SBLE mutants with altered activity and SBLE homologues

Abstract

The present invention relates to mutants of Starmerella bombicola SBLE enzyme that have modulated interesterification and / or transesterification activity compared to S. bombicola SBLE enzyme and homologues of S. bombicola SBLE enzyme such as S. kuoi LE enzyme, S. batistae LE enzyme and S. riodocensis LE enzyme, with interesterification and transesterification activity on bola glycolipids. The invention also provides genetically modified yeast strains comprising a mutant LE enzyme or a homologue of S. bombicola SBLE enzyme of the invention. The invention further provides uses and methods of the mutant LE enzymes, the homologues of S. bombicola SBLE enzyme and the modified yeast strains of the invention for the production of acidic glycolipids, lactonic glycolipids and / or glycolipid oligomers / polymers / esters. The invention further provides a three dimensional structure of S. bombicola SBLE enzyme and uses thereof for designing mutants with modulated hydrolytic and / or transesterification activity.

Description

[0001] SBLE mutants with altered activity and SBLE homologues

[0002] Technical field of the invention

[0003] The present invention broadly relates to the field of glycolipid biosynthesis. In particular, the invention provides mutants and homologues of lactone esterase (LE) enzymes, in particular the Starmerella bombicola lactone esterase (SBLE) enzyme, and uses thereof for the production of acidic glycolipids, lactonic glycolipids and / or glycolipid oligomers / polymers / esters. The invention further provides modified yeast strains comprising a mutant or homologue LE enzyme of the invention and uses thereof for the production of acidic glycolipids, lactonic glycolipids and / or glycolipid oligomers / polymers / esters. The invention also provides three dimensional structures of Starmerella bombicola SBLE enzyme and uses thereof for identifying or designing mutant LE enzymes with modulated enzymatic activity.

[0004] Background of the invention

[0005] The yeast S. bombicola is, among others, well-known in the art for its high production of sophorolipids (SLs). SLs consist of the disaccharide sophorose linked to a hydroxylated fatty acid. The wild type S. bombicola strain produces a mixture of acidic and lactonic SLs, which can be non-, mono- or diacetylated and mainly contain a C18:l fatty acid. The SL biosynthetic pathway was previously elucidated (Dierickx et.al., 2022).

[0006] All but one gene involved in the SL biosynthesis are found in one large subtelomeric gene cluster. The current understanding of the SL biosynthetic pathway involves five steps with mainly di-acetylated lactonic SLs as the final product (Dierickx et.al., 2022). The first step consists of (sub)terminal hydroxylation of a fatty acid by the action of a cytochrome P450 monooxygenase (Cyp52Ml) (Van Bogaert et al., 2009a). Subsequent glycosylation of the hydroxy fatty acid involves two glucosyltransferases. The first one (UgtAl) (Saerens et al., 2011a) is responsible for the transfer of a glucose molecule from UDP-glucose to the hydroxylated fatty acid yielding a glucolipid and UDP while the second one (UgtBl) (Saerens et al., 2011c) specifically transfers a second glucose molecule from UDP-glucose to the formed glucolipid (and not to the hydroxylated fatty acid). The SLs are subsequently acetylated by the action of an acetyltransferase (Atl) (Saerens et al., 2011b) and can be further lactonised by the action of a secreted lactone esterase (SBLE) (Ciesielska et al. 2014, WO 2013 / 092421) after secretion by a specific SL transporter (Mdr). In contrast to all the other genes involved in SL biosynthesis byS. bombicola, the sble gene is not located in the biosynthetic gene cluster and seems to be differently regulated. This last step is also (mainly) performed in the extracellular space as the SBLE protein possesses a secretion signal and is actively secreted and abundantly found in the extracellular space (Ciesielska et al. 2014). An SL transporter (Mdr) is also encoded within the biosynthetic gene cluster. Deletion of this gene results in a decrease of at least 90% of SL production (Van Bogaert et al., 2013).

[0007] A single S. bombicola atl deletion strain S. bombicola Aatl has been described by Saerens et al., (2011b) and was described to produce non-acetylated acidic and non-acetylated lactonic sophorolipids (SLs). Non-acetylated lactonic SLs were reported to be the most predominant structures in the mixture, in addition to minor amounts of open-ring / acidic SLs. A single S. bombicola sble deletion strain S. bombicola Asble was later described by Ciesielska et al. (2014) and reported to exclusively produce acidic SLs. The authors also suggested that the SLs are secreted by the yeast in the acidic form and are subsequently lactonized by the extracellularly secreted SBLE enzyme. Roelants et al. (2016) also reported production experiments with this strain, which was again reported to exclusively produce acidic SLs in a mixture of acetylated and non- acetylated congeners. Ciesielska et al. (2016) later investigated the mode of action of the SBLE enzyme through in vitro enzyme assays and reported it to catalyze the intramolecular esterification (lactonization) of acetylated acidic sophorolipids in an aqueous environment into acetylated lactonic sophorolipids. No lactonization activity was observed at all by the authors for non-acetylated acidic SLs, so acetylation was deemed to be essential for this esterification reaction to occur by SBLE. The acetylated acidic SLs used in the in vitro SBLE enzyme assays described in the abovementioned work were obtained from the Asble S. bombicola strain (Ciesielska et al. 2014) and HPLC and LC-MS analyses suggested that the extracted SLs used for these in vitro enzyme assays consisted of a mixture composed of 50 % non-acetylated acidic SLs, 17 % mono-acetylated acidic SLs, 31 % di-acetylated acidic SLs and 2 % contaminants as described by the authors in the art.

[0008] Subsequently, it was found that a combination of these two deletions in one strain (i.e. S. bombicola AatlAsble) unexpectedly resulted in the biosynthesis of bolaform / bola amphiphilic glycolipids of which the general formula is shown in Figure 1A and IB. More specifically, the authors reported the biosynthesis of non-acetylated bola sophorolipids (Figure 1C) (Van Bogaert et al., 2016 and WO2015 / 028278). Based on the previous findings and the proposed biosynthetic pathway, this strain should logically produce non-acetylated acidic sophorolipids. Surprisingly, in addition to these anticipated non-acetylated acidic SLs, also bola sophorolipids (74 % of the produced SLs) were obtained. These bola sophorolipids contain an additional sophorose molecule linked to the carboxyl function of the acidic sophorolipids as confirmed by LC-MS and NMR analysis. Bola sophorolipid biosynthesis was proven to be attributed to the promiscuous activity of both UDP-glucosyltransferases UgtAl and UgtBl from the sophorolipid biosynthetic pathway, found to also display activity towards the carboxyl group of non-acetylated intermediates. The absence of acetyl groups was hypothesized to trigger formation of bola glycolipid compounds starting from acidic sophorolipids as they were found to be produced by the AatlAsble strain and not by the Asble strain. The authors also hinted this as the potential reason why these bola sophorolipids are only detected in marginal amounts (< 0.1 % of produced sophorolipids) in cultures of the wild type Starmerella bombicola strain (Price at al., 2012) as the presence of the Atl enzyme in the wild type strain is giving rise to acetylated sophorolipids, which would thus hamper the formation of bolaform sophorolipids. LC-MS analysis of the produced sophorolipid mixture by the AatlAsble strain revealed the production of non-acetylated bola sophorolipids with variation in the incorporated fatty acid chain length and position of the hydroxyl group on the fatty acid (as is also the case for wild type sophorolipids). Fractionation on the sophorolipid mixture produced by this new strain was performed and NMR analysis confirmed the structure of non-acetylated bola sophorolipids as shown in Figure 1. Because this was an unexpected finding, the authors again investigated the glycolipid mixtures produced by both the single deletion strains described above. The Asble S. bombicola strain was confirmed to only produce acidic sophorolipids by the authors (Van Bogaert et al., 2016), while the Aatl S. bombicola strain was, upon reanalysis using an adapted glycolipid extraction protocol for more hydrophilic compounds, found to also produce non-acetylated bola sophorolipids in addition to the previously reported non-acetylated acidic and lactonic sophorolipids produced by this strain (Van Bogaert et al., 2016). The authors thus suggested again -as mentioned above- that the absence of acetylation seems to be a key factor triggering bola sophorolipid synthesis and suggested that this effect is enhanced by the absence of lactonic sophorolipid forms (where the carboxyl group is not freely available anymore) as better production efficiencies of bola sophorolipids seem to be obtained with the double deletion strain. Non-acetylated glycolipid compounds were hypothesized to allow a certain conformational orientation in the UgtAl and UgtBl enzymes, which would not be possible for the acetylated equivalents, thus resulting in further glycosylation of non-acetylated acidic sophorolipids resulting in non-acetylated bola sophorolipids.

[0009] It is however currently unknown that the actual substrate of SBLE are the bola sophorolipids, and not, as described in the art, non-, mono-, or di-acetylated acidic sophorolipids. Moreover, it is completely unknown that the SBLE enzyme is capable to perform a transesterification reaction, more specifically it is completely unknown that the SBLE enzyme has transesterification activity on bola sophorolipids / bola glucolipids giving rise to lactonic sophorolipids / glucolipids, respectively. It is also unknown that the SBLE enzyme is capable to perform a hydrolytic reaction, more specifically it is completely unknown that the SBLE enzyme has hydrolytic activity on bola sophorolipids / glucolipids giving rise to acidic sophorolipid / glucolipids, respectively. It is also unknown that mutations of SBLE can give rise to improved conversion of bola sophorolipids into a more uniform product, and, that yeast strains comprising a gene encoding for such mutant SBLE enzymes produce certain sophorolipids more uniformly. It is also unknown that SBLE enzymes of sophorolipid production yeasts are capable of performing an intermolecular transesterification reaction, more specifically it is completely unknown that so-called SBLE enzymes in sophorolipid producing yeast strains have intermolecular transesterification activity on bola sophorolipids and other esters, in particular acyl esters such as MAG, DAG and TAG esters.

[0010] It is also unknown that yeast strains which comprise a gene encoding for a mutant SBLE enzyme and one or more dysfunctional acetyltransferase enzymes (Atl, At2 or At3), encoded in the SL biosynthetic gene cluster, and responsible for sophorolipid acetylation, are capable to produce non-acetylated lactonic sophorolipids more uniformly. Moreover, it is currently unknown that a modified yeast strain which comprises a gene encoding for the Starmerella batistae SBLE enzyme produces (acetylated) acidic sophorolipids. Finally, the invention discloses the currently unknown crystal structure of the Starmerella bombicola SBLE enzyme.

[0011] Brief description of figures

[0012] Figure 1: A and B) Bolaform / bola amphiphilic glycolipids Ri = H or CO-CH3; R? = H or CO-CH3; R3 = H or CO-CH3; R4 — H or CO-CH3; Rs = an unsubstituted or hydroxy-substituted, unbranched, optionally one to three double or triple bonds containing, divalent organic moiety comprising 6 to 32 carbon atoms, R6= H, CH 3 or an unsubstituted or hydroxy-substituted, unbranched, optionally one to three double or triple bonds containing, organic moiety comprising 2 to 10 carbon atoms; n = 1 or 0; m = 1 or 0. C) Non-acetylated bola sophorolipids as reported to be produced by the S. bombicola AatlAsble strain and also by the S. bombicola Aatl strain. Only non-acetylated variants were reported to be produced by these strains by Van Bogaert et al., 2016.

[0013] Figure 2: Adapted sophorolipid biosynthesis pathway in S. bombicola. (1) Cyp52Ml, (2) UgtAl, (3) UgtBl, (4) Atl, (5) SBLE. UDP: uridine diphosphate, CoA: coenzyme A, (n)Ac: non-acetylated or acetylated.

[0014] Figure 3: A) Knock-out cassette for the replacement of the SBLE gene in S. bombicola with a URA3 auxotrophic marker (Example 2). upSBLE: upstream homologous region of the S. bombicola SBLE gene, tTK: tTK terminator, URA3: coding sequence of the S. bombicola URA3 gene, pURA: promoter of the S. bombicola URA3 gene, downSBLE: downstream homologous region of the S. bombicola SBLE gene. B) Knock-out cassette for the replacement of the SBLE gene in S. bombicola with a hygromycine resistance marker (Example 2). upSBLE: upstream homologous region of the S. bombicola SBLE gene, pGAPD787: promoter of the S. bombicola pGAPD gene, Hygro: coding sequence of the hygromycine resistance gene HygB, tTK: tTK terminator, downSBLE: downstream homologous region of the S. bombicola SBLE gene. C) Knock-in cassette for the insertion of the SBLE gene, or a gene or cDNA encoding an SLBE mutant or a homolog of S. bombicola SBLE enzyme as described herein, in S. bombicola with a ura3 marker at the URA3 locus. upURA: upstream homologous region of the S. bombicola LIRAS gene, pURA: promoter of the S. bombicola URA3 gene, URA3: coding sequence of the S. bombicola URA3 gene, tTK: tTK terminator, pGAPD787: promoter of the S. bombicola pGAPD gene, LE: coding sequence of the S. bombicola SBLE or a mutant or homolog thereof as described herein, tGAL: tGAL terminator, tURA: terminator of the S. bombicola URA3 gene, dURA3': downstream homologous region of of the S. bombicola URA3 gene. In Example 3 the coding sequence for S. bombicola SBLE enzyme was used. For Examples 4 to 16, the knock-in cassette was similar, whereby the coding sequence of a mutant or homolog of S. bombicola SBLE enzyme as described in the corresponding Example. D) Knock-out cassette for the replacement of the ATI gene in S. bombicola with a ura3 auxotrophic marker (Example 12). upATl: upstream homologous region of the S. bombicola ATI gene, tTK: tTK terminator, URA3: coding sequence for the S. bombicola URA3, pURA: promoter of the S. bombicola URA3 gene, downATl: downstream homologous region of the S. bombicola ATI gene. E) Knock-out cassette for the replacement of the AT2 gene in S. bombicola with a ura3 auxotrophic marker (Example 12). upAT2: upstream homologous region of the S. bombicola AT2 gene, tTK: tTK terminator, URA3: coding sequence for the S. bombicola URA3, pURA: promoter of the S. bombicola URA3 gene, downAT2: downstream homologous region of the S. bombicola AT2 gene. F) Knock-out cassette for the replacement of the AT3 gene in S. bombicola with a ura3 auxotrophic marker (Example 12). upAT3: : upstream homologous region of the S. bombicola AT3 gene, tTK: tTK terminator, URA3: coding sequence for the S. bombicola URA3, pURA: promoter of the S. bombicola URA3 gene, downAT3: downstream homologous region of the S. bombicola ATS gene. G) Cassette for the knock-in of the SBLE gene or a gene or cDNA encoding a mutant or homolog of S. bombicola SBLE in S. bombicola with a hygromycin antibiotic marker (Example 13, Example 14). upSBLE: upstream homologous region of the S. bombicola SBLE gene, pGKI: promoter of the S. bombicola GKI gene, LE: coding sequence of either the S. bombicola SBLE or a gene or cDNA encoding a mutant or homolog of S. bombicola SBLE, tScCYCl: ScCYCl terminator, pGAPD787: promoter of the S. bombicola pGAPD gene, Hygro: coding sequence of the hygromycine resistance gene HygB, , tTK: tTK terminator downSBLE: downstream homologous region of the S. bombicola SBLE gene. H) Knock-out cassette for the replacement of the AT3 gene in S. bombicola with a nourseothricin antibiotic marker (Example 5). upAT3: upstream homologous region of the S. bombicola AT3 gene, pGAPD787: promoter of the S. bombicola GAPD gene, Nat: coding sequence of the nourseothricin resistance gene NAT1, tTK: tTK terminator, downAT3: downstream homologous region of the S. bombicola AT3 gene. I) Knock-out cassette for the knock-out of the CYP52M1 gene in S. bombicola with a hygromycin antibiotic marker (Example 8). upCYP52Ml: upstream homologous region of the S. bombicola CYP52M1 gene, pGAPD787: promoter of the S. bombicola GAPD gene, Hygro: coding sequence of the hygromycine resistance gene HygB, tTK: tTK terminator downCYP52Ml: downstream homologous region of the S. bombicola CYP52M1 gene. J) Cassette for the knock-in of the Starmerella kuoi CYP52M1 gene in S. bombicola with a URA3 auxotrophic marker (Example 8). upCYP52Ml: upstream homologous region of the S. bombicola CYP52M1 gene, tGAL: tGAL terminator, pURA: promoter of the S. bombicola URA3 gene, URA3: coding sequence of the S. bombicola URA3 gene, tTK: tTK terminator, downCYP52Ml: downstream homologous region of the S. bombicola CYP52M1 gene.

[0015] Figure 4: UPLC-CAD chromatograms of end samples of shake flask experiments for A) the Aura3::0 ASble:: pGAPD_hygro_Ttk strain (ASBLE::hygro strain, negative control, Example 2), B) the Aura3::0 Asble:: tTK_Ura3_pUra3 strain( ASBLE::ura3 strain, negative control, Example 2), C) the Aura3:: pURA_URA3_tTK pGAPD_SBLE_tGAL Asble::pGAPD_hygro_Ttk strain (SBLE strain, positive control, Example 3) and D)-H) the SBLE mutant strains as indicated (Example 4) fed with 20 g / L oleic acid. Important peak groups are highlighted in the figures: 1: (acetylated) bola sophorolipids, 2: (acetylated) acidic sophorolipids, 3: (acetylated) lactonic sophorolipids.

[0016] Figure 5: UPLC-CAD chromatograms of end samples of shake flask experiments for the A) AuraS Aatl Aat2 Aat3::nat Asble::hygro strain (negative control, Example 5) compared to the B) Aura3::sble Aatl Aat2 Aat3::nat Asble::hygro strain (positive control, Example 6) and C-F) the SBLE mutant strains (Example 7) fed with 20 g / L oleic acid. Important peak groups are highlighted in the figures. 1: (nonacetylated) bola sophorolipids. 2: (non-acetylated) acidic sophorolipids. 3: (non-acetylated) lactonic sophorolipids.

[0017] Figure 6: UPLC-CAD chromatograms of end samples of shake flask experiments for the A) AsbleAura3::sble_kuoi strain (LE_kuoi, Example 8), B) the Aura3:: pURA_URA3_tTK pGAPD_StarmerellabatistaeLE_tGAL Asble::pGAPD_hygro_Ttk (LE_batistae, Example 9), C) the AsbleAura3::sble_riodocensis strain (LE_riodocensis, Example 10), D) the AsbleAura3::CALA strain (trCALA, Example 11), E) the Aura3::0 Aatl::0 Aat2::0 Aat3::pURA_URA3_tTK A sble::pGKI_StarmerellakuoiLE_tScCYCl_ pGAPD_hygro_Ttk strain (LE_kuoi_ATKO, Example 13), F) the Aura3::pURA_URA3_tTK _pGAPD_Starmerella kuoi LE_tGAL Aatl::0 Aat2::0 strain (LE_kuoi_AT3, Example 15), G) the Aura3::0 Acyp52ml::SkCYP52Ml_tGAL_pURA_URA3_tTK Asble::pGAPD_hygro_Ttk_pGKI_StarmerellakuoiSBLE_tScCYCl strain (KuoiLE_SkCYP, Example 8) and H) the Aura3::0 Acyp52ml::SkCYP52Ml_tGAL_pURA_URA3_tTK Asble::pGAPD_hygro_Ttk_pGKI_ StarmerellabatistaeSBLE_tScCYCl strain (BatistaeLE_SkCYP, Example 9), fed with 20 g / L oleic acid. Important peak groups are highlighted in the figures. Figure 7: SDS-PAGE analysis of the deglycosylation of rSBLE (produced by Pichia pastoris) with Endo H, reactions were incubated at room temperature at different time points. The rSBLE_DGH is heterogeneous and shows two bands with quite close MW between 37kDa and 50kDa, which was indicated by black arrow. M: molecular weight marker in kDa.

[0018] Figure 8: Structure of monomeric SBLE in C2221 space group. In panels a), b) and d), the Ca of SBLE is colored from blue at the N-terminus through the rainbow to red at the C-terminus. a): A trace of Ca is shown in divergent stereo view. N- and C-terminus were labeled, b): A cartoon view is presented from the same position, and the N- and C-terminus and the disulfide bridge of the crystal structure of SBLE are also labeled as in a), c): a surface model of SBLE is shown. The region in the white dashed box is the location of the active site pocket of this enzyme, d): A topology diagram for SBLE, a-helices and p- sheets are shown as cylinders and arrows, respectively. The N- and C-terminus are also labeled, and the active-site residues are marked.

[0019] Figure 9: Structure of SBLE in the P21212 space group with two molecules in non-crystallographic symmetry, molecule A and molecule B. The partial electron density of bola SS, a ligand mixed with protein for co-crystallization, is also observed at a similar position on the side of both molecules. Bola SS is displayed as stick and colored as yellow, a): Cartoon representation of the two molecules of SBLE. b): A Ca trace of two molecules is shown in divergent stereo view, c): Overlay of the two molecules of SBLE in the P21212 space group. A Ca trace (c)-l) and a cartoon view (c)-2) are shown, N- and C- terminus are labeled, d): A sphere mode of SBLE in the P21212 space group, it shows the significant and extensive contact of two molecules at the N-terminal interface.

[0020] Figure 10: Multi-angle laser light scattering (SEC-MALLS) analysis of rSBLE_DGH and the complex of this protein with the mixing of 3x molar excess bola SSs. Differential refractive index (d Rl, left vertical axis) is plotted against the determined molecular mass (right vertical axis). rSBLE_DGH (blue trace, concentration of 1.4mg-mL-l) and rSBLE_DGH complexed with 3x molar bola SSs (green trace, concentration of 3.9mg-mL-l). The molecular mass reported indicates the average molecular mass ± s.d. across the elution peak.

[0021] Figure 11. a): A view of the electron density of the ligand tetra-acetylated bola SS (C18:l, co) on the sides of molecule A and B in the P21212 space group is presented in the PH2FOFCWT map. b) A view of the electron density of the ligand tetra-acetylated bola SS (C18:l, co) on the sides of molecule A and B in the P21212 space group is presented in polder map with contour level of +3.0. c) View of the binding of bola SS with molecule A and B of SBLE in a surface mode. The region in the black dashed boxes in a zoomed-in view represents the binding between bola SS and the two molecules of SBLE. Residues in the binding cavity of two molecules are labeled and shown as sticks. The pale dashed lines in the zoomed-in view indicate the hydrogen bonding interactions between atoms.

[0022] Figure 12. Structure of the model after docking the substrate non-acetylated bola SL (C18:l, co-1) in the active site of monomeric SBLE based on the crystal structure of the C2221 space group. Some of the residues that contact the substrate are shown. The contact between the catalytic serine and the substrate is indicated with dashes and their distance is indicated. Important peak groups are highlighted in the figures. 1: (non-)acetylated bola sophorolipids. 2: (non-)acetylated acidic sophorolipids. 3: (non-)acetylated) lactonic sophorolipids.

[0023] Figure 13. HPLC-CAD chromatograms of reaction products from assays using purified enzyme A. WT using non-acetylated substrates, B. mutant S125P P129G using non-acetylated substrates. C. WT using acetylated substrates, D. mutant S125P P129G using acetylated substrates. Important peak groups are highlighted in the figures: 1: (non-)acetylated bola sophorolipids, 2: (non-)acetylated acidic sophorolipids, 3: (non-)acetylated) lactonic sophorolipids.

[0024] Figure 14. Detail of the HPLC-UV chromatograms of samples from (a) the negative control of reaction at 12h for the synthesis of SL oligomers / esters using bola SLs (code: INV-113) and rapeseed oil but without the addition of rSBLE, and the reaction at 12h (b) and 24h (c) after adding rSble. The two diacetylated lactonic SLs (Cig:i) used as references were indicated in both panel b and c, the two SL-di- glycerides that were produced after 12h of reaction but disappeared after 24h were indicated in panel b and the synthesized SL-triglyceride was indicated in panel b and c.

[0025] Figure 15. TLC analysis of end samples of shake flask experiments for A) the Asble Aura3::sble_kuoi strain (E5, LE_kuoi, Example 8), B) the Aura3::pURA_URA3_tTK pGAPD_StarmerellabatistaeLE_tGAL Asble::pGAPD_hygro_Ttk (F5, LE_batistae, Example 9) and C) the reference strain Aura3::pURA_URA3_tTK_pGAPD_SBLE_tGAL Asble::pGAPD_hygro_Ttk strain (SBLE strain, Example 3). The region where oligomeric / polymeric glycolipids and / or glycolipid esters are visible is indicated in a black rectangle.

[0026] Summary of the invention

[0027] The present invention is based on the finding that a known enzyme denominated as 'Starmerella bombicola lactone esterase (SBLE)' is able to catalyze intramolecular and intramolecular transesterification and hydrolysis reactions on bola amphiphilic glycolipid esters.

[0028] The inventors have found that mutations in the substrate binding pocket of the Starmerella bombicola SBLE enzyme allow for modulating the hydrolytic and / or transesterification activity of the SBLE enzyme. In an aspect, the invention provides a mutant of a lactone esterase (LE) enzyme, said mutant comprising at least one mutation in a substrate binding pocket of the LE enzyme, preferably wherein said substrate binding pocket consists of regions corresponding to the loop forming region T103-A113, the helix forming region S125-T131, the loop forming region S132-A136, the loop forming region D151- G162, the loop forming region G192-S194, the helix forming region G195-D208, the loop forming region G223-A228, the helix forming region W229-I235, the loop forming region D236-L242, the helix forming region K243-G256, the loop forming region A282-S298, the loop forming region G344-K349, the loop forming region P374-H378 and the helix forming region D379-D398 of SEQ ID NO:2.

[0029] In particular embodiments, the mutant LE enzyme comprises at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E; N126G; S125P and P129G; G244S, G244T, G244C, G244N, G244Q, G244W or G244Y; P290S, P290T, P290C, P290N, P290Q, P290F or P290Y; L128T or L128Y; Q288Y or Q288V; N287F; I248A or I248G; and L348D.

[0030] The inventors found that specific mutants of the SBLE enzyme are improved in their ability to perform a hydrolysis reaction compared to the SBLE enzyme, optionally while maintaining or decreasing transesterification activity compared to the SBLE enzyme, while other specific mutants of the SBLE enzyme are improved in their ability to perform a transesterification reaction compared to the SBLE enzyme, optionally while maintaining or decreasing the hydrolysis reaction. Yet other mutants of the SBLE enzyme were found to have improved hydrolytic activity and improved transesterification activity compared to the SBLE enzyme.

[0031] Specific mutants of the SBLE enzyme for which hydrolysis is improved, while transesterification is maintained or is decreased result in a more uniform product formation i.e. predominant formation of hydrolysis products with minor to no ester product formation. As an example: the predominant conversion (hydrolysis) of bola sophorolipids into acidic sophorolipids and sophorose, with minor to no ester formation (e.g. formation of lactonic sophorolipids and / or sophorolipid oligomers / polymers / esters). The invention further provides a method to more uniformly produce acidic sophorolipids using a modified yeast strain comprising a gene encoding such mutant SBLE enzyme. In such modified yeast strains, more uniform production of acidic sophorolipids is obtained as hydrolysis reactions are improved, while the parallel 'contaminating' formation of esters, such as lactonic sophorolipids or sophorolipid oligomers / polymers / esters is decreased or maintained. The invention further discloses a method to produce non-acetylated acidic sophorolipids via additionally rendering the acetyltransferase enzymes Atl, At2 and / or At3 dysfunctional in the modified yeast strains. In addition, the invention further discloses a method to more uniformly produce nonacetylated acidic sophorolipids using a modified yeast strain comprising a gene encoding for said mutant SBLE enzyme and further comprising a dysfunctional Atl, At2 and / or At3 enzyme. Specific mutants of SBLE, for which the transesterification reaction is improved, while hydrolysis is maintained or decreased, allow for a more uniform product formation of predominantly transesterification products with minor to no hydrolysis products. As an example, improved conversion of (non-acetylated) bola sophorolipids into (non-acetylated) lactonic sophorolipids and sophorose with limited or low formation of acidic sophorolipids is obtained using such SBLE mutant. The invention further provides a method to more uniformly produce (non-acetylated) lactonic sophorolipids using a modified yeast strain comprising a gene encoding for said mutant SBLE enzyme. In such modified yeast strain, more uniform production of ester products such as lactonic sophorolipids is obtained as transesterification reactions are improved compared, while the parallel 'contaminating' formation of hydrolysis products, such as acidic sophorolipids, is decreased or maintained. The invention further discloses a method to produce non-acetylated lactonic sophorolipids via additionally rendering the acetyltransferase enzymes Atl, At2 and / or At3 dysfunctional in the modified yeast strains. In addition, the invention further provides a method to more uniformly produce non-acetylated lactonic sophorolipids using a modified yeast strain comprising a gene encoding for said mutant SBLE enzyme and further comprising said dysfunctional Atl, At2 and / or At3.

[0032] Further aspects and embodiments are directed to:

[0033] A genetically modified yeast strain comprising a gene encoding a mutant LE enzyme of the invention, wherein said yeast strain is a naturally sophorolipid producing yeast strain.

[0034] Use of a mutant LE enzyme of the invention for the production of acidic glycolipids. In preferred embodiments, said mutant LE enzyme has improved hydrolytic activity compared to the non-mutated LE enzyme.

[0035] A method for the production of acidic glycolipids, said method comprising contacting bola glycolipids with a mutant LE enzyme of the invention. In preferred embodiments, said mutant LE enzyme has improved hydrolytic activity compared to the non-mutated LE enzyme.

[0036] Use of a modified yeast strain comprising a gene encoding a mutant LE enzyme of the invention to produce acidic glycolipids. In preferred embodiments, said mutant LE enzyme has improved hydrolytic activity compared to the non-mutated LE enzyme.

[0037] A method to produce acidic glycolipids, said method comprising culturing a modified yeast strain comprising a gene encoding a mutant LE enzyme of the invention to produce a broth comprising acidic glycolipids. In preferred embodiments, said mutant LE enzyme has improved hydrolytic activity compared to the non-mutated LE enzyme. Use of a mutant LE enzyme of the invention for the production of lactonic glycolipids. In preferred embodiments, said mutant LE enzyme has improved transesterification activity compared to the nonmutated LE enzyme.

[0038] A method for the production of lactonic glycolipids, said method comprising contacting bola glycolipids with a mutant LE enzyme of the invention. In preferred embodiments, said mutant LE enzyme has improved transesterification activity compared to the non-mutated LE enzyme.

