Mutant galactosidase permease and uses thereof

Abstract

The present invention relates to mutant galactoside permeases, in particular microbial host cells and fermentation processes employing mutant galactoside permeases that display reduced or abolished proton-coupled transport activity while retaining substrate translocation. These permease variants, when expressed at levels exceeding endogenous wild-type expression, enable cultivation and production processes, in particular in the presence of elevated lactose concentrations without the growth limitations typically associated with lactose-induced energetic collapse. The invention further relates to genetic constructs, metabolic pathways, and process strategies suitable for the production of lactose-based products, such as human milk oligosaccharides.

Description

[0001] NOVEL MUTANT GALACTOSIDASE PERMEASE AND USES THEREOF

[0002] TECHNICAL FIELD

[0003] The present invention relates to mutant galactoside permeases, in particular microbial host cells and fermentation processes employing mutant galactoside permeases that display reduced or abolished proton-coupled transport activity while retaining substrate translocation. These permease variants, when expressed at levels exceeding endogenous wild-type expression, enable cultivation and production processes, in particular in the presence of elevated lactose concentrations without the growth limitations typically associated with lactose-induced energetic collapse. The invention further relates to genetic constructs, metabolic pathways, and process strategies suitable for the production of lactose-based products, such as human milk oligosaccharides.

[0004] TECHNICAL BACKGROUND

[0005] Lactose is widely used as a carbon source, precursor substrate or glycosyl acceptor in microbial fermentation processes, including the industrial production of human milk oligosaccharides and other lactose-derived products. However, microorganisms that express the native lactose permease (LacY) frequently suffer from so-called lactose toxicity (lactose killing) when exposed to lactose-rich media, particularly under conditions of elevated lactose concentrations or when LacY expression is high. This toxicity results from proton-coupled lactose transport and constitutes a major bottleneck for robust large-scale fermentation processes relying on lactose as a feedstock.

[0006] An emerging scientific and industrial field, where lactose is used as precursor, is the fermentative biosynthesis of human milk oligosaccharides. Beyond the widely known “milk sugar” lactose, human breast milk contains a large group of other sugar compounds, collectively referred to as human milk oligosaccharides (HMO).

[0007] Industrial production and commercialization of HMOs is of increasing importance. For the breast-fed child HMOs are indigestible; the relevance of HMOs is due to their unique biological effects, hence they are also called functional sugars. HMOs have become key products for nutrition and therapeutic uses as nutraceutical, antimicrobial, anti-inflammatory and immune-modulatory agents. As a result, the industrial scale production of HMOs associated with high yields at low costs, is desirable.

[0008] To date, more than 150 distinct HMOs differing in structure have been identified. HMOs consist of combinations of five monosaccharide components, i.e. , D-glucose, D-galactose, N-acetyl-glucosamine, L-fucose and N-acetylneuraminic acid. As a matter of course, obtaining of HMOs from their natural source is restricted. Thus, large- scale industrial production is to be realized only by artificial synthesis. HMOs can be synthesized in vitro by chemical or enzymatic methods, however, with minor efficiency. Highly efficient production can be achieved by fermentative processes wherein oligosaccharides are produced by a host cell. Industrial scale fermentative production of individual HMOs made these prebiotic compounds accessible for nutritional and therapeutic uses, such as supplements for infant formula.

[0009] HMOs are modified forms of lactose, such as 2’-fucosyllactose, 3’-fucosyllactose, 3’- sialyllactose, 6’-sialyllactose, etc. (listed under point 22). During the fermentative process for the production of such HMOs, a host cell is typically cultivated in the presence of (i) a carbon source, and (ii) a galactoside (sugar), preferably lactose. Lactose is a naturally occurring common disaccharide composed of galactose and glucose, i.e., p-D-galactopyranosyl- (1— >4)- D-glucose.

[0010] In fermentative production of oligosaccharides, the phenomenon of lactose toxicity, also called lactose killing is of particular interest, since it inhibits growth of many organisms. Lactose killing was first described in 1961 by Von Hofsten (Von Hofsten, B, Biochim. Biophys. Acta 48, 164-171) in a mutant E. coli strain. During the many years since then, the phenomenon was studied in more detail and in general it can be defined by saying that lactose killing occurs when the lactose transporter gene is (inducibly or constitutively) overexpressed in the presence of lactose or other galactoside substrates (with variously strong effect).

[0011] Overexpression of the lactose transporter can occur even in wild type strains under specifically chosen growth conditions, or in recombinant strains where the lactose transporter gene is artificially overexpressed using molecular genetic methods, as it is usually the case in industrial processes. The natural lactose transporter gene lacY (galactoside permease) is part of the lac operon, which is transcriptionally induced by the presence of lactose. Via induction by lactose, expression of the lac operon reaches only about 1 / 3 of its maximum level, regulated by cellular feedback control. Further increase of expression can be achieved, for example, by growing the cells in a minimal medium maintaining a low concentration of lactose (Dykhuisen D. and Hartl D. Journal of Bacteriology 1978 Sept. 876-882), where competition for the substrate causes genetic changes in the cells. Other artificial ways to increase / acY-expression include deleting the lac-repressor gene lad, or replacing the lac promoter with other, stronger promoters.

[0012] Over several decades, mechanistic studies on the lactose permease (LacY) have demonstrated that the native transporter functions as a proton-lactose symporter. Work by Smirnova et al., Kaback and co-workers, Johnson et al., Franco et al. and others established that uncontrolled proton influx associated with elevated LacY expression can lead to collapse of the proton motive force and growth arrest in the presence of lactose or related galactosides. These studies predominantly focused on biophysical characterization of LacY transport and on dissecting the role of residues required for proton translocation.

[0013] Mutations in several residues involved in proton coupling were described in this literature. These mutants were generally regarded as deficient for physiological transport because classical uptake assays assessed uphill transport or intracellular accumulation. As a result, proton-uncoupled variants were not considered relevant for microbial cultivation or carbonbased fermentation processes, and the available literature did not explore their performance under industrial cultivation conditions or high lactose loads.

[0014] When the cells take up too much lactose too fast in an uncontrolled way, this depletes the membrane potential as well as the biochemical energy-restoring capacity of the cell, leading to an energetic crisis. The cells sense this adverse condition and respond to it by drastically saving energy. This involves rapid growth arrest as well as shutting down protein synthesis in general in a mechanism known as stringent response. These metabolic adjustments severely affect HMO production or any other fermentation process that uses lactose.

[0015] Since said phenomenon delays growth and inhibits metabolic processes, which consequently has a negative effect on the production of e.g. HMOs when lactose is supplied to the medium as a ready molecular building block, it is desirable to prevent lactose killing and concomitantly increase yields of HMOs during industrial scale production. Patent literature exists which has already attempted to solve the problem, which is shortly reviewed here.

[0016] WO 2012 / 112 777 A2 proposes a solution against lactose killing either by severely reducing lactose-uptake or by decoupling the growth phase from the production phase in order to obtain at first sufficient biomass. Subsequent to the growth phase, lactose is added in the second phase in order to produce the specific product. Even though the authors could achieve sufficient biomass with this method, the lactose toxicity is unfortunately not a mere growth arrest, but rather the deeper-lying energy crisis that effectively inhibits metabolic output in the production phase.

[0017] EP 3 218 509 B1 provides microorganisms being resistant to lactose killing by altering the expression level of a lactose transporter. More specifically, the expression level of said lactose transporter is reduced, thus, leading to a microorganism that is capable to withstand the lactose challenge but still retains at least 50 % of the lactose influx compared to a wildtype expression cassette of said lactose transporter. However, adverse effects on the growth of bacterial cells are still observed.

[0018] WO 2022 / 136 568 A1 takes the fact in consideration, that the lactose transporter also possesses regulatory functions exerted via protein-level interaction, for that it is desirable not to decrease the cellular abundance of the LacY protein. Instead, in contrast to the previous patent that manipulates lacY transcription level, this patent attempts to decrease lactose transport capacity by mutations that reduce the specific activity of the transporter enzyme. The authors disclose the expression of mutant LacY lactose permeases, with mutation(s) at position(s) 292, and / or 293, and / or 294 compared to the wild-type LacY in a microbial cell. The authors succeeded to reduce the effect of lactose killing, as well as claiming a modest 20% increase in HMO yield obtained from the microorganisms compared to the wild-type LacY-harboring strain.

[0019] Published studies on LacY variants, including work by Smirnova, Johnson, Franco and others, describe proton-coupling mutants primarily for mechanistic and structural analysis. These publications do not disclose microbial cultivation or fermentation performance of such mutants in lactose-containing media. Similarly, more recent patent literature in the field of human milk oligosaccharide production does not address proton-coupled lactose toxicity by deliberately modifying LacY proton coupling. In particular, none of these documents discloses the use of galactoside permease variants with reduced proton coupling that are overexpressed above endogenous wild-type levels for cultivation or production processes, for example carried out at elevated lactose concentrations.

[0020] The prior art does not describe or suggest the purposeful combination of (i) proton-uncoupled permease variants and (ii) expression levels exceeding the endogenous lacY expression. Even where mutant permeases are mentioned, they are not overexpressed and therefore do not solve lactose toxicity caused by high transporter abundance.

[0021] In light of the above, the present disclosure particularly addresses this object and provides enhanced yields of lactose-based products, like HMOs, without the adverse effects of lactose killing or without reducing the expression level or activity of the galactoside permease.

[0022] SUMMARY OF THE INVENTION

[0023] The present invention provides microbial host cells and corresponding methods that overcome limitations associated with proton-coupled lactose transport and the resulting lactose toxicity observed in conventional fermentation systems. By employing galactoside permease variants with reduced or abolished proton coupling in combination with enhanced expression levels, the invention enables stable cultivation and efficient production in lactose- containing media, including media with elevated lactose concentrations. The problem is solved by the subject matter of the independent claims. Further embodiments are described in the dependent claims.

[0024] In particular, the present disclosure provides a microbial host cell comprising a nucleic acid encoding a galactoside permease mutant having reduced or abolished proton-coupled galactoside transport, wherein the galactoside permease mutant is expressed in the host cell at a level exceeding the endogenous expression level of the corresponding wild-type galactoside permease.

[0025] Further the present invention provides a nucleic acid comprising: (a) a nucleotide sequence encoding a galactoside permease mutant having reduced or abolished proton-coupled galactoside transport; and (b) one or more regulatory or structural elements suitable for achieving overexpression of the mutant permease, selected from promoter sequences, ribosome binding sites, untranslated regions, transcriptional or translational enhancers, gene-dosage-increasing elements, or combinations thereof.

[0026] Further the present invention provides a vector comprising the nucleic acid of claim 2, optionally together with elements enabling multi-copy replication, chromosomal integration, or controlled induction of expression.

[0027] Further the present invention provides a use of a microbial host cell, a nucleic acid or a vector according to claim 1 , 2 or 3 for producing a lactose-based product in a fermentation process.

[0028] Further the present invention provides a process for producing a lactose-based product, comprising: (a) culturing a microbial host cell according to claim 1 in a medium comprising lactose; and (b) producing or recovering the lactose-based product from the culture medium and / or the host cell.

[0029] Suitably, overexpression may be achieved by one or more genetic or regulatory elements selected from promoter sequences, ribosome binding sites, transcriptional or translational enhancers, gene-dosage-increasing elements, derepression strategies, or combinations thereof.

[0030] Suitably, the overexpression may be achieved by a heterologous or synthetic promoter selected from PT7, Ptac, Ptrc, Pt5, PBAD, PlacllV5, Ptet, PJ23119, Pveg, P43 or functional variants thereof.

[0031] Suitably, the overexpression may be achieved by a multicopy plasmid or by multiple chromosomal insertions.

[0032] Suitably, the overexpression may be supported by an engineered ribosome binding site.

[0033] Suitably, lacl or another repressor of the lac operon may be deleted or inactivated.

[0034] Suitably, the galactoside permease mutant may retain substrate binding and galactoside transport activity in the absence of proton translocation.

[0035] Suitably, the mutant may comprise one or more amino acid substitutions that impair proton translocation while retaining substrate translocation.

[0036] Suitably, the mutation may be located in a residue contributing to proton translocation of LacY. Suitably, the host cell may be selected from Escherichia coli, Corynebacterium, Bacillus, or a yeast species.

[0037] Suitably, the host cell may further comprise a nucleic acid encoding one or more enzymes involved in the biosynthesis or modification of lactose-based products.

[0038] Suitably, the host cell may be cultured or is capable of being cultured in a medium comprising at least 10 g / L lactose, preferably at least 15 g / L, more preferably 20-60 g / L lactose.

[0039] Suitably, in the use the fermentation medium may comprise at least 10 g / L lactose, preferably at least 15 g / L, more preferably 20-60 g / L lactose.

[0040] Suitably, in the process the lactose concentration in step (a) may be at least 10 g / L, preferably at least 15 g / L, more preferably 20-60 g / L.

[0041] Suitably, in the host cell, when cultured in a medium comprising at least 10 g / L lactose, the overexpression of the galactoside permease mutant may provide a synergistic improvement in cell growth or product titer relative to the same host cell expressing the mutant at endogenous levels.

[0042] Suitably, for the use, wherein culturing the host cell in a medium comprising at least 10 g / L lactose together with overexpression of the galactoside permease mutant may provide a synergistic improvement in growth or product titer.

[0043] Suitably, for the process, wherein the combination of overexpression of the galactoside permease mutant and a lactose concentration of at least 10 g / L may provide a synergistic improvement in cell growth, yield or product titer relative to culturing a host cell expressing the mutant at endogenous levels.

[0044] Suitably, the process may yield a higher amount of a lactose-based product compared to a corresponding process using a host cell expressing the same permease mutant at endogenous levels.

[0045] Suitably, the process may be performed at industrial scale, defined as a working fermentation volume of at least 100 L, preferably at least 300 L, more preferably 1 ,000 L or more.

[0046] BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Fig. 1 : Representation of the amino acid sequence of Escherichia coli strain BL21 and Escherichia coli strain K12, substrain MG 1655, galactoside permease LacY, i.e., SEQ ID NO: 1 as in the attached sequence list.

[0048] Fig. 2: Representation of the nucleic acid sequence of Escherichia coli strain BL21 and Escherichia coli strain K12 galactoside permease LacY, i.e., SEQ ID NO: 2 as in the attached sequence list. Fig. 3 : Schematic overview illustrating lactose toxicity by Overexpression of LacY. Excessive lactose / H+ uptake caused by overexpression of LacY in the presence of high lactose concentrations leads to strong depletion of the proton-motive force, limiting cellular ATP generation. Because ATP is essential for cell growth and protein biosynthesis (PBS), this energy deficit broadly compromises the cell’s ability to synthesize proteins and complete cell division. In particular, the biosynthesis of the key enzyme FutC is notably reduced upon addition of lactose. Moreover, cells fail to divide normally while continuing to elongate, resulting in an extended cell morphology. The combined effect of energy limitation, impaired PBS, and consequently restricted cell growth ultimately led to very low 2'-FL production.

