).

[0293]

[0294] Example 37: Whole-cell biotransformation synthesis of lactose

[0295] The E. coli K-12 MG1655 strain described in Example 1 naturally expressing UDP-glucose-4-epimerase (GalE) with UniProt ID P09147 was transformed with expression plasmids comprising a combination of constitutive transcriptional units for sucrose synthase (SuSy) and for β-1,4-galactosyltransferase (GalT). The SuSy was either from Acidithiobacillus ferroxidans (with UniProt ID A0A059ZV61) or from Glycine max (with UniProt ID P13708) in combination with GalT from Neisseria meningitidis with UniProt ID Q51116 or from Pasteurella multocida with UniProt ID D0EAD4. For the SuSy and GalT enzymes from Glycine max different transcriptional units (TUs) were evaluated which differ in their promoter, 5'-untranslated region and / or terminator sequences as described in Example 1. The new strains were evaluated for lactose production in growth experiments according to the cultivation conditions provided in Example 1 with the strains being cultivated in minimal medium with 30 g / L sucrose, 10 g / L glucose and 10 g / L glycerol. The strains were grown in 96-well plates in four biological replicates. After 72 h of incubation the culture broth was harvested and analyzed for lactose formation as described in Example 1. The various combinations of the three co-expressed enzymes resulted in lactose production in this whole-cell biotransformation setup without a permeabilization step (Table 7).

[0296] Table 7. Lactose production in whole-cell biotransformation with strains expressing different combinations of sucrose synthase, β-1,4-galactosyltransferase and UDP-glucose-4-epimerase (normalized to the production of the Acidithiobacillus ferroxidans SuSy / Neisseria meningitidis (TU variant 1) combination).

[0297]

[0298] Example 38: Whole-cell biotransformation synthesis of lactose in a bioreactor

[0299] The E. coli K-12 MG1655 strain described in Example 1, which naturally expresses UDP-glucose 4-epimerase (GalE) with UniProt ID P09147, was transformed with an expression plasmid comprising a constitutive transcriptional unit for sucrose synthase from Glycine max (SuSy) with UniProt ID P13708 and a constitutive transcriptional unit for beta-1,4-galactosyltransferase (GalT) from Neisseria meningitidis with UniProt ID Q51116. Subsequently, the resulting strain was evaluated in a fed-batch process at bioreactor scale as described in Example 1. Glycerol, sucrose and glucose were added in the batch medium and sucrose and glucose were added via additional feeds during the fed-batch phase. The formation of lactose at the end of the process was analyzed as described in Example 1 and compared to the formation in microtiter plates. The titer in the bioreactor was almost 12 times the titer in the microtiter plates (Table 8).

[0300] Table 8. Lactose production in whole cell bioconversion with strains expressing sucrose synthase, beta-1,4-galactosyltransferase and UDP-glucose 4-epimerase (normalized to production in microtiter plates in the bioreactor)

[0301]

Claims

1. A method for producing lactose, characterized in that, The method includes using i) synthase and ii) UDP for the formation of UDP-glucose.

2. The method of claim 1, further comprising using UDP-glucose-4-epimerase to convert the UDP-glucose into UDP-galactose.

3. The method according to any one of claims 1 or 2, wherein the UDP-galactose is a galactose donor for the formation of lactose by β1,4-galactosyltransferase.

4. The method according to any one of claims 1 to 3, further comprising using β1,4-galactosyltransferase to convert glucose and UDP-galactose to form lactose and UDP.

5. The method according to any one of claims 1 to 4, wherein the UDP released by the β1,4-galactosyltransferase is recycled for use by the synthase to form UDP-glucose.

6. The method according to any one of claims 1 to 5, wherein the synthase further forms glucose or fructose.

7. The method according to any one of claims 1 to 6, wherein the synthase is sucrose synthase or trehalose synthase.

8. The method according to any one of claims 1 to 7, wherein the β1,4-galactosyltransferase uses glucose as an acceptor and UDP-galactose as a donor.

9. The method according to any one of claims 7 to 8, wherein fructose is converted into glucose.

10. The method according to any one of claims 1 to 9, wherein the method is a fermentation process, a biotransformation process, or an enzymatic process.

11. The method according to any one of claims 1 to 10, wherein the method is an enzymatic process, and wherein at least one enzyme is immobilized.

12. The method according to any one of claims 1 to 11, wherein the method is an enzymatic process, and wherein the reaction is carried out between 50°C and 80°C, preferably between 55°C and 70°C.

13. The method according to any one of claims 1 to 10, wherein the method is a fermentation process or biotransformation, characterized in that, Enzymatic conversion is carried out by at least one enzyme, which is produced by the cell that synthesizes the enzyme.