[0039] Use of a modified yeast strain comprising a gene encoding a mutant LE enzyme of the invention to produce lactonic glycolipids. In preferred embodiments, said mutant LE enzyme has improved transesterification activity compared to the non-mutated LE enzyme.

[0040] A method to produce lactonic glycolipids, said method comprising culturing a modified yeast strain comprising a gene encoding a mutant LE enzyme of the invention to produce a broth comprising lactonic glycolipids. In preferred embodiments, said mutant LE enzyme has improved transesterification activity compared to the non-mutated LE enzyme.

[0041] The invention further provides functional homologues of Starmerella bombicola SBLE enzyme, which have improved hydrolytic activity and / or improved intermolecular transesterification activity compared to the Starmerella bombicola SBLE enzyme. In embodiments, said homologue of Starmerella bombicola SBLE enzyme is a Starmerella kuoi LE enzyme, a Starmerella batistae LE enzyme or a Starmerella riodocensis LE enzyme.

[0042] The homologues of Starmerella bombicola SBLE enzyme of the invention were found to have improved hydrolytic activity compared to Starmerella bombicola SBLE enzyme, and are particularly useful for the production of acidic sophorolipids, in particular acetylated acidic sophorolipids, more particularly mono-acetylated or di-acetylated acidic sophorolipids. A further aspect is directed to use of a homologue of Starmerella bombicola SBLE enzyme of the invention for the production of acidic glycolipids. A related aspect is directed to a method for the production of acidic glycolipids, said method comprising contacting bola glycolipids with a homologue of Starmerella bombicola SBLE enzyme of the invention. Moreover, the invention provides a modified yeast strain comprising a homologue of Starmerella bombicola SBLE enzyme of the invention, wherein said yeast strain is a naturally sophorolipid producing yeast strain, and use of said modified yeast strain to produce acidic glycolipids. A related aspect is directed to a method for the production of acidic glycolipids, said method comprising culturing a modified yeast strain comprising a homologue of Starmerella bombicola SBLE enzyme of the invention to produce a broth comprising acidic sophorolipids. Via additionally rendering the acetyltransferase enzymes Atl, At2 and / or At3 dysfunctional or non- functional in these modified yeast strains, they can be used for the production of non-acetylated acidic sophorolipids.

[0043] The inventors have also shown that homologues of Starmerella bombicola SBLE enzyme of the invention show intermolecular transesterification activity between bola amphiphilic glycolipids and the alcohol group(s) in mono acyl glycerol esters and / or di acyl glycerol esters, resulting in the production of glycolipid oligomers / polymers / esters and saccharides. The invention further provides for use of a homologue of Starmerella bombicola SBLE enzyme of the invention and a modified yeast strain comprising a homologue of Starmerella bombicola SBLE enzyme of the invention for the production of glycolipid oligomers / polymers / esters. In related aspect, the invention provides a method for the production of glycolipid oligomers / polymers / esters, said method comprising contacting bola glycolipids and another ester, preferably an acyl ester, more preferably an acyl glycerol ester such as a mono acyl glycerol ester, a di acyl glycerol ester and / or a tri acyl glycerol ester, preferably a mono acyl glycerol ester and / or a di acyl glycerol ester, with a homologue of Starmerella bombicola SBLE enzyme of the invention. Another aspect is directed to a method for the production of glycolipid oligomers / polymers / esters, said method comprising culturing a modified yeast strain comprising a homologue of Starmerella bombicola SBLE enzyme of the invention to produce a broth comprising glycolipid oligomers / polymers / esters.

[0044] The present inventors have also determined a three-dimensional structure of S. bombicola SBLE enzyme. In particular, the inventors have produced crystals of S. bombicola SBLE enzyme of suitable quality for performing X-ray diffraction analysis following co-crystallization of S. bombicola SBLE enzyme and a bola sophoroside, an inert analog of a bola sophorolipid. Crystal structures were obtained following X-ray diffraction analysis. The present findings provide structural insights into the S. bombicola SBLE enzyme and its interaction with its substrate, enabling screening methods to identify or design mutants of S. bombicola SBLE enzyme with modulated enzymatic activity, in particular mutants with a modulated hydrolytic and / or transesterification activity.

[0045] Further aspects and embodiments are directed to:

[0046] A three-dimensional structure of Starmerella bombicola SBLE enzyme characterized by the atomic coordinates as defined in any one of Tables 9 to 11 or a subset thereof.

[0047] Use of a three dimensional structure represented by a set of atomic coordinates presented in any one of Tables 9 to 11 or a subset thereof, or atomic coordinates which deviate from those in any one of Tables 9 to 11 or a subset thereof by a root mean square deviation (RMSD) of residue over protein backbone atoms by no more than 3 A, for identifying or designing mutants of an SBLE enzyme with modulated hydrolytic and / or transesterification activity. A computer-implemented method for identifying or designing mutants of an SBLE enzyme with modulated hydrolytic and / or transesterification activity, said method comprising:

[0048] - employing a three dimensional structure of the SBLE enzyme represented by a set of atomic coordinates presented in any one of Tables 9 to 11 or a subset thereof, or atomic coordinates which deviate from those in any one of Tables 9 to 11 or a subset thereof by a root mean square deviation (RMSD) of residue over protein backbone atoms by no more than 3 A, for fitting or docking a three dimensional structure or atomic coordinates of a model substrate, in particular a tetra acetylated bolaglycolipid, more particularly a tetra acetylated bolasophorolipid, in said three dimensional structure of the SBLE enzyme to generate a three-dimensional computational representation of an enzyme-substrate complex; and

[0049] - identifying amino acid residues in the enzyme of said three-dimensional representation of the enzyme-substrate complex with unfavourable interactions with the substrate as candidate positions for enzyme mutagenesis; and

[0050] - identifying mutations of said candidate positions that form more favourable interactions with said substrate.

[0051] The invention further provides a computer program product comprising instructions which when the program is executed by a computer, cause the computer to carry out the steps of the aforementioned computer-implemented method of the invention; and a computer-readable storage medium, comprising a data storage material encoded with computer readable data wherein said data comprises the information needed for and configured to perform the aforementioned computer-implemented method of the invention.

[0052] In preferred embodiments of the aforementioned uses and methods, the subset is the subset of atomic coordinates of Table 11 defining the loops and helices of the S. bombicola SBLE enzyme that bind the substrate, in particular the subset of atomic coordinates of Table 11 defining the loops and helices corresponding to the loop forming region T103-A113, the helix forming region S125-T131, the loop forming region S132-A136, the loop forming region D151-G162, the loop forming region G192-S194, the helix forming region G195-D208, the loop forming region G223-A228, the helix forming region W229-I235, the loop forming region D236-L242, the helix forming region K243-G256, the loop forming region A282-S298, the loop forming region G344-K349, the loop forming region P374-H378 and the helix forming region D379-D398 of the amino acid sequence of SEQ. ID NO:2.

[0053] A method for producing mutants of an SBLE enzyme with modulated hydrolysis and / or transesterification activity, , said method comprising:

[0054] - identifying or designing mutants of the SBLE enzyme by the computer-implemented method of the invention, - generating mutants of the SBLE enzyme that comprise one or more of the identified mutations, and

[0055] - optionally testing the generated mutants of the SBLE enzyme for modulated hydrolysis and / or transesterification activity to identity mutants of the SBLE enzyme with modulated hydrolysis and / or transesterification activity.

[0056] A method for determining a crystal structure of an SBLE enzyme, said method comprising:

[0057] (a) co-crystallizing the SBLE enzyme and a substrate analog, in particular a bolaform glycoside, more particularly a bolaform sophoroside, to form a crystalline complex,

[0058] (b) subjecting the crystalline complex to X-ray diffraction, and

[0059] (c) determining the crystal structure based on said X-ray diffraction data.

[0060] A crystal of a S. bombicola SBLE enzyme comprising a structure characterized by the atomic coordinates as presented in any one of Tables 9-11 or a subset thereof.

[0061] Description of the invention

[0062] As used herein, the singular forms "a", "an", and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0063] The terms "comprising", "comprises" and "comprised of" as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass "consisting of" and "consisting essentially of", which enjoy well-established meanings in patent terminology.

[0064] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression "from... to..." or the expression "between... and..." or another expression.

[0065] The terms "about" or "approximately" as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers is itself also specifically, and preferably, disclosed.

[0066] Whereas the terms "one or more" or "at least one", such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g. any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0067] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0068] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0069] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e. also in the context of other aspects or embodiments of the invention, unless otherwise defined. For example, embodiments directed to products are also applicable to corresponding features of methods and uses.

[0070] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0071] Reference throughout this specification to "one embodiment", "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, alternative combinations of claimed embodiments are encompassed, as would be understood by those in the art.

[0072] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to standard handbooks as well as to the general background art referred to herein and to the further references cited therein.

[0073] The present invention builds on several recent findings considering the sophorolipid biosynthesis pathway in S. bombicola. We hereby disclose that the actual substrate of the SBLE enzyme are bola sophorolipids, and not, as described in the art, non-, mono-, or di-acetylated acidic sophorolipids. We also disclose that the SBLE enzyme is capable to perform a transesterification reaction, more specifically that the SBLE enzyme has transesterification activity on bola sophorolipids / bola glucolipids giving rise to lactonic sophorolipids / glucolipids, respectively. Moreover, we also disclose that the SBLE enzyme has hydrolytic activity on bola sophorolipids / glucolipids giving rise to acidic sophorolipid / glucolipids, respectively. Finally, we further disclose that yeast strains which comprise one or more dysfunctional acetyltransferase enzymes (Atl, At2 or At3) responsible for sophorolipid acetylation, are capable to produce non-acetylated lactonic sophorolipids.

[0074] Results obtained in recently performed biosurfactant production experiments with three S. bombicola strains developed and described in the past, i.e. Asble (Ciesielska et al. 2014, Roelants et al. 2016 and WO2013 / 092421), Aatl Asble (Van Bogaert et al., 2016 and WO2015 / 028278) and Aatl (Saerens et al. (2011b)) were unexpected and in contradiction with the art. First, masses corresponding to (acetylated) bola sophorolipids up to an acetylation degree of 4 were detected in data from mass spectrometric analysis of samples from the experiment with the Asble strain, in contrast to previous observations and reports where only acidic SLs were described to be produced. Second, acetylated (bola) sophorolipids up to an acetylation degree of 2 (mainly acetylation degree of 1) were detected in the samples from the experiments with the Aatl Asble strain and the Aatl strain, in contrast to previous observations and reports in the art. The Asble strain had been described to exclusively produce (acetylated) acidic SLs. The Aatl Asble strain and Aatl had been described not to produce any acetylated (bola) SLs, due to mutation of the atl gene, which has been described as the (only) enzyme responsible for acetylation of sophorolipids. The production of acetylated bola sophorolipids has thus not been described.

[0075] As the results were unexpected and in contradiction with the art, new S. bombicola strains (Asble_full, Aatl_f ul I and Asble_f ul I Aatl_f ull ) were generated by deleting the full ORFs / coding sequences and the glycolipid production profile of the new strains was evaluated after performing production experiments. Production samples were subjected to UHPLC-HRMS analysis. Indeed, also the Asble_full strain produces (acetylated) bola SLs. Moreover, fully acetylated bola SLs (tetra acetylated C18:l bola SLs) are produced in quite abundant amounts. This was completely unexpected as Van Bogaert et al. (2016) stated that the absence of acetyl groups triggers the formation of bola sophorolipids starting from acidic sophorolipids. For the katl_full strain, again acetylated glycolipid compounds were detected. However, lower acetylation degrees as described above for the Asble strain were observed. The production spectrum of the Aatl strain consists mainly of non-acetylated bola SL, monoacetylated bola SL, non-acetylated triglucolipids, non-acetylated acidic SL, non-acetylated glucolipid and non-acetylated and mono-acetylated lactonic SL. As the acetyltransferase from the SL biosynthetic gene cluster was fully removed in the novel Aatl_full strain and acetylated compounds were still observed, this indicates the activity of an unknown acetyltransferase active on SLs. Lastly, the Asble_full Aatl_full predominantly produces non-acetylated bola SLs, mono-acetylated bola SLs, mono-acetylated acidic SLs and non-acetylated glucolipids (GLs). In general, it is observed that lactonic SLs are only observed when the SBLE gene was not deleted. Furthermore, the Aatl strain produces predominantly bola SLs and lactonic SLs with lower acetylation degrees, while the Asble strain produces mainly bola SLs with higher acetylation degrees. The AatlAsble strain predominantly produces bola SLs with lower acetylation degrees. This indicates that the SBLE enzyme has a preference to perform a transesterification reaction on acetylated bola amphiphilic glycolipids. These findings are in contrast with what is described in the art (Van Bogaert et al. (2016), Van Renthergem et al. (2019), WO 2013 / 092421 and WO / 2021 / 229017); namely bola sophorolipids can only be produced as completely non-acetylated molecules, because deletion of the atl gene was described to be required to generate bola sophorolipids. The Atl enzyme was moreover described to be the only enzyme acetylating (bola) glycolipids in S. bombicola (Saerens et al. (2011b), Van Bogaert et al (2016)), so the described bola sophorolipids in the art did not contain any acetylgroups. The production spectrum of a Asble Augtbl strain consists mainly of non-acetylated bola glucolipids, non-acetylated bola acidic glucolipids and mono-acetylated bola acidic glucolipids. This finding is also in contrast to what is described in the art, namely that a Asble Augtbl Aatl strain would be required to produce bola glucolipids and that these bola glucolipids would be expected to be completely non-acetylated. Analysis of a Asble Augtbl Aatl however revealed that also acetylated bola glucolipids were detected.

[0076] The unexpected finding of bola SLs in ksble strains raised questions about the actual substrates of SBLE. Therefore, activity tests were performed with the recombinant SBLE (rSBLE) described in De Waele et al. (2018). For production, a HAC1 co-expressing strain of P. pastoris (syn. Komagataella phaffii) NRRL-Y-11430 transformed with the pPICZaB_rSBLEopt construct described in De Waele et al. (2018) was utilized. For purification of rSBLE, a two-step purification strategy was utilized by following the protocol described in De Waele et al. (2018). To determine the catalytic property of rSBLE, an HPLC- based activity assay was followed as described by De Waele et al. (2018) with some adaptations. Samples of acidic SLs from the catalytic assay were analyzed by HPLC on an Ettan™ LC system using a ZORBAX Eclipse Plus C18 Rapid Resolution column. Samples of bola SLs from the catalytic assay were analyzed by another HPLC analytic method on the same LC system using a Brownlee Spheri-5 RP-18 cartridge column. Compounds were identified via Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Additionally, to verify the conversion of bola SLs to lactonic SLs by rSBLE, LC-MS analysis was performed.

[0077] Activity tests were performed on different SL samples. First, the activity of SBLE, using the recombinantly produced enzyme rSBLE, towards acidic SLs was tested. The samples used as substrate were (1) di- acetylated acidic SLs (C18:l) co (2) di-acetylated acidic SLs (C18:l) mix co and co-1 and (3) nonacetylated acidic SLs (C18:l) mix co and co-1. The results showed that no corresponding lactonic SLs were detected after reaction of any of the three acidic SLs. As this was a surprising result, the reaction time and concentration of enzyme were increased to investigate whether this was due to a low E:S ratio or slower reaction. Again, no lactonic SLs were detected after the reaction indicating that the reaction time and enzyme concentration are not the crucial parameters in view of lactonization of acidic SLs. Additionally, all the samples mentioned previously were analyzed using MALDI-TOF MS to analyze the possible formation of polymers of di-acetylated acidic SLs. However, inspection of the mass spectra at higher m / Z rate did not provide any indication that polymerization occurred in the reaction. rSBLE was thus surprisingly not able to convert the three provided acidic SLs into lactonic SL.

[0078] We returned to an activity test using the original crude SL mixture that was used by Ciesielska et al. (2016) and was used during further investigations to test activity of rSBLE. This mixture was obtained from the ksble strain described by Ciesielska et al. (2014) and not purified / extracted. This crude SLs mixture was always expected to only contain acidic SLs based on the data in the art. However, as described above, the ksble strain was surprisingly found to produce a mixture of bola SLs and acidic SLs, both in acetylated and non-acetylated form. Indeed, when comparing the HPLC chromatogram of this old SL mixture with the new samples of acidic SLs with high homogeneity, it became clear that additional compounds were indeed present in this old crude sample that was used for initial SBLE activity assays. MALDI-TOF MS was performed to investigate the identity of these compounds. They were identified as mono-acetylated bola SLs and di-acetylated bola SLs. And indeed, exactly these compounds disappeared after incubation of this sample with rSBLE giving rise to the formation of di- acetylated lactonic SLs, whereas peaks corresponding to acidic SLs hardly diminished in intensity.

[0079] Based on the observation that upon incubating SBLE with a mixture of bola SLs and acidic SLs, lactonized SLs were obtained, in contrast to the assays with acidic SLs alone, it was argued that in fact bola amphiphilic glycolipids unexpectedly might be the actual substrate of the SBLE enzyme, which then would catalyze a transesterification reaction rather than a lactonizing esterification reaction. In order to confirm this, two types of bola SLs were tested of which the main compounds were: (1) tri-acetylated bola SLs and di-acetylated bola SLs in an approximately 1:1 ratio (code: INV- 113) and (2) non-acetylated (and minor amounts of mono-acetylated) bola SLs (code: INV_22). For acetylated bola SLs, our data showed that four lactonic SLs products were formed, in which diacetylated lactonic SLs were the most abundant product. The peaks corresponding to the main bola SLs disappeared or decreased significantly after the reaction. This was also the case for the sample of mainly non acetylated bola SLs present in the other sample of bola SLs (INV_22). The results of the activity assays thus further confirm that SBLE converts acetylated bola SLs (acetylation degree mono-, di-, tri- and tetra-) to form the corresponding lactonic SLs through a transesterification reaction. All peaks that decreased in intensity after reaction with rSBLE corresponded to bola SLs, whereas peaks corresponding to acidic SLs remained unchanged after reaction. For the sample of bola SLs containing mainly non-and mono- acetylated bola SLs, only minor amounts of lactonic SLs were produced by rSBLE, although the bola SLs decreased significantly. Herein, non-acetylated acidic SLs showed a significant accumulation, demonstrating hydrolysis of the ester bond of (non- acetylated) bola SLs, indicating that the enzyme has an additional hydrolysis activity for substrates with low to no acetylation degree.

[0080] A depiction of the adapted sophorolipid biosynthesis based on these surprising findings is shown in Figure 2. The biosynthesis of lactonic SLs by S. bombicola as it has been always described and depicted in the art, i.e. an internal esterification reaction of acidic SLs giving rise to lactonic SLs, is thus to be revisited. It was thus found that the biosynthesis of lactonic SLs is instead the result of a transesterification reaction of bola sophorolipids into lactonic sophorolipids. In Figure 2, the formation and conversion of tetra-acetylated bola SLs is shown, but as shown above, also bola SLs with a lower acetylation degree are converted into non-, mono- and di- acetylated lactonic and / or non-, mono- and di- acetylated acidic SLs.

[0081] From the observations explained above, it can also be concluded that the Atl enzyme is not the only enzyme acetylating glycolipids in S. bombicola. BlastP analysis was performed using the acetyltransferase protein sequence from the Atl enzyme against all translated ORFs from the S. bombicola genome. 71 hits were obtained, of which 2 were selected for further investigation. Several S. bombicola strains were created with combinations of Aatl, Aat2, Aat3 and Asble. The newly developed strains were evaluated for their production characteristics in shake flask (SF) experiments. Production samples were subjected to UHPLC-HRMS analysis. The SL production spectrum of the AatlAat2Aat3 strain mainly consists of non-acetylated bola SLs and non-acetylated lactonic SLs, but also nonacetylated acidic SLs. The AatlAat2Aat3Asble strain mainly produces non-acetylated bola sophorolipids. No acetylated sophorolipids / glycolipids or other acetylated (bola) amphiphilic glycolipids were produced anymore. In these analyses, the clear appearance of non-acetylated acidic SLs for AatlAat2Aat3 compared minor amounts in the strain AatlAat2Aat3Asble is in line with the in vitro data described above: SBLE will preferably perform a transesterification reaction for acetylated bola amphiphilic compounds, while a hydrolysis reaction exists alongside the transesterification reaction for non-acetylated bola amphiphilic compounds. This was unexpected as it was assumed that the acetyltransferase gene present in the SL biosynthetic gene cluster (atl) was solely responsible for acetylation of glycolipids in S. bombicola. We have found and shown that other previously unknown genes / enzymes (at2 / At2 and at3 / At3) present in the S. bombicola genome also have this acetylation activity on (bola) sophorolipids and glucolipids, but to a lower extent and with different specificity.

[0082] The present invention is based on the finding that mutations in the substrate binding pocket of an LE enzyme allow to modulate the activity of the LE enzyme, in particular the hydrolytic and / or transesterification activity of the LE enzyme.

[0083] In an aspect, the invention provides a mutant of an LE enzyme comprising at least one mutation in a substrate binding pocket of the LE enzyme. Such mutant has modulated enzymatic activity, in particular modulated hydrolytic and / or transesterification activity, compared to the non-mutated LE enzyme. In embodiments, the substrate binding pocket is defined by regions corresponding to the loop forming region T103-A113, the helix forming region S125-T131, the loop forming region S132-A136, the loop forming region D151-G162, the loop forming region G192-S194, the helix forming region G195-D208, the loop forming region G223-A228, the helix forming region W229-I235, the loop forming region D236-L242, the helix forming region K243-G256, the loop forming region A282-S298, the loop forming region G344-K349, the loop forming region P374-H378 and the helix forming region D379-D398 of the amino acid sequence of SEQ ID NO:2. In further embodiments, the mutant LE enzyme comprises at least one mutation in at least one region corresponding to a region selected from the group consisting of: the helix forming region S125-T131, the helix forming region K243-G256, the loop forming region A282-S298, the loop forming region G344-K349 and the helix forming region D379-D398 of the amino acid sequence of SEQ ID NO:2.

[0084] Certain mutants of the LE enzyme are capable of or show improved hydrolysis activity. An example of such improved hydrolysis is the improved conversion of bola amphiphilic glycolipids into acidic glycolipids and saccharides, while decreasing or maintaining transesterification activity. Other mutants of the LE enzyme are capable of or show improved transesterification activity. An example of such improved transesterification is the improved conversion of bola amphiphilic glycolipids into lactonic glycolipids and saccharides or the improved conversion of bola amphiphilic glycolipids and other esters, such as, but not limited to, acyl esters, preferably acyl glycerol esters such as mono acyl glycerol esters, di acyl glycerol esters and / or tri acyl glycerol esters, preferably a mono acyl glycerol ester and / or a di acyl glycerol ester, into glycolipid oligomers / polymers / esters and saccharides, while decreasing or maintaining hydrolysis activity. Certain mutants of the LE enzyme are capable of or show improved hydrolysis activity and improved transesterification activity.

[0085] "Transesterification" or a "transesterification reaction" generally refers to a process of displacement of the alcohol from an ester by another alcohol in a process similar to hydrolysis, but using an alcohol instead of water. In "intramolecular transesterification" as used herein the displaced alcohol is sophorose and / or glucose, and the other alcohol is another sophorose and / or glucose. In "intermolecular transesterification" as used herein, any alcohol is displaced by any other alcohol. "Hydrolysis" or a "hydrolysis reaction" refers to the process of the displacement of the alcohol from an ester by water.

[0086] The term "glycoside" generally refers to a molecule in which at least one carbohydrate molecule is covalently bound to at least one other molecule via a glycosidic bond.

[0087] "Glycolipids" as used herein refer to compounds wherein one or more carbohydrate molecule(s) is / are covalently bound to a lipid molecule, wherein preferably at least one of these lipids is an aliphatic carbon chain(s) of, preferably at least 6, carbon atoms that contains a carboxylic functionality, such as, e.g., fatty acids or hydroxyl fatty acids. A hydroxyl fatty acid is a fatty acid having a hydroxyl group. A glycolipid wherein the carbohydrate molecule is sophorose, is referred to herein as "sophorolipid" or "SL". The term "acidic glycolipids" as used herein refers to a glycolipid in which the carboxyl group is free, i.e. not covalently bound to another molecule, moiety, or functional group. The term "acidic glycolipid" also encompasses salt forms of the acidic glycolipid. The term "lactonic glycolipids" as used herein refers to a glycolipid in which the carboxyl group is bound to a carbohydrate within the glycolipid molecule. With "glycolipid oligomers / polymers / esters" is meant herein -but not limited to- intermolecular esters connecting multiple glycolipid molecules as such forming dimers, trimers, tetramers, etc. and also esters consisting of glycolipid molecules and di- and / or mono-acyl glycerol esters.

[0088] The term "bola glycoside" or "bolaform glycoside" or "bola amphiphilic glycoside" as used herein refers to a glycoside molecule containing at least two carbohydrates both bound to a hydrophobic aliphatic linker, in particular an aliphatic chain of carbon atoms, connecting the two carbohydrates. The linker may comprise an ester group or moiety. The terms 'bola amphiphilic glycolipids' and 'bola glycolipids' as used interchangeably in the present invention refers to molecules as described by WO2015 / 028278 and are in general compounds with the general formula as shown in Figure 1 (A and B), wherein Ri = H or CO-CH3; R? = H or CO-CH3; Rs = H or CO-CH3; R4 = H or CO-CH3; Rs = an unsubstituted or hydroxysubstituted, unbranched, optionally one to three double or triple bonds containing, divalent organic moiety comprising 6 to 32 carbon atoms, R6= H, CH 3 or an unsubstituted or hydroxy-substituted, unbranched, optionally one to three double or triple bonds containing, organic moiety comprising 2 to 10 carbon atoms; n = 1 or 0; m = 1 or 0. The bola glycolipids used herein preferably comprise an ester group or moiety.

[0089] The 'carbohydrate' in the acidic glycolipids, lactonic glycolipids, bolaform glycolipids and glycolipid oligomers / polymers / esters described herein can be any carbohydrate known in the art, but is preferably sophorose, glucose, mannose, rhamnose, xylose, arabinose, trehalose, cellobiose or lactose, more preferably sophorose or glucose. The type of carbohydrate can be tailored as known to the skilled person, e.g. through a proper selection of microbial strain (e.g. a natural sophorolipid producing yeast strain, e.g. Starmerella bombicola) and / or through further genetic modifications of the microbial strain. For example, upon knocking-out the glucosyltransferase UGTB1 gene in Starmerella bombicola, glucosides are produced instead of sophorosides; or upon replacing the glucosyltransferase UGTB1 by the glucosyltransferase UGT1 gene of U. maydis in Starmerella bombicola cellobiosides are produced instead of sophorosides (Roelants et al. 2013. Biotechnol Bioeng. 110:2494-2503).

[0090] The terms 'an SBLE enzyme' and 'a lactone esterase (LE) enzyme" are used interchangeably herein to refer to an enzyme that has transesterification and / or hydrolysis activity on bola amphiphilic glycolipid compounds, in particular it is capable of catalyzing one or more of the conversion of bola glycolipids / bola sophorolipids / bola glucolipids into lactonic glycol ipids / lactonic sophorolipids / lactonic glucolipids, respectively while releasing saccharides such as sophorose and / or glucose; and / or the conversion of bola glycolipids / bola sophorolipids / bola glucolipids into acidic glycolipids / acidic sophorolipids / acidic glucolipids, respectively while releasing saccharides such as sophorose and / or glucose; and / or the conversion of bola glycolipids / bola sophorolipids / bola glucolipids and another ester, preferably an acyl ester, more preferably an acyl glycerol ester such as a mono acyl glycerol ester, a di acyl glycerol ester and / or a tri acyl glycerol ester, preferably a mono acyl glycerol ester and / or a di acyl glycerol ester, into glycolipid / sophorolipid / glucolipid oligomers / polymers / esters while releasing saccharides such as sophorose and / or glucose and relates to the enzyme previously denominated as a 'lactonase' or 'Starmerella bombicola lactone esterase (SBLE)' and described in detail in WO2013 / 092421. The terms encompass fragments, variants and homologues of said S. bombicola SBLE enzyme. The terms encompass natural LE enzyme, as well as recombinantly, semi-synthetically or synthetically produced LE enzyme. Hence, for example, an LE enzyme can be present in or isolated from Starmerella bombicola or a sophorolipid-producing strain of the Wickerhamiella / Starmerella clade; or an LE enzyme can be recombinant, and / or can be, partly or entirely, chemically or biochemically synthesized.

[0091] As used herein, the term "isolated" or "purified" in association with a polypeptide or nucleic acid means that the polypeptide or nucleic acid is substantially or essentially free from components that normally accompany it in its native state. Thus, the term "isolated" includes polypeptides or nucleic acids taken from the original environment, for example, if it is naturally occurring. Isolated polypeptides include the naturally produced polypeptides contained in cell lysates, the polypeptides in purified or partially purified form, recombinant polypeptides, the polypeptides expressed or secreted by cells, and in heterologous host cells or cultures of the polypeptide. In connection with nucleic acids, the term isolated or purified indicates that the nucleic acid is not in its natural genomic background (e.g., in a vector, as an expression cassette, linked to a promoter, or artificially introduced into a host cell).

[0092] With "Starmerella bombicola SBLE enzyme" and "Starmerella bombicola LE enzyme" as used herein interchangeably is meant herein a polypeptide or protein comprising or consisting of the amino acid sequence given by SEQ ID NO: 2 or 13 and having the above described enzymatic activity for an SBLE enzyme, and (functional) fragments and variants thereof as described herein. The term encompasses natural Starmerella bombicola SBLE enzyme, as well as recombinantly, semi-synthetically or synthetically produced Starmerella bombicola SBLE enzyme. Hence, for example, a Starmerella bombicola SBLE enzyme can be present in or isolated from Starmerella bombicola; or Starmerella bombicola SBLE enzyme can be recombinant, and / or can be, partly or entirely, chemically or biochemically synthesised.

[0093] SEQ ID NO:2 provided herein represents the sequence of natural, wild-type Starmerella bombicola SBLE enzyme.