[0049] Fig.4 : Schematic overview illustrating how alleviation of lactose toxicity via proton-uncoupled lactose transport by the LacY-H322R variant enhances cellular 2’-FL production. When proton influx is uncoupled from lactose uptake through the overexpressed LacY-H322R variant, the proton-motive force is preserved, thereby restoring cellular ATP generation. The recovery of cellular energy metabolism enables the resumption of protein biosynthesis (PBS), normal cell growth, and completion of cell division even under high lactose conditions. As a result of this restored energy balance, the cellular capacity to produce 2’-FL is dramatically increased in LacY-H322R variants.

[0050] Experimental data shown in Figures 5 to 9 are based on flasks experiments.

[0051] Fig.5: Improved growth kinetics and 2’-FL production in proton symport-deficient LacY mutants. A) Graph illustrating the growth kinetics (OD600) over time (h) of bacterial cells expressing Escherichia coli wild-type lactose permease LacY, control mutant LacY (L293R) and proton symport-deficient mutants LacY (H322R) and LacY (E325A). B) Graph illustrating 2’FL-productivity (mg / L) over time (h) of bacterial strains expressing Escherichia coli wild-type lactose permease LacY, control mutant LacY (L293R), mutant LacY (H322R), and mutant LacY (E325A).

[0052] Fig. 6 : Inhibition of FutC biosynthesis is restored in LacY-H322R variants. A) 2'-FL-producing cells carrying the lacY gene under control of the strong tac promoter were cultivated in complex medium, and production was induced by IPTG addition. The cells were exposed to increasing lactose concentrations (0-5 g / L). SDS-PAGE analysis shows that elevated lactose levels - and the resulting increase in lactose / H+uptake - reduce overall protein biosynthesis, and in particular the a-1 ,2-fucosyltransferase FutC; B) When the native lacY gene is replaced with the H322R variant, biosynthesis of the key enzyme FutC is restored even at high lactose concentrations (12 g / L).

[0053] Fig. 7 : Proton-uncoupled lactose transport in the LacY-H322R improves cell growth, restores normal cell division and maximizes 2’-FL production. In LacY-H322R cells, uncoupling lactose transport from proton influx alleviates lactose toxicity: A) cell growth is improved in LacY-H322R, B) normal cell division is restored, resulting in rod-shaped cells (LacY-H322R) rather than elongated cells (LacY) in microscopic images, and consequently (C) 2’-FL production is dramatically increased in LacY-H322R.

[0054] Fig. 8: Increasing expression of the proton-uncoupled LacY-H322R uniporter overcomes the lactose-uptake bottleneck and maximizes 2’-FL production. The LacY-H322R mutation converts the native LacY symporter into a proton-uncoupled uniporter, enabling passive, gradient-driven lactose transport rather than proton-coupled active uptake. A) While low-level expression from native lac promoters (Plac, PlacllV5) is sufficient to restore cell growth (OD600), lactose uptake remains limiting, constraining B) 2’-FL synthesis (g / L). Stronger promoters (Ptac, Pt5) significantly enhance LacY- H322R expression by increasing lactose transport capacity and thereby enable maximal 2’-FL titers (g / L).

[0055] Fig. 9: Overexpression of LacY-H322R rescues growth and increases 2’-FL Yield. A) Overexpression of LacY under control of the strong tac promoter (Ptac, LacY) impairs cell growth in 2’-FL-producing strains compared to expression from the natural lac promoter (Plac, LacY), demonstrating pronounced lactose toxicity. This growth defect is eliminated in strains expressing the LacY-H322R variant under tac promoter control (Ptac, H322R). B) Under native LacY expression (Plac, LacY), cells exhibit robust growth, but limited lactose uptake constrains 2’-FL titers (g / L). Overexpression of the LacY-H322R variant (Ptac, H322R) leads to a dramatic increase in 2’-FL production.

[0056] Fig. 10: Comparison of the metabolic response of a 2'-FL production strain with A) and without B) the lacYH322 variant, described in this invention, on high lactose concentrations in the fermentation medium. Batchwise feeding of a lactose solution led to a final product concentration of 75 g / L A), respectively 18,5 g / L B) within approx. 65 hours.

[0057] Fig. 11 : Schematic overview of Down Sream Processing (DSP) steps.

[0058] Fig. 12: HILIC-Z - HPLC - ELSD chromatogram of a produced 2'-FL powder sample.

[0059] DETAILED DESCRIPTION

[0060] Subsequent investigations carried out by the inventors revealed that proton-coupled lactose transport represents a central limitation in lactose-based microbial processes. The inventors found that microbial host cells expressing galactoside permease mutants defective in proton translocation, but retaining substrate binding and translocation, can be cultivated under lactose concentrations that are inhibitory for strains expressing elevated levels of the wildtype permease. These mutants, which are defective in proton-substrate symport and therefor also named uniporters, do not generate the proton influx normally associated with lactose uptake.

[0061] The present inventors developed novel uses of mutant galactoside permeases according to the disclosure with unexpected advantageous properties when overexpressed above endogenous wild-type LacY levels. In particular, it was found that the over-expressed mutant galactoside permeases exhibit enhanced production of lactose-based products, in particular, oligosaccharides, specifically HMOs.

[0062] Galactoside permease mutants that exhibit reduced or abolished proton-coupled lactose symport may maintain substrate translocation in the absence of proton influx. These mutants include substitutions in residues known to contribute to proton translocation. When expressed above endogenous wild-type levels, such variants provide sufficient galactoside flux to support biosynthetic processes relying on lactose as carbon source, precursor, or acceptor substrate. At the same time, the absence of proton symport maintains the proton motive force and prevents energetic collapse during growth in lactose-containing media.

[0063] This functional combination — maintenance of substrate transport while avoiding proton influx — allows the host cell to avoid the physiological state associated with lactose-induced growth inhibition (lactose killing) traditionally observed in strains expressing elevated levels of wild-type LacY.

[0064] The most economic processes of producing HMO sugars utilize externally added lactose to the fermentation broth as a ready molecular building block. This method, however, poses a toxic effect on the production cells. Surprisingly, the mutant galactoside permeases according to the present invention can advantageously be used in the production of oligosaccharides since host cells according to the invention, that possess any of the mutant galactoside permease alleles are insensitive or much less sensitive to lactose killing, even when the permease is overexpressed. The use of the mutant galactoside permease variants of the present invention, preferably when combined with the overexpression of their genes, results in increased HMO levels compared to control cells. In particular, the advantages are observed when the mutant permease is expressed at levels exceeding endogenous wild-type LacY expression. These novel features could not have been expected.

[0065] The toxic effect of lactose increases with medium lactose concentration of up to about 5 g / L, where it reaches maximum toxicity with full growth arrest in shake flasks (in fermenter the dynamics is somewhat different). Surprisingly, cells comprising a galactoside permease according to the invention, as well as cells that over-express a mutant lacY allele according to the present invention can tolerate very high medium lactose concentrations, e.g., of up to 100 g / L, as additional benefit without any noticeable negative effect. This is very good for two reasons.

[0066] Firstly, lactose from concentrated solutions used for fed batch procedure is prone to precipitate in the feeding tube, frequently leading to clogging, therefore the preferred way to add lactose to the fermentation broth is in batches, whereby lactose concentration can temporarily reach higher levels, which the cells must then tolerate. Secondly, since the galactoside permease of the present invention reduce or inactivate the proton symport function of the lactose permease, they also abolish its active transport capability, turning it into a passive carrier. Consequently, the ability of the transporter, to accumulate lactose in the host cells to higher concentrations than in the medium is lost. Since substrate concentration is an important factor for all enzyme reactions, lowered cytoplasmic lactose levels might negatively influence HMO production. By passive carriers, the force that drives transport is the concentration gradient, in other words the transport process is running as long as the medium concentration of the transported substance is higher than that of the cytoplasm. Therefore, in order to support rapid lactose uptake, as well as to reach high intracellular lactose concentrations, the medium lactose concentration may beneficially be increased, when using the galactoside permease of the present invention, and this is fully applicable since toxicity has been abolished. As the actual experimental data support, according to the present invention, production of HMOs, such as 2’-fucosyl lactose (2’-FL), is increased in bacteria over-expressing the mutant galactoside permease.

[0067] Due to the improved lactose tolerance achieved by over-expressing the mutant galactoside permease according to the present invention any priming, pretreatment or accommodating the bacteria over-expressing said mutant galactoside permease to high lactose conditions, e.g., by cultivation in pre-culture medium with a reduced lactose content, is not required. Further, monitoring I controlling of the lactose content in the culture medium is not required during the fermentative production process of lactose-based products, in particular HMOs. Resultantly, the present invention simplifies and improves fermentation protocols for the production of lactose-based products, in particular HMOs.

[0068] Mutants

[0069] In a first aspect of the present the present invention refers to mutant galactoside permeases comprising amino acid sequences having at least 57% sequence identity to the amino acid sequence of SEQ ID NO: 1 and having at least one mutation, which provides a reduced or a lack of proton symporter activity.

[0070] As used herein the terms “mutant”, “variant” are used interchangeably and refer to any galactoside permease having reduced or abolished proton-coupled galactoside transport. As used herein, the term „proton-coupled galactoside transport” is equivalent to “proton symporter activity” and refers to the coupled translocation of a galactoside substrate together with a proton across the membrane, as characteristic for the wild-type LacY permease.

[0071] As used herein, the terms “reduced proton-coupled galactoside transport” and “abolished proton-coupled galactoside transport” refer to a functional state of a galactoside permease in which the mechanistic coupling between galactoside translocation and proton translocation is diminished or lost relative to the corresponding wild-type galactoside permease.

[0072] A reduction in proton-coupled transport means that, under otherwise comparable assay conditions, the permease variant exhibits a measurably lower proton influx associated with galactoside uptake than the wild-type permease, whereas an abolition of proton-coupled transport means that proton influx becomes undetectable or falls to background levels.

[0073] In both cases, the ability of the permease to bind and translocate galactosides is at least partially retained, such that galactoside uptake occurs predominantly or exclusively through a passive, facilitated or otherwise proton-uncoupled mechanism.

[0074] Unless stated otherwise, the comparison to the “wild-type” refers to the endogenous LacY protein of the respective microbial species, determined under identical substrate, pH, membrane potential and assay conditions.

[0075] Galactoside permease, also known as lactose permease, is a transmembrane protein encoded by the lacY gene of the lac operon in E. coli (but it has homologs in many organisms), which facilitates the passage of lactose across the cell membrane. Lactose permease, i.e., LacY, belongs to the family of the Major Facilitators, i.e., membrane proteins, such as uniporters, symporters, and antiporters, that facilitate the influx and efflux of specific small molecules. The transport mechanism is a symport that utilizes the inward directed electrochemical proton gradient across the cell membrane as its driving force for cotranslocation of p-galactosides and protons into the cell. LacY can transport lactose, melibiose, lactulose and the analogue methyl-1-thio-p,D-galactopyranoside (TMG), but no sucrose or fructose. Structurally, the majority of known secondary transporters (i.e. cotransporting a solute with another solute, usually an ion) are all related and can be classified into just two common folds (LacY-class and LeuT class) for one of which the archetype is the lactose permease. Despite significant divergence in their primary sequence, they show a high degree of conservation in their overall structure and at specific positions in their sequence. The currently known most divergent galactoside-permease to E. coli LacY has been found in Enterobacter cloacae, with just 57% sequence identity, annotated as MelY as melibiose permease, is still a functional lactose transporter.

[0076] The amino acid sequence of wild-type LacY as represented by SEQ ID NO: 1 , refers to the amino acid sequence of LacY of E. coli strain BL21 (NCBI accession No: CP010816.1 , locus: AJH09253.1) and E. coli strain K12 (NCBI accession No: CP025268.1 , locus: AUG15133.1 UniProtKB - P02920, last release date on May 29, 2024), shown in Figure 1 . The E. coli LacY consists of an amino acid chain of 417 amino acid residues in length, and has a calculated mass of 46,503 Da. The secondary structure of the E. coli wild-type LacY consists of 12 transmembrane helices organized into two bundles of six helices connected by a long flexible loop. A central hydrophilic cavity contains the lactose binding site and the amino acid residues involved in proton translocation. A more detailed topology of E. coli wild-type LacY is provided in Table 1. Table 1 : Topology of E. coli wild-type LacY corresponding to SEQ ID NO: 1 .

[0077] Some amino acid residues play an essential role in the transport of lactose through the protein, such as provided in Table 2. Table 2: Essential amino acid residues corresponding to the positions of the amino acid sequence of SEQ ID NO: 1 .

[0078] Further, mutagenic analysis of E. coli LacY revealed that certain amino acid residues are also relevant for LacY activity. D237 is ion-paired with K358 and they are known to play a structural role. For example, D237 carries a + charge, K358 carries a - charge. If the charges are swapped by a double mutation, or both of them are simultaneously mutated to hydrophobic amino acids, active transport is retained. However, loss of transport activity is created, when one of D237 and K358 remains charged while the other is mutated into a non-charged amino acid. A replacement of the leucine residues at the positions 292, 293, and / or 294 corresponding to the amino acid sequence of SEQ ID NO: 1 , with a basic amino acid residue selected from the group consisting of R, K and H, leads to a reduced transport activity for lactose as compared to the E. coli wild-type LacY, as provided in Table 3.

[0079] Table 3: Mutagenic results on transport activity of E. coli LacY.

[0080] In various embodiments, the mutant galactoside permease of the present disclosure, has at least 57%, at least 65%, at least 70%, at least 80%, preferably 85%, more preferably at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1. In additional embodiments, the mutant galactoside permease has at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 .

[0081] In additional and / or alternative embodiments, the mutant galactoside permease as disclosed herein, has the at least one mutation at a position that is involved in proton translocation of the galactoside permease according to SEQ ID NO: 1.

[0082] In particular embodiments, the mutant galactoside permease:

[0083] (i) has a modified proton symporter activity, preferably a reduced proton symporter activity, more preferably a lack of proton symporter activity; and / or

[0084] (ii) is not capable of mediating galactoside: proton symport with a 1 :1 stoichiometry; and / or

[0085] (iii) has a reduced proton translocation activity, preferably a lack of proton translocation activity, as comparted to the wild-type galactoside permease according to SEQ ID NO: 1.