14. The method according to any one of claims 1 to 10, wherein the method is a fermentation process, characterized in that, The lactose is produced by cells that have been genetically modified to produce all the enzymes required for lactose production.

15. The method according to any one of the preceding claims, wherein the carbon source for conversion to lactose is selected from the list of: sucrose, trehalose, and combinations of sucrose and glucose.

16. The method according to claim 15, used for enzymatically producing lactose using i) sucrose, ii) trehalose, or iii) sucrose and glucose as carbon sources, characterized in that, Lactose is produced by using i) sucrose, ii) trehalose, or iii) sucrose and glucose as carbon sources, and the reaction is catalyzed by a combination of multiple enzymes.

17. The method of claim 15, wherein the synthase is: a) trehalose synthase, and wherein i) trehalose is converted to UDP-glucose and glucose using the trehalose synthase, and ii) the UDP-glucose is converted to UDP-galactose, and iii) glucose and UDP-galactose are converted to lactose; b) sucrose synthase, and wherein i) sucrose is converted to UDP-glucose and fructose using the sucrose synthase, ii) the UDP-glucose is converted to UDP-galactose, and iii) fructose is converted to glucose, and iv) the UDP-galactose from step ii) and the glucose from step iii) are converted to lactose; or c) sucrose synthase, and the carbon source is sucrose and glucose, wherein i) sucrose is converted to UDP-glucose and fructose using the sucrose synthase, ii) the UDP-glucose is converted to UDP-galactose, and iii) the UDP-galactose from step ii) and the glucose provided with sucrose are converted to lactose.

18. The method according to any one of claims 3, 4 or 8, wherein the β1,4-galactosyltransferase is lactose synthase.

19. The method according to any one of the preceding claims, wherein the method further comprises the step of separating the lactose.

20. The method according to any one of the preceding claims, wherein the method further comprises the step of purifying the lactose.

21. The method according to any one of the preceding claims, wherein the method comprises the step of drying or crystallizing the lactose, preferably the drying step comprises any one or more of the following: spray drying, freeze drying, evaporation, precipitation, spray freeze drying, freeze spray drying, strip drying, belt drying, vacuum strip drying, vacuum belt drying, drum drying, roller drying, vacuum drum drying, vacuum roller drying, and stirred film drying.

22. The method according to any one of the preceding claims, wherein the biotechnologically synthesized lactose is purified by at least one of the following purification steps: microfiltration, centrifugation, ultrafiltration, nanofiltration, ion exchange, simulated moving bed, color removal, and preferably further includes concentration, spray drying and / or crystallization, wherein the ion exchange is preferably cation and / or anion exchange.

23. The method according to any one of claims 19 to 21, wherein the purity of lactose before purification is < 70%, < 60%, < 50%, < 40%, < 30%, < 20%, < 10% based on total solids, and / or the purity at the end of the method including purification is > 80% based on dry solids, preferably > 85% based on dry solids, more preferably > 90% based on dry solids, even more preferably > 95% based on dry solids, even more preferably > 97% based on dry solids, even more preferably > 98% based on dry solids, and most preferably > 99% based on dry solids.

24. The method according to any one of claims 19 to 22, wherein the purification of lactose synthesized by enzymatic means comprises the steps of microfiltration or ultrafiltration to recover the used enzyme.

25. The method according to any one of claims 19 to 23, wherein the purification of the biotechnologically synthesized lactose comprises the steps of centrifugation, microfiltration and / or ultrafiltration to remove biomass.

26. The method according to any one of claims 19 to 24, comprising i) a step of nanofiltration to remove / reduce monosaccharides and / or salts; and / or ii) a step of ion exchange to remove charged materials.

27. The method according to any one of claims 19 to 25, wherein the purified lactose has an ash content of less than 1% based on total solids, preferably less than 0.5% based on total solids, and preferably has one or more of the following: a) Lead content less than 0.1 mg / kg solids, preferably less than 0.02 mg / kg solids; b) Arsenic content less than 0.2 mg / kg solids, preferably less than 0.02 mg / kg solids; c) Cadmium content less than 0.1 mg / kg solids, preferably less than 0.01 mg / kg solids; or d) Mercury content less than 0.5 mg / kg solids, preferably less than 0.1 mg / kg solids.

28. The method according to any one of claims 19 to 26, wherein the purified lactose has at least one or more of the following: i) a protein content of less than 100 mg / kg dry solids, ii) a DNA content of less than 10 ng / g dry solids, and iii) an endotoxin content of less than 10,000 EU / g dry solids.

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