[0094] MLALFFSLAPLLSQALPLGYTAAPAESFYFWPENI SSLQAGEIFRKRELLTLPDI FDFGPNLEKWQVAYKTRLT DGNDSFSIASI FI PKNPSPELKLYSYQTFEDAVQLDCAPSYALEVGNKSSNYLPVTSNLSAI SRELEKGRHCI I P DHEGYI SGFFAGRQEGYAGLDGIRAARNYLNGTNETPIGI FGYSGGAQATAWIVDLHDEYAPDLNFVGTVSGGTL VDAWGTFQYIDYPKVYLKGSILIMYTGLFSGYPAQFEVIWPYIEPVIQENMLLLRLAPNDCNQSPILQGYNNSIM AGIHVDLPEFPASKYI FQHESLLANYSWPVSTPKFPRYMYHGGSDELAKLSLVEQYVDQQWNTGANLTFWYPG LLHDETAYRGFDAAMDWLDAQLDSGYLPPVNSTHT ( SEQ ID NO : 2 )

[0095] The nucleic acid sequence as depicted by SEQ ID NO:1 corresponds to the open reading frame which encodes for the polypeptide sequence of the SBLE enzyme depicted by the sequence of SEQ ID NO:2.

[0096] As used herein, the terms 'an SBLE enzyme' and 'a Starmerella bombicola SBLE enzyme' encompass polypeptide comprising or consisting of the amino acid sequence given by SEQ ID NO:2, or a fragment thereof retaining the enzymatic activity, i.e. the transesterification and / or hydrolysis activity on (acetylated) bola amphiphilic glycolipid compounds, more specifically the conversion of (acetylated) bola sophorolipids / (acetylated) bola glucolipids into (acetylated) lactonic sophorolipids / (acetylated) lactonic glucolipids respectively while releasing (acetylated) saccharides such as (acetylated) sophorose and / or (acetylated) glucose and / or the conversion of (acetylated) bola sophorolipids / (acetylated) bola glucolipids into (acetylated) acidic sophorolipids / (acetylated) acidic glucolipids while releasing (acetylated) saccharides such as (acetylated) sophorose and / or (acetylated) glucose and / or the conversion of (acetylated) bola sophorolipids / (acetylated) bola glucolipids and another ester, preferably an acyl ester, more preferably an acyl glycerol ester such as a mono acyl glycerol ester, a di acyl glycerol ester and / or a tri acyl glycerol ester, preferably a mono acyl glycerol ester and / or a di acyl glycerol ester, into (acetylated) sophorolipid / glucolipid oligomers / polymers / esters while releasing (acetylated) saccharides such as (acetylated) sophorose and / or (acetylated) glucose, or a variant thereof having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity with SEQ ID NO: 2 and retaining or having said enzymatic activity.

[0097] SEQ ID NO:13 provided herein represents the sequence of recombinantly produced Starmerella bombicola SBLE enzyme.

[0098] AGLPLGYTAAPAESFYFWPENI SSLQAGEI FRKRELLTLPDI FDFGPNLEKWQVAYKTRLTDGNDSFSIASI FI PKNPSPELKLYSYQTFEDAVQLDCAPSYALEVGNKSSNYLPVTSNLSAI SRELEKGRHCI I PDHEGYI SGFFAGR QEGYAGLDGIRAARNYLNGTNETPIGI FGYSGGAQATAWIVDLHDEYAPDLNFVGTVSGGTLVDAWGTFQYIDYP KVYLKGSILIMYTGLFSGYPAQFEVIWPYIEPVIQENMLLLRLAPNDCNQSPILQGYNNSIMAGIHVDLPEFPAS KYI FQHESLLANYSWPVSTPKFPRYMYHGGSDELAKLSLVEQYVDQQWNTGANLTFWYPGLLHDETAYRGFDA AMDWLDAQLDSGY ( SEQ ID NO : 13 )

[0099] The terms 'an SBLE enzyme' and 'a Starmerella bombicola SBLE enzyme' as used herein also relate to a polypeptide comprising or consisting of the amino acid sequence given by SEQ ID NO:13 and having the described enzymatic activity for said SBLE enzyme, i.e. the enzymatic activity described for the natural S. bombicola enzyme, or a fragment thereof retaining the above described enzymatic activity for said SBLE enzyme, or a variant thereof having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity with SEQ ID NO:13 and having said enzymatic activity.

[0100] (Functional) variants, fragments and homologues as described herein typically have or retain the same activity of protein X, such as the enzymatic activity, to a similar, but not necessarily the same, degree, compared to the wild type, i.e. natural, activity of said protein X. For example, a (functional) variant, fragment or homologue of protein X may have an enzymatic activity that is at least 10%, 20%, 30% or 40%, preferably at least 50%, 60%, 70%, 80%, 85%, 90% or 95%, or 100%, or 101%, 102%, 103% 104%, 105%, 110%, 115%, 120%, 130%, 140%, 150% or more compared to the activity of protein X.

[0101] The 'variant' or 'variants' may differ from the protein X, such as the proteins as depicted by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19 or any fragment or homologue thereof, only in conservative substitutions and / or modifications, such that the ability of the activity is retained. A "conservative substitution" is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of protein chemistry would expect the nature of the protein to be substantially unchanged. In particular, a "conservative amino acid substitution" may refer to a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In general, the following groups of amino acids represent conservative changes: (1) Ala, Pro, Gly, Glu, Asp, Gin, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Vai, He, Leu, Met, Ala, Phe; (4) Lys, Arg, His; and (5) Phe, Tyr, Trp, His.

[0102] The 'variant' or 'variants' may also (or alternatively) be proteins that differ from the protein X in the deletion or addition of amino acids that have minimal influence on the enzymatic activities as defined above, secondary structure or hydropathic nature of the enzyme.

[0103] Furthermore, the term 'variant' or 'variants' also refers to any glycosylated form of the protein X, such as a glycosylated form of the protein as depicted by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19 or any (functional) fragment or homologue thereof, or modified in any other way. A non-limitative list of such protein modifications: acetylation, acylation, ADP-ribosylation, amidation, covalent attachment, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, selenoylation, transfer-RNA mediated addition of amino acids to proteins, such as arginylation, and ubiquitination, ... The term 'fragment' as used herein in connection to a protein, such as an enzyme, for example as in "a fragment of protein X", refers to any fragment of protein X that retains the activity of protein X, such as the enzymatic activity (functional fragment). A fragment of a protein X preferably means a protein or peptide sequence which comprises or consists of an amount of consecutive amino acid residues from said protein X and wherein said amount of consecutive amino acid residues is at least 50.0 %, 60.0 %, 70.0 %, 80.0 %, 81.0 %, 82.0 %, 83.0 %, 84.0 %, 85.0 %, 86.0 %, 87.0 %, 88.0 %, 89.0 %, 90.0 %, 91.0 %, 92.0 %, 93.0 %, 94.0 %, 95.0 %, 95.5%, 96.0 %, 96.5 %, 97.0 %, 97.5 %, 98.0 %, 98.5 %, 99.0 %, 99.5 %, 100 %, preferably at least 80.0 %, more preferably at least 85.0 %, even more preferably at least 90.0 % or at least 95.0 %, of the full-length of said protein X and which retains the activity of protein X, such as the enzymatic activity (functional fragment). Such fragment can -for example- be a protein with a deletion of 10% or less of the total number of amino acids at the C- and / or N-terminus. For example, the term 'fragment' refers to a protein or peptide or polypeptide containing fewer amino acids than the amino acid sequence as depicted by SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 13 and that retains said enzymatic activity described for said SBLE enzyme.

[0104] The term "homolog" or "homologue" as used herein in connection to a protein, such as an enzyme, for example as in "a homolog of protein X" refers to the fact that the protein differs from protein X in its sequence, but that has the activity of protein X, such as the enzymatic activity, and originates from another species, i.e. is a naturally occurring sequence. A homolog of protein X can be identified by the skilled person by pairwise search methods such as BLAST and checking of the corresponding activity. Homologues as defined herein may have at least 20%, preferably at least 25% or 30% such as at least 31%, 32%, 33%, 34% or 35% sequence identity with protein X. There are two types of homologues: orthologues and paralogues. Orthologues are defined as homologous expressed from genes in different organisms, i. e. the genes share a common ancestor coincident with the speciation event that generated them. Paralogues are defined as homologous expressed from genes in the same organism derived from a gene / chromosome / genome duplication, i.e. the common ancestor of the genes occurred since the last speciation event. In certain embodiments, the homologues of S. bombicola SBLE enzyme are orthologues. Hence, orthologues and paralogues, or any protein in other genera or species than the strains from which SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 19 are derived which encode for polypeptides having the described activities, are part of the present invention.

[0105] The term 'an SBLE enzyme' as used herein thus also encompasses a polypeptide comprising an amino acid sequence given by SEQ ID NO: 6 and having the above described enzymatic activity for an SBLE enzyme, in particular the transesterification and / or hydrolysis activity on bola amphiphilic glycolipid compounds, or a fragment thereof retaining the above described enzymatic activity for an SBLE enzyme, or a variant thereof having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity with SEQ ID NO: 6 and having said enzymatic activity. The nucleic acid sequence as depicted by SEQ ID NO: 5 corresponds to the open reading frame which encodes for the polypeptide sequence of an SBLE enzyme of the present invention as depicted by the sequence of SEQ ID NO: 6.

[0106] Similarly, the term 'an SBLE enzyme' as used herein also encompasses a polypeptide comprising an amino acid sequence given by SEQ ID NO: 8 and having the above described enzymatic activity for an SBLE enzyme, or a fragment thereof retaining the above described enzymatic activity for an SBLE enzyme, or a variant thereof having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity with SEQ ID NO: 8 and having said enzymatic activity. The nucleic acid sequence as depicted by SEQ ID NO: 7 corresponds to the open reading frame which encodes for the polypeptide sequence of an SBLE enzyme of the present invention as depicted by the sequence of SEQ ID NO: 8.

[0107] Similarly, the term 'an SBLE enzyme' as used herein also encompasses a polypeptide comprising an amino acid sequence given by SEQ ID NO:114 and having the above described enzymatic activity for an SBLE enzyme, or a fragment thereof retaining the above described enzymatic activity for an SBLE enzyme, or a variant thereof having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity with SEQ ID NO:114 and having said enzymatic activity. The nucleic acid sequence as depicted by SEQ ID NO: 113 corresponds to the open reading frame which encodes for the polypeptide sequence of an SBLE enzyme of the present invention as depicted by the sequence of SEQ ID NO:114.

[0108] In an aspect, the invention provides homologues, in particular functional homologues, of S. bombicola SBLE enzyme. Homologues of S. bombicola SBLE enzyme may be identified in other Starmerella species or other glycolipid producing yeast species. In embodiments, the (functional) homologue has improved hydrolytic activity compared to the S. bombicola SBLE enzyme. In embodiments, the (functional) homologue has improved transesterification activity, in particular improved intermolecular transesterification activity, compared to the S. bombicola SBLE enzyme. In embodiments, the (functional) homologue has improved hydrolytic activity and improved transesterification activity, in particular improved intermolecular transesterification activity, compared to the S. bombicola SBLE enzyme. In particular embodiments, the homologue of S. bombicola SBLE enzyme is selected from the group comprising or consisting of a Starmerella kuoi SBLE enzyme, Starmerella batistae SBLE enzyme and Starmerella riodocensis SBLE enzyme.

[0109] The term 'Starmerella batistae SBLE enzyme' or 'Starmerella batistae LE enzyme' as used interchangeably herein refers to a polypeptide or protein comprising or consisting of the amino acid sequence given by SEQ ID NO: 8 and having the described enzymatic activity for an SBLE enzyme, and fragments and variants thereof as described herein. In particular, the term encompasses variants having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity, with SEQ ID NO:8 and retaining the described SBLE activity, variants that differ from the protein depicted by SEQ ID NO:8 only in conservative substitutions and / or modifications, such that the activity of the protein is retained and glycosylated forms of the protein depicted by SEQ ID NO:8, or the protein of SEQ ID NO:8 modified in another way as described elsewhere herein. The term also encompasses proteins or peptides or polypeptides containing fewer amino acids than the amino acid sequence depicted by SEQ ID NO:8 and that retain said enzymatic activity described for said Starmerella batistae SBLE enzyme of SEQ ID NO:8 ((functional) fragments). The nucleic acid sequence as depicted by SEQ ID NO:7 corresponds to the open reading frame which encodes for the polypeptide sequence of the Starmerella batistae SBLE enzyme depicted by the sequence of SEQ ID NO:8. The term encompasses natural Starmerella batistae SBLE enzyme, as well as recombinantly, semi-synthetically or synthetically produced Starmerella batistae SBLE enzyme. Hence, for example, a Starmerella batistae SBLE enzyme can be present in or isolated from Starmerella batistae; or Starmerella batistae SBLE enzyme can be recombinant, and / or can be, partly or entirely, chemically or biochemically synthesised.

[0110] MKHPLLSLSFLLSALLAATAQDVQAVPNDSFYDPPTNLSDYNYGDVIRRRSIQQKFWQKATIKEI IQVQYKTRLT SGEDSWSI STVLVPSNPGAKKQLYSYQVFEDAVQLDCAPSWTIKNGILSPGFGGITNAVADSIEGQVDQGRYVW PDHEGAESAFFGGRQEGYAGLDGIRAAINAVDLPKDTGWI FGYSGGAHATAWMVNQWESYAKDINLIGAAYGGT LVDAYQQIVYDDHYESLVKGSVASLIAGLLVAYPDVNSSLWSHITPDIQTAIVQMRIQPGGCDSAAFLTNFQGTV RAGINVNLSSFEPTKRMFQQETLLANLSSI PI PAPKFPRYIYHGSADEVAPYELIKEYVDEQESKGANITFKTYD GYGHVQTAFNGLQDAENWINSI FASN ( SEQ ID NO : 8 )

[0111] The term 'Starmerella kuoi SBLE enzyme' or 'Starmerella kuoi LE enzyme' as used interchangeably herein refers to a polypeptide or protein comprising or consisting of the amino acid sequence given by SEQ ID NO:6 and having the described enzymatic activity for an SBLE enzyme, and fragments and variants thereof as described herein. In particular, the term encompasses variants having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity, with SEQ ID NO:6 and retaining the described Sble activity, variants that differ from the protein depicted by SEQ ID NO:6 only in conservative substitutions and / or modifications, such that the activity of the protein is retained and glycosylated forms of the protein depicted by SEQ ID NO:6, or the protein of SEQ ID NO:6 modified in another way as described elsewhere herein. The term also encompasses proteins or peptides or polypeptides containing fewer amino acids than the amino acid sequence depicted by SEQ ID NO:6 and that retain said enzymatic activity described for said Starmerella kuoi SBLE enzyme of SEQ ID NO:6 ((functional) fragments). The nucleic acid sequence as depicted by SEQ ID NO:5 corresponds to the open reading frame which encodes for the polypeptide sequence of the Starmerella kuoi SBLE enzyme depicted by the sequence of SEQ ID NO:6. The term encompasses natural Starmerella kuoi SBLE enzyme, as well as recombinantly, semi-synthetically or synthetically produced Starmerella kuoi SBLE enzyme. Hence, for example, a Starmerella kuoi SBLE enzyme can be present in or isolated from Starmerella kuoi; or Starmerella kuoi SBLE enzyme can be recombinant, and / or can be, partly or entirely, chemically or biochemically synthesised.

[0112] MFSLILPLI I SCLSLVLAQGTGARPSDPFYDAPDNLGSYKNGEVFRRRSI PLTLDLGVKVSGWQVAYKTQLADG GDSYSI STI FKPENPNSRIELFSYQI FEDAVQLDCAPSWTFKEGFFTPGVSVIDNNVTLAFSTQLEQGRYVWPD HEGAVSAFFCGRQEGYAGLDGIRAAINELNATNDNTPVIMYGYSGGAHATAWMVNVQDYYAPDLNVIAAAYGGTL VDALGELNYADI PSDPVKGSVAAMYTGLLVGYPAQNETIWSHISQYIQNTILQLRWTPNLCSSQVLLQNYNKSIR ENIDVDIATFPAFVEI FKNESLLTSYSGQPVATPKYPRYIYHGTNDEIAPYNLTLSYINQQKASGGNIDWWYNG LGHIEAGFAGENAAYDWIDQQFDNYH ( SEQ ID NO : 6 )

[0113] The term "Starmerella riodocensis SBLE enzyme" or "Starmerella riodocensis LE enzyme" as used interchangeably herein refers to a polypeptide or protein comprising or consisting of the amino acid sequence given by SEQ ID NO:114 and having the described enzymatic activity for an SBLE enzyme, and fragments and variants thereof as described herein. In particular, the term encompasses variants having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity, with SEQ ID NO:114 and retaining the described Sble activity, variants that differ from the protein depicted by SEQ ID NO:114 only in conservative substitutions and / or modifications, such that the activity of the protein is retained and glycosylated forms of the protein depicted by SEQ ID NO:114, or the protein of SEQ ID NO:114 modified in another way as described elsewhere herein. The term also encompasses proteins or peptides or polypeptides containing fewer amino acids than the amino acid sequence depicted by SEQ ID NO:114 and that retain said enzymatic activity described for said S. riodocensis SBLE enzyme of SEQ ID NO:114 ((functional) fragments). The nucleic acid sequence as depicted by SEQ ID NO:113 corresponds to the open reading frame which encodes for the polypeptide sequence of the S. riodocensis SBLE enzyme depicted by the sequence of SEQ ID NO:114. The term encompasses natural S. riodocensis LE enzyme, as well as recombinantly, semi-synthetically or synthetically produced S. riodocensis SBLE enzyme. Hence, for example, a S. riodocensis SBLE enzyme can be present in or isolated from S. riodocensis; or S. riodocensis SBLE enzyme can be recombinant, and / or can be, partly or entirely, chemically or biochemically synthesised.

[0114] MTLLWVSVLLSFFSIVWAYEQPPQSVPNDPFYDVPPNLDQYKPGDI IRRREI SQKFWVDANI SKIVEVAYKTNLA LGGDTWSIATI ITPGNPDPNLKLYSYQFFEDAVQLSCAPSWSLKNGLFSPGI SSWNSLTGTVGSQLRAGRHVW PDHEGYI SAFFSGRQEGKAGLDGIRAALNELNASVSDTPVALYGYSGGGHATAWMVNLQPDYAPELNIVGAAYGG VLIDALKELQWTDQKGAEAKGSVAALLTGLLLGNPPANESLWSHFSPYLKTKIYQLRTI PNFCSSELLWLDSDDT IQSNIDVDIATFPPAVQMFKKETLLSNYSSI PI PTPKFPRYIYQGVDDTLAPYYLVKQYVDQQLATGANIEFVPY PGLDHI PVAFKGLDAAMDWIDQVFDKTD ( SEQ ID NO : 114 )

[0115] The term 'CALA' relates to the enzyme which is previously denominated as a 'Candida antarctica lipase A' or a 'Moesziomyces antarcticus lipase A' and is described in detail in Maria et al. (2005) and Ericsson et al. (2008). The CALA enzyme thus relates to a polypeptide comprising an amino acid sequence given by SEQ ID NO: 10, or a fragment thereof retaining the enzymatic activity, i.e. the capacity for esterification as well as for hydrolysis of a wide variety of compounds, or a variant thereof having at least 34 % sequence identity with SEQ ID NO: 10 and having said enzymatic activity. The nucleic acid sequence as depicted by SEQ ID NO: 9 corresponds to the open reading frame which encodes for the polypeptide sequence of the CALA enzyme as depicted by the sequence of SEQ ID NO: 10.

[0116] As used herein, the terms "identity" and "identical" and the like are used interchangeably with the terms "homology" and "homologues" and the like herein and refer to the sequence similarity between two polymeric molecules, e.g., between two nucleic acid molecules or polypeptides. The percentage of amino acid sequence identity may be determined by alignment of the two sequences and identification of the number of positions with identical amino acids divided by the number of amino acids in the shorter of the sequences x 100.

[0117] Throughout this disclosure, specific residues and positions are represented with respect to SEQ ID NO:2. If in this disclosure an amino acid residue is indicated by a residue position, such description corresponds to the specific position within SEQ ID NO:2. For example, recitation of "Ala382" or "A382" indicates that the identified residue corresponds to the alanine residue at 382nd position in SEQ ID NO:2. Such nomenclature applies even if a mutant of SEQ ID NO: 13 is identified. For example, the phrase "A382 within SEQ ID NO: 13" indicates that the identified residue corresponds to the alanine residue at 382nd position in SEQ ID NO:2 even if SEQ ID NO:13 contains less amino acids than SEQ ID NO:2. Sequence alignment of SEQ ID NO:2 with SEQ ID NO:13 can be performed to identify the referenced position. As used herein, the term corresponding to is used to mean enumerated positions within the reference protein, e .g ., wild-type S. bombicola SBLE enzyme of SEQ ID NO:2, and those positions in the queried protein (e.g. a homologue of S. bombicola SBLE enzyme) that align with the positions on the reference protein. Thus, when the amino acid sequence of a subject SBLE enzyme, e.g., SEQ ID NOs: 6, 8 or 114, is aligned with the amino acid sequence of the reference SBLE enzyme, e .g ., SEQ ID NO:2, the amino acids in the subject SBLE enzyme sequence that "corresponds to" certain enumerated positions of the reference SBLE enzyme sequence are those that align with these positions of the reference SBLE enzyme sequence, but are not necessarily in these exact numerical positions of the reference SBLE enzyme sequence.

[0118] Mutant SBLE enzymes

[0119] The term 'mutant' refers to a polypeptide comprising at least one change in its polypeptide sequence. Such change can be spontaneous and / or induced, undertaken by any method in the art known by a skilled person (e.g. via targeted mutagenesis of the respective nucleic acid coding sequence), such as, but not limited to, one or several insertion(s), one or several deletion(s), one or several point mutation(s) and / or one or several amino acid change(s). Such a mutant may or may not retain the protein's or peptide's or polypeptide's enzymatic activity. The mutant can have an abolished, impaired, reduced or delayed activity of said protein or peptide or polypeptide compared to the original activity of the protein or peptide or polypeptide or can have an improved, an accelerated or an enhanced activity of said protein or peptide or polypeptide compared to its original activity. For example, the polypeptide comprising an amino acid sequence given by SEQ ID NO: 4 is a mutant of the SBLE enzyme of the present invention as depicted by the sequence SEQ ID NO:2. The nucleic acid sequence as depicted by SEQ ID NO:3 corresponds to the open reading frame which encodes for the polypeptide depicted by the sequence SEQ ID NO:4.

[0120] In particular embodiments, the mutant SBLE enzyme has at least 80%, more preferably at least 85%, even more preferably at least 90%, and yet more preferably at least 95% such as at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the non-mutated, original protein, preferably calculated over the entire length of the sequence. It is understood that the mutant proteins or enzymes described herein may have either or both non-conservative or essential amino acid substitutions, which do have a substantial effect on protein activity and conservative or non-essential amino acid substitutions, which do not have a substantial effect on the protein activity.

[0121] Disclosed herein is a mutant of the SBLE enzyme from S. bombicola comprising at least one mutation at one of the following residues: Asnl26, Prol29, Serl32, Leul34, Phe 159, Phel60, Pro212, Leu 225, Thr231, Phe232, Ile235, Val240, Leu242, Gly244, Met249, Thr251, Ser255, Pro258, Phe261, Glu262, Trp265, Met276, Leu277, Arg280, Asn287, Gln288, Ser289, Pro290, Gly302, Phe317, Glu 347, Leu348, Asp379, Ala382 and in the region I235-K243 and G344-L351. Also disclosed herein is a mutant of S. bombicola SBLE enzyme comprising any one of the mutations of Table A or an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table A. Table A

[0122] In embodiments, the mutant LE enzyme comprises at least one mutation, preferably a substitution, at a position corresponding to one of the following positions of SEQ ID NO:2: position 382, position 126, positions 125 and 129; position 244, position 290, position 128, position 288, position 287, position 248 and / or position 348. In embodiments, the mutant LE enzyme is a mutant of S. bombicola SBLE enzyme comprising an amino acid sequence or polynucleotide coding sequence set forth in any one of SEQ ID NO:115-168, and 173.

[0123] In embodiments, the mutant LE enzyme comprises at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E; N126G; S125P and P129G; G244S, G244T, G244C, G244N, G244Q, G244W or G244Y; P290S, P290T, P290C, P290N, P290Q, P290F or P290Y; L128T or L128Y; Q288Y or Q288V; N287F; I248A or I248G; and L348D. In embodiments, the aforementioned mutant LE enzyme comprises an amino sequence set forth in any one of the corresponding SEQ. ID NOs in Table B.

[0124] Table B

[0125] In further embodiments, the mutant LE enzyme comprises at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ. ID NO:2: G244S, G244C or G244Q;

[0126] P290S, P290T, P290C, P290N or P290Q; L128Y; Q288Y; and I248A or I248G. In further embodiments, the aforementioned mutant LE enzyme comprises an amino sequence set forth in any one of the corresponding SEQ ID NOs in Table B.

[0127] In embodiments, the mutant LE enzyme has improved hydrolytic activity compared to the original (i.e. non-mutated) LE enzyme. Mutant LE enzymes with improved hydrolytic activity may comprise at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E; N126G; S125P and P129G; G244S, G244T, G244C, G244N, G244Q, G244W or G244Y; P290S, P290T, P290C, P290N, P290Q, P290F or P290Y; L128T; Q288Y or Q288V; N287F; I248A or I248G.; and L348D. The aforementioned mutant LE enzymes with improved hydrolytic activity may comprise an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme comprise at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E; N126G; S125P and P129G; G244S, G244T, G244C, G244N, G244Q, G244W or G244Y; P290S, P290T, P290C, P290N, P290Q, P290F or P290Y; L128T; Q288Y or Q288V; N287F; I248A or I248G; and L348D. In embodiments, the aforementioned mutant LE enzyme comprises an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B.

[0128] In further embodiments, the present invention provides mutants of an LE enzyme, in particular mutants with improved hydrolytic activity, which are capable of improved conversion of (acetylated) bola sophorolipids and (acetylated) bola glucolipids into respectively (acetylated) acidic sophorolipids and (acetylated) acidic glucolipids while (acetylated) saccharides are released from the reaction. Mutant LE enzymes capable of improved conversion of acetylated bola sophorolipids and acetylated bola glucolipids into respectively acetylated acidic sophorolipids and acetylated acidic glucolipids and acetylated saccharides may comprise at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E; N126G; S125P and P129G; G244S, G244T, G244C, G244N, G244Q, G244W or G244Y; P290S, P290T, P290C, P290N, P290Q, P290F or P290Y; L128T; Q288Y or Q288V; N287F; and I248A or I248G; or may comprise an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme comprises at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E; N126G; S125P and P129G; G244S, G244T, G244C, G244N, G244Q, G244W or G244Y; P290S, P290T, P290C, P290N, P290Q, P290F or P290Y; L128T; Q288Y or Q288V; N287F; and I248A or I248G. In embodiments, the aforementioned mutant LE enzyme comprises an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme comprises at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E; N126G; S125P and P129G; G244S, G244T, G244C, G244N, G244Q, G244W or G244Y; P290S, P290T, P290C, P290N, P290Q, P290F or P290Y; L128T; Q288Y; N287F; and I248A or I248G. In embodiments, the aforementioned mutant LE enzyme comprises an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme comprises at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E; S125P and P129G; and Q288V. In embodiments, the aforementioned mutant LE enzyme comprises an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B.

[0129] In further embodiments, the present invention provides mutants of an LE enzyme, in particular mutants with improved hydrolytic activity, which are capable of improved conversion of nonacetylated bola sophorolipids and non-acetylated bola glucolipids into respectively non-acetylated acidic sophorolipids and non-acetylated acidic glucolipids while non-acetylated saccharides are released from the reaction. Mutant LE enzymes capable of improved conversion of non-acetylated bola sophorolipids and non-acetylated bola glucolipids into respectively non-acetylated acidic sophorolipids and non-acetylated acidic glucolipids and non-acetylated saccharides may comprise at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: S125P and P129G; G244S or G244C; P290S, P290T, P290C, P290N or P290Q; Q288Y or Q288V; I248A or I248G; and L348D; or may comprise an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme comprises at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: S125P and P129G; G244S or G244C; P290S, P290T, P290C, P290N or P290Q; Q288Y or Q288V; I248A or I248G; and L348D. In embodiments, the aforementioned mutant LE enzyme comprises an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme comprises at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244S or G244C; P290S, P290T, P290C, P290N or P290Q; Q288Y; and I248A or I248G. In embodiments, the aforementioned mutant LE enzyme comprises an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme comprises at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: S125P and P129G; Q288V; and L348D. In embodiments, the aforementioned mutant LE enzyme comprises an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B.

[0130] In further embodiments, the present invention provides mutants of an LE enzyme, in particular mutants with improved hydrolytic activity, which are capable of improved conversion of non- acetylated bola sophorolipids and non-acetylated bola glucolipids into respectively non-acetylated acidic sophorolipids and non-acetylated acidic glucolipids while non-acetylated saccharides are released from the reaction and capable of improved conversion of acetylated bola sophorolipids and acetylated bola glucolipids into respectively acetylated acidic sophorolipids and acetylated acidic glucolipids while acetylated saccharides are released from the reaction. Such mutant LE enzymes may comprise at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: S125P and P129G; G244S or G244C; P290S, P290T, P290C, P290N or P290Q; Q288Y or Q288V; and I248A or I248G; or may comprise an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme comprises at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: S125P and P129G; G244S or G244C; P290S, P290T, P290C, P290N or P290Q; Q288Y or Q288V; and I248A or I248G. In embodiments, the aforementioned mutant LE enzyme comprises an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B.

[0131] In particular embodiments, the mutant LE enzyme has improved hydrolytic activity and decreased or maintained transesterification activity compared to the original (i.e. non-mutated) LE enzyme. Mutant LE enzymes with improved hydrolytic activity and decreased or maintained transesterification activity may comprise at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244C; P290S, P290T, P290C, P290N or P290Q; and I248A or I248G. In embodiments, the mutant LE enzyme comprises at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244C; P290S, P290T, P290C, P290N or P290Q; and I248A or I248G. In embodiments, the aforementioned mutant LE enzyme comprises an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B.