[0086] In various embodiments, the mutant galactoside permease displays an altered membrane topology as compared to the membrane topology of the wild-type galactoside permease according to SEQ ID NO: 1. Preferably mutations in the transmembrane helix IX and / or X may lead to a decoupled galactoside:proton symport activity. Alternatively, mutations in the transmembrane helix VIII and / or VII may lead to a decoupled galactoside:proton symport activity. Without being bound by theory, it is assumed that the initial proton acceptor is E269, that forms a H-bond with W151. Ligand-binding causes amino acid side chain displacements and shifts E269 to a more hydrophilic environment, allowing its deprotonation to H322 through water transmission. Deprotonated E269 stabilizes with the hydroxyl group at 03 oxygen of the bound galactoside substrate as well as by forming a salt bridge with R144 that also participates in substrate binding. Protonated H322 moves closer to E325 and transfers the proton onto it. Neutralized E325 shifts into a hydrophobic area between the transporter surface and the lipid bilayer of the membrane, and this intramolecular movement seems to be a key mechanistic element that via force transmission by intramolecular linkages drives the transport of the galactoside substrate. The galactoside substrate dissociates on the cytoplasmic side of the membrane leading to further dynamics within the molecule, whereby E325 gets closer to and deprotonated by the R302 base, through which it returns to its initial state and the cycle can start over.

[0087] Neutral substitutions at E325, like E325A, are considered to stabilize the transporter in this normally transitory conformation, keeping the transport channel open and allowing free passive exchange of galactosides between the two sides of the membrane, whereas proton symport is blocked. In addition to that, amino acid positions within the LacY amino acid sequence which are involved in proton translocation have been identified. Unexpectedly, (non-human) host cells expressing the galactosidase permease mutants of the present invention, comprising amino acid substitutions at the identified positions, provide for improved tolerance against lactose killing.

[0088] In various embodiments, the mutant galactoside permease: a) is capable of substrate binding and / or has no mutation in the substrate-binding site; and / or b) has I maintains substrate specificity for galactosides; and / or c) exhibits I maintains galactoside translocation activity.

[0089] In various embodiments, the mutant galactoside permease does not require (full) proton translocation for the binding of galactoside(s). The galactoside according to the present invention is for example lactose.

[0090] In various embodiments, the mutant galactoside permease as disclosed herein has at least one mutation at positions corresponding to any of positions 31 to 400 of SEQ ID NO: 1. For example, in various embodiments, the mutant galactoside permease as disclosed herein has the at least one mutation at one or more positions corresponding to any of positions 295 to 334 of SEQ ID NO: 1. In various embodiments, the mutant galactoside permease as disclosed herein has the at least one mutation at one or more position of Table 4.

[0091] Table 4: Exemplary mutation positions of the mutant galactoside permease as disclosed herein corresponding to the positions of the amino acid sequence of SEQ ID NO: 1. In various embodiments, the mutant galactoside permease as disclosed herein has at least one mutation at a positions corresponding to 322, 325, 302 329, 31 , 64, 68, 115, 126, 144, 147, 237, 240, 269, 319, 334, 346, 348, 358, 35, 76, 131 , 140, 151 , 177, 184, 350, 380, 400, of SEQ ID NO: 1. In various embodiments, the mutant galactoside permease as disclosed herein has the at least one mutation at one or more positions in transmembrane helix IX and / or transmembrane helix X of galactoside permease according to SEQ ID NO: 1. For example, the mutant galactoside permease as disclosed herein has the at least one mutation selected from 322, 325, 319 and 334 in transmembrane helix X corresponding to the galactoside permease according to SEQ ID NO: 1 and / or 302 in transmembrane helix IX corresponding to the galactoside permease according to SEQ ID NO: 1. For example, the mutant galactoside permease as disclosed herein has the at least one mutation selected from 322, 325, 319 and 334 in helix X corresponding to the galactoside permease according to SEQ ID NO: 1.

[0092] In various embodiments, the mutant galactoside permease has at least one mutation at one or more positions corresponding to any of positions 322, 325, 302, 269 and / or 237 of the amino acid sequence of SEQ ID NO: 1 , preferably at the position corresponding to position 322 of the amino acid sequence of SEQ ID NO: 1.

[0093] In various embodiments, the mutant galactoside permease has a mutation at two or more positions corresponding to any of positions 322, 325, 302, 269 and / or 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least two mutation at positions corresponding to 322 and 325 of the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least two mutation at positions corresponding to 322 and 302 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least two mutation at positions corresponding to 325 and 302 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least two mutation at positions corresponding to 322 and 269 of the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least two mutation at positions corresponding to 322 and 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least two mutation at positions corresponding to 325 and 269 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least two mutation at positions corresponding to 325 and 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least two mutation at positions corresponding to 302 and 269 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least two mutation at positions corresponding to 302 and 237 of the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least two mutation at positions corresponding to 237 and 269 of the amino acid sequence of SEQ ID NO: 1.

[0094] In various embodiments, the mutant galactoside permease has a mutation at three or more positions corresponding to any of positions 322, 325, 302, 269 and / or 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least three mutation at positions corresponding to 322, 325 and 302 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least three mutation at positions corresponding to 322, 325 and 269 of the amino acid sequence of SEQ ID NO: 1.

[0095] For example, the mutant galactoside permease has at least three mutation at positions corresponding to 322, 325 and 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least three mutation at positions corresponding to 302, 325 and 269 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least three mutation at positions corresponding to 302, 325 and 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least three mutation at positions corresponding to 302, 322 and 269 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least three mutation at positions corresponding to 302, 322 and 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least three mutation at positions corresponding to 322, 269 and 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least three mutation at positions corresponding to 325, 269 and 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least three mutation at positions corresponding to 302, 269 and 237 of the amino acid sequence of SEQ ID NO: 1 .

[0096] In various embodiments, the mutant galactoside permease has a mutation at four or more positions corresponding to any of positions 322, 325, 302, 269 and / or 237 of the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least four mutation at positions corresponding to 322, 325, 302 and 269 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least four mutation at positions corresponding to 322, 325, 302 and 237 of the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least four mutation at positions corresponding to 322, 325, 269 and 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least four mutation at positions corresponding to 322, 302, 269 and 237 of the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least four mutation at positions corresponding to 325, 302, 269 and 237 of the amino acid sequence of SEQ ID NO: 1 . In various embodiments, the mutant galactoside permease has at least five mutations at the five positions corresponding to any of positions 322, 325, 302, 269 and 237 of the amino acid sequence of SEQ ID NO: 1 .

[0097] Said positions 322, 325 in transmembrane helix X and / or 302 in transmembrane helix IX are involved in proton translocation of the galactoside permease according to SEQ ID NO: 1 , as shown in Tables 1 and 2.

[0098] In various embodiments, the at least one mutation is at least one amino acid substitution. For example, the amino acid substitution is a substitution of the native amino acid at the respective position with any other amino acid of the 19 alternative amino acids. For example, the amino acid substitution is selected from the group consisting of A (Ala, alanine), R (Arg, arginine), N (Asn, asparagine), D (Asp, aspartic acid), C (Cys, cysteine), E (Glu, glutamic acid), Q (Gin, glutamine), G (Gly, glycine), H (His, histidine), I (lie, isoleucine), L (Leu, leucine), K (Lys, lysine), M (Met, methionine), F (Phe, phenylalanine), P (Pro, proline), S (Ser, serine), T (Thr, threonine), W (Trp, tryptophan), Y (Tyr, tyrosine), and V (Vai, valine).

[0099] In various embodiments, the at least one amino acid substitution comprises a substitution of

[0100] (i) a basic amino acid residue against a basic amino acid residue; and / or

[0101] (ii) a basic amino acid residue against a polar amino acid residue; and / or

[0102] (iii) a basic amino acid residue against a non-polar amino acid residue; and / or

[0103] (iv) a basic amino acid residue against an acidic amino acid residue; and / or

[0104] (v) an acidic amino acid residue against a non-polar amino acid residue; and / or

[0105] (vi) an acidic amino acid residue against a polar amino acid residue; and / or

[0106] (vii) an acidic amino acid residue against an acidic amino acid residue; and / or

[0107] (viii) an acidic amino acid residue against a basic amino acid residue.

[0108] In various embodiments, the basic residue is an amino acid selected from the group consisting of histidine (His, H), arginine (Arg, R), and lysine (Lys, K).

[0109] In various embodiments, the polar amino acid residue is an amino acid residue selected from the group consisting of serine (Ser, S), tyrosine (Tyr, Y), asparagine (Asn, N), and glutamine (Gin, Q).

[0110] In various embodiments, the non-polar amino acid residue is an amino acid selected from the group consisting of alanine (Ala, A), cysteine (Cys, C), leucine (Leu, L), methionine (Met, M), and phenylalanine (Phe, F).

[0111] In various embodiments, the acidic amino acid residue is aspartic acid (Asp, D). In various embodiments, the amino acid substitution at the position corresponding to position 322 of the amino acid sequence of SEQ ID NO: 1 is a substitution of histidine against an amino acid residue selected from any one of Y, Q, C, R, K, N, A, M, F, or D. In preferred embodiments, the amino acid substitution at the position corresponding to position 322 of the amino acid sequence of SEQ ID NO: 1 is a substitution of histidine against an amino acid residue selected from any one of Q, C, R, K, or N. In more preferred embodiments, the amino acid substitution at the position corresponding to position 322 of the amino acid sequence of SEQ ID NO: 1 is a substitution of histidine against an amino acid residue selected from any one of R or K.

[0112] In various embodiments, the amino acid substitution at the position corresponding to position 325 of the amino acid sequence of SEQ ID NO: 1 is a substitution of glutamic acid against an amino acid residue selected from any one of C, D, A, Q, K, S, or H. In preferred embodiments, the amino acid substitution at the position corresponding to position 325 of the amino acid sequence of SEQ ID NO: 1 is a substitution of glutamic acid against an amino acid residue selected from any one of C, D, or A. In more preferred embodiments, the amino acid substitution at the position corresponding to position 325 of the amino acid sequence of SEQ ID NO: 1 is a substitution of glutamic acid against an amino acid residue selected from any one of C or A.

[0113] In various embodiments, the amino acid substitution at the position corresponding to position 302 of the amino acid sequence of SEQ ID NO: 1 is a substitution of arginine against an amino acid residue selected from any one of K, H, L, A, S, or C. In preferred embodiments, the amino acid substitution at the position corresponding to position 302 of the amino acid sequence of SEQ I D NO: 1 is a substitution of arginine against an amino acid residue selected from any one of K, H, L, A, or S. In more preferred embodiments, the amino acid substitution at the position corresponding to position 302 of the amino acid sequence of SEQ ID NO: 1 is a substitution of arginine against an amino acid residue selected from any one of K or H.

[0114] In various embodiments, the amino acid substitution at the position corresponding to position 269 of the amino acid sequence of SEQ ID NO: 1 is a substitution of glutamic acid against an amino acid residue selected from any one of Q, C, or D. For example, the amino acid substitution at the position corresponding to position 269 of the amino acid sequence of SEQ ID NO: 1 is a substitution of glutamic acid against aspartic acid (D).

[0115] In various embodiments, the amino acid substitution at the position corresponding to position 237 of the amino acid sequence of SEQ ID NO: 1 is a substitution of aspartic acid against an amino acid residue selected from any one of C, A, or K. For example, the amino acid substitution at the position corresponding to position 269 of the amino acid sequence of SEQ ID NO: 1 is a substitution of aspartic acid against glutamic acid cysteine (C). In various embodiments, the amino acid substitution at the position corresponding to position 334 of the amino acid sequence of SEQ ID NO: 1 is a substitution of phenylalanine against cysteine (C).

[0116] In various embodiments, the amino acid substitution at the position corresponding to position 319 of the amino acid sequence of SEQ ID NO: 1 is a substitution of lysine against cysteine (C).

[0117] In other particularly preferred embodiments, the mutant galactoside permease comprises at least one of the following amino acid substitutions:

[0118] (i) H322R at the position corresponding to position 322 of the amino acid sequence of SEQ ID NO: 1 ; and / or

[0119] (ii) E325A at the position corresponding to position 325 of the amino acid sequence of SEQ ID NO: 1 ; and / or

[0120] (iii) R302K at the position corresponding to position 302 of the amino acid sequence of SEQ ID NO: 1 ; and / or

[0121] (iv) E269D at the position corresponding to position 269 of the amino acid sequence of SEQ ID NO: 1 ; and / or

[0122] (v) D237C at the position corresponding to position 237 of the amino acid sequence of SEQ ID NO: 1.

[0123] In various embodiments, the mutant galactoside permease is a mutant P-galactoside permease, preferably a mutant lactose permease.

[0124] In a further aspect, a nucleic acid sequence encoding the mutant galactoside permease according to the present invention is provided. A nucleic acid sequence encoding the wildtype lacY gene is represented by SEQ ID NO: 2. The nucleic acid sequence encoding the mutant galactoside permease is a nucleotide which encodes any one of the mutant galactoside permease described in the present disclosure.

[0125] In various embodiments, the nucleic acid sequence encoding the mutant galactoside permease comprising the amino acid substitution at the position 322 corresponding to the amino acid position represented by SEQ ID NO: 1 , comprises a codon selected from the group consisting of CGG, CGA, CGC, CGT, AGA, AGG instead of a codon encoding the histidine residue, i.e. , CAC or CAT.

[0126] In various embodiments, the nucleic acid sequence encoding the mutant galactoside permease comprising the amino acid substitution at the position 325 corresponding to the amino acid position represented by SEQ ID NO: 1 , comprises a codon selected from the group consisting of GCT, GCC, GCA, GCG instead of a codon encoding the glutamic acid residue, i.e., GAG or GAA.

[0127] In various embodiments, the nucleic acid sequence encoding the mutant galactoside permease comprising the amino acid substitution at the position 302 corresponding to the amino acid position represented by SEQ ID NO: 1 , comprises a codon selected from the group consisting of AAG, AAA instead of a codon encoding the arginine residue, i.e., CGG, CGA, CGC, CGT, AGG or AGA.

[0128] In various embodiments, the mutant galactoside permease has at least two mutations selected from H322R, E325A, R302K, E269D and / or D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least the two mutation H322R and E325A, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the two mutation H322R and R302K, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the two mutation E325A and R302K, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the two mutation H322R and E269D, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the two mutation H322R and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the two mutation E325A and E269D, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the two mutation E325A and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the two mutation R302K and E269D, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the two mutation R302K and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least the two mutation E269D and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 .