[0132] In embodiments, the mutant LE enzyme has improved transesterification activity compared to the original (i.e. non-mutated) LE enzyme. These mutant LE enzymes may be capable of improved conversion of (non-acetylated) bola sophorolipids and / or (non-acetylated) bola glucolipids into respectively (non-acetylated) lactonic sophorolipids and (non-acetylated) lactonic glucolipids while releasing (non-acetylated) saccharides such as (non-acetylated) sophorose or (non-acetylated) glucose. Mutant LE enzymes with improved transesterification activity may comprise at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244S or G244Q; L128Y; and Q288Y; or may comprise an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme comprises at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244S or G244Q; L128Y; and Q288Y. In embodiments, the aforementioned mutant LE enzyme comprises an amino acid sequence set forth in any one of the corresponding SEQ ID NOs in Table B. In further particular embodiments, the mutant LE enzyme has improved transesterification activity and decreased or maintained hydrolysis activity compared to the original (i.e. non-mutated) LE enzyme. Mutant LE enzymes with improved transesterification activity and decreased or maintained hydrolytic activity may comprise at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244Q and L128Y; or may comprise an amino acid sequence as set forth in the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme may comprise at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244Qand L128Y. In embodiments, the aforementioned mutant LE enzyme may comprise an amino acid sequence as set forth in the corresponding SEQ ID NOs in Table B. In other further particular embodiments, the mutant LE enzyme has improved transesterification activity and improved hydrolysis activity compared to the original (i.e. non-mutated) LE enzyme. Mutant LE enzymes with improved transesterification activity and improved hydrolytic activity may comprise at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244S and Q288Y; or may comprise an amino acid sequence as set forth in the corresponding SEQ ID NOs in Table B. In embodiments, the mutant LE enzyme may comprise at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244S and Q288Y. In embodiments, the aforementioned mutant LE enzyme may comprise an amino acid sequence as set forth in the corresponding SEQ ID NOs in Table B.

[0133] In certain embodiments, mutants of the SBLE enzyme are capable of improved conversion of (acetylated) bola sophorolipids and (acetylated) bola glucolipids into respectively (acetylated) lactonic sophorolipids and (acetylated) lactonic glucolipids while releasing (acetylated) saccharides. More specifically, such (acetylated) saccharides may be (acetylated) sophorose or (acetylated) glucose.

[0134] In certain embodiments, a mutant of the SBLE enzyme is capable of improved conversion of acetylated bola sophorolipids into (acetylated) acidic sophorolipids and (acetylated) sophorose.

[0135] The term ''improved conversion' as used herein refers to an improvement of the transesterification or hydrolysis activity on esters, more specifically on (acetylated and / or non-acetylated) bola amphiphilic glycolipid ester compounds, more specifically the conversion of (acetylated and / or non-acetylated) bola sophorolipids / (acetylated and / or non-acetylated) bola glucolipids into (acetylated and / or non- acetylated) lactonic sophorolipids / (acetylated and / or non-acetylated) lactonic glucolipids respectively or the conversion of (acetylated and / or non-acetylated) bola sophorolipids / (acetylated and / or non- acetylated) bola glucolipids into (acetylated and / or non-acetylated) acidic sophorolipids / (acetylated and / or non-acetylated) acidic glucolipids respectively, or of the intermolecular transesterification activity on bola amphiphilic glycolipid ester compounds and other esters, such as, but not limited to acyl esters, such as mono acyl glycerol esters and di acyl glycerol esters. The improvement may be an improvement with 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more, or an improvement with 60%, 70%, 80%, 90% or more, or even an improvement with 100%, 200%, 300%, 400%, 500% or more, when compared to the transesterification or hydrolysis activity of the original enzyme, or compared to a S. bombicola SBLE enzyme. The term ''improved transesterification' as used herein refers to an improvement of the transesterification activity on esters, more specifically on (acetylated and / or non-acetylated) bola amphiphilic glycolipid ester compounds, more specifically the conversion of (acetylated and / or nonacetylated) bola sophorolipids / (acetylated and / or non-acetylated) bola glucolipids into (acetylated and / or non-acetylated) lactonic sophorolipids / (acetylated and / or non-acetylated) lactonic glucolipids respectively, or of the intermolecular transesterification activity on bola amphiphilic glycolipid ester compounds and other esters, such as, but not limited to acyl esters, such as mono acyl glycerol esters and di acyl glycerol esters. The improvement may be an improvement with 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more, or an improvement with 60%, 70%, 80%, 90% or more, or even an improvement with 100%, 200%, 300%, 400%, 500% or more, when compared to the transesterification activity of the original enzyme, or compared to a S. bombicola SBLE enzyme.

[0136] The term '’decreased transesterification' as used herein refers to a decrease of the transesterification activity on esters, more specifically on (acetylated and / or non-acetylated) bola amphiphilic glycolipid ester compounds, more specifically the conversion of (acetylated and / or non-acetylated) bola sophorolipids / (acetylated and / or non-acetylated) bola glucolipids into (acetylated and / or non- acetylated) lactonic sophorolipids / (acetylated and / or non-acetylated) lactonic glucolipids respectively, or of the intermolecular transesterification activity on bola amphiphilic glycolipid ester compounds and other esters, such as, but not limited to acyl esters, such as mono acyl glycerol esters and di acyl glycerol esters. The decrease may be a decrease with 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more, or a decrease with 60%, 70%, 80%, 90% or more when compared to the transesterification activity of the original enzyme, or compared to a S. bombicola SBLE enzyme.

[0137] The term '’improved hydrolysis' as used herein refers to an improvement of the hydrolysis activity on esters, more specifically on (acetylated and / or non-acetylated) bola amphiphilic glycolipid compounds, more specifically the conversion of (acetylated and / or non-acetylated) bola sophorolipids / (acetylated and / or non-acetylated) bola glucolipids into (acetylated and / or non-acetylated) acidic sophorolipids / (acetylated and / or non-acetylated) acidic glucolipids, respectively. The improvement may be an improvement with 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more, or an improvement with 60%, 70%, 80%, 90% or more, or even an improvement with 100%, 200%, 300%, 400%, 500% or more, when compared to the hydrolysis activity of the original enzyme, or compared to a S. bombicola SBLE enzyme.

[0138] The term 'decreased hydrolysis' as used herein refers to a decrease of the hydrolysis activity on esters, more specifically on (acetylated and / or non-acetylated) bola amphiphilic glycolipid ester compounds, more specifically the conversion of (acetylated and / or non-acetylated) bola sophorolipids / (acetylated and / or non-acetylated) bola glucolipids into (acetylated and / or non-acetylated) acidic sophorol i pids / (acety lated and / or non-acetylated) acidic glucolipids, respectively. The decrease may be a decrease with 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more, or a decrease with

[0139] 60%, 70%, 80%, 90% or more when compared to the hydrolysis activity of the original or wild-type enzyme, in particular the original or wild-type SBLE enzyme, or compared to a S. bombicola SBLE enzyme.

[0140] Suitable assays to determine the hydrolytic activity and transesterification activity are known to the skilled person and involve, for instance, high-performance liquid chromatography (HPLC)-mass spectrometry (MS)analysis or HPLC analysis with CAD detection of the reaction products, for example as detailed in the experimental section. Hydrolytic and transesterification activity may further be determined by calculating peak areas corresponding to the glycolipids of interest (e.g. peak area for acidic glycolipids for hydrolytic activity and peak area for lactonic glycolipids for transesterification activity) as known to the skilled person, e.g. using Chromeleon 7, and normalizing the calculated peak areas by the sum of the peak areas for acidic, lactonic and bola sophorolipids, as detailed in the experimental section.

[0141] Biocatalytic use of mutant SBLE enzymes and homologues of S. bombicola SBLE enzyme

[0142] Moreover, the present invention relates to the use of a mutant of the SBLE enzyme, in particular a mutant LE enzyme with improved hydrolytic activity as described herein, for the improved conversion compared to the wild type SBLE enzyme such as the SBLE enzyme of SEQ ID NO:2 or SEQ ID NO:13 of

[0143] (acetylated and / or non-acetylated) bola sophorolipids into (acetylated and / or non-acetylated) acidic sophorolipids while releasing (acetylated and / or non-acetylated) saccharides. It also relates to the use of a mutant of the SBLE enzyme, in particular a mutant LE enzyme with improved transesterification activity as described herein, more particularly a mutant LE enzyme with improved intramolecular transesterification activity, for the improved conversion compared to the wild type SBLE enzyme such as the SBLE enzyme of SEQ ID NO:2 or SEQ ID NO:13 of (acetylated and / or non-acetylated) bola sophorolipids into (acetylated and / or non-acetylated) lactonic sophorolipids while releasing

[0144] (acetylated and / or non-acetylated) saccharides. Further disclosed herein is use of a mutant of the SBLE enzyme for the improved conversion compared to the wild type SBLE enzyme such as the SBLE enzyme of SEQ. ID NO:2 or SEQ ID NO:13 of (acetylated and / or non-acetylated) bola sophorolipids / (acetylated and / or non-acetylated) bola glucolipids and another ester, preferably an acyl ester, more preferably an acyl glycerol ester such as a mono acyl glycerol ester, a di acyl glycerol ester and / or a tri acyl glycerol ester, preferably a mono acyl glycerol ester and / or a di acyl glycerol ester, into (acetylated and / or non- acetylated) sophorolipid / glucolipid oligomers / polymers / esters while releasing saccharides such as

[0145] (acetylated and / or non-acetylated) sophorose and / or (acetylated and / or non-acetylated) glucose. Moreover, the present invention relates to the use of a mutant of the SBLE enzyme, in particular a mutant LE enzyme with improved hydrolytic activity as described herein, for the improved conversion compared to the wild type SBLE enzyme of (acetylated and / or non-acetylated) bola sophorolipids into (acetylated and / or non-acetylated) acidic sophorolipids and (acetylated and / or non-acetylated) sophorose and / or (acetylated and / or non-acetylated) glucose. It also relates to the use of a mutant of the SBLE enzyme, in particular a mutant LE enzyme with improved transesterification activity as described herein, for the improved conversion compared to the wild type SBLE enzyme of (acetylated and / or non-acetylated) bola sophorolipids into (acetylated and / or non-acetylated) lactonic sophorolipids while releasing (acetylated and / or non-acetylated) sophorose and / or (acetylated and / or non-acetylated) glucose.

[0146] In certain embodiments of the aforementioned uses, the bola sophorolipids are acetylated bola sophorolipids. In certain embodiments of the aforementioned uses, the bola sophorolipids are non- acetylated bola sophorolipids.

[0147] A related aspect is directed to a method for the production of (acetylated and / or non-acetylated) acidic glycolipids, preferably (acetylated and / or non-acetylated) acidic sophorolipids, said method comprising contacting (acetylated and / or non-acetylated) bola glycolipids, preferably (acetylated and / or non-acetylated) bola sophorolipids, with a mutant LE enzyme as described herein, in particular a mutant LE enzyme with improved hydrolytic activity as described herein. Also provided herein is a method for the production of (acetylated and / or non-acetylated) lactonic glycolipids, preferably (acetylated and / or non-acetylated) lactonic sophorolipids, said method comprising contacting (acetylated and / or non-acetylated) bola glycolipids, preferably (acetylated and / or non-acetylated) bola sophorolipids, with a mutant LE enzyme as described herein, in particular a mutant LE enzyme with improved transesterification activity as described herein, more particularly a mutant LE enzyme with improved intramolecular transesterification activity. Also provided herein is a method for the production of (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters, preferably (acetylated and / or non-acetylated) sophorolipid oligomers / polymers / esters, said method comprising contacting (acetylated and / or non-acetylated) bola glycolipids, preferably (acetylated and / or non- acetylated) bola sophorolipids, and one or more other esters, such as, but not limited to acyl esters, preferably acyl glycerol esters such as mono acyl glycerol esters, di acyl glycerol esters and / or tri acyl glycerol esters, preferably mono acyl glycerol esters and / or di acyl glycerol esters, with a mutant LE enzyme as described herein.

[0148] In certain embodiments of the aforementioned methods, the bola glycolipids are acetylated bola glycolipids. In certain embodiments of the aforementioned methods, the bola glycolipids are non- acetylated bola glycolipids. A further aspect relates to use of a homologue of S. bombicola SBLE enzyme as described herein, in particular a Starmerella batistae LE enzyme, a Starmerella kuoi LE enzyme or a Starmerella riodocensis LE enzyme, for the production of (acetylated and / or non-acetylated) acidic glycolipids such as (acetylated and / or non-acetylated) acidic sophorolipids, in particular for the conversion of (acetylated and / or non-acetylated) bola sophorolipids into (acetylated and / or non-acetylated) acidic sophorolipids while releasing (acetylated and / or non-acetylated) saccharides such as (acetylated and / or non-acetylated) sophorose and / or (acetylated and / or non-acetylated) glucose. Another aspect relates to use of a homologue of S. bombicola SBLE enzyme as described herein, in particular a Starmerella batistae LE enzyme, a Starmerella kuoi LE enzyme or a Starmerella riodocensis LE enzyme, for the production of (acetylated and / or non-acetylated) lactonic glycolipids such as (acetylated and / or non-acetylated) lactonic sophorolipids, in particular for the conversion of (acetylated and / or non-acetylated) bola sophorolipids into (acetylated and / or non-acetylated) lactonic sophorolipids while releasing (acetylated and / or non-acetylated) saccharides such as (acetylated and / or non- acetylated) sophorose and / or (acetylated and / or non-acetylated) glucose. Further disclosed herein is use of a homologue of S. bombicola SBLE enzyme as described herein, in particular a Starmerella batistae LE enzyme, a Starmerella kuoi LE enzyme or a Starmerella riodocensis LE enzyme, for the production of (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters such as (acetylated and / or non-acetylated) sophorolipid / glucolipid oligomers / polymers / esters, in particular for the conversion of (acetylated and / or non-acetylated) bola sophorolipids / (acetylated and / or non- acetylated) bola glucolipids and one or more other esters, such as, but not limited to acyl esters, preferably acyl glycerol esters such as mono acyl glycerol esters, di acyl glycerol esters and / or tri acyl glycerol esters, preferably mono acyl glycerol esters di acyl glycerol esters, into (acetylated and / or non-acetylated) sophorolipid / glucolipid oligomers / polymers / esters while releasing (acetylated and / or non-acetylated) saccharides.

[0149] A related aspect is directed to a method for the production of (acetylated and / or non-acetylated) acidic glycolipids, preferably (acetylated and / or non-acetylated) acidic sophorolipids, said method comprising contacting (acetylated and / or non-acetylated) bola glycolipids, preferably (acetylated and / or non-acetylated) bola sophorolipids, with a homologue of S. bombicola SBLE enzyme as described herein, in particular a Starmerella batistae LE enzyme, a Starmerella kuoi LE enzyme or a Starmerella riodocensis LE enzyme. Also provided herein is a method for the production of (acetylated and / or non-acetylated) lactonic glycolipids, preferably (acetylated and / or non-acetylated) lactonic sophorolipids, said method comprising contacting (acetylated and / or non-acetylated) bola glycolipids, preferably (acetylated and / or non-acetylated) bola sophorolipids, with a homologue of S. bombicola SBLE enzyme as described herein, in particular a Starmerella batistae LE enzyme, a Starmerella kuoi LE enzyme or a Starmerella riodocensis LE enzyme. Also provided herein is a method for the production of (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters, preferably (acetylated and / or non-acetylated) sophorolipid oligomers / polymers / esters, said method comprising contacting (acetylated and / or non-acetylated) bola glycolipids, preferably (acetylated and / or non- acetylated) bola sophorolipids, and one or more other esters, such as, but not limited to acyl esters, such as mono acyl glycerol esters and di acyl glycerol esters, with a homologue of S. bombicola SBLE enzyme as described herein, in particular a Starmerella batistae LE enzyme, a Starmerella kuoi LE enzyme or a Starmerella riodocensis LE enzyme.

[0150] In certain embodiments of the aforementioned uses and methods, the bola sophorolipids are acetylated bola sophorolipids. In certain embodiments of the aforementioned methods, the bola sophorolipids are non-acetylated bola sophorolipids.

[0151] The mutant LE enzyme or the homologue of S. bombicola SBLE enzyme used herein may be a recombinantly produced and optionally purified LE enzyme.

[0152] A mutant LE enzyme may be prepared, for example, by introducing a mutation as described herein into a gene or cDNA coding for a wild-type LE enzyme as described herein through oligonucleotide sitespecific mutagenesis as known to the skilled person, and expressing said mutant gene or cDNA in a suitable host cell as known to the skilled person. For example, the gene or cDNA encoding a mutant LE enzyme as described herein can be cloned into a nucleic acid expression cassette, which can be transfected into a host cell for (site-directed) insertion into the genome of the host cell via genomic recombination.

[0153] A homologue of S. bombicola SBLE enzyme may be produced, for example, by (over)expressing the homologue of S. bombicola SBLE enzyme in a suitable host cell as known to the skilled person. For example, a gene or cDNA encoding the homologue of S. bombicola SBLE enzyme can be cloned into a nucleic acid expression cassette, which can be transfected into a host cell for (site-directed) insertion into the genome of the host cell via genomic recombination.

[0154] As used herein, the term "nucleic acid expression cassette" refers to nucleic acid molecules that contain a desired coding sequence, such as a gene or cDNA encoding a mutant LE enzyme or an S. bombicola SBLE enzyme homologue as described herein, and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host cell. The term "operably linked" as used herein refers to the arrangement of the various nucleic acid molecule elements relative to each such that the elements are functionally connected and are able to interact with each other. Regulatory sequences are selected to direct the expression of the coding sequence in the host cell, and include promoters, enhancers, and other expression control elements as known to the skilled person. For example, a nucleic acid expression cassette as used herein may comprise a nucleic acid molecule comprising a coding sequence, such as a gene or cDNA encoding a mutant LE enzyme or an S. bombicola SBLE enzyme homologue as described herein, a promoter for driving expression of the coding sequence, and a nucleic acid sequence encoding a terminator. A nucleic acid expression cassette as used herein may further comprise specific host cell sequences for genomic recombination.

[0155] Suitable host cells for recombinant production of a mutant LE enzyme or a S. bombicola SBLE enzyme homologue include, without limitation, bacterial cells, yeast cells, fungal cells, plant cells and animal cells. Suitable bacterial host cells include, without limitation, Escherichia spp. cells, Bacillus spp. cells, Streptomyces spp. cells, Erwinia spp. cells, Klebsiella spp. cells, Serratia spp. cells, Pseudomonas spp. cells, and Salmonella spp. cells. Suitable yeast host cells include species within Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g. Pichia pastoris), Hansenula (e.g. Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, Zygosaccharomyces and the like. Saccharomyces cerevisiae, S. carlsbergensis and K. lactis are non-limiting examples of yeast hosts. Animal host cells suitable for use with the invention include insect cells and mammalian cells (e.g. derived from Chinese hamster (e.g. CHO), and human cell lines, such as HeLa). Exemplary insect cell lines include, but are not limited to, Sf9 cells, baculovirus-insect cell systems.

[0156] The recombinant host cell can then be cultured under conditions sufficient to allow (over)expression of the coding sequence. Cell-free extracts can then be generated using known methods such as by lysing the host cells using detergents or by sonication. The LE enzymes can be isolated or purified from the cell-free extracts using known methods, or the cell-free extracts can be used as such. Alternatively, a secretion signal sequence can be operably linked to the coding sequence. The secreted LE enzymes can then be separated from the culture medium and optionally be purified using known methods without the need for obtaining cell-free extracts.

[0157] Next, (acetylated and / or non-acetylated) bola glycolipids such as (acetylated and / or non-acetylated) bola sophorolipids and optionally other esters, such as, but not limited to acyl esters, preferably acyl glycerol esters such as mono acyl glycerol esters, di acyl glycerol esters and / or tri acyl glycerol esters, preferably mono acyl glycerol esters and / or di acyl glycerol esters, can be added to the cell-free extracts or (optionally purified) mutant LE enzymes or S. bombicola SBLE enzyme homologues and maintained under conditions to allow hydrolysis or transesterification of the (acetylated and / or non- acetylated) bola glycolipids or transesterification of the (acetylated and / or non-acetylated) bola glycolipids and optionally the other esters, to produce respectively, (acetylated and / or non-acetylated) acidic glycolipids and (acetylated and / or non-acetylated) saccharides, (acetylated and / or non- acetylated) lactonic glycolipids and (acetylated and / or non-acetylated) saccharides, or (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters and (acetylated and / or non-acetylated) saccharides. (Acetylated and / or non-acetylated) bola glycolipids such as (acetylated and / or nonacetylated) bola sophorolipids can, for example, be produced according to the method described in WO2023213677, in particular using a naturally sophorolipid producing yeast strain such as Starmerella bombicola that has been genetically modified to have a nonfunctional SLBE enzyme or homologue thereof, and optionally a nonfunctional glucosyltransferase that is responsible for the second glucosylation step in the sophorolipid biosynthetic pathway UGTB1 or a homologue thereof or a nonfunctional acetyltransferase enzymes Atl, At2 or At3 that is responsible for the acetylation of glycolipids or a homologue thereof. A non-limiting example of an acyl ester, in particular an acyl glycerol ester, is rapeseed oil.

[0158] The (acetylated and / or non-acetylated) bola glycolipids may be incubated with the cell-free extracts or (purified) mutant LE enzymes or S. bombicola SBLE enzyme homologues for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22 or 24, 48 hours, preferably for between about 1 hour and about 48 hours, more preferably for between about 1 hour and about 24 hours, such as for about 1, 2, 4, 8, 12 or 24 hours, at a temperature from 20°C to 33°C, preferably from 25°C to 30°C, more preferably from 28°C to 30°C such as at about 30°C. During incubation the mixture may be agitated or shaked.

[0159] The produced (acetylated and / or non-acetylated) acidic glycolipids, (acetylated and / or non- acetylated) lactonic glycolipids or (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters can then optionally be separated and purified using known techniques.

[0160] Modified yeast strains

[0161] A further aspect of the invention is directed to a recombinant or genetically modified yeast strain comprising a mutant LE enzyme as described herein or a homologue of S. bombicola SBLE enzyme as described herein. The term 'a modified yeast strain' as used herein relates to a yeast strain modified in any way so that it comprises a gene or cDNA encoding a mutant SBLE enzyme or a homologue of S. bombicola SBLE enzyme, such as a Starmerella batistae SBLE enzyme, a Starmerella kuoi SBLE enzyme or a Starmerella riodocensis SBLE enzyme as described herein. The term 'a modified yeast strain' also relates to a yeast strain modified in any way so that it comprises a gene or cDNA encoding a mutant SBLE enzyme or a homologue of S. bombicola SBLE enzyme and (a) dysfunctional Atl, At2 and / or At3 enzyme(s). The term 'a modified yeast strain' also relates to a yeast strain modified in any way so that it comprises a gene or cDNA encoding a mutant SBLE enzyme or a homologue of S. bombicola SBLE enzyme and a gene or cDNA encoding Starmerella kuoi CYP52M1 cytochrome oxidase such as the Starmerella kuoi CYP52M1 cytochrome oxidase of SEQ. ID NO: 171.

[0162] In preferred embodiments, the modified yeast strain is a naturally sophorolipid producing yeast strain, more preferably a yeast strain selected from the group consisting of Starmerella bombicola (previously Candida) (Spencer et al., 1970), Starmerella apicola (Gorin et al., 1961) (previously Candida), which was initially identified as T. magnolia, Wickerhamiella domericqiae (Chen et al., 2006), Pseudohyphozyma bogoriensis sp. (previously Rhodotorula or Candida) (Tulloch et al., 1968), Starmerella batistae (Konishi et al., 2008) (previously Candida), Starmerella (previously Candida) floricola (Imura et al., 2010), Starmerella (previously Candida) batistae, Candida riodocensis, Candida tropicalis, Starmerella stellata (previously Candida) and Candida sp. NRRL Y-27208 (Kurtzman et al., 2010), Starmerella kuoi (Kurtzman, 2012) (previously Candida), Starmerella powelii (previously Candida), Candida gropengiesseri, Candida magnoliae, Candida antarctica, Pseudozyma antarctica, Candida tropicalis, Candida lipolytica and any other SL producing strain (of the Wickerhamiella / Starmerella clade (W / S clade) as defined by Goncalves et al. (2020) or the Starmerella clade) or the group consisting of Starmerella bombicola (previously Candida) (Spencer et al., 1970), Starmerella apicola (Gorin et al., 1961) (previously Candida), which was initially identified as T. magnolia, Wickerhamiella domericqiae (Chen et al., 2006), Pseudohyphozyma bogoriensis sp. (previously Rhodotorula or Candida) (Tulloch et al., 1968), Starmerella batistae (Konishi et al., 2008) (previously Candida), Starmerella (previously Candida) floricola (Imura et al., 2010), Starmerella (previously Candida) batistae, Candida riodocensis, Candida tropicalis, Starmerella stellata (previously Candida) and Candida sp. NRRL Y-27208 (Kurtzman et al., 2010), Starmerella kuoi (Kurtzman, 2012) (previously Candida), Candida gropengiesseri, Candida magnoliae, Candida antarctica, Pseudozyma antarctica, Candida tropicalis, Candida lipolytica and any other SL producing strain (of the Starmerella clade). In particular embodiments, the modified yeast strain is a strain belonging to a genus selected from the group comprising or consisting of Wickerhamiella and Starmerella, in particular a strain belonging to a species selected from the group comprising or consisting of Wickerhamiella infanticola, Wickerhamiella sorbophila, Wickerhamiella galacta, Wickerhamiella hasegawae, Wickerhamiella pararugosa, Wickerhamiella cacticola, Wickerhamiella occidentalis, Wickerhamiella versatilis, Wickerhamiella domercqiae, Starmerella gropengiesseri, Starmerella tilneyi, Starmerella geochares, Starmerella vaccinii, Starmerella magnoliae, Starmerella sorbosivorans, Starmerella bacillaris, Starmerella davenportii, Starmerella apicola, Starmerella ratchasimensis, Starmerella riodocensis, Starmerella kuoi and Starmerella bombicola. In particular embodiments, the modified yeast strain is a strain belonging to the Starmerella genus. In further particular embodiments, the modified yeast strain is a Starmerella bombicola strain.

[0163] In particular embodiments, the modified yeast strain may be genetically modified by introducing one or more mutations as described herein into an endogenous LE encoding gene of the yeast strain. In other embodiments, the yeast strain may be genetically modified by transforming the yeast strain with a recombinant nucleic acid encoding a mutant LE enzyme as described herein or a homologue of S. bombicola SBLE enzyme as described herein such as a nucleic acid expression cassette described above. In further embodiments, the modified yeast strain no longer contains an endogenous (unmutated) SBLE gene. The genetically modified yeast strain may comprise further genetic modifications, such as, e.g., a dysfunctional or non-functional acetyltransferase enzyme, preferably a dysfunctional or non-functional acetyltransferase enzyme Atl, At2 and / or At3 as described elsewhere herein, and / or a gene or cDNA encoding Starmerella kuoi CYP52M1 cytochrome oxidase.

[0164] In general, methods for generating a modified yeast strain as described herein involve standard genetic modifications, for which well-established methods are available to the skilled person. For example, genetic engineering of a yeast strain containing a recombinant nucleic acid encoding a mutant SBLE enzyme or a homologue of S. bombicola SBLE enzyme as taught herein may be accomplished in one or more steps via the design and construction of appropriate nucleic acid expression cassettes, such as a nucleic acid expression as described elsewhere herein, or vectors containing such nucleic acid expression cassettes, and transfection or transformation of the yeast strain with those nucleic acid expression cassettes or vectors, followed by the selection of a yeast strain capable of expressing the mutant LE enzyme or the homologue of S. bombicola SBLE enzyme. The nucleic acid expression cassette may be inserted into the genome of the yeast strain via homologous recombination or genomic recombination.

[0165] Suitable genetic engineering methods for introducing a mutation in an endogenous gene are known to the skilled person, including, for example, but not limited to, using so-called molecular scissors (nucleases) (e.g. TALEN, CRISPR / Cas9 and the like), or by using vectors containing specific sequences for homologous recombination and site-directed insertion.

[0166] In addition, methods for transfecting or transforming yeasts are well known to a skilled person and any such method can be used herein. For example, electroporation and / or chemical (such as calcium chloride- or lithium acetate-based) transformation methods as known in the art can be used. For example, electroporation may be conducted as detailed in the Examples section.

[0167] Numerous vectors are known to practitioners skilled in the art and any such vector may be used; selection of an appropriate vector is a matter of choice. Typically, a vector may comprise a nucleic acid expression cassette as described elsewhere herein and may further contain restriction sites of various types for linearization or fragmentation. The vector may further include an origin of replication that is required for maintenance and / or replication in a specific cell type or types.

[0168] Nucleic acid expression cassettes and vectors as used herein also preferably contain one or more selection marker gene cassettes. A selectable marker gene cassette typically includes a promoter and transcription terminator sequence, operatively linked to a selectable marker gene. Suitable markers may be selected from markers that confer antibiotic resistance or markers that complement auxotrophic deficiencies of a host cell, in particular a yeast as described herein. For example, the selection marker may confer resistance to an antibiotic such as hygromycin or hygromycin B (such as the hyg or hygB gene), zeocin / phleomycin (such as the ble gene), kanamycin or G418 (such as the nptll or aphVIII genes), spectinomycin (such as the aadA gene), neomycin (such as the aphVIII gene), blasticidin (such as the bsd gene), nourseothricin (such as the NAT1 gene), puromycin (such as pac gene) and paromomycin (such as the aphVIII gene). An example of auxotrophic deficiency is uracil deficiency (e.g. URA3 gene). Cells that are orotidine-5'-phosphate decarboxylase negative (ura3-) cannot grow on media lacking uracil. Thus a functional URA3 gene can be used as a selection marker on a host cell having a uracil deficiency, and successful transformants can be selected on a medium lacking uracil. Only cells transformed with the functional URA3 gene are able to synthesize uracil and grow on such medium. If the wild-type strain does not have a uracil deficiency, an auxotrophic mutant having the deficiency must be made in order to use URA3 as a selection marker for the strain. Methods for accomplishing this are well known in the art.

[0169] Successful transformants can be selected for in known manner, by taking advantage of the attributes contributed by the marker gene, or by other characteristics contributed by the introduced recombinant nucleic acid. Screening can also be performed by PCR or Southern analysis to confirm that the desired modifications have taken place, to confirm copy number and to identify the point of integration of coding sequences into the host genome.

[0170] In embodiments, the modified yeast strain is further genetically engineered to comprise a dysfunctional acetyltransferase enzyme, preferably a dysfunctional acetyltransferase enzyme Atl, At2 and / or At3, more preferably a dysfunctional acetyltransferase enzyme Atl, even more preferably a dysfunctional acetyltransferase enzyme Atl, At2 and At3. In further embodiments, the modified yeast strain is further genetically engineered to comprise a non-functional acetyltransferase enzyme, preferably a non-functional acetyltransferase enzyme Atl, At2 and / or At3, more preferably a nonfunctional acetyltransferase enzyme Atl, even more preferably a non-functional acetyltransferase enzyme Atl, At2 and At3.

[0171] In embodiments, the modified yeast strain is further genetically engineered to comprise a gene or cDNA encoding Starmerella kuoi CYP52M1 cytochrome oxidase such as the Starmerella kuoi CYP52M1 cytochrome oxidase of SEQ. ID NO: 171, optionally said modified yeast strain comprises a dysfunctional or non-functional Starmerella bombicola CYP52M1 cytochrome oxidase.

[0172] Uses of modified strains The present invention further relates to the finding that yeasts strains which comprise a gene or cDNA encoding for a mutant SBLE enzyme, in particular a mutant LE enzyme with improved hydrolytic activity as described herein, produce acidic sophorolipids more uniformly. In addition, the invention further discloses a method to produce (acetylated and / or non-acetylated) acidic glycolipids, in particular (acetylated and / or non-acetylated) acidic sophorolipids, more uniformly via modifying yeast strains so they comprise a gene or cDNA encoding for a mutant of the SBLE enzyme, in particular a mutant LE enzyme with improved hydrolytic activity as described herein. A further aspect provides for use of a modified yeast strain comprising a mutant LE enzyme, in particular a mutant LE enzyme with improved hydrolytic activity, for the production of (acetylated and / or non-acetylated) acidic glycolipids, in particular (acetylated and / or non-acetylated) acidic sophorolipids. A related aspect provides a method for the production of (acetylated and / or non-acetylated) acidic glycolipids, said method comprising culturing a modified yeast strain comprising a mutant LE enzyme, in particular a mutant LE enzyme with improved hydrolytic activity, in a culture medium so as to allow the production of (acetylated and / or non-acetylated) acidic glycolipids, in particular (acetylated and / or non-acetylated) acidic sophorolipids, wherein said modified yeast strain has been genetically engineered to (over)express a gene or cDNA encoding a mutant LE enzyme, in particular a mutant LE enzyme with improved hydrolytic activity as disclosed herein.

[0173] The present invention also relates to the surprising finding that yeasts strains which comprise a gene or cDNA encoding for a mutant SBLE enzyme, in particular a mutant LE enzyme with improved transesterification activity, produce lactonic sophorolipids more uniformly. In addition, the invention further discloses a method to produce (acetylated and / or non-acetylated) lactonic glycolipids, in particular (acetylated and / or non-acetylated) lactonic sophorolipids, more uniformly via modifying yeast strains so they comprise a gene or cDNA encoding for a mutant of the SBLE enzyme, in particular a mutant LE enzyme with improved transesterification activity as described herein. A further aspect provides for use of a modified yeast strain comprising a mutant LE enzyme, in particular a mutant LE enzyme with improved transesterification activity, for the production of (acetylated and / or non- acetylated) lactonic glycolipids, in particular (acetylated and / or non-acetylated) lactonic sophorolipids. A related aspect provides a method for the production of (acetylated and / or non-acetylated) lactonic glycolipids, said method comprising culturing a modified yeast strain comprising a mutant LE enzyme, in particular a mutant LE enzyme with improved transesterification activity as described herein in a culture medium so as to allow the production of (acetylated and / or non-acetylated) lactonic glycolipids, in particular (acetylated and / or non-acetylated) lactonic sophorolipids, wherein said modified yeast strain has been genetically engineered to (over)express a gene or cDNA encoding a mutant LE enzyme, in particular a mutant LE enzyme with improved transesterification activity as described herein.

[0174] Further disclosed herein is use of a modified yeast strain comprising a mutant LE enzyme as described herein for the production of (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters, in particular (acetylated and / or non-acetylated) sophorolipid oligomers / polymers / esters. Further disclosed herein is a method for the production of (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters, in particular (acetylated and / or non-acetylated) sophorolipid oligomers / polymers / esters, said method comprising culturing a modified yeast strain comprising a mutant LE enzyme as described herein in a culture medium so as to allow the production of (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters, in particular (acetylated and / or non- acetylated) sophorolipid oligomers / polymers / esters, wherein said modified yeast strain has been genetically engineered to (over)express a gene or cDNA encoding a mutant LE enzyme as described herein.

[0175] The term 'more uniformly' refers to a broth being produced which comprises a higher amount of a certain glycolipid, sophorolipid or glucolipid type, such as, but not limited to, one or more acidic glycolipids, sophorolipids or glucolipids, or one or more lactonic glycolipids, sophorolipids or glucolipids, or one or more glycolipid, sophorolipid or glucolopid oligomers / polymers / esters, compared to a broth produced by a wild type Starmerella bombicola or a yeast strain that has been genetically engineered to comprise a gene or cDNA encoding a wild type Starmerella bombicola SBLE enzyme.

[0176] The invention further provides for use of a modified yeast strain comprising a homologue of S. bombicola SBLE enzyme as described herein for the production of (acetylated and / or non-acetylated) acidic glycolipids, preferably (acetylated and / or non-acetylated) acidic sophorolipids. The invention also provides a method to produce (acetylated and / or non-acetylated) acidic glycolipids, in particular (acetylated and / or non-acetylated) acidic sophorolipids, via modifying a yeast strain so that it comprises a gene or cDNA encoding a homologue of S. bombicola SBLE enzyme as described herein. A further aspect provides a method for the production of (acetylated and / or non-acetylated) acidic glycolipids, preferably (acetylated and / or non-acetylated) acidic sophorolipids, said method comprising culturing a modified yeast strain comprising a homologue of S. bombicola SBLE enzyme as described herein, in a culture medium so as to allow the production of (acetylated and / or non- acetylated) acidic glycolipids, in particular (acetylated and / or non-acetylated) acidic sophorolipids, wherein said modified yeast strain has been genetically engineered to (over)express a gene or cDNA encoding a homologue of S. bombicola SBLE enzyme as described herein. The invention further provides for the use of a modified yeast strain comprising a homologue of S. bombicola SBLE enzyme as described herein for the production of (acetylated and / or non-acetylated) lactonic glycolipids, preferably (acetylated and / or non-acetylated) lactonic sophorolipids. The invention also provides a method to produce (acetylated and / or non-acetylated) lactonic glycolipids, in particular (acetylated and / or non-acetylated) lactonic sophorolipids, via modifying a yeast strain so that it comprises a gene or cDNA encodinga homologue of S. bombicola SBLE enzyme as described herein. A further aspect provides a method for the production of (acetylated and / or non-acetylated) lactonic glycolipids, preferably (acetylated and / or non-acetylated) lactonic sophorolipids, said method comprising culturing a modified yeast strain comprising a homologue of S. bombicola SBLE enzyme as described herein in a culture medium so as to allow the production of (acetylated and / or non- acetylated) lactonic glycolipids, in particular (acetylated and / or non-acetylated) lactonic sophorolipids, wherein said modified yeast strain has been genetically engineered to (over)express a gene or cDNA encoding a homologue of S. bombicola SBLE enzyme as described herein.

[0177] Further disclosed herein is use of a modified yeast strain comprising a homologue of S. bombicola SBLE enzyme as described herein for the production of (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters, in particular (acetylated and / or non-acetylated) sophorolipid oligomers / polymers / esters. Also disclosed herein is a method for the production of (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters, in particular (acetylated and / or non-acetylated) sophorolipid oligomers / polymers / esters, said method comprising culturing a modified yeast strain comprising a homologue of S. bombicola SBLE enzyme as described herein in a culture medium so as to allow the production of (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters, in particular (acetylated and / or non-acetylated) sophorolipid oligomers / polymers / esters, wherein said modified yeast strain has been genetically engineered to (over)express a gene or cDNA encoding a homologue of S. bombicola SBLE enzyme as described herein.

[0178] Starmerella batistae SBLE enzyme and uses thereof

[0179] In particular embodiments, the present invention provides for the use of a modified yeast strain which comprise a gene or cDNA encoding for the Starmerella batistae SBLE enzyme, or a variant or (functional) fragment thereof, to produce acidic sophorolipids. In embodiments, the invention discloses a method to produce acidic sophorolipids via modifying yeast strains so they comprise a gene or cDNA encoding for the Starmerella batistae SBLE enzyme, or a variant or (functional) fragment thereof. Embodiments also relate to the use of a modified yeast strain which comprises a gene or cDNA encoding for the Starmerella batistae SBLE enzyme, or a variant or (functional) fragment thereof, to produce mono-acetylated or di-acetylated acidic sophorolipids. Further embodiments are directed to a method to produce mono-acetylated or di-acetylated acidic sophorolipids via modifying yeast strains so they comprise a gene or cDNA encoding for the Starmerella batistae SBLE enzyme, or a variant or (functional) fragment thereof. In further embodiments of the aforementioned methods and uses, the modified yeast strain comprising a gene or cDNA encoding a Starmerella batistae LE enzyme has further been genetically modified to comprise a gene or cDNA encoding Starmerella kuoi CYP52M1 cytochrome oxidase such as the Starmerella kuoi CYP52M1 cytochrome oxidase of SEQ. ID NO: 171, and optionally to comprise a dysfunctional or non-functional Starmerella bombicola CYP52M1 cytochrome oxidase.

[0180] In particular embodiments, the present invention provides for the use of a modified yeast strain which comprise a gene or cDNA encoding for the Starmerella batistae SBLE enzyme, or a variant or (functional) fragment thereof, and a dysfunctional or non-functional acetyltransferase, in particular a dysfunctional or non-functional acetyltransferase enzyme Atl, At2 and / or At3, more particularly a dysfunctional or non-functional acetyltransferase enzyme Atl and At 2, or dysfunctional or nonfunctional acetyltransferase enzyme Atl, At2 and At3 to produce non-acetylated acidic sophorolipids. In embodiments, the invention discloses a method to produce acidic sophorolipids via modifying yeast strains so they comprise a gene or cDNA encoding for the Starmerella batistae SBLE enzyme, or a variant or (functional) fragment thereof, and a dysfunctional or non-functional acetyltransferase, in particular a dysfunctional or non-functional acetyltransferase enzyme Atl, At2 and / or At3, more particularly a dysfunctional or non-functional acetyltransferase enzyme Atl and At 2, or dysfunctional or non-functional acetyltransferase enzyme Atl, At2 and At3.

[0181] Embodiments are provided herein related to the use of a modified yeast strain which comprises a gene or cDNA encoding for the Starmerella batistae SBLE enzyme, or variant or (functional) fragment thereof, to produce oligomeric- and / or polymeric sophorolipid molecules and / or oligomeric and / or polymeric esters composed of sophorolipids and esters, such as, but not limited to acyl esters such as mono-acyl glycerol esters (MAG esters) and di-acyl glycerol esters (DAG esters).

[0182] Starmerella kuoi SBLE enzyme and uses thereof

[0183] In particular embodiments, the present invention provides for the use of a modified yeast strain which comprise a gene or cDNA encoding for a Starmerella kuoi LE enzyme to produce acidic sophorolipids. In embodiments, the invention discloses a method to produce acidic sophorolipids via modifying yeast strains so they comprise a gene or cDNA encoding for a Starmerella kuoi LE enzyme. Embodiments also relate to the use of a modified yeast strain which comprises a gene or cDNA encoding for a Starmerella kuoi LE enzyme to produce mono-acetylated or di-acetylated acidic sophorolipids. Further embodiments are directed to a method to produce mono-acetylated or di-acetylated acidic sophorolipids via modifying a yeast strain so that it comprise a gene or cDNA encoding for a Starmerella kuoi LE enzyme. In further embodiments of the aforementioned methods and uses, the modified yeast strain comprising a gene or cDNA encoding a Starmerella kuoi LE enzyme has further been genetically modified to comprise a gene or cDNA encoding Starmerella kuoi CYP52M1 cytochrome oxidase such as the Starmerella kuoi CYP52M1 cytochrome oxidase of SEQ. ID NO: 171, and optionally to comprise a dysfunctional or non-functional Starmerella bombicola CYP52M1 cytochrome oxidase.

[0184] In particular embodiments, the present invention provides for the use of a modified yeast strain which comprise a gene or cDNA encoding for the Starmerella kuoi SBLE enzyme, or a variant or (functional) fragment thereof, and a dysfunctional or non-functional acetyltransferase, in particular a dysfunctional or non-functional acetyltransferase enzyme Atl, At2 and / or At3, more particularly a dysfunctional or non-functional acetyltransferase enzyme Atl and At2, or dysfunctional or non-functional acetyltransferase enzyme Atl, At2 and At3 to produce non-acetylated acidic sophorolipids. In embodiments, the invention discloses a method to produce acidic sophorolipids via modifying yeast strains so they comprise a gene or cDNA encoding for the Starmerella kuoi SBLE enzyme, or a variant or (functional) fragment thereof, and a dysfunctional or non-functional acetyltransferase, in particular a dysfunctional or non-functional acetyltransferase enzyme Atl, At2 and / or At3, more particularly a dysfunctional or non-functional acetyltransferase enzyme Atl and At2, or dysfunctional or nonfunctional acetyltransferase enzyme Atl, At2 and At3.

[0185] Particular embodiments relate to the use of a modified yeast strain which comprises a gene or cDNA encoding for the Starmerella kuoi SBLE enzyme, or a variant or (functional) fragment thereof, to produce oligomeric and / or polymeric sophorolipid molecules and / or oligomeric and / or polymeric esters composed of sophorolipids and esters, such as, but not limited to acyl esters such mono-acyl glycerol esters (MAG esters) and di-acyl glycerol esters (DAG esters).

[0186] Starmerella riodocensis LE enzyme and uses thereof

[0187] In particular embodiments, the present invention provides for the use of a modified yeast strain which comprise a gene or cDNA encoding for a Starmerella riodocensis LE enzyme to produce acidic sophorolipids, in particular mono-acetylated or di-acetylated acidic sophorolipids. In embodiments, the invention discloses a method to produce acidic sophorolipids, in particular mono-acetylated or diacetylated acidic sophorolipids, via modifying yeast strains so they comprise a gene or cDNA encoding for a Starmerella riodocensis LE enzyme.

[0188] The modified yeast strains as described herein are cultured under conditions suitable for the production of (acetylated and / or non-acetylated) acidic glycolipids, (acetylated and / or nonacetylated) lactonic glycolipids and / or (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters by the modified yeast strains. In particular this implies "conditions sufficient to allow (over)expression" of the gene or cDNA encoding the mutant LE enzyme or the homologue of S. bombicola SBLE enzyme, which means any condition that allows the yeast strain to (over)produce the mutant LE enzyme or the homologue of S. bombicola SBLE enzyme.

[0189] The conditions suitable for the production of (acetylated and / or non-acetylated) acidic glycolipids, (acetylated and / or non-acetylated) lactonic glycolipids or (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters, may involve conventional culture conditions suitable for growth of the yeast strain as known to the skilled person. Culturing may be carried out under aerobic conditions, and general methods such as agitated culture and shaking culture can be applied. The culturing temperature may be from 20°C to 33°C, preferably from 25°C to 30°C, more preferably from 28°C to 30°C such as at about 30°C. The culturing time may be for at least 1, 2, 3, 4, 5 or 6 days, preferably for at least 7, 8, 9 or 10 days such as for about 240 hours, or for at least 15, 20, 25 or 30 days such as for 1, 2 or 3 months or more. In particular embodiments, cultivating the yeast strain may be in a suitable culture medium for at least 1, 2, 3, 4, 5 or 6 days, preferably for at least 7, 8 or 9 days, more preferably for about 10 days or more, at a temperature of about 30°C.

[0190] Suitable culture media include any culture medium suitable for growth of the yeast strain as known to the skilled person and may include any conventional medium containing a carbon source, a nitrogen source, an inorganic salt, and if necessary, organic trace nutrients such as amino acids and vitamins. The carbon source and the nitrogen source included in the culture medium may be any type of material that can be metabolized by the yeast strain to be cultured. Exemplary, non-limiting, carbon sources include carbohydrates such as glucose, glycerol, fructose, sucrose, maltose, mannose, galactose, starch hydrolysate, and malt; organic acids such as acetic acid and citric acid; and alcohols such as ethanol. These carbon sources can be used singly or in combination of two or more kinds thereof. Examples of the nitrogen source include yeast extracts, ammonia; ammonium salts such as ammonium sulfate, ammonium carbonate, ammonium chloride, ammonium phosphate, and ammonium acetate; and nitrates. The culture medium may be any of a synthetic medium and a natural medium.

[0191] In particular embodiments, the culture medium may comprise one or more of a fatty acid, a fatty acid alkyl ester, an alkane, an alkene, an alkyne, an alcohol, a triacylglycerol, a diacylglycerol, a monoacylglycerol, and a fat or oil.

[0192] In further embodiments, methods are provided for producing (acetylated and / or non-acetylated) acidic glycolipids, (acetylated and / or non-acetylated) lactonic glycolipids or (acetylated and / or non- acetylated) glycolipid oligomers / polymers / esters, which, in addition to the step described above, may further comprise the step of recovering (acetylated and / or non-acetylated) acidic glycolipids, (acetylated and / or non-acetylated) lactonic glycolipids or (acetylated and / or non-acetylated) glycolipid oligomers / polymers / esters from the modified yeast strain or the culture medium. Suitable purification can be carried out by methods known to the person skilled in the art such as by using lysis methods, extraction, ion exchange, electrodialysis, ultrafiltration, nanofiltration, etc.

[0193] The acidic glycolipids, lactonic glycolipids or glycolipid oligomers / polymers / esters produced by the methods and uses disclosed herein can be acetylated, non-acetylated or a mixture of acetylated and non-acetylated acidic glycolipids, lactonic glycolipids or glycolipid oligomers / polymers / esters. Acetylated acidic glycolipids, lactonic glycolipids and glycolipid oligomers / polymers / esters may have an acetylation degree of 1, 2, 3, 4 or more. With the term 'an acetylation degree' is meant the number of glucose moieties present in the glycolipids, sophorolipids or glucolipids that are acetylated.

[0194] The present invention is also based on the surprising finding that yeasts strains which comprise a gene encoding for such a mutant SBLE enzyme as described herein, in particular a mutant LE enzyme with improved hydrolytic activity as described herein, and which comprise a dysfunctional acetyltransferase enzyme Atl, At2 and / or At3 produce non-acetylated acidic sophorolipids more uniformly. In addition, the invention further discloses a method to produce non-acetylated acidic sophorolipids more uniformly via rendering acetyltransferase enzymes Atl, At2 and / or At3 dysfunctional in yeasts strains which comprise a gene encoding for such a mutant SBLE enzyme. The present invention is also based on the surprising finding that yeasts strains which comprise a gene encoding for such another mutant SBLE enzyme as described herein, in particular a mutant LE enzyme with improved transesterification activity, and a dysfunctional acetyltransferase enzyme Atl, At2 and / or At3 produce non-acetylated lactonic sophorolipids more uniformly. In addition, the invention further discloses a method to produce non-acetylated lactonic sophorolipids more uniformly via rendering acetyltransferase enzymes Atl, At2 and / or At3 dysfunctional in yeasts strains which comprise a gene encoding for such another mutant SBLE enzyme.

[0195] In particular embodiments of the methods, the produced acidic glycolipids, the lactonic glycolipids or glycolipid oligomers / polymers / esters are non-acetylated, wherein the modified yeast strain is further genetically engineered to comprise a dysfunctional acetyltransferase enzyme, preferably a dysfunctional acetyltransferase enzyme Atl, At2 and / or At3, more preferably a dysfunctional acetyltransferase enzyme Atl, even more preferably a dysfunctional acetyltransferase enzyme Atl, At2 and At3. In further particular embodiments, the modified yeast strain is further genetically engineered to comprise a non-functional acetyltransferase enzyme, preferably a non-functional acetyltransferase enzyme Atl, At2 and / or At3, more preferably a non-funtional acetyltransferase enzyme Atl, even more preferably a nonfunctional acetyltransferase enzyme Atl, At2 and At3. The term 'dysfunctional' in the present invention refers to an enzyme or a fragment or a variant thereof, as described herein, which is not functioning 'normally', and / or, has no or an impaired activity. The term thus refers to an enzyme which is: a) not functional because it is not present, b) still present but non-functional or c) still present but with a weakened or reduced activity, whereby a weakened or reduced activity is an activity that is significantly less (p < 0.05) than 90%, 80%, 70%, 60% or 50%, 40% or 30%, preferably less than 20%, more preferably less than 10%, even more preferably less than 5% such as less than 4%, 3%, 2% or 1% of the activity of the corresponding wild-type enzyme. Situation a) wherein said enzyme or a fragment or a variant thereof is not functional because it is not present, situation b) is still present but non-functional or situation c) is still present but with a weakened or reduced activity, can be obtained through any known means to avoid, reduce and / or silence the transcription and / or translation of the nucleic acid sequence encoding said enzyme or through any known means to impair enzyme activity. For example, but not limited to, by knock-out; by insertion of a nucleic acid fragment containing a marker gene or any other nucleotide fragment in the target gene resulting impaired transcription or translation of the nucleic acid sequence encoding said enzyme; through the usage of CRISPR; through homologous recombination; through siRNA; through CRISPRi; through the use of riboswitches; through recombineering; through ssDNA mutagenesis; through RNAi, miRNA, or asRNA; through mutating the enzyme or the nucleic acid sequence encoding said enzyme; through transposon mutagenesis; by disruption of (the function of) a necessary regulator / activator protein; through interference with the cellular synthesis of the target enzyme or of an activator / regulator; through the use of one or more aptamers; through the use of one or more ribozymes; through the use of antibodies, amino acids, peptides or any small molecules that interfere with transcription, translation, the synthesis of an active enzyme or enzyme activity; through the use of an oligoribonucleotide sequence such a dsRNA used to initiate RNA interference (RNAi) or an antisense nucleic acid; through the introduction of point mutations; through the usage of truncated, modified or mutated enzymes; through the usage of inhibitors or antibodies; through mutation (spontaneous, induced and / or directed, point mutation, deletion, frameshift, insertion or any other type of mutation); ... or any other means known to a skilled person. Situation a), b) or c) can also be obtained through any known means to affect the gene encoding for the enzyme or a fragment or a variant thereof. Also here, the gene can be d) not functioning because it is not present, e) still present but not functioning or f) still present but with a weakened, reduced or altered activity. Situation d) wherein said gene thereof is not functioning because it is not present, situation e) is still present but not functioning or situation f) is still present but with a weakened or reduced activity, can be obtained through any known means to avoid, reduce, alter and / or silence the transcription and / or translation of the nucleic acid sequence encoding said enzyme or a fragment or a variant thereof. For example, but not limited to, by knock-out; by insertion of a nucleic acid fragment containing a marker gene or any other nucleotide fragment in the target gene resulting impaired transcription or translation of the nucleic acid sequence encoding said enzyme; by promoter engineering, by removal of the promoter, by switching promoters, by Kozak sequence engineering, by removal of the Kozak sequence, by switching Kozak sequences, through RBS (ribosomal binding site) engineering, by removal of the RBS, by switching RBS sequences, by UTR (untranslated region) engineering, by removal of the UTR, by switching UTR sequences, through the usage of CRISPR; through homologous recombination; through siRNA; through CRISPRi; through the use of riboswitches; through recombineering; through ssDNA mutagenesis; through RNAi, miRNA, or asRNA; through mutating the nucleic acid sequence encoding said enzyme; through transposon mutagenesis; by disruption of (the function of) a necessary regulator / activator protein; through interference with the cellular synthesis of the target enzyme or of an activator / regulator; through the use of one or more aptamers; through the use of one or more ribozymes; through the use of antibodies, amino acids, peptides or any small molecules that interfere with transcription, translation or the synthesis of an active enzyme; through the use of an oligoribonucleotide sequence such a dsRNA used to initiate RNA interference (RNAi) or an anti-sense nucleic acid; through the introduction of point mutations; through the usage of truncated, modified or mutated enzymes; through the usage of inhibitors or antibodies; through mutation (spontaneous, induced and / or directed, point mutation, deletion, frameshift, insertion or any other type of mutation); ... or any other means known to a skilled person.

[0196] The term 'removed' in the context of a gene means in general a gene or a fragment or a variant thereof, which is, in whole or in part, removed from the genomic DNA. Such a removal can be obtained through any known means, for example, but not limited to, by knock-out of the coding sequence through homologous recombination, by knock-out of the gene through homologous recombination; by knockout of the coding sequence through the use of CRISPR technology, by knock-out of the gene through the use of CRISPR technology; or any other means known to a skilled person.

[0197] The term 'an acetyltransferase enzyme 1 (Atl)' refers to the enzyme previously described in detail in W02012 / 080116 and by Saerens et al. (2011b) and Saerens et al. (2015). This Atl enzyme is referred to herein as 'Atl' and is responsible for acetylation of glycolipids produced by S. bombicola (Saerens et al. (2015)). An Atl enzyme as used to herein thus refers to a polypeptide comprising or consisting of an amino acid sequence given by SEQ ID NO: 15, or a fragment thereof retaining the enzymatic activity (i.e. 'the acetylation of (bola) amphiphilic glycolipid compounds, more specifically the acetylation of (bola) sophorolipids and / or (bola) glucolipids) (i.e. a protein or peptide or polypeptide containing fewer amino acids than the amino acid sequence depicted by SEQ ID NO:15, such as e.g. a deletion of 10% or less amino acids of the total number of amino acids, preferably at the C- and / or N- terminus, and that retains said enzymatic activity described for said Atl enzyme), or a variant thereof as described herein, such as a variant having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity, with SEQ ID NO: 15 and having said enzymatic activity; or variant that differs from the protein depicted by SEQ ID NO:15 only in conservative substitutions and / or modifications, such that the ability of the activity is retained; or a glycosylated form of the protein depicted by SEQ ID NO:15, or the protein SEQ ID NO:15 modified in another way as described elsewhere herein.

[0198] MWNSSKDPQNKGMTPRKEIDQEMVSWAKKNLKNTPGNENYEKMVSGVPYNPYDPDLMFRALATSEKVREFNTIA SESRTFSNHAAYIKKVEILKDTFGQTKDIVWLTAPFSVDFGFNI SVGEHFYANFNVCFLDSAPI I FGDEVIVGPN TTFVTATHPI SPEKRARRIYALPIKVGNNVWIGANVTVLPGVTIGDGSTIAAGAWREDVPPRTWGGVPARILK HI PEEDPDEAEGEELEFLLPVEMNVNTANQKV ( SEQ ID NO : 15 )

[0199] The nucleic acid sequence as depicted by SEQ ID NO: 14 corresponds to the open reading frame which encodes for the polypeptide sequence of the Atl enzyme depicted by the sequence of SEQ ID NO:15.

[0200] The term 'an acetyltransferase enzyme 2 (At2)' relates to an enzyme responsible for the acetylation of glycolipids produced by S. bombicola. In particular, an At2 enzyme as used herein refers to a polypeptide comprising or consisting of an amino acid sequence given by SEQ ID NO: 17, or a fragment thereof retaining the enzymatic activity (i.e. 'the acetylation of (bola) amphiphilic glycolipid compounds, more specifically the acetylation of (bola) sophorolipids and / or (bola) glucolipids) (i.e. a protein or peptide or polypeptide containing fewer amino acids than the amino acid sequence depicted by SEQ ID NO:17, such as e.g. a deletion of 10% or less amino acids of the total number of amino acids, preferably at the C- and / or N-terminus, and that retains said enzymatic activity described for said At2 enzyme), or a variant thereof as described herein, such as a variant having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity, with SEQ ID NO: 17 and having said enzymatic activity; or variant that differs from the protein depicted by SEQ ID NO:17 only in conservative substitutions and / or modifications, such that the ability of the activity is retained; or a glycosylated form of the protein depicted by SEQ ID NO:17, or the protein SEQ ID NO:17 modified in another way as described elsewhere herein.

[0201] MPSGAPRIEYNWDLIKWARENLSHLPVDDDNYHRMI SGLPYEATRTDYSRHRIESHELLLEYLNMKLKDFATLEK YNQARADLLSKVFGSMGTNCFIEQHLFVDYGCNIKVGNNFYANNNLTMLDCSVIEIGDNVFFGPNVTITTASHPL ESKPRAEGVEFAFNVKIGNNVWIGSNAWLPGVTIGDDVWAAGAWNKDVPPSVWGGVPAKILKQIQN ( SEQ ID NO : 17 )

[0202] The nucleic acid sequence as depicted by SEQ ID NO:16 corresponds to the open reading frame which encodes for the polypeptide sequence of the At2 enzyme depicted by the sequence of SEQ ID NO:17. The term 'an acetyltransferase enzyme 3 (At3)' relates to an enzyme responsible for the acetylation of glycolipids produced by S. bombicola. In particular, an At3 enzyme as used herein refers to a polypeptide comprising or consisting of an amino acid sequence given by SEQ ID NO:19, or a fragment thereof retaining the enzymatic activity (i.e. 'the acetylation of (bola) amphiphilic glycolipid compounds, more specifically the acetylation of (bola) sophorolipids and / or (bola) glucolipids) (i.e. a protein or peptide or polypeptide containing fewer amino acids than the amino acid sequence depicted by SEQ ID NO:19, such as e.g. a deletion of 10% or less amino acids of the total number of amino acids, preferably at the C- and / or N-terminus, and that retains said enzymatic activity described for said At3 enzyme), or a variant thereof as described herein, such as a variant having at least 34 % sequence identity, preferably having at least 51-70 % sequence identity, more preferably having at least 71-90% sequence identity or most preferably having at least 91, 92, 93, 94, 95, 96, 97, 98 or 99 % sequence identity, with SEQ ID NO: 19 and having said enzymatic activity; or variant that differs from the protein depicted by SEQ ID NO:19 only in conservative substitutions and / or modifications, such that the ability of the activity is retained; or a glycosylated form of the protein depicted by SEQ ID NO:19, or the protein SEQ ID NO:19 modified in another way as described elsewhere herein.