[0129] In various embodiments, the mutant galactoside permease has at least three mutations selected from H322R, E325A, R302K, E269D and / or D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least the three mutation H322R, E325A and R302K, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the three mutation H322R, E325A and E269D, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the three mutation H322R, E325A and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least the three mutation R302K, E325A and E269D, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the three mutation R302K, E325A and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the three mutation R302K, H322R and E269D, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the three mutation R302K, H322R and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: I .For example, the mutant galactoside permease has at least the three mutation H322R, E269D and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least the three mutation E325A, E269D and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the three mutation R302K, E269D and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1.

[0130] In various embodiments, the mutant galactoside permease has at least four mutations selected from H322R, E325A, R302K, E269D and / or D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least the four mutation H322R, E325A, R302K and E269D, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the four mutation H322R, E325A, R302K and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the four mutation H322R, E325A, E269D and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. For example, the mutant galactoside permease has at least the four mutation H322R, R302K, E269D and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1 . For example, the mutant galactoside permease has at least the four mutation E325A, R302K, E269D and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1.

[0131] In various embodiments, the mutant galactoside permease has at least five mutations selected from H322R, E325A, R302K, E269D and D237C, wherein the amino acid positions correspond to the amino acid sequence of SEQ ID NO: 1. In additional embodiments, the nucleic acid sequence which encodes the mutant galactoside permease according to the present disclosure, has a sequence identity of at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% to the nucleotide sequence of SEQ ID NO: 2.

[0132] In various embodiments, the nucleic acid sequence encoding the mutant galactoside permease according to the invention, is stably integrated into the genomic DNA of the host cell.

[0133] The term “encodes” and “codes for” refers broadly to any process whereby the information in a polymeric macromolecule is used to direct the production of a second molecule that is different from the first. The second molecule may have a chemical structure that is different from the chemical nature of the first molecule. For example, in some aspects, the term “encode” describes the process of semi-conservative DNA replication, where one strand of a double-stranded DNA molecule is used as a template to encode a newly synthesized complementary sister strand by a DNA-dependent DNA polymerase. In other aspects, a DNA molecule can encode an RNA molecule (e.g., by the process of transcription that uses a DNA- dependent RNA polymerase enzyme). Also, an RNA molecule can encode a polynucleotide, as in the process of translation. When used to describe the process of translation, the term “encode” also extends to the triplet codon that encodes an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, e.g., by the process of reverse transcription incorporating an RNA-dependent DNA polymerase. In another aspect, a DNA molecule can encode a polypeptide, where it is understood that “encode” as used in that case incorporates both the processes of transcription and translation.

[0134] The terms “nucleic acid”, “nucleotide”, and “polynucleotide” as used herein are used interchangeably and refer to a single or double-stranded polymer of deoxyribonucleotide bases or ribonucleotide bases read from the 5’ to the 3’ end and include double stranded DNA (dsDNA), single stranded DNA (ssDNA), single stranded RNA (ssRNA), double stranded RNA (dsRNA), genomic DNA, complementary DNA (cDNA), complementary RNA (cRNA), recombinant DNA or recombinant RNA and derivatives thereof, such as those containing modified backbones. Preferably, a polynucleotide, particularly to be stably integrated into the genome of the host cell, is a DNA or cDNA. Polynucleotides according to the invention can be prepared in different ways (e.g. by chemical synthesis, by gene cloning, etc.) and can take various forms (e.g. linear or branched, single or double stranded, or a hybrid thereof, primes, probes etc.) The term “nucleotide sequence” or “nucleic acid sequence” refers to both the sense and antisense strands of a nucleic acid as either individual single strands or in the duplex. “Nucleic acid sequence”, “gene” or “sequences” can be introduced into a target cell directly or preferably by using an “expression vector”. Methods used to construct vectors are well known to the person skilled in the art and described in various publications. In particular, techniques for constructing suitable vectors, including a description of functional components such as promoters, enhancers, termination and polyadenylation signals, selection markers, origin of replication, and in case of eukaryotic host cells splicing signals, are reviewed in considerable details in (Sambrook J, et al., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press) and references cited therein. Vectors may include but are not limited to plasmid vectors, phagemids, cosmids or artificial / mini-chromosomes (e.g. ACE). The eukaryotic expression vectors will typically contain also prokaryotic sequences that facilitate the propagation of the vector in bacteria such as an origin of replication and antibiotic resistance genes for selection in bacteria. A variety of eukaryotic and prokaryotic expression vectors, containing a cloning site into which a polynucleotide can be operably linked, are well known in the art and some are commercially available from companies such as pET (Novagen / Merck Biosciences), pQE (Qiagen), pBAD (Invitrogen / Thermo Fisher Scientific), pF vectors (Promega). Usually, expression vectors also comprise an expression cassette encoding a selectable marker, allowing selection of host cells carrying said expression marker.

[0135] The term “stable integration” or “stably integrated” as used in the patent refers to a polynucleotide being introduced into a host cell genome, as opposed to transiently introduced polynucleotides that remain separate from the genomic DNA of the host cell. Stable integration may occur by homologous recombination or other types of recombination. Stable integration may comprise a step of transient introduction of a polynucleotide into a host cell.

[0136] According to a further aspect, a vector comprising the nucleic acid sequence according to the present invention is provided.

[0137] According to a further aspect according to the invention a vector is provided comprising the nucleic acid encoding a galactoside permease mutant having reduced or abolished proton- coupled galactoside transport.

[0138] A “vector” is a nucleic acid that can be used to introduce a polynucleotide into a cell. One type of vector is a “plasmid”, which refers to a linear or circular double stranded DNA molecule into which additional nucleic acid sequences can be ligated. Preferably, the vector is a prokaryotic vector or a eukaryotic vector integrating into the genome of a (non-human) host cell and culturing under selective pressure, and thereby are replicated along with the host genome. A vector can be used to direct the expression of a polynucleotide in a cell.

[0139] In various embodiments, the vector comprises an expression cassette comprising the nucleic acid sequence according to the invention. The term “expression” as used herein refers to transcription and / or translation of a nucleic acid sequence within a host cell. The level of expression of a gene product in a host cell may be determined on the basis of either the amount of corresponding RNA that is present in the cell, or the amount of the polypeptide encoded by the selected sequence. For example, RNA transcribed from a selected sequence can be quantified by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization to cellular RNA or by PCR, such as qPCR. Proteins encoded by a selected sequence can be quantitated by various methods, e.g. by photometric measurement, by ELISA, by Western Blotting, by radioimmunoassay, by immunoprecipitation, by assaying for the biological activity of the protein.

[0140] As used herein, the term “expression cassette” refers to the part of a vector comprising one or more genes encoding for an RNA or a protein and the sequences controlling their expression. Thus, it comprises a promoter sequence, an open reading frame a 3’- untranslated region, typically containing a polyadenylation site. Preferably, the vector is an expression vector comprising one or more gene(s) encoding for the recombinant protein. It may be part of a vector, typically an expression vector including a plasmid or a viral vector. It may also be integrated into the genome by random or targeted integration, such as by homologous recombination. An expression cassette is prepared using cloning techniques and does therefore not refer to a natural occurring gene structure.

[0141] A “promoter” or “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3’ direction) coding sequence. A promoter sequence is bounded at its 3’ terminus by the transcription initiation site and extends upstream in 5’ direction. For example, a promoter is about 50 - 3000, about 100 - 2500, about 200 - 2000, about 300 - 1500, or about 350 - 1000 base long. A promoter sequence comprises a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of the RNA polymerase. Prokaryotic promoters contain a Pribnow box. Eukaryotic promoters often, but not always, contain “TATA” boxes and “CAT” boxes. Promoter sequences often contain additional consensus sequences recognized by proteins involved in regulating expression of the respective gene. Regulation of gene expression by a promoter can occur by enhancing or inhibiting binding of a regulatory protein. Enhancing or inhibiting the binding or a regulatory protein can occur by many different means, including but not limited to, base modifications (i.e., methylation) or protein modification (i.e., phosphorylation). For example, the promoter is a promoter which overexpresses the mutant galactoside permease as disclosed herein. Overexpression as used herein refers to an expression of the mutant galactoside permease as disclosed herein which is higher than the expression of the galactoside permease under the wild-type (native) promoter. Overexpression results in a higher number of transcripts and / or protein of the T1 mutant galactoside permease as disclosed herein compared to expression under the wildtype (native) promoter.

[0142] In preferred embodiments, the (non-human) host cell overexpresses the mutant galactoside permease, wherein the at least one or more mutation is at positions corresponding to positions 322, 325, 302, 269 and / or 237 of the amino acid sequence of SEQ ID NO: 1 .

[0143] In various embodiments the (non-human) host cell contains a nucleic acid sequence encoding the mutant galactoside permease as disclosed herein. The nucleic acid sequence is for expression of the mutant galactoside permease according to the invention. In various embodiments the nucleotide sequence for expression of the mutant galactoside permease is operably linked to expression control sequences.

[0144] In preferred embodiments, the (non-human) host cell is a microbial cell, preferably a bacterial cell or a yeast cell, more preferably a bacterial cell.

[0145] The term “organism”, “(non-human) host cell” or “cell” as indicated herein refers to a microbial host cell. The microbial cell may be a prokaryotic cell or a eukaryotic cell. Suitable microbial host cells include bacterial cells, yeast cells, archaebacterial cells, and fungal cells.

[0146] In preferred embodiments, the prokaryotic cell is a bacterial cell selected from bacteria of a genus selected from the group consisting of Bacillus, Bifidobacerium, Clostridium, Corynebacterium, Enterococcus, Lactobacillus, Lactococcus, Micrococcus, Micromonospora, Pseudomonas, Rhodococcus, and Sporolactobacillus. In additional and / or alternative embodiments, the bacterial species are Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, Bacillus thermophilus, Bacillus laterosporus, Bacillus megaterium, Bacillus mycoides, Bacillus pumilus, Bacillus lentus, Bacillus cereus, Bacillus circulans, Bifidobacterium longum, Bifidobacterium infantis, Bifidobacterium bifidum, Citrobacter freundii, Clostridium cellulolyticum, Clostridium ljungdahlii, Clostridium autoethanogenum, Clostridium acetobutylicum, Corynebacterium glutamicum, Enterococcus faecium, Entercoccus thermophiles, Escherichia coli, Erwinia herbicola (Pantoea agglomerans), Lactobacillus acidophilus, Lactobacillus salivarius, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus rhamnosus, Lactobacillus bulgaricus, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus casei, Lactobacillus reuteri, Lactobacillus jensenii, Lactobacillus lactis, Pantoea citrea, Pectobacterium carotovorum, Proprionibacterium freudenreichii, Pseudomonas fluorescens, Pseudomonas aeruginosa, Streptococcus thermophiles, and Xanthomonas campestris.

[0147] In various embodiments, the eukaryotic cell is a yeast cell, preferably a yeast cell selected from the group consisting of Saccharomyces sp., in particular Saccharomyces cerevisiae, Saccharomycopsis sp., Pichia sp., more preferably Pichia pastoris, Hansenula sp., Kluyveromyces sp., Yarrowia sp., Rhodotorula sp., and Schizosaccharomyces sp.. In various embodiments, the (non-human) host cell comprises a nucleic acid sequence encoding a glycosyltransferase, preferably a fucosyltransferase. For example, the host cell comprises a nucleic acid sequence encoding an exogenous glycosyltransferase, preferably a fucosyltransferase. For example, the host cell comprises a nucleic acid sequence encoding an endogenous glycosyltransferase, preferably a fucosyltransferase.

[0148] Glycosyltransferases transfer a monosaccharide moiety from a nucleotide activated monosaccharide as donor substrate to an acceptor molecule. The glycosyltransferase may be selected from the group consisting of fucosyltransferases, siayltransferases, mannosyltransferases, N-acetylglucosaminyltransferases, N-acetylgalactosaminyl- transferases, and galactosyltransferases. The acceptor molecule may be selected from the group consisting of lactose and oligosaccharides bearing a lactose moiety at their reducing end.

[0149] According to another aspect, use of the mutant galactoside permease or the nucleic acid sequence, or the vector, or the (non-human) host cell according to the present disclosure for the production of lactose-based products, for example, (human milk) oligosaccharides is provided.

[0150] As used herein, the term “lactose-based product” refers to any compound, material or energy carrier which is produced, directly or indirectly, through a biological or biochemical process in which lactose, a lactose analogue, a lactose-derived substrate, or a lactose-containing feedstock serves as a carbon source, energy source, metabolic substrate, precursor, cosubstrate or structural component. This term includes, without limitation: (a) oligosaccharides and glycans such as human milk oligosaccharides (HMOs), including fucosylated, sialylated, galactosylated, phosphorylated or otherwise modified lactose derivatives; (b) extended lactose-containing carbohydrates and conjugates thereof; (c) fermentation-derived metabolites produced from lactose, such as organic acids, alcohols, esters, or gases; and (d) bioenergy products including biogas, biomethane, biosyngas or other gaseous fermentation products generated from lactose-containing substrates.

[0151] The term “oligosaccharide” as used herein refers to a saccharide molecule consisting of three to twenty monosaccharide residues, wherein each of said monosaccharide residues is bound to at least one other of said monosaccharide units by a glycosidic linkage. The oligosaccharide may be a linear chain of monosaccharide residues or a branched chain of monosaccharide residues.

[0152] In an additional and / or alternative embodiment, the oligosaccharide is a human milk oligosaccharide.

[0153] As used herein, “lactose toxicity” refers to a detrimental physiological response of a microbial host cell to lactose-rich media, typically caused by proton-coupled lactose import mediated by the wild-type LacY permease. Lactose toxicity manifests as reduced growth rate, loss of viability, membrane depolarisation, collapse of the proton motive force, or failure to sustain fermentation, and is alleviated or absent in cells expressing galactoside permease mutants with reduced or abolished proton coupling.

[0154] Expressions and definitions provided elsewhere herein with regards to other aspects of the present invention equally apply here. Definitions, embodiments, examples etc. herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0155] The use of galactoside permease variants that exhibit reduced or abolished proton-coupled transport, when expressed at levels exceeding the endogenous wild-type permease, results in a distinct technical effect during microbial cultivation and product formation. The increased expression level of such permease variants provides a higher rate of galactoside translocation into the cell, thereby supplying an elevated intracellular concentration of lactose or related galactosides as carbon source, precursor, or acceptor substrate. This increased substrate availability supports higher metabolic fluxes, leading to increased biomass formation and improved yields of lactose-derived products. In several cultivation settings, the use of proton-uncoupled permease variants further results in reduced formation of undesired by-products that are typically associated with disturbed energy homeostasis under conditions of wild-type permease overexpression.

[0156] Because proton-uncoupled variants do not generate excessive proton influx during substrate uptake, the cellular energy state remains stable, which contributes to more robust fermentation performance. This robustness can manifest in more consistent growth kinetics, improved tolerance to elevated lactose concentrations, and greater reproducibility under industrial fermentation conditions. These combined effects enable fermentation processes with higher productivities, longer cultivation times, or higher product titers, and support the use of lactose-rich media in industrial manufacturing of lactose-based or lactose-derived products.