[0203] MLPATEIDRELVQWARENLPNLPQSTHYDKQI SGMLIKPKWSSMVHETKMKQLTRDYDSINLNHFSSVAKYFEAR TSFIQKHLLGKTGKRVYLESPVHINHGYNI SVGENFYCNFNCIFLDWSI IRIGDNVAIGPNCTLSCINHPLSGDD RKNGAGLYAFPI FIDDNVWIGANCVILSGIHVAEGSWAAGSWTKSVPPHVIVAGNPAKI IAKATDRRLRAAAE DSSSPESSDAEESYMFITKTADP ( SEQ ID NO : 19 )

[0204] The nucleic acid sequence as depicted by SEQ ID NO: 18 corresponds to the open reading frame which encodes for the polypeptide sequence of the At3 enzyme depicted by the sequence of SEQ ID NO:19.

[0205] Crystal structure

[0206] The present inventors have also determined a three-dimensional structure of S. bombicola SBLE enzyme. In particular, the inventors have produced crystals of S. bombicola SBLE enzyme of suitable quality for performing X-ray diffraction analysis following co-crystallization of S. bombicola SBLE enzyme and a bola sophoroside, an inert analog of a bola sophorolipid. Crystal structures were obtained following X-ray diffraction analysis. The present findings provide structural insights into the S. bombicola SBLE enzyme and its interaction with its substrate, enabling screening methods to identify or design mutants of S. bombicola SBLE enzyme with modulated enzymatic activity, in particular mutants with a modulated hydrolytic and / or transesterification activity.

[0207] Accordingly, a further aspect of the present invention provides a crystal structure of an SBLE enzyme, in particular S. bombicola SBLE enzyme as described herein, more particularly S. bombicola SBLE enzyme comprising or consisting of the amino acid sequence set forth in SEQ ID NO:13, defined by the atomic coordinates as depicted in any one of Tables 9 or 10, or a subset thereof. Two crystal structures were obtained, one displaying a monomeric structure, one displaying a dimeric structure in which a substrate analogue is binding between the two monomers.

[0208] The term "crystal structure" as used herein is a three-dimensional description of ordered arrangements or structures of elements such as atoms, ions, or molecules in a crystalline material. Crystal structure refers to a protein crystal structure obtained by protein crystallography, the process of forming a protein crystal by experimentation, unless stated otherwise. In a typical protein crystallization process, proteins are dissolved in an aqueous environment comprising a sample solution until supersaturation is obtained. Different approaches have been described in detail in the art and include as non-limiting examples vapor diffusion, batch, microdialysis and liquid-liquid diffusion. The term "supersaturation" refers to a condition of a solution that contains more of a dissolved material than can be dissolved by the solvent under normal conditions and has been defined in the art as a nonequilibrium condition in which some quantity of the macromolecule in excess of the solubility limit, under specific chemical and physical conditions, is nonetheless present in solution (McPherson and Gavira, Struct Biology Commun, 2014). Protein crystals thus also compose a large amount of solvent molecules such as the non-limiting example of water. Due to the different methodologies for preparing a protein crystal, these crystals further comprise a varying range of buffers, salts, small binding proteins, and precipitation agents which can vary substantially in concentration. Typical crystals have a size of between 20 pm to multiple mm. A crystal optimal for X-ray diffraction analysis is ideally free of cracks and other defects. Once a protein crystal is obtained, different techniques such as X-ray diffraction, cryo-electron microscopy, or nuclear magnetic resonance are suitable to determine the protein crystal structure.

[0209] Further disclosed herein is a method for determining a crystal structure of an SBLE enzyme, said method comprising:

[0210] (a) co-crystallizing the SBLE enzyme and a substrate analog, in particular a bolaform glycoside, more particularly a bolaform sophoroside, to form a crystalline complex,

[0211] (b) subjecting the crystalline complex to X-ray diffraction, and

[0212] (c) determining the crystal structure based on said X-ray diffraction data.

[0213] The crystals may be grown by any suitable method known to the skilled person.

[0214] In certain embodiments, the method further comprises a step of producing the SBLE enzyme in a host cell and recovering the SBLE enzyme from the host. The SBLE enzyme may be a variant of a homologue of S. bombicola SBLE enzyme as described elsewhere herein. The host cell may be of any suitable cell type, including bacterial cells, yeast cells, plant cells and animal cells as described elsewhere herein. Preferably, the recombinant SBLE enzyme is subjected to a deglycosylation step prior to crystallization. Also disclosed herein is a crystal of an SBLE enzyme, in particular S. bombicola SBLE enzyme as described herein, more particularly S. bombicola SBLE enzyme comprising or consisting of the amino acid sequence set forth in SEQ ID N:13, comprising a structure characterized by the atomic coordinates as presented in any one of Tables 9-11 or a subset thereof. Optionally, the crystal is obtained by crystallizing a protein comprising SEQ ID NO:2 or 13, in particulars, bombicola SBLE enzyme as defined by SEQ ID NO:13, in a solution supplemented with a substrate analog, in particular bola sophoroside.

[0215] Furthermore, the present invention provides for the use of a crystal structure as defined above to design or identify mutants of an SBLE enzyme, in particular S. bombicola SBLE enzyme, in particular mutants with a modulated hydrolytic and / or transesterification activity. Also provided herein is the use of a docking model wherein a substrate molecule is fitted in an active site of the SBLE enzyme, to design or identify mutants of the SBLE enzyme, in particular mutants with a modulated a modulated hydrolytic and / or transesterification activity. Such model may be generated using a computer by employing a three dimensional structure of an SBLE enzyme, in particular S. bombicola SBLE enzyme, such as a three dimensional structure represented by a set of atomic coordinates presented in Table 9 or 10 or a subset thereof, for fitting or docking a three dimensional structure or atomic coordinates of a model substrate, in particular a tetra acetylated bolaglycolipid, more particularly a tetra acetylated bolasophorolipid, in said three-dimensional structure of said SBLE enzyme, in particular in an active site of said three-dimensional structure, and generating a three-dimensional representation of an enzyme-substrate complex. For example, the enzyme-substrate complex may be represented as a three dimensional structure represented by a set of atomic coordinates presented in Table 11.

[0216] The structural information obtained from the structures, in particular the crystal structures described herein, and the model, in particular the docking model and the one generated by molecular dynamics simulation, can be used to define how the substrates are interacting with certain amino acid residues of the enzyme, in particular the S. bombicola SBLE enzyme. These structures and models can be used to design mutants that result in the use of alternative substrates or that result in different reaction types such as improved hydrolysis / improved transesterification / improved conversion. In particular, the structural information provided herein is useful in designing potential mutant SBLE enzymes with modulated enzymatic activity, in particular mutants with modulated hydrolytic and / or transesterification activity, more particularly mutants with improved hydrolytic activity and / or improved transesterification activity. Actual mutants may be identified from among potential mutants generated following design and model work performed in silico as shown in the examples.

[0217] Accordingly, a further aspect relates to use of a three dimensional structure represented by a set of atomic coordinates presented in any one of Tables 9 to 11 or a subset thereof, or atomic coordinates which deviate from those in any one of Tables 9 to 11 or a subset thereof by a root mean square deviation (RMSD) of residue over protein backbone atoms by no more than 3 A, for identifying or designing mutants of an SBLE enzyme with modulated hydrolytic and / or transesterification activity, in particular mutants of S. bombicola SBLE enzyme as defined herein with modulated hydrolytic and / or transesterification activity.

[0218] A related aspect is directed to a computer-implemented method for identifying or designing mutants of an SBLE enzyme with modulated hydrolytic and / or transesterification activity, in particular mutants of S. bombicola SBLE enzyme as defined herein, said method comprising:

[0219] - employing a three dimensional structure of the SBLE enzyme represented by a set of atomic coordinates presented in any one of Tables 9 to 11 or a subset thereof, or atomic coordinates which deviate from those in any one of Tables 9 to 11 or a subset thereof by a root mean square deviation (RMSD) of residue over protein backbone atoms by no more than 3 A, for fitting or docking a three dimensional structure or atomic coordinates of a model substrate, in particular a tetra acetylated bolaglycolipid, more particularly a tetra acetylated bolasophorolipid, in said three dimensional structure of the SBLE enzyme, in particular in an active site of said three dimensional structure to generate a three-dimensional computational representation of an enzyme-substrate complex; and

[0220] - identifying amino acid residues in the enzyme of said three-dimensional representation of the enzyme-substrate complex with unfavourable interactions with the substrate as candidate positions for enzyme mutagenesis; and identifying mutations, in particular amino acid substitutions, of said candidate positions that form more favourable interactions with said substrate.

[0221] Said computer-implemented method typically comprises: a step of receiving input from a user; said input for example being the indication of the desired effect of the mutations in the enzyme sought for, such as: modulated hydrolytic and / or transesterification activity, in particular improved hydrolytic activity, or improved transesterification activity; a step of calculating the fitting or molecular docking of one or more randomly generated mutant enzyme sequences, employing a three dimensional structure of the SBLE enzyme represented by a set of atomic coordinates presented in any one of Tables 9 to 11 or a subset thereof, or atomic coordinates which deviate from those in any one of Tables 9 to 11 or a subset thereof by a root mean square deviation (RMSD) of residue over protein backbone atoms by no more than 3 A; a decision making step based on molecular fitting or docking, e.g. a comparison step with the fitting or docking result of a three dimensional structure or atomic coordinates of a model substrate, in particular a tetra acetylated bolaglycolipid, more particularly a tetra acetylated bolasophorolipid, in said three dimensional structure of the SBLE enzyme, more particularly in an active site of said three dimensional structure; a step of providing output wherein the decision is provided to a user or stored on a computer-readable storage medium.

[0222] In another aspect, the invention provides for a computer system configured to perform said computer implemented method.

[0223] In embodiments, said docking or fitting can be performed using known software programs such as (but not-limited to): Molecular Similarity program of QUANTA (Molecular Simulations Inc., San Diego, CA), Autodock, GOLD, MOE-Dock or Glide.

[0224] In embodiments, the decision making is done based on the assessment as to what extent a molecular structure bears similarity to the structure defined by the atomic coordinates, or a subset of atomic coordinates described herein in any one of Tables 9-11. This permit extensive comparison between different structures, different conformations of the same structure, and different parts of the same structure. The method of comparison typically involves a step of calculating one or more optimal translations and rotations required such that the RMSD of the fit over the specified pairs of equivalent atoms is an absolute minimum, such as maximally 3A, preferably of 2.5 A or less, preferably of 2 A or less, more preferable of 1.5 A or less, even more preferably 1 A or less.

[0225] Said molecular docking encompasses the prediction of a binding and / or preferred orientation of one molecule to a second molecule when bound to each other to form a stable complex. Hence, it is understood that molecular docking method or software predicts the behavior of molecules in binding sites of target proteins. Molecular docking methods or software that allow assessing of specificity of a candidate molecule or candidate compound against a particular target have been described in the art. Exemplary molecular docking methods or software allow searching for complementarities between shape and / or electrostatics of binding sites surfaces and ligands. A molecular docking process can be separated into two major steps: searching and scoring. Numerous examples of different docking tools and programs have been described and are thus known to a skilled person (Pagadala et al., Biophys Rev, 2017). Two main popular molecular docking approaches have been described, a first being molecular docking relying on shape complementarity or geometric matching, and a second one relying on simulating the docking process whereby ligand-protein pairwise interaction energies are calculated.

[0226] In another aspect, the invention provides for a computer program product comprising instructions which when the program is executed by a computer, cause the computer to carry out the steps of a computer implemented method as described herein. In another aspect, the invention provides for a computer-readable storage medium, comprising a data storage material encoded with computer readable data wherein said data comprises the information needed for and configured to perform said computer implemented method.

[0227] Said computer-readable storage medium can further comprise computer readable data wherein said data comprises the results of the decision making step for each generated mutant sequence.

[0228] The computer program product according, or the computer-readable storage medium can further comprise a database containing the results of the decision making step for each generated mutant sequence.

[0229] A further aspect is directed to a method for producing mutants of an SBLE enzyme with modulated hydrolysis and / or transesterification activity, as defined herein, said method comprising identifying or designing mutants of the SBLE enzyme by a method as described herein, and generating mutants of the SBLE enzyme that comprise one or more of the identified mutations. In further embodiments, the method may further comprise steps of testing the generated mutants of the SBLE enzyme for modulated hydrolysis and / or transesterification activity and identifying mutants of the SBLE enzyme with modulated hydrolysis and / or transesterification activity.

[0230] In particular embodiments, the mutants of the SBLE enzyme are mutants with modulated enzymatic activity, in particular mutants with improved hydrolytic activity and / or improved transesterification activity.

[0231] The term "atomic coordinates" as used herein refers to a position of an atom in space, typically expressed by a set of X, Y, and Z Cartesian coordinates and the chemical element each atom represents. Atomic coordinates for a certain protein structure are typically combined in atomic coordinate data files, which can have various data formats, including the formats of Tables 9-11 as enclosed in this specification. Other non-limiting data formats include Protein Data Bank (PDB) format or various text formats. Minor variations in the atomic coordinates are envisaged, and the claims have been formulated with the intent of encompassing such variations. In certain embodiments, the atomic coordinates further contain additional information. It is evident to a skilled person that a three- dimensional rigid body rotation or a translation of said atomic coordinates does not alter the structure of the molecule. It is evident that, since the atomic coordinates disclosed herein are a relative collection of points delineating a three-dimensional structure, a distinct set of coordinates may define a similar or identical three-dimensional structure. In view hereof, multiple computer analysis tools and programs have been developed to assess whether a molecular structure bears similarity to the structured defined by the atomic coordinates, or a subset of atomic coordinates described herein in any one of Tables 9-11. By means of illustration and not limitation, a suitable software application for conducting such analyses is the Molecular Similarity program of QUANTA (Molecular Simulations Inc.,

[0232] San Diego, CA), Autodock, GOLD, MOE-Dock or Glide. The Molecular Similarity program and consorts permit extensive comparison between different structures, different conformations of the same structure, and different parts of the same structure. The method of comparison typically involves a step of calculating one or more optimal translations and rotations required such that the RMSD of the fit over the specified pairs of equivalent atoms is an absolute minimum. Therefore, atomic coordinates of S. bombicola SBLE enzyme or S. bombicola SBLE enzyme-SS complex, or fragments leading to the atomic coordinates in any one of Tables 9-11 by translations and / or rotations are within the scope of the present invention.

[0233] "RMSD", "root-mean-square deviation", or "root-mean-square deviation of atomic positions" as used herein is indicative for a quantitative measurement of similarity between two or more protein structures, more specifically the measure of the average distance between the (backbone) atoms of superimposed proteins. The RMSD value is commonly calculated by the formula: RMSD = wherein 5 is the distance between atom I and the mean position of the N equivalent atoms, or alternatively a reference structure. When calculating the RMSD for backbone, heavy atoms values are calculated for C, N, O, and Ca or solely for Ca. As a RMSD value represents a distance, the value is commonly expressed in the art in A (Angstrom). 1 A corresponds to IO10m, or 0.1 nanometer.

[0234] A skilled person appreciates that a lower RMSD indicates smaller structural differences between the compared structures, or between a structure and a reference structure. In certain embodiments, the atomic coordinates used in the method deviate by no more than 2.5 A, preferably no more than 2 A, more preferable no more than 1.5 A, even more preferably no more than 1 A from the atomic coordinates of Table 9, 10, or 11.

[0235] The methods and uses described herein may be performed using the atomic coordinates presented in any one of Table 9 (which in their totality represent a monomeric form of S. bombicola SBLE enzyme),

[0236] Table 10 (which in their totality represent a dimeric form of S. bombicola SBLE enzyme) or Table 11

[0237] (which in their totality represent a model of an S. bombicola SBLE enzyme bound to a tetra acetylated bolasophorolipid, also referered to herein as S. bombicola SBLE enzyme-substrate complex), or may alternatively be performed using a subset thereof. The term "subset" as defined herein indicates a portion of the atomic coordinates of any one of Tables 9-11. By means of illustration and not limitation, a possible subset in the context of the invention is the subset of coordinates of Table 11 defining the

[0238] S. bombicola SBLE enzyme part of the S. bombicola SBLE enzyme-substrate complex. An alternative possible subset is the subset of coordinates of Table 10 defining one S. bombicola SBLE enzyme part of the S. bombicola SBLE enzyme dimer. Yet an alternative possible subset is the subset of coordinates of Table 11 defining the loops and helices of the S. bombicola SBLE enzyme in the (S. bombicola) SBLE enzyme-substrate complex that bind the substrate, in particular the loops and helices corresponding to the loop forming region T103-A113, the helix forming region S125-T131, the loop forming region S132-A136, the loop forming region D151- G162, the loop forming region G192-S194, the helix forming region G195-D208, the loop forming region G223-A228, the helix forming region W229-I235, the loop forming region D236-L242, the helix forming region K243-G256, the loop forming region A282-S298, the loop forming region G344-K349, the loop forming region P374-H378 and the helix forming region D379-D398 of SEQ ID NO:2. The size of the subset is not particularly limiting, however a skilled person appreciates that the performance of the uses and methods described herein benefits from increasing sizes of said subset derived from Tables 9-11. By means of illustration and not limitation, the subset may comprise 20%, preferably 40%, preferably 50%, preferably 60% preferably 70%, preferably 80%, preferably 90% of the atomic coordinates presented in Tables 9-11. In preferred embodiments, the methods described herein are computer-implemented methods. In further embodiments, the computer comprising an inputting device, a processor, a user interface, and an outputting device. “In silico analysis” as defined herein is indicative for an analysis performed on a computing system or by use of a computer simulation system that is guided by a set of specific instructions such as a molecular docking computer program or tool. Further disclosed herein are: (1) A mutant of the SBLE enzyme from S. bombicola comprising at least one of the following mutations: - A382E - S132D or S132E - L134F or L134W - F159D or F160D or L225D or L348D, or a mutation of F159, F160, L225 or L348 to a glutamic acid - P212D or P212E - T231H - I235S or L242S or another mutation of I235 or L242 to a polar amino acid residue such as asparagine, glutamine or cysteine - V240S or another mutation of V240 to a polar amino acid residue such as asparagine, glutamine or cysteine - F261S or W265S or another mutation of F261 or W265 to a polar amino acid residue such as asparagine, glutamine or cysteine (2) Use of a mutant SBLE enzyme according to (1) to improve a hydrolysis reaction, or, to improve a hydrolysis reaction and to decrease a transesterification reaction. (3) Use of a mutant SBLE enzyme according to (1) for improved conversion of bola sophorolipids into acidic sophorolipids and sophorose. (4) Use of a modified yeast strain which comprises a gene encoding for a mutant SBLE enzyme according to (1) to produce acidic sophorolipids more uniformly. (5) A method to more uniformly produce acidic sophorolipids, said method comprising the conversion of (a) suitable substrate(s) with a suitable microbial strain to produce a broth comprising acidic sophorolipids, wherein said microbial strain has been modified to comprise a gene encoding for a mutant SBLE enzyme according to (1) and wherein said microbial strain is preferably a naturally SL producing fungal strain, more preferably a yeast selected from the group consisting of Starmerella (Candida) bombicola, Starmerella (Candida) apicola, Starmerella (Candida) magnoliae, Candida gropengiesseri, Starmerella (Candida) batistae, Starmerella (Candida)floricola, Candida riodocensis, Candida tropicalis, Starmerella (Candida) stellata, Starmerella (Candida) kuoi, Candida sp. NRRL Y- 27208, Pseudohyphozyma (Rhodotorula, Candida) bogoriensis sp., Wickerhamiella domericqiae and a sophorolipid-producing strain of the Starmerella clade. (6) A mutant of the SBLE enzyme from S. bombicola comprising at least one of the following mutations: - N126F or N287F or Q288F or S289F or another mutation of N126 or N287 or Q288 or S289 to a hydrophobic amino acid residue such as isoleucine, leucine, valine or tryptophan - P129F or G302F or another mutation of P129 or G302 to a hydrophobic amino acid residue such as isoleucine, leucine, valine or tryptophan - F159L or another mutation of F159 to a hydrophobic amino acid such as isoleucine, valine or tryptophan - F232S or another mutation of F232 to a polar amino acid residue such as asparagine, glutamine or cysteine - I235F or L242F or G244 or another mutation of I235, L242 or G244 to an aromatic amino acid such as phenylalanine, tryptophan or tyrosine - M249G or M249A, or another mutation of M249 to a small amino acid residue such as serine - P290L - Mutations in the loops I235-K243 or G344-L351 that result in the transformation of the loop into a helical structure, for example into the sequences TDDGIFAGI or IPDEIVPYQ, respectively. (7) Use of a mutant SBLE enzyme according to (6) to improve a transesterification reaction, or, to improve a transesterification reaction and to decrease a hydrolysis reaction.

[0239] (8) Use of a mutant SBLE enzyme according to (6) for improved conversion of (non-acetylated) bola sophorolipids into lactonic sophorolipids and sophorose.

[0240] (9) Use of a modified yeast strain which comprises a gene encoding for a mutant SBLE enzyme according to (6) to produce (non-acetylated) lactonic sophorolipids more uniformly.

[0241] (10) A method to more uniformly produce lactonic sophorolipids, said method comprising the conversion of (a) suitable substrate(s) with a suitable microbial strain to produce a broth comprising lactonic sophorolipids, wherein said microbial strain has been modified to comprise a gene encoding for a mutant SBLE enzyme according to (6) and wherein said microbial strain is preferably a naturally SL producing fungal strain, more preferably a yeast selected from the group consisting of Starmerella (Candida) bombicola, Starmerella (Candida) apicola, Starmerella (Candida) magnoliae, Candida gropengiesseri, Starmerella (Candida) batistae, Starmerella (Candida) floricola, Candida riodocensis, Candida tropicalis, Starmerella (Candida) stellata, Starmerella (Candida) kuoi, Candida sp. NRRL Y- 27208, Pseudohyphozyma (Rhodotorula, Candida) bogoriensis sp., Wickerhamiella domericgiae and a sophorolipid-producing strain of the Starmerella clade.

[0242] (11) Use of a mutant SBLE enzyme according to (6) for improved conversion of bola sophorolipids into non-acetylated lactonic sophorolipids and sophorose.

[0243] (12) Use of a modified yeast strain which comprises a gene encoding for a mutant SBLE enzyme according to (6) to produce non-acetylated lactonic sophorolipids more uniformly.

[0244] (13) A method according to (10) wherein said lactonic sophorolipids are non-acetylated lactonic sophorolipids.

[0245] (14) Use of a modified yeast strain which comprises a gene encoding for a mutant SBLE enzyme according to (6) and which has further been modified to have (a) dysfunctional acetyltransferase enzyme(s) Atl, At2 and / or At3 to produce non-acetylated lactonic sophorolipids more uniformly.

[0246] (15) A method to more uniformly produce non-acetylated lactonic sophorolipids, said method comprising the conversion of (a) suitable substrate(s) with a suitable microbial strain to produce a broth comprising non-acetylated lactonic sophorolipids, wherein said microbial strain has been modified according to (12) or (14) and wherein said microbial strain is preferably a naturally SL producing fungal strain, more preferably a yeast selected from the group consisting of Starmerella (Candida) bombicola, Starmerella (Candida) apicola, Starmerella (Candida) magnoliae, Candida gropengiesseri, Starmerella (Candida) batistae, Starmerella (Candida) floricola, Candida riodocensis, Candida tropicalis, Starmerella (Candida) stellata, Starmerella (Candida) kuoi, Candida sp. NRRL Y- 27208, Pseudohyphozyma (Rhodotorula, Candida) bogoriensis sp., Wickerhamiella domericgiae and a sophorolipid-producing strain of the Starmerella clade.

[0247] (16) Use of the the Starmerella batistae SBLE enzyme to produce acidic sophorolipids.

[0248] (17) Use of the the Starmerella batistae SBLE enzyme to produce mono-acetylated or di-acetylated acidic sophorolipids.

[0249] (18) Use of the Starmerella batistae SBLE enzyme for the improved production of sophorolipid oligomers.

[0250] (19) Use of a modified yeast strain which comprises a gene encoding for the Starmerella batistae SBLE enzyme to produce acidic sophorolipids.

[0251] (20) Use of a modified yeast strain which comprises a gene encoding for the Starmerella batistae SBLE enzyme to produce mono-acetylated or di-acetylated acidic sophorolipids.

[0252] (21) Use of a modified yeast strain which comprises a gene encoding for the Starmerella batistae SBLE enzyme to produce sophorolipid oligomers.

[0253] (22) A method for the production of acidic sophorolipids, said method comprising the conversion of (a) suitable substrate(s) with a suitable microbial strain to produce a broth comprising acidic sophorolipids, wherein said microbial strain has been modified according to (19), (20) or (21) and wherein said microbial strain is preferably a naturally SL producing fungal strain, more preferably a yeast selected from the group consisting of Starmerella (Candida) bombicola, Starmerella (Candida) apicola, Starmerella (Candida) magnoliae, Candida gropengiesseri, Starmerella (Candida) batistae, Starmerella (Candida) floricola, Candida riodocensis, Candida tropicalis, Starmerella (Candida) stellata, Starmerella (Candida) kuoi, Candida sp. NRRL Y-27208, Pseudohyphozyma (Rhodotorula, Candida) bogoriensis sp., Wickerhamiella domericgiae and a sophorolipid-producing strain of the Starmerella clade.

[0254] (23) A method according to (22) wherein said acidic sophorolipids are mono-acetylated or di- acetylated acidic sophorolipids.

[0255] (24)The crystal structure of the rSBLE enzyme as defined by the atomic coordinates as depicted in Table

[0256] 9 to 11.

[0257] (25)Use of the crystal structure according to (24) to design mutants of the SBLE enzyme. Examples

[0258] Example 1

[0259] Material and methods in vivo SBLE mutants and homologues evaluation

[0260] Strains and cultivation methods

[0261] Cloning experiments and plasmid maintenance were performed with Escherichia coli top 10 cells. E. coli cells were grown in Lysogeny-Broth medium (37°C, 10 g / l trypton, 5 g / l yeast extract, 5 g / l sodium chloride and if required 15 g / l agar; Sigma-Aldrich) supplemented with 100 mg / L ampicillin (MP Biomedicals), 50 pg / mL kanamycin (Sigma Aldrich) or 25 pg / mL chloramphenicol (Sigma Aldrich) when applicable. Wild type S. bombicola (WT; ATCC 22214) and a URA3 auxotrophic mutant strain (PT36) were used during this study (Lodens et al. (2020)). The PT36 strain was used to make an SBLE knockout strain using a hygromycin resistance containing knock-out cassette. This strain was then used to make the different knock-in strains comprising SBLE variants by using knock-in cassettes with URA3 selection. Solid synthetic dextrose with complete supplement mixture without uracil (6.7 g / L Yeast nitrogen base without amino acids (Sigma-Aldrich), 20 g / L glucose (Cargill), 20 g / L agar Noble (Difco), 0.77 g / L complete supplement mixture without uracil (MP biomedicals)) yeast extract peptone dextrose supplemented with hygromycin (20 g / L glucose (Cargill), yeast extract (DSM), 20 g / L bactopepton (BD biosciences), agar (Biokar Diagnostics), 1 g / L Hygromycine B (Sigma-Aldrich)) or yeast extract peptone dextrose supplemented with nourseothricin (20 g / L glucose (Cargill), yeast extract (DSM), 20 g / L bactopepton (BD biosciences), agar (Biokar Diagnostics), 0.75 g / L nourseothricin (Jena Bioscience)) were used for selection for positive deletion mutants after transformation with a URA3 auxotrophic, r a hygromycine resistance marker or a nourseothricin resistance marker, respectively.

[0262] For the glycolipid production experiments, the production medium as described by Lang et al. (2000) was used. Precultures (5 mL) were inoculated from cryovials (1%) and incubated for 48 h (30°C, 200 rpm). Subsequently, shake flasks (n=3) containing 10 mL production medium were inoculated (1%) from precultures. Shake flasks were incubated for 240h (30°C, 200 rpm). 20 g / L oleic acid (Sigma- Aldrich) or rapeseed oil was supplemented after 48h of cultivation.

[0263] Analytical techniques

[0264] Sample preparation was performed on SF broth samples. Firstly, 70% EtOH (3:1, v / v) or 100% EtOH (2:1, v / v) was added to the sample and vigorously vortexed for 5 min. Subsequently, a centrifugation step was performed (5 min, 15 000 rpm) on which the supernatant was filtered through a PES filter (0.2μm, sartorius). If needed, samples were diluted with a 3:1 mixture of 70% ethanol and MQ water.

[0265] In-house SL standards were analysed together with the production samples.

[0266] For the analysis of product samples using HPLC-MS, SLs were separated using an Agilent 1100 series HPLC (Agilent) with Kinetex C18 150x4.6 mm 5 pm solid core column (Phenomenex), which was operated at 35°C and a flow rate of 1.5 mL / min. A default injection volume of 2 μL was applied. A binary gradient was used for the mobile phase consisting of 0.1% formic acid in mQ (eluent A) and acetonitrile (eluent B). The ratio of eluent B increased from 20% to 80% in 30 minutes and was kept at 80% for 5 minutes. The subsequent mass spectrometry analysis was performed using a G1956B single quadrupole MS detector (Agilent) equipped with a heated electrospray ionization (HESI) source, running in negative ion mode. The parameters were set to detect mass-to-charge (m / z) ratio in the range of 200 - 1200 atomic mass units (amu), and the spectra were analyzed using the Spectrus Processor software (ACDLabs).

[0267] An Acquity H-Class UPLC (Waters) was used in combination with the Acquity ELSD (Waters). Samples were diluted appropriately and filtered over a 13 mm PTFE syringe filter with a pore size of 0.2 pm (Novolab). For the UPLC analysis of SLs, an Acquity UPLC CSH C18 column (130 A, 1.7 μm, 2.1 mm x 50 mm) was used. A sample volume of 2 μL was injected, the column was kept at 35°C and a flow rate of 0.6 mL / min was applied for 10 min / sample. A binary gradient elution system was applied, consisting of 0.5% acetic acid in mQ water (eluent A) and 100% acetonitrile (eluent B) and performed as follows: during the first 6.8 min, the concentration of eluent B increases from 5% to 95%, after which it decreases again to 5% in 1.8 min. For the remaining 1.4 min of the sample run, the concentration of B is maintained at 5%. For the subsequent detection by the ELSD, the nebulizer was cooled to 12°C and the drift tube was kept at a temperature of 50°C. The gain was set at a value of 100.

[0268] A Vanquish Flex Duo UHPLC (Thermo Fisher Scientific) was used in combination with a Charged Aerosol Detector (CAD) (Thermo Fisher Scientific). For the UHPLC analysis of sophorolipids, a ZORBAX RR Eclipse Plus C18 column (95 A, 3.5 μm, 4.6 mm x 100 mm, Agilent) was used with CAD. A sample volume of 5 μL was injected, the column was kept at 35°C and a flow rate of 1.0 mL / min was applied for 30 min / sample. A binary gradient elution system was applied, consisting of 0.1% trifluoroacetic acid in mQ water (eluent A) and 0.1% trifluoracetic acid in acetonitrile (eluent B) and performed as follows: during the first 22.5 min, the concentration of eluent B increases from 20% to 95%, after which it is maintained at 95% for 2 min. Next, the concentration of eluent B decreases to 0% in 3 min and it maintained at 0% for 1 min. For the remaining 1.5 min of the sample run, the concentration of B increased to 20% again. For the subsequent detection by the CAD, the evaporator temperature was set at 35°C. Peak areas were calculated by using Chromeleon 7 (Thermo Fisher Scientific). The ratio acidic sophorolipids to total sophorolipids, the ratio lactonic sophorolipids to total sophorolipids and the ratio bola sophorolipids to total sophorolipids was calculated as follows: the peak area corresponding to either acidic sophorolipids, lactonic sophorolipids or bola sophorolipids was divided by the sum of the peak areas for acidic, lactonic and bola sophorolipids and multiplied by 100.

[0269] The percentages of improvement or decrease in the production of acidic (hydrolysis), lactonic (transesterification) and (acetylated) bolaform sophorolipids by the mutant strains were calculated as follows: the ratio of either lactonic, acidic or bola sophorolipids to total sophorolipids of a specific mutant enzyme was divided by the corresponding ratio obtained with the wild type enzyme and multiplied by 100. The wild type enzyme was used as reference and values were set at 100 %.

[0270] Thin Layer Chromatography was used as a semi-quantitative method to monitor oligomeric sophorolipid production. The stationary phase used were Reverse phase 18 TLC Silica gel 60 F254 Alu plates (Supelco). The mobile phase consisted of a solvent mixture of methanol and water (95 / 5, v / v). 1 μL sample was spotted at 1 cm from the bottom of the plate. The TLC plate was put in a TLC chamber which was filled with 1 cm mobile phase. When the solvent front reached 1 cm from the top of the plate, the TLC plate was taken out and let to dry to evaporate the mobile phase. Next, the plate was dipped in 10% H2SO4 solution and dried by using a heat gun until spots started to appear. The most hydrophobic substrates are visible at the bottom of the plate.

[0271] Molecular methods

[0272] Circular polymerase extension cloning (CPEC) pieces and the linear deletion cassettes were amplified with Primestar® according to the manufacturer's instructions. Plasmids were assembled with CPEC or with Golden Gate via a modified version of the VErsatile Genetic Assembly System (VEGAS) (Kuijpers et al., Microb. Cell Fact. 12, 47 (2013); Mitchell et al., Nucleic Acids Res. 43, 6620-6630 (2015)). Colony PCR was performed on E. coli and S. bombicola according to De Graeve et al. (2019). CPEC was performed with Q5® Hifi DNA polymerase according to the manufacturer's instructions and as described in Quan and Tian (2011). Plasmids were transformed into TOPIO E. coli cells through heat shock. Linear deletion cassettes were transformed via electroporation in S. bombicola according to De Graeve et al. (2019). Sequencing of CPEC assembled plasmids was performed by Macrogen Inc. Oligonucleotides were purchased at IDT DNA Technologies.

[0273] Different insertion cassettes were constructed for subsequent gene insertion in S. bombicola (Figure 3).

[0274] The original SBLE originates from the S. bombicola genome, and the A382 mutant and other mutants were made starting from this gene. The Starmerella kuoi and Starmerella batistae and Starmerella riodocensis homologs hereof were identified by BLAST and amplified fom their respective genomes. trCALA is a truncated version of CALA lipase without the lid and was fused with the signal peptide of the original SBLE gene. The hygromycin B selection marker (Hygro) and the terminator of the Herpes simplex virus tyrosine kinase (tTK) terminator that were used, are described by (Van Bogaert et al., 2008). The nourseothricin selection marker (Nat) that was used, was obtained from plasmid pYL16 (WERNER BioAgents GmbH, Germany). In order to make the insertion cassettes, the coding DNA sequence was fused with the pGAPD promoter (SEQ ID NO: 11) and the tGAL terminator (SEQ ID NO: 12) from S. bombicola flanked by the URAB gene and homologous regions to enable insertion by homologous recombination on the URAB locus of the Starmerella bombicola genome as described by Lodens et al. (2018). Alternatively, insertion cassettes with the pGKI promoter (SEQ ID NO: 111) from S. bombicola and the tScCYCl terminator (SEQ ID NO: 112) from Saccharomyces cerevisiae, flanked by the homologuous regions on the sides of the SBLE gene and Hygro in between pGAPD and tGAL were used for recombination on the SBLE locus of the Starmerella bombicola genome. The URA3 marker could be recycled with 5-fluoroorotic acid as described by Lodens et al. (2018)

[0275] Example 2

[0276] The knock-out cassettes as shown Figure 3A and B were amplified from their respective plasmids and used for transformation into S. bombicola PT36 strains according to the methods described in Example 1. The cassette as shown in Figure 3A was used to create the control strain 'ASBLE::ura' (genotype: Aura3::0 Asble:: tTK_Ura3_pUra3), while the cassette as shown in Figure 3B was used to create the control strain 'ASBLE::hygro' (genotype: Aura3::0 ASble:: pGAPD_hygro_Ttk). After successful verification of the gene deletions, the newly developed strains were evaluated for their production characteristics in shake flask (SF) experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results are pictured in Figure 4A and B. Both knock-out strains predominantly produce (acetylated) bola sophorolipids.

[0277] Example 3

[0278] The knock-in cassette as shown Figure 3C was amplified from its respective plasmid and used for transformation into S. bombicola ASBLE::hygro strains according to the methods described in Example 1 and with the strain of Example 2. The cassette as shown in Figure 3C was used to create the control strain 'SBLE' (genotype:Aura3::pURA_URA3_tTK_pGAPD_SBLE_tGAL Asble::pGAPD_hygro_Ttk), whereby the SBLE coding sequence as depicted by SEQ ID NO: 1 was used. After successful verification of the knock-in, the newly developed strains were evaluated for their production characteristics in shake flask (SF) experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results are pictured in Figure 4C. The novel strain showed a very comparable production pattern compared to the S. bombicola wild type strain i.e. di-acetylated lactonic sophorolipids were produced. Together with example 2 this proves that (acetylated) bola sophorolipids are the substrate of SBLE, which converts them into (di-acetylated) lactonic sophorolipids and (acetylated) sophorose.

[0279] Example 4

[0280] The knock-in cassettes as shown Figure 3C was amplified from its respective plasmid and used for transformation into the S. bombicola ASBLE::hygro strain of Example 2 according to the methods described in Example 1. The cassettes as shown in Figure 3C were used to create the strain 'SBLE_A382E' (genotype: Aura3::0 ASble::pUra3_Ura3_tTK_pGAPD787_SBLE_A382E_tGAL) and other strains indicated in Table 1, with the coding sequences for the mutant SBLE enzymes indicated. After successful verification of the knock-in, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results are pictured in Figure 4 D)-H) and displayed in Table 1. Here, the ratio acidic sophorolipids to total sophorolipids, the ratio lactonic sophorolipids to total sophorolipids and the ratio bola sophorolipids to total sophorolipids are mentioned to determine if changes in hydrolysis and / or transesterification activity are observed.

[0281] Moreover, in Table 2, the percentages of improvement or decrease in the production of acidic (hydrolysis), lactonic (transesterification) and (acetylated) bolaform sophorolipids by the mutant strains are mentioned. For clarity, it is emphasised that for the experiments described in Example 4, the strain has none of its acetyl transferases knocked out, so the substrate of the SBLE mutants are mainly acetylated bola sophorolipids.

[0282] The SBLE_N126G, the SBLE_S125P_P129G, the SBLE_G244S, the SBLE_G244T, the SBLE_G244C, the SBLE_G244N, the SBLE_G244Q, the SBLE_L128Y, the SLBE_Q288Y, the SBLE_G244Y, SBLE_P290Y and the SBLE_N287F overexpression strains were constructed as described in material and methods. The strains produced a mixture including acetylated bola SL, di-acetylated acidic SL and di-acetylated lactonic SL. The results in Table 1 and 2 indicate that the ratio acidic SL to total SLs increased compared to the reference strain meaning that an improvement in hydrolysis activity was seen for the mutant enzymes compared to the wild type enzyme. These results demonstrate that the activity of the mutant enzyme shifted to higher hydrolysis activity compared to the wild type enzyme. In most cases the increased hydrolysis activity was associated with a transesterification activity similar to the wild type (Fig. 4E), while in others the transesterification activity was decreased (e.g. SBLE_G244T and SBLE_G244Y) (Fig. 4F).

[0283] The SBLE_A382E, SBLE_P290S, the SBLE_P290T, the SBLE_P290C, the SBLE_P290N, the SBLE_P290Q, the SBLE_G244W, the SBLE_P290F, the SBLEJ248A and SBLEJ248G overexpression strains were constructed as described in material and methods. The strains produced a mixture including acetylated bola SL, di-acetylated acidic SL and di-acetylated lactonic SL. The results in Table 1 and 2 indicate that the ratio acidic SL to total SLs clearly increased compared to the reference strain meaning that an increased hydrolysis activity is seen for the mutant enzymes compared to the wild type enzyme as described in Example 3. These results demonstrate that the change of a proline (P) at position 290 of the enzyme to five polar amino acids resulted in a shift to higher hydrolysis activity in the mutant enzymes compared to the wild type enzyme. These results also demonstrate that the change of an isoleucine (I) at position 248 of the enzyme to either alanine (A) or glycine (G) resulted in a shift to higher hydrolysis activity in the mutant enzymes compared to the wild type enzyme in the strain background as described in Example 3. In some cases the increased hydrolysis activity was associated with a transesterification activity similar to the wild type (Fig. 4G), while in others the transesterification activity was decreased (e.g. SBLEJ248A) (Fig. 4H). In one case (SBLE_A382E), the transesterification activity was almost lost (Fig. 4D).

[0284] Table 1: Ratio's of acidic, lactonic and bolaform sophorolipids produced by the mutant strains.

[0285] Table 2: Percentage of improvement or decrease in the production of acidic (hydrolysis), lactonic (transesterification) and (acetylated) bolaform sophorolipids by strains with a mutant LE enzyme compared to those with a wild type LE enzyme.

[0286] Example 5

[0287] The knock-out cassettes as shown Fig. 3B, 3D, 3E and 3H were amplified from their respective plasmids and used for transformations according to the methods described in Example 1. Using these cassettes and with URA3 recovery in between, the reference strain 'AAT1::O AAT2::0 AAT3::nat ASBLE::hygro' with genotype Aura3::0 Aatl::0 Aat2::0 Aat3::pGAPD787_nat_tTK Asble::pGAPD_hygro_Ttk was created. After successful verification of the gene deletions, the newly developed strain was evaluated for its production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results are pictured in Fig. 5A. The strain produced mainly non-acetylated bola SLs as all 3 acetyltransferase genes coding for acetyltransferases involved in the SL biosynthetic pathway, were knocked out, as well as its SBLE gene.

[0288] Example 6

[0289] The knock-in cassettes as shown in Fig. 3D, 3E and 3F were amplified from their respective plasmids and used for transformation into the S. bombicola PT36 strain according to the methods described in Example 1 to create the control strain with genotype Aatl::0 Aat2::0 Aat3::0. After successful verification of the knock-outs, the strain was evaluated for its production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results are pictured in Fig. 5B. The novel strain produced a mixture including non-acetylated bola sophorolipids, non-acetylated acidic sophorolipids and non-acetylated lactonic sophorolipids. Together with Example 3 this proves that (non-acetylated) bola sophorolipids are the substrate of SBLE, which converts them into (non-acetylated) lactonic sophorolipids and (non- acetylated) sophorose.

[0290] Example 7

[0291] The knock-in cassette as shown Fig. 3C was amplified from its respective plasmid and used for transformation into S. bombicola AAT1::O AAT2::0 AAT3::nat ASBLE::hygro strain of Example 5 according to the methods described in Example 1. The cassette as shown in Fig. 3C was used to create the strains in Table 3, with the coding sequences for the mutant SBLE enzymes indicated. After successful verification of the knock-ins, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results are displayed in Figure 5 C)-F) and in Table 3. Here the ratio acidic sophorolipids to total sophorolipids, the ratio lactonic sophorolipids to total sophorolipids and the ratio bola sophorolipids to total sophorolipids are mentioned, to determine if changes in hydrolysis and / or transesterification activity occur. Moreover, in Table 4, the percentages of improvement or decrease in the production of acidic (hydrolysis), lactonic (transesterification) and bolaform sophorolipids by the mutant strains are mentioned. For clarity, it is emphasised that for the experiments described in this Example 7, the strain has all of its acetyl transferases involved in the SL biosynthetic pathway knocked out, so the substrate of the SBLE mutants are mainly non-acetylated bola sophorolipids.

[0292] The SBLE_G244S, the SBLE_G244Q, the SBLE_L128Y and the the SBLE_Q288Y overexpression strains were constructed as described in material and methods. The strains produced a mixture including nonacetylated bola SL, non-acetylated acidic SL and non-acetylated lactonic SL. The results in Table 3 and 4 indicate that the ratio lactonic SL to total SLs increased compared to the reference strain meaning that an increased transesterification activity was seen for the mutant enzymes compared to the wild type enzyme described in Example 6. In some cases the increased transesterification activity was associated with a hydrolysis activity similar to the one obtained with the wild type enzyme (Fig. 5F), while in others the hydrolysis activity was also increased (e.g. SBLE_G244S and SBLE_Q288Y) (Fig. 5C).

[0293] The SBLE_G244C, the SBLE_P290S, the SBLE_P290T, the SBLE_P290C, the SBLE_P290N, the SBLE_P290Q, the SBLEJ248A and the SBLEJ248G overexpression strains were constructed as described in material and methods. The strains produced a mixture including non-acetylated bola SL, non-acetylated acidic SL and non-acetylated lactonic SL. The results in Table 3 and 4 indicate that the ratio acidic SL to total SLs increased compared to the reference strain meaning that an increased hydrolysis activity was seen for the mutant enzyme compared to the wild type enzyme as described in Example 6. These results demonstrate that the change of a proline (P) at position 290 of the enzyme to five polar amino acids resulted in a shift to higher hydrolysis activity in the mutant enzymes compared to the wild type enzyme described in Example 6. These results also demonstrate that the change of an isoleucine (I) at position 248 of the enzyme to either alanine (A) or glycine (G) resulted in a shift to higher hydrolysis activity in the mutant enzymes compared to the wild type enzyme described in Example 6. In some cases the increased hydrolysis activity was associated with a transesterification activity similar to the one obtained with the wild type enzyme (Fig. 5E), while in others the transesterification activity was decreased (e.g. SBLE_P290T) (Fig. 5D). Table 3: Ratio's of acidic, lactonic and bolaform sophorolipids produced by the mutant strains which have all of their acetyl transferases involved in the SL biosynthetic pathway knocked out.

[0294] Table 4: Percentage of improvement or decrease in the production of acidic (hydrolysis), lactonic (transesterification) and (acetylated) bolaform sophorolipids by the mutant strains which have all of their acetyl transferases involved in the SL biosynthetic pathway knocked out.

[0295] Example 8

[0296] The knock-in cassette as shown in Figure 3C was amplified from its respective plasmid and used for transformation into the S. bombicola ASBLE::hygro strain of Example 2 according to the methods described in Example 1. The cassette as shown in Figure 3C was used to create the control strain 'LE_kuoi' (genotype: Aura3::pURA_URA3_tTK_pGAPD_StarmerellakuoiLE_tGAL

[0297] Asble::pGAPD_hygro_Ttk), whereby the Starmerella kuoi SBLE coding sequence as depicted by SEQ ID NO:5 was inserted at the URA3 locus. After successful verification of the knock-in, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1.

[0298] The results are pictured in Figure 6A. This strain produced acidic SLs and bola-SLs, with a majority of the bola SLs. In comparison with the Starmerella bombicola SBLE of Example 3 there was a clear increase in hydrolysis of the produced bola SL substrates in order to produce di-acetylated acidic SLs. The presence of bola SLs indicates that only partial hydrolysis took place and might be caused by the fact that Starmerella kuoi produces mainly Q-hydroxylated SLs whereas Starmerella bombicola produces 0-1 hydroxylated SLs and therefore the Starmerella kuoi LE might be less active on the 0-1 hydroxylated SLs (Price et al. 2012). There was also a smaller lacton peak present, indicating that on some of the bola congeners transesterification by the S. kuoi LE took place. As this is the second lacton peak (see Example 3 with lactonic SL production by the wild type S. bombicola SBLE), this most likely corresponds to sophorolipids with a C18:0 tail. The saturated nature might influence the flexibility of the substrate and therefore the ease to fit into the active site of the enzyme.

[0299] The hypothesis of the hydroxylation preferences was verified with the construction of a strain in which the Starmerella bombicola CYP52M1 cytochrome oxidase (SEQ ID NO: 169), responsible for the Q-l fatty acid hydroxylation was replaced by the Starmerella kuoi CYP52M1 cytochrome oxidase (SEQ ID NO: 171), performing Q-hydroxylation on fatty acids. Hereto the knock-in cassette as shown Fig. 31 was amplified from its respective plasmid and used for transformation into the S. bombicola PT36 strain described in Example 1 according to the methods described in Example 1. The cassette as shown in Fig. 31 was used to create the strain 'CYP::hygro' (genotype: Aura3::0 Acyp52ml::pGAPD_hygro_Ttk) to create a knock-out mutant of the cytochrome oxidase. The knock-in cassette as shown Fig. 3J was amplified from its respective plasmid and used for transformation into this 'CYP::hygro' strain described in this example according to the methods described in Example 1. The cassette as shown in Fig. 3J was used to create the strain 'CYP::SkCYP' (genotype: Aura3::0 Acyp52ml::SkCYP52Ml_tGAL_pURA_URA3_tTK) whereby the Starmerella kuoi CYP52M1 coding sequence as depicted in SEQ ID NO: 5 was inserted at the Starmerella bombicola CYP52M1 locus. The knock-in cassette as shown Fig. 3G was amplified from its respective plasmid and used for transformation into the 'CYP::SkCYP' strain described in this example according to the methods described in Example 1 to create the strain 'KuoiLE_SkCYP' (genotype: Aura3::0 Acyp52m l::SkCYP52M l_tGAL_pU RA_U RA3_tTK

[0300] Asble::pGAPD_hygro_Ttk_pGKI_StarmerellakuoiSBLE_tScCYCl) whereby the Starmerella kuoi LE coding sequence as depicted in SEQ ID NO: 5 was inserted at the Starmerella bombicola SBLE locus and the original SBLE gene was knocked out. After successful verification of the changes, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid or rapeseed oil was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results with oleic acid are pictured in Fig. 6G. The 'KuoiLE_SkCYP' strain produced mainly di-acetylated acidic SLs and almost no bola SLs substrate is left. There was still production of lactonic SLs as was the case before. This proves the hypothesis that the hydroxylation preferences play an important part in the activity of the SBLE homologs, as in this case there is a very clear hydrolysis of the Q-hydroxylated SLs.

[0301] When rapeseed oil was used as substrate an improved production of oligomeric / polymeric glycolipids or glycolipid esters was observed (Fig. 15).

[0302] Example 9

[0303] The knock-in cassette as shown Figure 3C was amplified from its respective plasmid and used for transformation into the S. bombicola ASBLE::hygro strain of Example 2 according to the methods described in Example 1. The cassette as shown in Figure 3C was used to create the strain 'L.E_batistae' (genotype: Aura3::pURA_URA3_tTK_pGAPD_StarmerellabatistaeLE_tGAL Asble::pGAPD_hygro_Ttk), whereby the Starmerella batistae LE coding sequence as depicted by SEQ ID NO: 7 was inserted at the URA3 locus. After successful verification of the knock-in, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid or rapeseed oil was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results with oleic acid are pictured in Figure 6B.

[0304] This strain produced mainly di-acetylated acidic SLs. In comparison with the Starmerella bombicola LE of Example 3 there was a clear shift towards hydrolysis on the produced bola SL substrates in order to produce di-acetylated acidic SLs. The hydrolysis reaction was way more efficient than the one described with the Starmerella kuoi LE of Example 8, possibly due to the hydroxylation preferences. The knock-in cassette as shown Fig. 3G was amplified from its respective plasmid and used for transformation into the 'CYP::SkCYP' strain described in example 8 according to the methods described in Example 1. The cassette as shown in Fig. 3G was used to create this strain 'BatistaeLE_SkCYP' (genotype: Aura3::0 Acyp52ml::SkCYP52Ml_tGAL_pURA_URA3_tTK

[0305] Asble::pGAPD_hygro_Ttk_pGKI_StarmerellabatistaeSBLE_tScCYCl) whereby the Starmerella batistae LE coding sequence as depicted in SEQ ID NO: 7 was inserted at the Starmerella bombicola SBLE locus and the original SBLE gene was knocked out. This strain in which the cytochrome oxidase was switched still produced mainly di-acetylated acidic SLs (Fig. 6H, just like the strain with the S. bombicola cytochrome oxidase. This indicates that for the S. batistae LE both O and 0-1 hydroxylation were well accepted by the enzyme to perform the hydrolysis reaction. In comparison with the 'KuoiLE_SkCYP' strain described in Example 8, there were almost no lactonic SLs produced.

[0306] When rapeseed oil was used as substrate an improved production of oligomeric / polymeric glycolipids or glycolipid esters was observed, in the strain 'EE_batistae' (Fig. 15) and in the strain 'BatistaeLE_SkCYP'.

[0307] Example 10

[0308] The knock-in cassette as shown Fig. 3C was amplified from its respective plasmid and used for transformation into the S. bombicola ASBLE::hygro strain of Example 2 according to the methods described in Example 1. The cassette as shown in Fig. 3C was used to create the strain 'EE_riodocensis' (genotype: Aura3::pURA_URA3_tTK_pGAPD_StarmerellariodocensisLE_tGAL

[0309] Asble::pGAPD_hygro_Ttk), whereby the Starmerella riodocensis LE coding sequence as depicted by SEQ ID NO: 113 was inserted at the URA3 locus. After successful verification of the knock-in, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results are pictured in Fig. 6C.

[0310] This strain produced mainly di-acetylated acidic SLs. In comparison with the Starmerella bombicola LE of Example 3 there was a clear shift towards hydrolysis on the produced bola SL substrates in order to produce di-acetylated acidic SLs. The production spectrum was very similar to the one with the Starmerella batistae LE of Example 9, except that there also was a peak around minute 18.75, corresponding with a lacton SL congener. This indicates that also some transesterification was taking place. This peak was also observed with the Starmerella kuoi LE, but the hydrolysis reaction there was way more limited as quite a lot of bola SL substrate was still present, in contrast to here. Example 11

[0311] The knock-in cassette as shown Figure 3C was amplified from its respective plasmid and used for transformation into into the S. bombicola ASBLE::hygro strain of Example 2 according to the methods described in Example 1. The cassette as shown in Figure 3C was used to create the control strain 'trCALA' (genotype:Aura3::pURA_URA3_tTK_pGAPD_trCALA_tGAL Asble::pGAPD_hygro_Ttk), whereby the SBLE coding sequence as depicted by SEQ ID NO: 9 wasinserted at the URAB locus. After successful verification of the knock-in, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results are pictured in Figure 6D. This strain produced mainly bola SLs s, like the knock out mutants of Example 2. This indicates that the trCALA enzyme does not have activity on the bola SLs produced by Starmerella bombicola.

[0312] Example 12

[0313] The knock-out cassettes as shown Fig. 3D, BE and 3F were amplified from their respective plasmids and used for transformations according to the methods described in Example 1. Using these cassettes and with URAB recovery in between, the strains with genotypes Aura3::0 Aatl::0 Aat2::0 and Aura3::0 Aatl::0 Aat2::0 Aat3::pURA_URA3_tTK were created. The strain with three knock-outs produces mainly non-acetylated bola SLs (WO / 2023 / 213677).

[0314] Example 13

[0315] The knock-in cassette as shown Fig. 3G was amplified from its respective plasmid and used for transformation into the S. bombicola Aura3::0 Aatl::0 Aat2::0 Aat3::pURA_URA3_tTK strain of Example 12, according to the methods described in Example 1. The cassette as shown in Fig. 3G was used to create the strain 'LE_ / <uo / _ATKO' (genotype: Aura3::0 Aatl::0 Aat2::0 Aat3::pURA_URA3_tTK Asble::pGKI_StarmerellakuoiLE_tScCYCl_ pGAPD_hygro_Ttk), whereby the Starmerella kuoi LE coding sequence as depicted by SEQ ID NO: 5 was inserted at the URA3 locus. After successful verification of the knock-in, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results are pictured in Fig. 6E.

[0316] This strain produced mainly non-acetylated acidic SLs. In comparison with the Starmerella kuoi LE of Example 8, there was a clear shift towards production of non-acetylated rather than di-acetylated acidic SLs and no bola SL substrate was left. The lacton peak was also gone. This shows that the hydrolysis reaction was further improved when the available substrates were non-acetylated bola SLs.

[0317] Example 14

[0318] The knock-in cassette as shown Fig. 3G was amplified from its respective plasmid and used for transformation into the S. bombicola Aura3::0 Aatl::0 Aat2::0 Aat3::pURA_URA3_tTK strain of Example 12, according to the methods described in Example 1. The cassette as shown in Fig. 3G was used to create the strain 'LE_bot7Stoe_ATKO' (genotype: Aura3::0 Aatl::0 Aat2::0 Aat3::pURA_URA3_tTK Asble::pGKI_StarmerellabatistaeLE_tScCYCl_pGAPD_hygro_Ttk), whereby the Starmerella batistae LE coding sequence as depicted by SEQ ID NO: 7 was inserted at the URAB locus. After successful verification of the knock-in, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1.

[0319] This strain produced mainly the non-acetylated acidic SLs. In comparison with the Starmerella batistae LE of Example 9, there was a clear shift towards production of non-acetylated rather than di-acetylated acidic SLs. This shows that the hydrolysis reaction was improved when the available substrate were non-acetylated bola SLs. The production characteristics of this strain were very comparable with the results of the Starmerella kuoi LE in the acetyl transferase KO strain of Example 13 (Fig. 6E.).

[0320] Example 15

[0321] The knock-in cassette as shown Fig. 3C was amplified from its respective plasmid and used for transformation into the S. bombicola Aura3::0 Aatl::0 Aat2::0 strain of Example 12 in order to create the strain 'LE_ / <uo / _AT3' (genotype: Aura3::pURA_URA3_tTK_pGAPD_StarmerellakuoiLE_tGAL Aatl::0 Aat2::0), whereby the Starmerella kuoi LE coding sequence as depicted by SEQ ID NO: 5 was inserted at the URAB locus. After successful verification of the knock-in, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. The results are pictured in Fig. 6F.

[0322] This strain produced mainly non-acetylated acidic SLs. In comparison with the Starmerella kuoi LE of Example 8, there was a clear shift towards production of non-acetylated rather than di-acetylated acidic SLs, just like in Example 13. This shows that the hydrolysis reaction was further improved when the available substrate were non-acetylated bola SLs. Example 16

[0323] The knock-in cassette as shown Fig. 3C was amplified from its respective plasmid and used for transformation into the S. bombicola Aura3::0 Aatl::0 Aat2::0 strain of Example 12 in order to create the strain 'LE_bot7Stoe_AT3' (genotype: Aura3::pURA_URA3_tTK_pGAPD_StarmerellabatistaeLE_tGAL Aatl::0 Aat2::0), whereby the Starmerella batistae LE coding sequence as depicted by SEQ. ID NO: 7 was inserted at the URA3 locus. After successful verification of the knock-in, the newly developed strains were evaluated for their production characteristics in shake flask experiments according to the methods described in Example 1. 20 g / L oleic acid was supplemented after 48h of cultivation. Production samples were subjected to analysis according to the methods described in Example 1. This strain produced mainly non-acetylated acidic SLs. In comparison with the Starmerella batistae LE of Example 9, there was a clear shift towards production of non-acetylated rather than di-acetylated acidic SLs, just like in Example 14 (Fig. 6F). This shows that the hydrolysis reaction was further improved when the available substrate were non-acetylated bola SLs.

[0324] The results of the S. bombicola SBLE enzyme homologs are summarized in Table 5.

[0325] Table 5: Summary of the results obtained with SBLE homologs.

[0326] Example 17

[0327] Material and methods

[0328] Materials

[0329] Most chemicals were purchased from Chem-Lab (Zedelgem, Belgium) and Sigma-Aldrich (New Jersey, U.S.), unless indicated otherwise. Bola sophorosides (SSs) (Table 6) were obtained from the Amphistar NV and were produced according to WO / 2021 / 229017

[0330] Table 6

[0331] Production of recombinant SBLE

[0332] For recombinant SBLE (rSBLE) production, a HAC1 co-expressing strain of P. pastoris (syn. Komagataella phaffii) NRRL-Y-11430 (Claes et al., 2016) transformed with the pPICZaB_rSBLEopt construct described in De Waele et al. (2018), was utilized in this research. The strain was grown in buffered glycerolcomplex medium (BMGY) in 3L baffled shake flasks containing 500mL medium for 48h at 28°C, 250rpm. Then, the induction of gene expression was performed in buffered-methanol complex (BMMY) medium for 48 hours at 16°C, 250rpm. Every 12h, 1% methanol was added for continuous stimulation of protein production. Both BMGY and BMMY consist of 1% (w / v) yeast extract (Lab M), 2% (w / v) peptone (BD), lOOmM phosphate buffer at pH 6.0 and 1.34% (w / v) yeast nitrogen base (YNB, Formedium) with 1% (v / v) glycerol or 1% (v / v) methanol as sole carbon source respectively. Finally, the culture containing the produced rSBLE was centrifuged (5000g, lOmin) to collect the supernatant for protein purification. Of note, the rSBLE protein sequence differs from the native SBLE sequence due to replacement of the signal sequence and addition of a C-terminal histidine tag as described by De Waele et al. (2018) (SEQ ID NO: 13).