[0157] Overexpression of the galactoside permease mutant may be achieved by any suitable genetic or regulatory strategy known in the art, and the invention is not limited to a particular expression system. Examples of suitable approaches include, without limitation:

[0158] (i) Promoter-based overexpression: The use of strong heterologous, synthetic, inducible or constitutive promoters, such as PT7, Ptac, Ptrc, PBAD, PlacllV5, PAPL, PAPR, Ptet, promoters of the Anderson series (e.g. PJ23119, PJ23105), or strong industrial promoters such as Pveg or P43 in Bacillus, or Pgap, Peftu, Peno in Corynebacterium. Promoter variants, mutated or optimized promoter sequences, and chimeric promoters capable of increasing transcriptional strength are likewise encompassed.

[0159] (ii) Copy-number-based overexpression:

[0160] Expression of the mutant permease from a high-copy-number plasmid (e.g. pUC-derived, pBR322-derived, ColE1-derived plasmids), medium-copy plasmids (e.g. pET vectors), or from multiple chromosomally integrated copies. Multiple insertions may be achieved by sitespecific integration systems, recombination-based methods, or CRISPR-mediated tandem integration.

[0161] (iii) Derepression and endogenous control removal:

[0162] Overexpression may also be achieved through removal, reduction or inactivation of native repressors, such as deletion or functional inactivation of lacl, or manipulation of regulatory components of the lac operon or related regulatory networks. Additional regulatory deletions or knockdowns (e.g. gaIR, galS, purR) may further enhance expression in certain host backgrounds.

[0163] (iv) Post-transcriptional control:

[0164] Increased expression may be supported by engineering the ribosome binding site (RBS), for example by using synthetic Shine-Dalgarno sequences, RBS-Calculator-optimized sequences, or translational enhancer elements. Overexpression may further be facilitated by modifying the 5'-untranslated region (5'-UTR), incorporating stabilizing structures such as stem-loop motifs to increase mRNA stability, or use of strong transcription terminators to ensure defined transcript lengths.

[0165] (v) Post-translational and membrane-insertion enhancement:

[0166] Because the galactoside permease is a membrane protein, overexpression can additionally be promoted by co-expression of membrane insertase or chaperone systems, such as YidC, SecYEG, Ffh / FtsY, GroEL / GroES, or by modifications to the membrane lipid composition or biosynthesis pathways that stabilize insertion or retention of permease molecules in the membrane.

[0167] (vi) Auto-induction and dynamic control systems: Overexpression may also be obtained using auto-induction media, lactose- or rhamnose- triggered induction circuits, or other metabolic or quorum-sensing-based promoter systems enabling high-level expression without external inducers.

[0168] (vii) Combined strategies:

[0169] In some embodiments of the invention, two or more of the above strategies may be used in combination, such as a strong promoter placed upstream of an optimized RBS on a high- copy plasmid or in a multi-copy chromosomal context, optionally together with repressor removal or chaperone co-expression.

[0170] All of these approaches, individually or in combination, are suitable for achieving expression levels of the galactoside permease mutant that exceed endogenous wild-type LacY expression levels and thereby fall within the meaning of ‘overexpression’ as used herein.”

[0171] As used herein, the term “overexpression” refers to an expression level of the galactoside permease mutant that exceeds the endogenous expression level of the corresponding wildtype permease under otherwise comparable physiological conditions. Overexpression may result from increased transcription, increased translation, enhanced mRNA stability, elevated gene dosage or reduced repression, or a combination thereof. Unless indicated otherwise, overexpression includes any expression level that is measurably higher than native LacY expression arising from the chromosomal lac operon in the same host background.

[0172] In one embodiment of the invention, overexpression of the galactoside permease mutant is achieved by a heterologous or synthetic promoter. Suitable promoters include, for example, PT7, Ptac, Ptrc, PBAD, PlacUV5, Ptet, PJ23119, Pveg, P43, or functional variants or derivatives thereof capable of driving elevated transcription levels in the host cell.

[0173] In another embodiment, overexpression is achieved by increasing the gene dosage, for example by expressing the mutant permease from a multicopy plasmid or by providing multiple chromosomal insertions of the corresponding gene, obtained by site-specific integration or recombination-based methods.

[0174] In a further embodiment, overexpression is supported or enhanced by an engineered ribosome binding site (RBS), for example a Shine-Dalgarno sequence or synthetic RBS optimized for high translational efficiency.

[0175] In yet another embodiment, overexpression is achieved or facilitated by deletion or inactivation of lacl or another repressor of the lac operon, thereby relieving transcriptional repression and enabling elevated expression of the mutant galactoside permease. As used herein, “increased transcription” refers to an elevated rate or extent of mRNA synthesis from the nucleic acid encoding the galactoside permease mutant, compared to transcription from the endogenous lac operon of the corresponding wild-type permease under otherwise identical conditions. Increased transcription may result from the use of stronger or heterologous promoters, transcriptional enhancers, derepression of native regulatory elements or combinations thereof.

[0176] As used herein, “increased gene dosage” refers to any increase in the number of copies of a nucleic acid encoding the galactoside permease mutant relative to the single-copy chromosomal wild-type gene. Increased gene dosage may be achieved by high-copy or multicopy plasmids, tandem chromosomal integrations, site-specific recombination, or other genetic arrangements resulting in a higher copy number of the mutant permease gene within the host cell.

[0177] As used herein, “derepression” refers to the removal, reduction or functional inactivation of a regulatory element that normally suppresses transcription of the galactoside permease gene. Derepression includes, for example, deletion, mutation or inhibition of “lad” or other repressors acting on the lac operon, thereby permitting elevated transcription of the mutant permease under conditions in which the wild-type gene would remain partially or fully repressed.

[0178] Expressions and definitions provided elsewhere herein with regards to other aspects of the present invention equally apply here. Definitions, embodiments, examples etc. herein, for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0179] Nucleic acids, vectors, host cells and their uses

[0180] The invention further encompasses nucleic acids, expression constructs and host cells suitable for implementing the above-described permease mutants and their overexpression in microbial production systems.

[0181] In particular, the invention provides nucleic acids encoding the disclosed galactoside permease mutants together with regulatory or structural genetic elements that enable increased expression of the permease, such as promoters, ribosome binding sites, untranslated regions, transcriptional or translational enhancers, or genetic elements increasing gene dosage. Vectors comprising such nucleic acids, including plasmid-based and chromosomally integrative constructs, likewise form part of the invention, as do microbial host cells equipped with these constructs and capable of expressing the mutant permease at elevated levels.

[0182] Hence, according to a further aspect of the invention a vector comprising a nucleic acid which encodes the disclosed galactoside permease mutant and further comprises one or more genetic elements suitable for achieving overexpression of the mutant permease is provided.

[0183] The invention further relates to the use of such nucleic acids, vectors and host cells for recombinant production processes in which enhanced permease expression provides improved robustness, transport properties or metabolic performance.

[0184] In a preferred embodiment the additional genetic elements refer to promotor sequences.

[0185] Furthermore, the nucleic acids, vectors and host cells provided herein address limitations that have remained unconsidered in earlier studies on galactoside permeases. Previous work on permease variants has primarily focused on mechanistic analysis, substrate specificity or structural aspects, and did not contemplate the deliberate engineering of permeases with reduced or abolished proton coupling for use in recombinant production systems. In particular, earlier approaches did not disclose or suggest the combination of such permease variants with expression strategies designed to achieve expression levels exceeding endogenous wild-type levels. By enabling stable high-level expression of proton-uncoupled permeases in microbial hosts, the nucleic acids and vectors of the present invention provide an expression framework that was neither described nor predictable from prior work and that enables applications not previously accessible.

[0186] According to a further aspect of the invention the use of such nucleic acids and vectors encoding / comprising an overexpressed galactoside permease mutant with reduced or abolished proton-coupled galactoside transport for producing a lactose-based product in a fermentation process is provided.

[0187] In one embodiment the galactoside permease mutant retains galactoside transport activity sufficient to support production of a lactose-based product.

[0188] In one embodiment the host cell exhibits reduced lactose toxicity relative to a host cell overexpressing the wild-type galactoside permease when cultured in the presence of lactose.

[0189] In one embodiment reduced lactose toxicity is determined by increased growth rate, higher final cell density, or reduced loss of viability.

[0190] In one embodiment the host cell is cultured or is capable of being cultured in a medium comprising at least 10 g / L lactose, preferably at least 15 g / L, more preferably 30-90 g / L lactose. In one embodiment the galactoside permease mutant comprises one or more amino acid substitutions that impair proton translocation while retaining substrate translocation. In one embodiment the mutation is located in a residue contributing to proton translocation of LacY.

[0191] In one embodiment wherein overexpression is achieved by a heterologous promoter, a multicopy plasmid, chromosomal integration in multiple copies, or combinations thereof.

[0192] In one embodiment the host cell is selected from Escherichia coli, Corynebacterium, Bacillus, or a yeast species.

[0193] In one embodiment the host cell further comprises a nucleic acid encoding one or more enzymes involved in the biosynthesis or modification of lactose-based products.

[0194] Host cells according to the invention may further comprise nucleic acids encoding one or more enzymes involved in the biosynthesis, extension or modification of lactose-based products. Such enzymes can form part of a metabolic or enzymatic pathway that converts lactose or lactose-derived intermediates into target oligosaccharides, glycans or other lactose-based products.

[0195] The additional nucleic acids may encode, for example, glycosyltransferases, epimerases, fucosyltransferases, sialyltransferases, galactosyltransferases, acetyltransferases or other enzymes that act on lactose or on lactose-containing acceptor substrates. They may also encode auxiliary enzymes providing activated sugar donors (such as GDP-fucose, CMP- sialic acid or UDP-galactose) or enzymes enabling precursor formation, recycling or cofactor regeneration.

[0196] In some embodiments, the host cell comprises a genetic module or pathway assembled from multiple such enzymes, allowing for the stepwise or simultaneous synthesis of complex human milk oligosaccharides or other lactose-derived molecules. The integration of these biosynthetic capabilities with the overexpressed proton-uncoupled permease provides a production system optimized both for substrate uptake and for efficient downstream conversion, enabling robust performance in fermentation processes relying on lactose as a feedstock.

[0197] In one embodiment the lactose-based product is selected from a monosaccharide-, disaccharide-, trisaccharide-, oligosaccharide-, glycan-, glycolipid- or glycopeptide-based product.

[0198] The invention further relates to the use of the disclosed microbial host cells for the production of lactose-based products. By combining permease mutants with reduced or abolished proton-coupled galactoside transport with elevated expression levels, the host cells of the invention are capable of taking up lactose or lactose-derived substrates in a manner that avoids proton-driven toxicity.

[0199] As a result, such host cells can be employed in biotechnological or industrial fermentation processes in which lactose serves as a carbon source, precursor substrate or glycosyl acceptor. These uses include, for example, the recombinant production of human milk oligosaccharides and other lactose-derived molecules, but also extend to processes generating metabolites or energy carriers from lactose-containing feedstocks.

[0200] The use of the overexpressed, proton-uncoupled permease mutants therefore provides improved robustness, sustained growth and enhanced productivity compared with systems relying on the wild-type permease.

[0201] According to a further aspect of the invention the use of a microbial host cell comprising an overexpressed galactoside permease mutant with reduced or abolished proton-coupled galactoside transport for producing a lactose-based product in a fermentation process is provided.

[0202] In one embodiment the fermentation medium comprises at least 10 g / L lactose, preferably at least 15 g / L, more preferably 30-90 g / L lactose.

[0203] Expressions and definitions provided elsewhere herein with regards to other aspects of the present invention equally apply here. Definitions, embodiments, examples etc. herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0204] Methods

[0205] According to a further aspect of the invention a process for producing a lactose-based product is provided, comprising: a) culturing a microbial host cell comprising an overexpressed galactoside permease mutant that has reduced or abolished proton-coupled galactoside transport, in a medium comprising lactose; and b) producing and obtaining the lactose-based product from the culture medium and / or the host cell.

[0206] According to a further aspect of the invention a process for producing a lactose-based product is provided, comprising: a) culturing a microbial host cell according to the invention comprising an overexpressed galactoside permease mutant that has reduced or abolished proton-coupled galactoside transport, in a medium comprising lactose; and b) producing and obtaining the lactose-based product from the culture medium and / or the host cell. The technical effect associated with the use of proton-uncoupled galactoside permease variants becomes particularly pronounced when these variants are expressed at levels exceeding the endogenous wild-type permease while the host cells are cultivated in media comprising elevated lactose concentrations. The simultaneous presence of a strongly expressed, proton-uncoupled permease and optionally a high external lactose concentration results in a markedly increased net uptake of lactose or related galactosides, thereby supplying the cell with an enhanced intracellular substrate pool. Because these permease variants do not induce significant proton influx during substrate translocation, the cellular energy state remains stable even under lactose concentrations at which overexpression of the wild-type permease causes collapse of the proton motive force. The combination of (i) elevated lactose levels and (ii) increased expression of proton-uncoupled permease variants therefore allows higher metabolic fluxes, sustained growth and viability, improved formation of lactose-derived products, and more robust fermentation performance under conditions that are inhibitory for strains expressing the wild-type permease.

[0207] Suitable cultivation media may comprise lactose concentrations that exceed those commonly employed in standard laboratory or industrial fermentation processes. In certain implementations, lactose concentrations of at least 10 g / L or 15 g / L may be used. In other implementations, the process may employ concentrations of 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L or 80 g / L lactose. In further embodiments, the medium may comprise lactose concentrations of 90-100 g / L or even 100-150 g / L, for example 120 g / L or 140 g / L, depending on the reactor configuration, feeding strategy, osmotic tolerance, and metabolic activity of the host cell. A person skilled in the art would be aware that experiments / cultivations carried out in flasks, i.e. in smaller volumes, may or have to be performed with lower lactose concentrations than corresponding cultivations at larger scale. The skilled person would further recognize that the lactose tolerance of the production organism strongly depends on oxygen supply, which is significantly more limited in flasks than in bioreactors, such that higher lactose concentrations can be employed in bioreactors without adversely affecting growth and / or productivity. It must also be borne in mind that in the shaking flask, an optical density of only about 20 max. is generally achieved because ofe limited oxygen conditions, no pH regulation, etc. In the fermentation process, an optical density of 100 and above can be achieved by specifically controlling various parameters. This results in different ratios of cell density to lactose concentration in shaking flasks and fermenters.

[0208] Concentrations above 150 g / L or in the range of 150-250 g / L may be used in specialized processes, including highly concentrated feeds or processes relying on stepwise substrate dilution. These ranges accommodate both moderate and exceptionally high lactose concentrations and allow the system to be adapted to industrial fermentation regimes that rely on high substrate availability, long process times, or high productivity.

[0209] The ability of proton-uncoupled permease variants to maintain transport without affecting the proton motive force enables stable cultivation at elevated lactose concentrations. Suitable processes may therefore include batch, fed-batch or continuous fermentations in which the lactose concentration is at least 10 g / L, preferably at least 15 g / L, more preferably between 20 and 70 g / L. These cultivation conditions permit increased formation of lactose-derived products and stable operation of biosynthetic pathways relying on lactose uptake.

[0210] It was observed that such host cells can be cultured in media containing higher lactose concentrations, including concentrations of at least 10 g / L and, in certain processes, 30-90 g / L lactose. Under these conditions, strains expressing wild-type LacY at high levels showed reduced viability and loss of metabolic activity, while strains expressing proton-uncoupled variants maintained growth and product formation.

[0211] The synergistic effect is optional and not required for the core mechanism.

[0212] As used herein, “elevated lactose concentrations” refers to lactose levels in fermentation or cultivation media that exceed those typically tolerated by host cells expressing the wild-type lactose permease. Elevated concentrations include, for example, at least 10 g / L lactose, preferably at least 15 g / L, more preferably between 30 and 90 g / L, or any concentration at which wild-type cells exhibit lactose toxicity while the mutant permease-expressing cells remain viable.

[0213] Accordingly, in one embodiment the lactose concentration in step (a) is at least 10 g / L, preferably at least 15 g / L, more preferably 30-90 g / L.

[0214] The overexpression of permease mutants with reduced or abolished proton-coupled transport provides a robust and scalable basis for industrial fermentation processes. Elevated permease levels ensure efficient substrate uptake even at high cell densities, while the uncoupled transport mechanism prevents the proton-motive-force collapse and growth arrest typically observed upon overexpression of the wild-type permease.

[0215] In addition, the combination of overexpression with elevated lactose concentrations enables a synergistic improvement in process stability and productivity, because passive or low- coupled lactose transport becomes increasingly effective at higher substrate levels. This allows lactose-rich feedstocks, including industrial waste streams or high-load feeding strategies, to be used without inducing toxicity.

[0216] Together, these features support large-scale, long-duration fermentations, reduce process interruptions, and enable high-titer production of lactose-based products under conditions that were previously incompatible with wild-type permease expression. In one embodiment the process is performed at industrial scale, defined as a fermentation volume of at least 100 liters, preferably at least 300 liters, more preferably 1 ,000 liters or more.

[0217] The term “industrial scale” refers to fermentation or bioprocessing operations performed at production-relevant volumes, typically equal to or exceeding 100 L working volume, including pilot scale (100-300 L), demonstration scale (300-1 ,000 L) and full production scale (>1 ,000 L). The term also encompasses processes configured for commercial manufacture, irrespective of vessel type, provided that the process conditions and control systems correspond to those used in industrial fermentation.

[0218] In one embodiment the process yields a higher amount of a lactose-based product compared to a corresponding process using a host cell expressing the same permease mutant at endogenous levels.

[0219] Host cells according to the invention exhibit increased yield of lactose-based products compared to host cells expressing the same permease mutant at endogenous levels.

[0220] In a further aspect, a process for the production of (human milk) oligosaccharide is provided, comprising:

[0221] (a) culturing (non-human) host cell according to the present invention in the presence of (i) an exogenous carbon source and (ii) a galactoside (sugar), preferably lactose, and under conditions that allow the host cell to produce a (human milk) oligosaccharide, and

[0222] (b) obtaining the (human milk) oligosaccharide from the culture medium and / or the (non-human) host cell.

[0223] In a further aspect, a process for the production of (human milk) oligosaccharide is provided, comprising:

[0224] (a) culturing (non-human) host cell according to the present invention comprising an overexpressed galactoside permease mutant that has reduced or abolished proton-coupled galactoside transport in the presence of (i) an exogenous carbon source and (ii) a galactoside (sugar), preferably lactose, and under conditions that allow the host cell to produce a (human milk) oligosaccharide, and

[0225] (b) obtaining the (human milk) oligosaccharide from the culture medium and / or the (non-human) host cell. For example, a process for the production of (human milk) oligosaccharide according to the present invention does not require a pre-culturing of the (non-human) host cell in a culture medium having reduced content of galactoside (sugar), preferably lactose, in order to prime the (non-human) host cell. For example, the process for the production of (human milk) oligosaccharide according to the present invention does not require a controlled feeding of the galactoside (sugar), preferably lactose, to the culture medium. For example, the process for the production of (human milk) oligosaccharide according to the present invention does not require to monitor / control the content of the galactoside (sugar), preferably lactose, in the culture medium.

[0226] In various embodiments, culturing is for example performed without pre-cultivation the of (non-human) host cells in culture medium having a reduced content of galactoside (sugar), preferably lactose. Further, culturing is for example performed without monitoring I controlling the content of the content of the galactoside (sugar), preferably lactose, during the production process.

[0227] In various embodiments, culturing is for example performed under conditions having a high content of galactoside (sugar), preferably lactose. For example, culturing is performed in culture medium having a concentration of galactoside (sugar), preferably lactose, of at least 50, at least 60, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125 or at least 130 g / L. For example, culturing is performed in culture medium having a concentration of galactoside (sugar), preferably lactose, concentration of about 20 - 130 g / L, 25 - 110 g / L, 30 - 100 g / L, or 35 - 90 g / L.

[0228] The term “exogenous” as used herein refers to any compound, amino acid sequence and / or nucleic acid sequence that has an external origin, (i.e. , not derived from the native host-cell). “Exogenous” as used herein may refer to any external origin, such as any compound, amino acid sequence and / or nucleic acid sequence derived from different species, genus, family, order, class, phylum and / or domain. For example, “exogenous” as used herein refers to any compound, amino acid sequence and / or nucleic acid sequence that is added to a (non- human) host cell. For example, “exogenous” as used herein refers to any compound and / or nucleic acid sequence that is added to the culture medium of the (non-human) host cell prior to and / or during cultivation of the (non-human) host cell. Usually, the compound as such is added to the culture medium. The compound can also be produced by a cell that is present in and / or has been added to the culture medium.

[0229] The term “endogenous” as used herein refers to any compound, amino acid sequence and / or nucleic acid sequence that has a native origin, (i.e., internally derived from the native hostcell). For example, “endogenous” as used herein refers to any compound, amino acid sequence and / or nucleic acid sequence that is native to the (non-human) host cell, i.e., not added to the (non-human) host cell.

[0230] The “medium” or “culture medium” described herein, relates to any solution containing necessary substrates for an organism to grow. These substrates include, but are not limited to, nitrogen sources such as ammonium salts, nitrate salts, yeast extract, pepton, casamino, and / or amino acids, phosphor sources such as but not limited to phosphate salts, elements such as but not limited to copper, cobalt, iron, selenium, iodium, molybdate, magnesium, calcium, potassium, sodium, zinc nickel, manganese, and / or, boric acid and / or vitamins such as but not limited to thiamine, pantothenic acid, and / or niacin and / or an exogenous carbon sources. Exogenous carbon sources comprise, but are not limited to, glycerol, maltose, glucose, fructose, sucrose, fucose, mannose, sialic acid, starch, cellulose, polyols, such as but not limited to mannitol, xylitol, sorbitol, organic acids, such as but not limited to lactate, succinate, acetate, and / or, pentoses, such as but not limited to xylose and arabinose. In preferred embodiments, the exogenous carbon source is glycerol and / or glucose. For example, the exogenous carbon source is glucose.

[0231] The term “obtaining” as used herein relates to isolation or extraction of the synthesized oligosaccharides from the host cells in the culture medium. In general, this is performed by methods well known to the person skilled in the art. Typically, cells are harvested by centrifugation followed by subsequent separation of the supernatant from the cell harvest.

[0232] In various embodiments, the process is a (fed) batch or a continuous process. The term “fed batch process” relates to an operational technique in biotechnological processes, where one or more substrates are fed (supplied) to the bioreactor during cultivation and in which the products remain in the bioreactor until the end of the feeding run. The concentration of fed- substrate is controlled in the culture medium at arbitrarily desired levels.

[0233] The term “continuous process” relates to a flow production method used to produce materials without interruption. Materials, such as dry bulk or fluids are processed continuously in motion, undergoing chemical reactions or subject to mechanical or heat treatment. Continuous processing is contrasted with a fed batch production.

[0234] In various embodiments, the process for the production of HMOs is a process for improvement of the efficacy and reliability of producing HMOs in or by the (non-human) host cell, wherein the host cell is cultivated in the presence of exogenous lactose.

[0235] In preferred embodiments, the process for production of HMOs is a large-scale and / or industrial production process. The terms “large-scale”, “industrial scale”, “industrial” indicate the production of the oligosaccharide using microbial fermentation in a volume of fermentation broth in the full range of 100 L, 500 L, 1000 L, 5000 L, 10,000 L, 50,000 L, 100,000 L or 200,000 L.

[0236] In a preferred embodiment the human milk oligosaccharide and / or oligosaccharide is selected from the group consisting of 2’-fucosyllactose (2’-FL), 3-fucosyllactose (3-FL), 2’, 3- difucosyllactose (DFL), lacto-ZV-triose II, lacto-ZV-tetraose (LNT), lacto-ZV-neo-tetraose (LNnT), lacto-ZV-fucopentaose I (LNFP-I), lacto-N-neofucopentaose I (LNnFP-l), lacto-A / - fucopentaose II (LNFP-II), lacto-ZV-fucopentaose III (LNFP-III), lacto-ZV-fucopentaose V (LNFP-V), lacto- / V-neofucopentaose V (LNnFP-V), lacto- / V-hexaose (LNH), lacto- / V- neohexaose (LNnH), para-lacto-N-hexaose (paraLNH), para-lacto-N-neohexaose (paraLNnH), difucosyl-lacto-N-neohexaose (DF-LNnH), lacto- / V-difucosylhexaose I, lacto- / V- difucosylhexaose II, para-lacto- / V-fucosylhexaose (paraLNH), fucosyl-lacto- / V-sialylpentaose a (F-LST- a), fucosyl-lacto- / V-sialylpentaose b (F-LST-b), fucosyl-lacto- / V-sialylpentaose c (F- LST-c), disialyl-lacto- / V-fucopentaose (DS-LNFP), 3-fucosyl-3'-sialyllactose (3F-3’-SL), 3- fucosyl-6’-sialyllactose (3F-6’-SL), lacto- / V-neodifucohexaose I (LNnDFH I), 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), sialyl-lacto- / V-tetraose a (LST-a), sialyllacto- / V-tetraose b (LST- b), sialyllacto- / V-tetraose c (LST-c), disialyllacto- / V-tetraose (DS-LNT), disialyl-lacto- / V- fucopentaose V (DS-LNFP V), lacto-N-neodifucohexaose I (LNnDFH I), 3’-galactosyllactose (3’-GL), 6’-galactosyllactose (6’-GL), or derivatives thereof.

[0237] The general embodiments “comprising of” or “comprised of” encompass the more specific embodiment “consisting of”. Furthermore, singular and plural forms are not used in a limiting way. As used herein, the singular forms “a”, “an” and “the” designate both the singular and the plural, unless expressly stated to designate the singular only.

[0238] The term “corresponding to the sequence” or “corresponds to the sequence”, as used herein includes the defined sequence of Escherichia coli having the sequence defined of nucleotides of SEQ ID NO: 2. The skilled person will understand that genomic sequences of microbial cells vary and may therefore not be identical with sequences obtained from LacY of E. coli strain BL21 (NCBI accession No: CP010816.1 , locus: AJH09253.1) and E. coli strain K12 (NCBI accession No: CP025268.1 , locus: AUG15133.1) and as shown in SEQ ID NO: 2. However, using sequence alignment, the skilled person would know how to identify the sequence in a specific microbial cell corresponding to the sequence as defined in SEQ ID NO: 1. Such corresponding sequence would have at least 57% identity with the sequence defined in SEQ ID NO: 1 , preferably at least 80% identity with the sequence defined in SEQ ID NO: 1 or is identical with SEQ ID NO: 1. The corresponding sequence may also contain recombinant insertions, which is not to be considered for determining the corresponding sequence. The term “protein” is used interchangeably with “amino acid sequence” or “polypeptide” and refers to polymers of amino acids of any length. These terms also include proteins that are post-translationally modified through reactions that include, but are not limited to, phosphorylation, glycosylation, acetylation, or for eukaryotic host cells also protein processing. Modifications and changes, for example fusions to other proteins, amino acid sequence substitutions, deletions, or insertions, can be made in the structure of a polypeptide while the molecule maintains its biological functional activity. For example, certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence and a protein can be obtained with the same or advantageous properties. The term “polypeptide” typically refers to a sequence of more than 10 amino acids and the term “peptide” means sequences with up to 10 amino acids in length. However, the terms may be used interchangeably.

[0239] The term “genomic DNA”, or “genome” is used interchangeably and refers to the heritable genetic information of a host organism. For eukaryotic host cells, the genomic DNA comprises the DNA of the nucleus (also referred to as chromosomal DNA) but also of other cellular organelles (e.g., mitochondria).

[0240] The term “gene” as used herein refers to a DNA locus of heritable genomic sequence which affects an organism's traits by being expressed as a functional product or by regulation of gene expression. Genes and polynucleotides may include regions that regulate their expression, such as transcription initiation, translation, and transcription termination. Thus, included are regulatory elements such as a promoter.

[0241] The term “enhancement’, “enhance”, “enhanced”, “increase” or “increased”, as used herein, generally means an increase by at least about 21 % as compared to a control cell, for example an increase by at least about 30%, or at least about 40%, or at least about 50%, or at least about 75%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 10-800% as compared to a control cell. As used herein, a “control cell” is for example a wild-type host cell. For example, a “control cell” is a host cell comprising a mutant lacY gene.

[0242] In an industrial setting, the term ‘lactose killing’ typically refers to the phenomenon of growth inhibition or growth arrest of an organism that is grown in an environment in which lactose or galactosidase is not used as the actual carbon source. Typical carbon sources comprise, but are not limited to, glycerol, maltose, glucose, fructose, sucrose, fucose, mannose, sialic acid, starch, cellulose, polyols, such as mannitol, xylitol, sorbitol, organic acids, such as lactate, succinate, acetate, and / or, pentoses, such as xylose and arabinose. Expressions and definitions provided elsewhere herein with regards to other aspects of the present invention equally apply here. Definitions, embodiments, examples etc. herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments listed above can be applied for use in any of the aspects of the invention.

[0243] Further embodiments according to the invention are:

[0244] [1] A mutant of a galactoside permease that possesses lactose transport activity and carries at least one mutation, which provides a reduced or a lack of proton symporter activity, wherein the mutation is in at least at one position which corresponds to a conserved position selected from positions 322, 325, 302, 237, 269, 329, 31 , 64, 68, 115, 126, 144, 147, 240, 319, 334, 346, 348, 358, 35, 76, 131 , 140, 151 , 177, 184, 350, 380, and 400 of SEQ ID NO: 1.

[0245] [2] The mutant galactoside permease of [1], which has at least 57%, preferably 80%, more preferably 85%, even more preferably at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1 .

[0246] [3] The mutant galactoside permease of [1] or [2], wherein the at least one mutation is at a position that is involved in proton translocation of the (wild-type) galactoside permease according to SEQ ID NO: 1.

[0247] [4] The mutant galactoside permease of any one of [1] to [3], wherein the mutant galactoside permease:

[0248] (i) has a modified proton symporter activity, preferably a reduced proton symporter activity, more preferably a lack of proton symporter activity; and / or

[0249] (ii) is not capable of mediating galactoside: proton symport with a 1 :1 stoichiometry; and / or

[0250] (iii) has a reduced proton translocation activity, preferably a lack of proton translocation activity, as compared to the wild-type galactoside permease according to SEQ ID NO: 1.

[0251] [5] The mutant galactoside permease of any one of [1] to [4], wherein the mutant galactoside permease:

[0252] (a) is capable of substrate binding and / or has no mutation in the substrate-binding site; and / or

[0253] (b) has / maintains substrate specificity for galactosides; and / or (c) exhibits I maintains galactoside translocation activity.

[0254] [6] The mutant galactoside permease of [5], wherein the mutant galactoside permease does not require (full) proton translocation for the binding of galactoside(s).

[0255] [7] The mutant galactoside permease of any one of [1] to [6], wherein the at least one mutation is at a position corresponding to any of positions 237 to 334 of SEQ ID NO: 1, preferably at one or more positions corresponding to positions 322, 325, 302, 237, and 269, of the amino acid sequence of SEQ ID NO:1, preferably at the position corresponding to position 322 of the amino acid sequence of SEQ ID NO:1.

[0256] [8] The mutant galactoside permease of any one of [1] to [7], wherein the at least one mutation is at a position in helix IX and / or helix X of the galactoside permease according to amino acid sequence of SEQ ID NO:1; preferably, wherein the at least one mutation is at a position in transmembrane helix IX selected from 322, 325, 334, 319, and / or in transmembrane helix X at 302 of the amino acid sequence of SEQ ID NO: 1.

[0257] [9] The mutant galactoside permease of any one of [1] to [8], wherein the at least one mutation is at least one amino acid substitution.

[0258]

[0010] The mutant galactoside permease of [9], wherein the at least one amino acid substitution comprises a substitution of

[0259] (i) a basic amino acid residue against a basic amino acid residue; and / or

[0260] (ii) a basic amino acid residue against a polar amino acid residue; and / or

[0261] (iii) a basic amino acid residue against a non-polar amino acid residue; and / or

[0262] (iv) a basic amino acid residue against an acidic amino acid residue; and / or

[0263] (v) an acidic amino acid residue against a non-polar amino acid residue; and / or

[0264] (vi) an acidic amino acid residue against a polar amino acid residue; and / or

[0265] (vii) an acidic amino acid residue against an acidic amino acid residue; and / or

[0266] (viii) an acidic amino acid residue against a basic amino acid residue.

[0267]

[0011] The mutant galactoside permease of [9] or

[0010] , wherein:

[0268] (a) the amino acid substitution at the position corresponding to position 322 of the amino acid sequence of SEQ ID NO: 1 is a substitution of histidine against an amino acid residue selected from any one of Y, Q, C, R, K, N, A, M, F, or D, preferably a substitution of histidine against an amino acid residue selected from any one of Q, C, R, K, or N, more preferably a substitution of histidine against an amino acid residue selected from any one of R or K; and / or

[0269] (b) the amino acid substitution at the position corresponding to position 325 of the amino acid sequence of SEQ ID NO: 1 is a substitution of glutamic acid against an amino acid residue selected from any one of C, D, A, Q, K, S, or H, preferably a substitution of glutamic acid against an amino acid residue selected from any one of C, D, or A, more preferably a substitution of glutamic acid against an amino acid residue selected from any one of C or A; and / or

[0270] (c) the amino acid substitution at the position corresponding to position 302 of the amino acid sequence of SEQ ID NO: 1 is a substitution of arginine against an amino acid residue selected from any one of K, H, L, A, S, or C, preferably a substitution of arginine against an amino acid residue selected from any one of K, H, L, A, or S, more preferably a substitution of arginine against an amino acid residue selected from any one of K or H; and / or

[0271] (d) the amino acid substitution at the position corresponding to position 269 of the amino acid sequence of SEQ ID NO: 1 is a substitution of glutamic acid against aspartic acid; and / or

[0272] (e) the amino acid substitution at the position corresponding to position 237 of the amino acid sequence of SEQ ID NO: 1 is a substitution of aspartic acid against cysteine.

[0273]

[0012] The mutant galactoside permease of

[0011] , wherein the mutant galactoside permease comprises at least one of the following amino acid substitutions:

[0274] (i) H322R at the position corresponding to position 322 of the amino acid sequence of SEQ ID NO: 1 ; and / or

[0275] (ii) E325A at the position corresponding to position 325 of the amino acid sequence of SEQ ID NO: 1 ; and / or

[0276] (iii) R302K at the position corresponding to position 302 of the amino acid sequence of SEQ ID NO: 1 ; and / or

[0277] (iv) E269D at the position corresponding to position 269 of the amino acid sequence of SEQ ID NO: 1 ; and / or

[0278] (v) D237C at the position corresponding to position 237 of the amino acid sequence of SEQ ID NO: 1 .

[0013] The mutant galactoside permease of any one of [1] to

[0012] , wherein the mutant galactoside permease is a mutant P-galactoside permease, preferably a mutant lactose permease.

[0279]

[0014] A nucleic acid sequence encoding the mutant galactoside permease of any one of [1] to

[0013] ,

[0280]

[0015] A vector comprising the nucleic acid sequence of

[0014] ,

[0281]

[0016] A (non-human) host cell, preferably a microbial host cell, comprising the nucleic acid sequence of

[0014] , or the vector of

[0015] ,

[0282]

[0017] The (non-human) host cell of

[0016] , which is a bacterial cell or a yeast cell.

[0283]

[0018] The (non-human) host cell of

[0016] or

[0017] , wherein the host cell comprises an exogenous nucleic acid sequence encoding a glycosyltransferase, preferably a fucosyltransferase.

[0284]

[0019] Use of the mutant galactoside permease of any one of [1] to

[0013] , or the nucleic acid sequence of

[0014] , or the vector of

[0015] , or the (non-human) host cell of any one of

[0016] to

[0018] , for the production of (human milk) oligosaccharides.

[0285]

[0020] A process for the production of (human milk) oligosaccharide, comprising:

[0286] (a) culturing (non-human) host cell of any one of

[0016] to

[0018] in the presence of (i) an exogenous carbon source and (ii) a galactoside (sugar), preferably lactose, and under conditions that allow the host cell to produce a (human milk) oligosaccharide, and

[0287] (b) obtaining the (human milk) oligosaccharide from the culture medium and / or the (non-human) host cell.

[0288]

[0021] The process of

[0020] , wherein the process is a (fed) batch or a continuous process.

[0289]

[0022] The process of

[0020] or

[0021] , wherein the oligosaccharide is selected from the group consisting of 2’-fucosyllactose (2’-FL), 3’-fucosyllactose (3’-FL), 2’, 3-difucosyllactose (DFL), lacto-ZV-triose II, lacto-ZV-tetraose (LNT), lacto-ZV-neo- tetraose (LNnT), lacto- / V-fucopentaose I (LNFP-I), lacto-N-neofucopentaose I (LNnFP-l), lacto-A / - fucopentaose II (LNFP-II), lacto-ZV-fucopentaose III (LNFP- III), lacto- / V-fucopentaose V (LNFP-V), lacto-ZV-neofucopentaose V (LNnFP-V), lacto- / V-hexaose (LNH), lacto- / V-neohexaose (LNnH), para-lacto-N-hexaose (paraLNH), para-lacto-N-neohexaose (paraLNnH), difucosyl-lacto-N-neohexaose (DF-LNnH), lacto- / V-difucosylhexaose I, lacto- / V-difucosylhexaose II, para- lacto- / V-fucosylhexaose (paraLNH), fucosyl-lacto- / V-sialylpentaose a (F-LST- a), fucosyl-lacto-ZV-sialylpentaose b (F-LST-b), fucosyl- lacto- / V-sialylpentaose c (F-LST-c), fucosyl-lacto-ZV-sialylpentaose c, disialyl-lacto- / V- fucopentaose, 3-fucosyl-3'-sialyllactose (3F-3’-SL), 3-fucosyl-6’-sialyllactose (3F-6’- SL), lacto- / V-neodifucohexaose I, 3'-sialyllactose (3-SL), 6'-sialyllactose (6-SL), sialyl- lacto- / V-tetraose a (LST-a), sialyllacto-ZV-tetraose b (LST-b), sialyllacto-ZV-tetraose c (LST-c), disialyllacto-ZV-tetraose (DS-LNT), Disialyl-lacto- / V-fucopentaose (DS-LNFP V), lacto-N-neodifucohxaose I (LNnDFH I), 3’-galactosyllactose (3’- GL), 6’- galactosyllactose (6’-GL), or derivatives thereof.

[0290] EXAMPLES

[0291] 1. Material and Methods

[0292] 1.1 Reagents

[0293] Kits:

[0294] Plasmid extraction kit (any supplier, we used E.Z.N.A Plasmid DNA Mini Kit I from Omega Bio-Tek)

[0295] PCR product purification kit (any supplier, we used Thermo Scientific GeneJET PCR Purification Kit from Thermo Fisher Scientific)

[0296] Extraction of DNA fragments from agarose gel (any kit supplier, we used Thermo Scientific GeneJET Gel Extraction Kit from Thermo Fisher Scientific)

[0297] DNA-modifying enzymes: restriction endonucleases, T4 ligase, T4 polynucleotide kinase, Antarctic phosphatase, Taq polymerase, Q5 or any other high-fidelity DNA polymerase (we purchased all these enzymes from New England Biolabs)

[0298] Antibiotics: ampicillin (any supplier, Carl Roth) kanamycin (any supplier, Carl Roth) chloramphenicol (any supplier, PanReac AppliChem)

[0299] Electrophoresis buffer components and reagents:

[0300] Agarose (PeqGOLD Agarose, universal from VWR) TRIS base (Carl Roth)

[0301] EDTA (Na2EDTA, Carl Roth)

[0302] Acetic acid (100%, Rotipuran, Carl Roth) Bromophenol blue (Carl Roth)

[0303] Additional general chemicals and materials:

[0304] Ethanol (Carl Roth)

[0305] Na-acetate (Carl Roth) Primers (Eurofins Genomics) media components (suppliers are specified in the media section)

[0306] Isopropyl-p-D-thiogalactoside (IPTG) (Gerbu)

[0307] 1.2 Molecular biologic methods

[0308] Standard methods, descriptions can be found in any protocol books:

[0309] Plasmid DNA isolation I genomic DNA isolation I DNA fragment isolation were carried out according to the manufacturer’s instructions

[0310] Enzymatic DNA modifications: restriction digest, ligation, phosphorylation and dephosphorylation of DNA ends were all carried out according to the manufacturer’s instructions

[0311] PGR (polymerase chain reaction)

[0312] Agarose gel electrophoresis

[0313] Preparation of electrocompete nt E. coli cells and transformation by electroporation:

[0314] Genome modification in E. coir.

[0315] Deletions and insertions in the E. coli genome were carried out with the A-RED system using plasmids and protocols provided in the publication of Datsenko KA & Wanner BL, Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5. (doi: 10.1073 / pnas.120163297). Plasmids described in the paper were ordered from the Coli Genetic Stock Center at Yale university.

[0316] 1.3 Strains and plasmids

[0317] Strains (Escherichia coli)

[0318] NEB10-beta (New England Biolabs) genotype: A(ara-leu) 7697 araD139 fhuA AlacX74 galK16 galE15 e14- <p80dlacZAM15 recA1 relA 1 endA 1 nupG rpsL (Str^) rph spoT1 A(mrr-hsdRMS-mcrBC)

[0319] BL21 (available from multiple suppliers) genotype: F~ ompT hsdSB(rB“, ms-) gal dem

[0320] Plasmids (i pBluescript II SK (-) (available from multiple suppliers) pKD46, pDK3, pKD4, pKD13, pCP20 (Datsenko KA & Wanner BL 2000) pET21a (available from multiple suppliers) Generating a 2’-FL-producing strain with reduced lactose sensitivity

[0321] A genetically engineered derivative of the E. coli strain BL21 , that was capable of producing the HMO sugar 2’-FL, was prepared. This strain was based on overexpression of the fucose biosynthesis genes manC, manB, gmd and wcag, which were placed under control of the tac promoter. For a-1 ,2-fucosyltransferase activity, futC from Helicobacter pylori was expressed vector-based from pET21a under control of the T5 promotor. To improve precursor supply for 2’-FL production, the lacZYA operon was knocked out, blocking lactose degradation. Native level expression of the lactose transporter, lacY, represents a bottleneck for efficient 2’-FL production, therefore / acYwas also overexpressed. By native level of lacY expression lactose toxicity occurs only under specific conditions that don’t usually occur during a standard fermentation, but overexpression of the lacY gene augmented lactose toxicity to a level where it became quite pronounced and strongly influenced growth and cellular health and by causing lysis in a significant proportion of the cells, it also negatively influenced further downstream processing of the material. In order to reduce lactose toxicity in the lacY overexpressing strain, the above-mentioned point mutations H322R or E325A, or the L293R mutation for reference (from patent WO 2022 / 136568), were introduced into the lacY expression cassette first in a plasmid by PCR-based site-directed mutagenesis, using synthetic oligonucleotide mismatch primers. From this plasmid template the expression cassettes were amplified by PCR using primers that carried additional 50 base long homology regions to drive genome integration into the E. coli genome by A-RED-mediated homologous recombination to selected target sites, according to the publication Datsenko and Wanner 2000. Genome integration in individual clones was confirmed by PCR analysis of the integration site flanks as well as by sequencing the genomic insert. All mutant lacY overexpression cassettes were integrated to the same genomic locus, therefore the only difference between all lines are the single respective point mutations in their lacY expression cassettes.

[0322] 1.4 Media

[0323] Synthetic medium for shake flask cultures

[0324] NH4)2-H -citrate 1.00 g / L

[0325] (NH4)2-SO42.68 g / L

[0326] NaH2PO4xH2O 8.00 g / L

[0327] K2HPO429.20 g / L

[0328] Na2SO4x10H2O 4.54 g / L

[0329] NH4CI 0.50 g / L

[0330] LB powder Lennox 2g / L (all from Carl Roth) Glycerol 50 g / L (with adjustment to glycerol concentration of the stock:

[0331] 58 g / L) (Glycerol 86.5% from Chemsolute / T.H. Geyer)

[0332] Autoclave then add filter sterilized supplements:

[0333] MgSO40.49 g / L (1 : 1000 from stock) Carl Roth

[0334] Thiamine 0.01 g / L (1 :1000 from stock) Carl Roth

[0335] Trace element solution 3.0 mL / L (3:1000 from stock) (see below)

[0336] Trace element solution (TES)

[0337] CaCI2-2H2O 0.50 g / L Carl Roth

[0338] CUSO4-5H2O 0.16 g / L Carl Roth

[0339] Ammonium iron (III) citrate (18%) 1.8 g / LCarl Roth

[0340] MnSO4-H2O 0.10 g / L Carl Roth

[0341] ZnSO4-7H2O 0.18 g / L Carl Roth

[0342] Lactose was added to the cells upon induction of expression at different concentrations up to 20 g / L from a stock solution of 250 g / L that was sterilized by filtration. (Lactose from Carl Roth)

[0343] 1.5 Cultivation conditions

[0344] 2’-FL production strains containing the above described / acY-mutant expression cassettes were inoculated in 3 replicates into 25 mL culture media in 250 mL-sized shake flasks to a starting ODeoo of 0.1 and grown in growth chambers (Labwit ZWYR-D2402) at 30°C with 250 rpm agitation. Once cell density reached an approximate ODeoo of 0.6 lactose was added to the concentration of 12.5 g / L (36.5 mM) and protein expression was induced with 0.5 mM IPTG. Afterwards the cultures were grown for 72 hours without adding any further materials. Samples were taken at decided time points for following growth by OD600 and for HPLC quantification of the product 2’-FL and residual glycerol and lactose.

[0345] 1.6 Sampling methodology

[0346] A sample of 2 mL was taken at any decided time point from the cell suspension and centrifuged at 16,000 g at room temperature. Next the sample was heated to 95 °C for 5 min to precipitate proteins from the solution. All precipitates were then separated by centrifugation at 16,000 g for 5 min at room temperature. To 500 pL of the supernatant 500 pL deionized water was added as well as an additional 500 pL acetonitrile. Afterwards the sample was observed for 30-60 minutes to control if additional precipitation occurs. The sample is then clarified by 5 min centrifugation at 16,000 g at room temperature. Thereafter the sample was filtered using a 2 mL syringe connected to a 0.2 pm syringe filter (Altmann Analytik, PVDF, 13 mm, 0.2 pm, product no.: AG5191-5924) into the HPLC sample vials.

[0347] 1.7 Analytical methods

[0348] 2’-FL, rest lactose and rest glycerol were quantified by HPLC measurement. We used Agilent 1260 Infinity II MCT, vial sampler, quat pump and 1290 Infinity II ELSD detector. For the separation we used Infinitylab Poroshell 120 HILIC-Z column.

[0349] 1.8 Protein Expression and SDS-PAGE Analysis

[0350] For the analysis of protein biosynthesis, 2'-FL producing E. coli strains overexpressing the genes for either LacY or the LacY-H322R variant under control of the Tac promoter were used. Precultures were grown overnight in 10 mL LB medium at 30 °C and 250 rpm with appropriate antibiotics. The following day, multiple cultures were inoculated in 10 mL LB tubes at a starting optical density (OD600) of 0.3. When cultures reached an OD600 of 1.0, protein expression and 2’-FL production were induced in LacY cells by addition of 0.5 mM IPTG (control without IPTG addition). Lactose was added at final concentrations between 0 and 5 g / L (control: IPTG, but no lactose addition). In another experiment, cultures overexpressing the genes for either LacY or LacY-H322R under control of the Tac promotor were induced with 0.5 mM IPTG and 12 g / L lactose or were left untreated as non-induced controls. After induction, cells were further cultured for 24 h at 30 °C and 250 rpm.

[0351] After 24 h of cultivation, GD600 values were measured and adjusted to 5.0 to normalize biomass between samples. 100 pl of cells were harvested by centrifugation at 12,000 rpm for 1 min and resuspended in 1 * Laemmli SDS loading buffer. Samples were boiled for 5 min at 95 °C and stored at -20 °C. For each condition, 15-20 pL of the protein extract were loaded onto SDS-PAGE gels. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970). Gels consisted of a 5% stacking gel (0.125 M Tris-HCI, pH 6.8, 0.1% SDS) and a 12% resolving gel (0.375 M Tris-HCI, pH 8.8, 0.1% SDS). Electrophoresis was performed at constant voltage until the dye front reached the lower edge of the gel. Following electrophoresis, the gels were stained overnight with Coomassie solution (40% methanol, 10% acetic acid, 0.16% Coomassie Brilliant Blue R-250) and subsequently destained in 20% acetic acid until a clear background and distinct protein bands were obtained.

[0352] 1.9 Fermentation Within the fermentation process, a lactose solution is used as lactose source. A BG - medium is used as medium for microbial growth.

[0353] The fermentation process usually exhibits two phases:

[0354] 1) Growth phase: Batch fermentation until glycerol is consumed (after approx. 21 h); 2) Production phase: Fed-Batch fermentation with Lactose supplementation and feeding

[0355] (glycerol and supplements) after induction; possibly 2ndand 3rdlactose supplementation.

[0356] An overview of the materials, equipment and specific parameters used in the fermentation process is provided below: The Batch BG Medium features the following composition:

[0357]

[0358] The Trace Element Solution (TES) features the following composition:

[0359] The feeding solution features the following composition: Lactose solution for feeding purposes

[0360] For batch supplementation or feeding of lactose to the fomentation medium, lactose solution with a lactose concentration of 250 (fee) or 360 g / L (batch) is used. The lactose solution is sterile filtrated using microfiltration with a pore size of < 0.2 pm. The resulting lactose solution composition is as follows:

[0361] The pH value of the fermentation broth is actively adjusted by adding base. The following table provides an overview of further fermentation parameters: Preculture 50 mL LB Medium + Ampicillin are inoculated in a 500 mL unbaffled Erlenmeyer flask at 30 °C and 250 rpm for 8 h or until an ODeoo of approximately 3.0 is reached.

[0362] Feeding strategy

[0363] After reaching an ODeoo of 20-30 (after approx. 21 h fermentation time), the lactose solution is added to the (fed-)batch medium until reaching a final concentration of approx. 65 g / L. The production phase is initiated by induction with IPTG.

[0364] The glycerol feeding process is started and adapted to glycerol consumption of the culture and oxygen saturation throughout the fermentation.

[0365] When the lactose concentration in the fermentation broth reaches approx. 30 g / L, additional acid lactose solution is added to the fermentation broth up to a lactose concentration of approx. 65 g / L, and the fermentation process is continued.

[0366] The fermentation process demonstrates stable and efficient bioconversion, with key parameters — such as pH, temperature, and substrate conversion rates — maintained within optimal ranges. Using batch-wise feeding of a lactose solution, final product concentrations of 75 g / L for the strain carrying lacY H322R and 18,5 g / L for the strain with the native lacY were achieved within approx. 65 hours. Comparative evaluation of production strains with and without the lacY H322R mutation clearly shows the performance advantage conferred by this variant, reflected in increased growth and productivity of the production strain, as illustrated in Figure 10.

[0367] 1.10 Downstream Processing (DSP)

[0368] After fermentation, the fermentation broth contains 30 - 50% biomass. In a representative manufacturing example, a lactase-digested fermentation broth containing 2’-fucosyllactose was subjected to the following downstream purification sequence (Figure 11).

[0369] 1. Flocculation and Solid-Liquid Separation

[0370] The fermentation broth was first treated by adding an aqueous ferric sulfate solution to induce flocculation of suspended biomass and colloidal matter. The flocculated mixture was subsequently centrifuged, generating a biomass pellet and a 2-FL-containing supernatant. The pellet was optionally washed, and the corresponding wash supernatants were combined with the initial supernatant to obtain a pooled supernatant fraction.

[0371] 2. Microfiltration (MF) - Prefiltration Step

[0372] The pooled supernatant was then subjected to a prefiltration step using a microfiltration membrane. In a representative configuration, a membrane with an approximate molecular weight cut-off of 800 kDa was employed. This step served to remove residual fine particulates, colloids, and remaining high-molecular-weight impurities. The resulting clarified MF permeate was directed to the subsequent ultrafiltration step.

[0373] 3. Ultrafiltration (UF)

[0374] The microfiltered permeate was processed by ultrafiltration diafiltration using membranes with a nominal molecular weight cut-offs of 10 and 3 kDa. Retentates were optionally washed through multiple diavolumes (DV’s), and the corresponding permeates were collected. The final UF permeate was pooled (“UF permeate pool”) for further refinement.

[0375] 4. Nanofiltration (NF)

[0376] The UF permeate pool was subjected to nanofiltration diafiltration using a membrane with a molecular weight cutoff in the range of 150-300 Da. The NF permeate was removed, while the NF retentate and permeates from diavolume steps were combined to generate the final NF retentate.

[0377] 5. Decolorization

[0378] To remove colour bodies and related impurities, the NF retentate was contacted with activated carbon and subsequently filtered to remove the carbon. In alternative process variants, the activated-carbon step may be omitted.

[0379] 6. Ion-Exchange Purification

[0380] The decolorized filtrate was sequentially passed through an anion- or cation-exchange resin to remove charged contaminants. The purified stream was then sterile-filtered using a 0.2-pm membrane.

[0381] 7. Final Formulation

[0382] The sterile-filtered 2’-FL-containing solution was dried by spray-drying to produce a stable powder product.

[0383] Figure 12 shows a chromatogram of carbohydrate composition in the produced 2'-FL powder, measured by HILIC-Z HPLC with an ELS detector.

Claims

CLAIMS1. A microbial host cell comprising a nucleic acid encoding a galactoside permease mutant having reduced or abolished proton-coupled galactoside transport, wherein the galactoside permease mutant is expressed in the host cell at a level exceeding the endogenous expression level of the corresponding wild-type galactoside permease.

2. A nucleic acid comprising: (a) a nucleotide sequence encoding a galactoside permease mutant having reduced or abolished proton-coupled galactoside transport; and (b) one or more regulatory or structural elements suitable for achieving overexpression of the mutant permease, selected from promoter sequences, ribosome binding sites, untranslated regions, transcriptional or translational enhancers, gene-dosage-increasing elements, or combinations thereof.

3. A vector comprising the nucleic acid of claim 2, optionally together with elements enabling multi-copy replication, chromosomal integration, or controlled induction of expression.

4. Use of a microbial host cell, a nucleic acid or a vector according to claim 1 , 2 or 3 for producing a lactose-based product in a fermentation process.

5. A process for producing a lactose-based product, comprising: (a) culturing a microbial host cell according to claim 1 in a medium comprising lactose; and (b) producing or recovering the lactose-based product from the culture medium and / or the host cell.

6. The microbial host cell according to claim 1 , wherein overexpression is achieved by one or more genetic or regulatory elements selected from promoter sequences, ribosome binding sites, transcriptional or translational enhancers, gene-dosage-increasing elements, derepression strategies, or combinations thereof.

7. The microbial host cell according to any one of claims 1 or 6, wherein the overexpression is achieved by a heterologous or synthetic promoter selected from PT7, Ptac, Ptrc, Pt5, PBAD, PlacUV5, Ptet, PJ23119, Pveg, P43 or functional variants thereof.

8. The microbial host cell according to any one of claims 1—7, wherein overexpression is achieved by a multicopy plasmid or by multiple chromosomal insertions.

9. The microbial host cell according to any one of claims 1-8, wherein overexpression is supported by an engineered ribosome binding site.

10. The microbial host cell according to any one of claims 1-9, wherein lacl or another repressor of the lac operon is deleted or inactivated.

11. The microbial host cell according to any one of claims 1-10, wherein the galactoside permease mutant retains substrate binding and galactoside transport activity in the absence of proton translocation.

12. The microbial host cell according to any one of claims 1-11 , wherein the mutant comprises one or more amino acid substitutions that impair proton translocation while retaining substrate translocation.

13. The microbial host cell according to any one of claims 1-12, wherein the mutation is located in a residue contributing to proton translocation of LacY.

14. The microbial host cell according to any one of claims 1-13, wherein the host cell is selected from Escherichia coli, Corynebacterium, Bacillus, or a yeast species.

15. The microbial host cell according to any one of claims 1-14, further comprising a nucleic acid encoding one or more enzymes involved in the biosynthesis or modification of lactose- based products.

16. The microbial host cell according to any one of claims 1-15, wherein the host cell is cultured or is capable of being cultured in a medium comprising at least 10 g / L lactose, preferably at least 15 g / L, more preferably 20-60 g / L lactose.

17. The use according to claim 4, wherein the fermentation medium comprises at least 10 g / L lactose, preferably at least 15 g / L, more preferably 20-60 g / L lactose.

18. The process according to claim 5, wherein the lactose concentration in step (a) is at least 10 g / L, preferably at least 15 g / L, more preferably 20-60 g / L.

19. The microbial host cell according to any one of claims 1-18, characterized in that, when cultured in a medium comprising at least 10 g / L lactose, the overexpression of the galactoside permease mutant provides a synergistic improvement in cell growth or product titer relative to the same host cell expressing the mutant at endogenous levels.

20. Use according to claim 4, wherein culturing the host cell in a medium comprising at least 10 g / L lactose together with overexpression of the galactoside permease mutant provides a synergistic improvement in growth or product titer.

21. A process according to claim 5, wherein the combination of overexpression of the galactoside permease mutant and a lactose concentration of at least 10 g / L provides a synergistic improvement in cell growth, yield or product titer relative to culturing a host cell expressing the mutant at endogenous levels.

22. A process according to any one of claims 5, 18 or 21 , wherein the process yields a higher amount of a lactose-based product compared to a corresponding process using a host cell expressing the same permease mutant at endogenous levels.

23. The process according to any one of claims 5, 18, 21 or 22, wherein the process is performed at industrial scale, defined as a working fermentation volume of at least 100 L, preferably at least 300 L, more preferably 1 ,000 L or more.

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