[0333] Protein purification

[0334] For purification of rSBLE, a two-step purification strategy was utilized following the protocol described in De Waele et al. (2018). In brief, for the first step, purification was done on an AKTA Purifier system (GE Healthcare). Before sample loading, 0.01% (w / v) reduced glutathione and 2mM (final concentration) of magnesium sulfate were added in the supernatant, after which the pH was adjusted to 7.5. After filtering the sample through a Steritop® Filter Unit (EMD Millipore) or VacuCap® (VWR) with a pore size of 0.22μm, the filtrate was subsequently loaded on a HisTrap™ HP column (5mL, Cytiva) previously equilibrated with binding buffer of 50mM Na2HPO4, pH 7.5, 500mM NaCI at a flow rate of SmL-min1. Following sample loading, the column was washed with binding buffer until the UV (280nm) absorbance reached a steady baseline. Then, a stepwise elution was performed using 20- and 200mM, respectively, imidazole in binding buffer. The 2 eluted fractions were mixed and immediately desalted via a buffer exchange using 25mM Tris-HCI, pH 7.5, 150mM NaCI and Amicon® Ultra-15 centrifugal filter devices (Merck) with a lOkDa cut-off, and concentrated to ImL eventually. In the second step, the ImL concentrated IMAC fraction was injected onto a HiLoad® 16 / 600 Superdex® 200pg column (GE Healthcare) equilibrated with the desalting buffer (25mM Tris-HCI, pH 7.5, 150mM NaCI) and eluted with the same buffer. The fractions containing rSBLE were concentrated to l.OmL using Amicon® Ultra-15 centrifugal filter devices (Merck) with a lOkDa cut-off. The concentration of rSBLE was determined using the Thermo Scientific™ Coomassie (Bradford) Protein Assay Kit and using the Bio-Rad Microplate Reader model 680 (595nm). The protein was stored at -80°C for further experiments.

[0335] Protein deglycosylation

[0336] For the deglycosylation of rSBLE using Peptide-N-Glycosidase F (PNGase F), both denaturing and nondenaturing conditions were tested. For the denaturing reactions, 20pg of rSBLE was mixed with lμL Glycoprotein Denaturing Buffer (lOx stock in PNGase F kit, New England Biolabs) and the same SEC purification buffer of rSBLE, as described above, to create a total reaction volume of lOμL. Subsequently, rSBLE was denatured by heating the mixture at 100°C for 10 minutes. After chilling on ice, 2μL GlycoBuffer 2 (lOx stock in PNGase F kit, New England Biolabs), 2μL 10% NP-40 (in PNGase F kit, New England Biolabs) and 6μL SEC purification buffer of rSBLE were added to the mixture to make a total reaction volume of 20μL. The reaction was initiated by the addition of lμL PNGase F (500 units-μL-1, New England Biolabs) and incubated at 37°C for 1 hour. Deglycosylation of rSBLE was verified by SDS-PAGE after mixing with 2x Laemmli buffer. With regard to the non-denaturing reactions, lOpg of rSBLE was mixed 2μL GlycoBuffer 2 (lOx stock in PNGase F kit, New England Biolabs) and SEC purification buffer of rSBLE to a reaction volume of 20μL. Afterwards, lμL PNGase F (500 units-μL-1, New England Biolabs) was added, and the reaction was then incubated at 37°C or room temperature to examine the effect of temperature on the deglycosylation of rSBLE. The deglycosylation samples were also mixed with 2x Laemmli buffer prior to SDS-PAGE analysis.

[0337] SDS-PAGE

[0338] Protein samples were prepared by diluting in 2x Laemmli buffer, samples were then separated through

[0339] SDS-PAGE by loading on a Mini-PROTEAN precast gel (12% polyacrylamide) (Bio-Rad) and using a Tris- Glycine-SDS (TGS) running buffer (Bio-Rad), and the gel was subjected to 150V for approximately one hour. The Precision Plus Protein™ Unstained Standard (Bio-Rad) was used as a molecular weight marker. Gels were stained with Coomassie brilliant blue G for a minimum of 3 hours or using SimplyBlue™ SafeStain solution (Invitrogen™) for approximately 1 hour. Afterwards, the gels were destained using 30% (v / v) methanol or Mill i-Q water for at least 1 hour, depending on the staining method. The gels were scanned using a GS-800 calibrated densitometer (Bio-Rad) and visualized via the Quantity One software package (Bio-Rad).

[0340] Enzymatic activity assays

[0341] An HPLC-based activity assay was followed as described by De Waele et al. (2018) with some adaptations. In brief, lOpg of purified deglycosylated rSBLE was added to 500μL of reaction buffer, containing 5mM of substrate and 50mM sodium citrate at pH 3.5. The crude acidic SL mixture (ASL mix) described in (Ciesielska et al., 2016) was used as substrate in the assay. The mixture was incubated for 2 hours at 30°C and 1400rpm after which the reaction was stopped using 1500μL 100% (v / v) ethanol. The reaction in which rSBLE was replaced by the same amount of buffer (25Mm Tris, 150mM NaCI, pH 7.5) used for protein purification was prepared as negative control in the assay. After concentrating the sample using a SpeedVac vacuum centrifuge (Thermo Savant, Holbrook, NY) to 250μL, lOOμL of the samples were analyzed by reverse phase HPLC using an Ettan™ LC system (GE Healthcare) implementing a ZORBAX Eclipse Plus C18 Rapid Resolution 4.6mmxl00mm column (Agilent) using a gradient separation with UV absorption detection (207nm). The gradient started at 30% ACN and linearly increased to 50% in 15min, after which the gradient increased linearly from 50% ACN until 80% in 30min. The elution solvent was kept at 80% ACN for 5min and was then returned to 30% ACN in lmin. A flow rate of O.lSmL-min1was applied. For those peaks that significantly differed in the chromatograms of samples before and after reaction, fractions were collected for MALDI-TOF MS analysis. Note that this method is slightly different from those described in (De Waele et al., 2018) and although HPLC profiles look similar, retention times cannot be directly compared.

[0342] Trypsin digestion and MALDI-TOF MS

[0343] The bands of interest on SDS-PAGE gel were excised and the stain was completely removed using 50% acetonitrile (ACN) (Biosolve) / 200mM NH4HCO3 at 30°C. Afterwards, gel bands were first submerged in lOmM DTT / lOOmM NH4HCO3 for 1 hour at 56°C for a reduction step, and then subjected to an alkylation step with 55mM iodoacetamide (IAA) / 100mM NH4HCO3 for 45 minutes at room temperature in the dark. Then, the gel bands were washed with lOOmM NH4HCO3 and dehydrated with 100% ACN, twice each. After the last dehydration step, gel bands were dried in a SpeedVac vacuum centrifuge (Thermo Savant, Holbrook, NY). lOμL diluted trypsin (Promega) (0.002pg-μL-1in 50mM NH4HCO3) was then added to the dried bands and samples were incubated on ice for 45 minutes. This was followed by the addition of 50μL 50mM NH4HCO3 in the samples and the proteins were digested at 37°C for overnight. The next day, peptides were extracted twice using 60% ACN (Biosolve) / 0.1% formic acid (HCOOH) solution. The dried peptides were then resuspended in 12μL of 50% ACN / 0.1% trifluoroacetic acid (TFA) solution. lμL of peptides suspension, mixed with a saturated a-cyano-4- hydroxycinnamic acid solution in a 1:1 ratio, was spotted onto an Opti-TOF 384 Well MALDI Plate Insert for MALDI-TOF MS analysis with the MALDI TOF / TOF 4800 Plus (ABSciex). Identification was done by peptide fragment fingerprint analysis using Mascot Server (Matrixscience), searching against the Starmerella bombicola protein database.

[0344] Multi-angle laser light scattering (SEC-MALLS)

[0345] Multi-angle laser light scattering (MALLS) analysis was carried out following the protocol described in (Verschueren et al., 2019) with some adjustments. In brief, lOOμL of protein sample was loaded onto a Superdex® 200 Increase 10 / 300 GL column (GE Healthcare) with a running buffer of 25mM Tris, pH 7.5 and 150mM NaCI at 0.5mL-min-1, coupled to an online UV-detector (Shimadzu), a multi-angle light scattering miniDAWN TREOS instrument (Wyatt) and a Optilab T-rEX refractometer (Wyatt) at 25°C. The protein concentration and molecular mass were determined by a refractive index increment (dn / dc) value of 0.185mL-g-1, and the data analysis was performed using the ASTRA6 software (Wyatt). Correction for band broadening was applied using parameters derived from BSA (2mg-mL-1, Pierce) loaded under identical running conditions. To obtain information about the protein after the addition of the ligand of bola SSs (molar ratio: protein / ligand=l / 3), the ligand with same molarity was also added into the running buffer.

[0346] Crystallization and structure determination

[0347] Following SEC purification, protein samples were concentrated to a final concentration of 4 to lOmg-mL-1using Amicon® Ultra-15 centrifugal filter devices (Merck) with a 10 kDa cut-off. The protein concentration was determined using the Thermo Scientific™Coomassie (Bradford) Protein Assay Kit and the Bio-Rad Microplate Reader model 680 (595nm). For the co-crystallization experiments with ligand, the concentrated protein was mixed with a 3-fold molar excess of the ligand of interest followed by overnight incubation at 277K to promote the formation of the protein-ligand complexes prior to setting up the crystallization screens (Hassell et al., 2007). Nanoliter-scale vapour diffusion crystallization experiments in sitting-drop format were set up at 277K or 287K in 96-well triple drop plates (SwissSci) using commercial sparse-matrix crystals screens (Molecular Dimensions, Hampton Research) and a Mosquito liquid handling crystallization robot (TTP Labtech). Promising hits were further optimized using gradient optimization in 96-well (SwissSci) and / or 24-well (Hampton Research) crystallization plates. The detailed process and results of crystallization trials are described in the results section, and the optimized conditions are listed in Table 7. Crystals were cryoprotected using a quick soak in mother liquid supplemented with the respective cryo-solution (Table 7) and, when appropriate, ligands prior to being cryo-cooled by plunging into liquid nitrogen. X-ray diffraction of crystals were performed at 100 K at synchrotron radiation facilities PETRA III Hamburg (microfocus beamlines P13, P14). All data were indexed, integrated and scaled using the XDS suite (Kabsch, 2010) and merged using Pointless and Aimless (Evans, 2011; Evans & Murshudov, 2013) from the CCP4 suite (Winn et al., 2011), and the data quality was analyzed by Phenix.xtriage (Zwart et al., 2005). To obtain the initial phases, molecular replacement was performed in Phaser (Mccoy et al., 2007) using the AlphaFold (Jumper et al., 2021) predicted structure of rSBLE as a search model. The refinement of coordinates and atomic displacement parameters was carried in Phenix (Afonine et al., 2012; Liebschner et al., 2019) and autoBuster (https: / / www.globalphasing.com / buster / ), and the model (re)building was done manually in Coot (Emsley et al., 2010). Model and map validation tools in Coot and the Phenix suite, the CCP4 suite and the PDB_REDO server (Joosten et al., 2014) were used to guide improvement and to validate the quality of crystallographic models during the entire the workflow.

[0348] Table 7 Figures containing structural models were prepared in PyMOL Molecular Graphics System (version

[0349] 2.5.4, Schrodinger, https: / / pymol.Org / 2 / ). Structures were compared and superimposed using DALI (Holm, 2022). Data were plotted using Origin 2023 software.

[0350] Results

[0351] Deglycosylation of rSBLE using Endo H endoglucosidase

[0352] Since the high degree of glycosylation is unfavourable for protein crystallization, the removal of most glycans from rSBLE was considered necessary for the preparation of protein samples. With reference to previous studies (Ciesielska et al., 2016), the N-glycans of rSBLE were first digested with Endo H, the most commonly used N-glycosidase. Unfortunately, the deglycosylated rSBLE (rSBLE_DGH) obtained was still heterogeneous as two protein bands were observed on SDS-PAGE, indicating two products with close molecular weights (MWs) around 45kDa (Figure 7). This indicates that even after digestion with Endo H the recombinant product was still heterogeneous, probably due to the different level of glycosylation between the two protein forms. Despite the heterogeneous character of this protein solution, we used it already for some initial crystallization efforts.

[0353] The importance of bola sophoroside as an additive for rSBLE crystallization

[0354] The crystallization of deglycosylated rSBLE generated using Endo H (rSBLE_DGH) was performed first. After SEC purification, the protein sample was concentrated to 5.5 mg-mL-1. The commercial sparse- matrix crystallization screens were then set up and carried out through vapor diffusion method in sitting drop geometry. In 96-well triple-drop plates (SwissSci), lOOnL of the concentrated protein solution was mixed with lOOnL mother liquor of reservoir using a Mosquito liquid handling crystallization robot (TTP Labtech). The crystallization drops were equilibrated against 45μL reservoir and plates were incubated at 287K (14°C) to grow crystals. A first hit appeared in "Proplex Screen" with the condition consisting of 0.1M Mg-acetate, 0.1M Na-acetate pH 4.5, 8% (w / v) Polyethylene glycol (PEG) 8000 (Table 7). This condition was further optimized in a 24-well plate by mixing lμL protein with lμL mother liquor also in sitting drop format. The crystals grown in the optimized condition (0.1M Mg- acetate, 0.1M Na-acetate, pH 5.0, 9% (w / v) PEG8000) were cryoprotected in mother liquor containing 30% (v / v) PEG400 prior to being cryocooled in liquid nitrogen. This was followed by X-ray diffraction of selected crystals. Unfortunately, none of the crystals displayed diffraction, indicating that the quality of frozen crystals is insufficient for diffraction by X-ray.

[0355] In order to improve the quality of crystals obtained ultimately, co-crystallization experiments with the ligand bola sophorosides (SSs), an inert analog of bola sophorolipids (SLs) was conducted using the protein sample of rSBLE_DGH. A 3-fold molar excess of bola SSs was complexed with the protein solution of concentrated rSBLE_DGH (4.6mg-mL-l). After overnight incubation at 277K (4°C), commercial sparse-matrix crystallization screens were set up also in sitting-drop format via vapordiffusion method as described previously. Diffraction-quality crystals appeared in following two conditions: (1) lOmM spermine tetrahydrochloride, lOmM spermidine trihydrochloride, lOmM 1,4- diaminobutane dihydrochloride, lOmM DL-ornithine monohydrochloride, 0.1M MOPSO / Bis-Tris, pH

[0356] 6.5, 12.5% (w / v) PEG 4000, 20% (w / v) 1,2,6-hexanetriol, and (2) 0.1M sodium HEPES, pH 7.0, 20% (w / v) PEG8000 (see Table 7). In the drop of condition 1, 200nL mother liquor was mixed with lOOnL protein complex, while in the drops of condition 2, lOOnL protein complex was added to 100 / 200nL mother liquor. The single crystals resulting from condition 1 were cryoprotected in mother liquor with the addition of bola SSs, the single crystals grown in condition 2 were cryoprotected by 25% (v / v)

[0357] PEG400 or ethylene glycol with bola SSs, and then flash frozen in liquid nitrogen for storage until data collection. The collected X-ray diffraction data (Table 8) showed that the crystals obtained under both conditions diffracted well and a different space group was obtained for each condition respectively. The high-resolution structures of rSBLE were obtained with a maximum resolution of 1.90A and 2.05A respectively. The positive results indicated that the ligand binds to rSBLE_DGH, thereby improving the stability of the protein and facilitating crystal packing.

[0358] Table 8

[0359] General features of the rSBLE crystal structures

[0360] Two different space groups, C2221 and P21212, in the orthorhombic crystal form were obtained under the two different crystallization conditions described above (Table 8). At first, one monomer is in the crystallographic asymmetric unit of the C2221 space group (Figure 8), while the asymmetric unit of the P21212 space group contains two rSBLE molecules (Table 8 and Figure 9). In the P21212 space group, the two monomers, named as A and B, show significant and extensive contact at the N-terminal interface (Figure 9d), and they can be superimposed by a certain rotation (Figure 9c): polar (omega / phi / kappa) = 92.1645 / 1.4959 / 178.5926 and euler (alpha / beta / gamma) =

[0361] 160.4894 / 175.4481 / 16.5189, obtained from Coot (Emsley et al., 2010). Further analysis applying PISA (https: / / www.ebi.ac.uk / pdbe / pisa / ) revealed that the two monomers formed a stable dimer with an interface area of 1367A2. We also performed a multi-angle laser light scattering (SEC-MALLS) analysis for the protein sample of rSBLE_DGH before and after the addition of 3x molar bola SS (Figure 10). The main fractions of both protein samples observed were monomer with molecular mass of 41.6 ± 0.7kDa and 47.2 + l.OkDa, respectively. This suggests that at the concentration of rSBLE_DGH in the protein solution mixture used for crystallization, the protein is not able to form a stable dimer after the addition of bola SSs. This indicates that the dimer is formed as a result of the relatively high concentration of protein reached during vapour diffusion in the sitting drop prior to crystallization. As a matter of fact, the addition of bola SSs to rSBLE_DGH also resulted in a small degree of aggregation of this protein since a small peak in the chromatogram with molecular mass higher than 150kDa appeared, while only a negligible amount of dimer (molecular mass in 60-90kDa) was observed in the protein sample before the addition of the ligand (Figure 10). The final model of rSBLE in the C2221 space group includes residues 4 to 398 of the 405 residues in amino acid sequence of rSBLE. The electron density of the three N-terminal residues of rSBLE is not clearly visible. Therefore, amino acids from the N-terminal cloning site Alai and Gly2 as well as Leu3 are missing. Similarly, the electron density for the C-terminal cloning site amino acid Asp399 and the C -terminal 6xHistag is also missing. This might be attributed to the flexibility of both the N- and C- termini. In comparison, the electron density of Leu3 for the two monomers in the asymmetric unit of the P21212 space group is apparently present, albeit not 100% complete. In addition, Asp399 of monomer A is also visible in terms of its electron density. The two molecules (A and B) in the asymmetric unit of the P21212 space group are highly similar, the root mean square deviation (RMSD) between them is 0.3A for Ca atoms (residues 3-398) calculated by DALI (Holm, 2022). Moreover, the monomer in the C2221 space group is also not significantly different from the two molecules (A and B) in the P21212 space group with the RMSD values in Ca atoms of 0.3A and 0.4A, respectively (residues 4-398).

[0362] SBLE shows the typical a / p hydrolase fold consisting of a discrete a-helix lid and a a / p hydrolase domain, which is consistent with the previously predicted structure (Ciesielska et al., 2016) using Phyre2.0 and the AlphaFold model used for the molecular replacement in structure determination of rSBLE. The structure of this enzyme contains 13 a-helices and 9 p-strands (Figure 8d), of which the a- helix lid is composed of 5 a-helices, from a6 to alO (residues Ala215 to Gln305) and inserted between P7 and P8, which is one less than the AlphaFold model mentioned above. A short a-helix (Gln275- Leu279) predicted by AlphaFold at low confidence is absent in the actual structure of SBLE where it is substituted by a flexible loop. The RMSD between the AlphaFold model of SBLE and its crystal structure is 1.7A calculated also by DALI, which is identical to the RMSD between the structure of SBLE and CALA. In the case of SBLE, therefore, the results showed that there is a clear discrepancy between the AlphaFold model and the actual structure of the protein. The coordinates of the crystal structures of the two structures are in Table 9 (monomeric) and Table 10 (dimeric form), respectively.

[0363] The bola SS ligand was added to the protein to serve as an inactive substrate analogue. We checked for electron density for bola SS in the electron density map in each of the two space groups. The electron density of sophorose head of bola SS was partially found in both molecules of the P21212 space group (Figure 11a, b). Surprisingly, the binding site of bola SSs is not at the active site, but in the cavity between the monomers, consisting of nine residues: Thr238, Ser242, Pro245, Phe248, Glu249, Trp252, Met263, Leu264 and Arg267 (numbering according to rSBLE, SEQ. ID NO: 13). Trp 252 seems to be involved in aromatic / n contacts with a glucose unit of the sophorose. This binding cavity shows a negative electrostatic potential usually observed in carbohydrate-binding proteins, and might explain the binding of sophorose head of bola SS at this cavity. The binding mode of bola SSs is most likely to be one molecule of bola SSs per two molecules of rSBLE (Figure lie), as such forming a bridge between the monomers in the dimer in the P21212 space group. The formed dimer probably stabilized the protein and allowed SBLE packing correctly in the crystals, and thereby improved the X-ray diffraction quality of crystals. In the electron density map of the monomer in the C2221 space group, only a small fraction of unknown electron density is observed around the same area, but we cannot distinguish whether this site is truly occupied by a glucose from the sophorose head of bola SS. Although this binding mode of bolaSS to bridge two rSBLE monomers may be a crystallization effect, the binding of the sophorose head into the cavity is strong and we postulate that it may serve as a model for adhesion of rSBLE on sophorolipid micellar or lamellar structures.

[0364] Active site, active site pocket, disulfide bridge and substrate binding

[0365] The consensus lipase active site motif GXSXG, is found in the rSBLE sequence at location 179-183 (GYSGG, SEQ ID NO: 13). Serl81 in rSBLE corresponds to Serl94 in SBLE (SEQ ID NO: 2) was previously predicted to form the catalytic triad with Asp346 and His378 (Ciesielska et al., 2014). Indeed, SBLE was completely deficient for its activity after site-directed mutagenesis (S194A). The crystal structure confirms the predicted catalytic triad in rSBLE, it is constituted of Serl81, Asp333 and His365 (SEQ ID NO: 13), of which Serl81 is located in a sharp bend, the well-known nucleophilic elbow, between P6 strand and a5 helix. Asp333 is situated in the loop after P8 strand and before all helix, and His365 is positioned at the beginning of al2 helix. Like typical lipases (e.g. CALA), the structure of SBLE also contains two main segments, i.e., the a / p hydrolase domain and the a-helix lid (Figure 8b). A large cavity (Figure 8c) is formed between the a / p hydrolase domain and the lid to serve as an active site pocket around the catalytic triad. This cavity is stabilized by a disulfide bridge between Cys99 and Cys273, corresponding to Cysll2 and Cys286 in the sequence of SBLE. These cysteines are conserved compared to CALA and the existence of this disulfide bridge was predicted previously (Ciesielska et al., 2016). As in CALA, this cavity is quite large explaining that bulky substrates, in case of SBLE the bola SLs, can bind into the active site. The active site region in SBLE exhibits a highly negative electrostatic potential, which is often observed in carbohydrate-binding proteins (Hermoso et al., 2004). This could accommodate the bola SLs' sophorose head.

[0366] Relationships to other structures

[0367] A number of similar structures were revealed by a structure alignment of rSBLE (monomer in C2221 space group) using DALI (Holm, 2022). The most similar structures are, as expected from primary structure comparison, from the CALA (Moesziomyces antarcticus lipase A) family: CALA (Pdb id: 2VEO / 3UGG) and lipase UM03410 from Ustilago maydis (Pdb id: 3ZPX). The RMSD values between rSBLE and the two lipases are 1.7A and 1.8A, respectively. For the rest of similar structures, the RMSD values are higher than 3.0A. Compared to the catalytic triad in both lipase structures, the catalytic triad of SBLE can be almost superimposed on the triad of CALA and lipase UM03410. The typical conserved active site motif (GXSXG) for lipases is GYSGG in all three enzymes. In addition, the residues Glyl85 and Asp95, forming the oxyanion hole in CALA, are quite conserved at the equivalent positions in both rSBLE (Glyl82 and Asp93) and lipase UM03410 (Glyl85 and Asp95).

[0368] As predicted, the most striking difference between SBLE and CALA is that SBLE shows an absence of approximately 40 amino acids at the C-terminus of CALA containing the active-site flap of CALA formed by residues Gly426 to Gly436 (P strands: piO-pil), which was considered to flip out to contact the lipid interface during interfacial activation. In addition, SBLE has a larger cavity in the active site pocket than CALA and lipase UM03410, this allows the possibility for bulky substrates to enter the active-site pocket and make contact with the active sites.

[0369] Compared to the lid domain of CALA, SBLE lacks one a-helix (a9 in CALA). This a-helix, positioned above the active site pocket in CALA, is replaced by a flexible loop in SBLE. In addition, helix a7 is slightly distorted at its N-terminal side. In CALA, this part of the molecule contains Phe233 that is part of the hydrophobic residues to stabilize the lipid substrate, but contains here a serine, presumably leaving room for larger substrates alike the sophorolipids.

[0370] Determination of glycosylation sites

[0371] Natural SBLE shows a high degree of glycosylation (Ciesielska et al., 2014), most likely as a result of extensive N-linked glycosylation. Nine potential N-linked glycosylation sites are present in the SBLE sequence. The Asn-Xaa-Ser / Thr consensus motifs for N-glycosylation, is found at Asn34, Asn78, Asnl22, Asnl33, Asnl81, Asn296, Asn325, Asn367 and Asn406 (corresponding to the residues of Asn21, Asn65, Asnl09, Asnl20, Asnl68, Asn283, Asn312, Asn354 and Asn393 in rSBLE). The crystal structure was obtained on the rSBLE treated with Endo H, which leaves a single N-acetylglucosamine (NAG) bound to the protein. Indeed, in the structure of rSBLE obtained from the monomeric species (C2221 space group), at seven of the consensus sequence asparagines (Asn65, Asnl09, Asnl68, Asn283, Asn312, Asn354 and Asn393 in rSBLE), there is an electron density that can be attributed to the presence of a NAG residue group. The electron density of NAG is very clear at all Asn sites, except for Asn312 where there is only a partial electron density. Unexpectedly, from the electron density map we also have strong evidence for O-linked glycosylation at the C-terminal residue Thr397 of rSBLE, on which a mannose a-1,2 mannose (Man a-1,2 Man) can be fitted. In the structure derived from the dimeric species (P21212 space group), eight N-glycosylation sites are clearly visible. Only for Asn21 no evidence for N-glycosylation is obtained, similar as for the monomeric structure indicating that this Asn21 is not glycosylated, although it is well exposed to the surface. Curiously, the molecule B in the dimeric structure is devoid of the electron density of NAG at residue Asn283 compared to the molecule A. The electron density revealing O-linked glycosylation (Man a-1,2 Man) at residue Thr397 was also observed for both molecules in the P21212 space group.

[0372] Example 18

[0373] Methods used to design and create mutants of rSBLE.

[0374] Computational methods

[0375] The crystal structure of rSBLE did not reveal how exactly the natural substrate, i.e a bola SL is recognized and converted. Indeed, the substrate analogue, bola sophoroside unexpectedly bound to a site different from the active site (Example 17). Therefore, a computational approach was followed to perform a docking of tetra acetylated bola SL into the active site of rSBLE. Using GROMACS (release 2021.5) molecular dynamics simulations of 2 ns of the structure of rSBLE was performed to mimic the flexibility of the structure. Then using the program Autodock Vina we performed several docking simulations of the structure of a tetra acetylated bola sophorolipid. From the results, we further analyzed the conformations with the lowest binding energy, taking into account a topology that would allow the nucleophilic attack of the active site Serl81 to the substrate ester bond according to the known mechanism of serine hydrolases. We used OpenMM program to run MD simulations of SBLE or its mutants with acetylated or non-acetylated sophorolipid to investigate the binding of the substrate.

[0376] The construction of models of the 3D structure of the homologous proteins in S. kuoi, S. batistae. and S. apicola was done using the service of ModWeb version r273, and alphaFold. Then superposition of these models, SBLE and CalA (RCSB: 2veo) was done with the tool PDBeFOLD from EMBL-EBI to compare the fold allowing to investigate if there are any features that could affect the binding of the sophorolipid.

[0377] Production of mutants of rSBLE

[0378] The vector pPICZaB_SBLE_Pstl_Sall used to integrate the SBLE gene into the AOX1 region of a host Pichia pastoris was mutated by site directed mutagenesis to incorporate the mutations S125P_P129G, L128Y P129Q, F160I, L225D, I237Q, L242Y, S245N, S245F, M249E, Q288Y, Q288V-S289V-P290L- P290_l291_insT, L348D, D379V, A382E and A382F into the SBLE gene. The mutations were performed using the kit Pfu Tubo DNA Polymerase from Agilent Technologies, Inc.

[0379] The standard method was optimized by performing a step of 7 cycles of the reactions with either the forward or reverse primer. After which the two reactions with the primer reverse or forward were combined for another 12 cycles. The composition was consisting of distilled water (dH2O) 16.3 pl, 10x Cloned Pfu reaction buffer 2.5 pl, dimethylsulfoxide (DMSO) 2.5 pl, MgSO4 20 mM 1.2 pl, dNTPs (25 mM each dNTP) 0.5 pl, DNA template (100 ng / pl) 1.0 pl, primer reverse or forward (100 ng / pl) 0.5 pl, PfuTurbo DNA polymerase (2.5 U / pl) 0.5 pl for a total of 25 pl.

[0380] The PCR reaction started with a step at 95°C for 2 min, then the cycles were compromised of 30 s at 95°C, 20 s at 55°C and 8 min at 72°C, after the cycles a last step of amplification for 10 min at 72 °C was performed.

[0381] The PCR was digested with 1 pl of Dpnl (New England Biolabs®) at 37°C overnight. The vector was transformed into E. coli DH5a electrocompetent cells. The colonies were growth in low salt Lysogeny- Broth medium supplemented with 200 pg / ml of zeocin, and plasmid extraction was performed following the nucleospin protocol Macherey-Nagel™.

[0382] The incorporation of the mutations in the vector were confirmed by sequencing.

[0383] The vectors were then linearized (5 pg) by digesting with 1 pl of Pmel (New England Biolabs®) at 37°C for 2 hrs. Then, 0.5 pl the linearized DNA was transformed into P. pastoris electrocompetent cells (with a Haclp overexpression). The electroporation was performed at 1.5 kV, the cells were allowed to recover in 1 ml of sorbitol for two hours (30°C) without shaking, then 1 ml of YPD was added for 2 more hours. Finally, the cells were spread into low salt YPD agar plates with zeocin 200 pg / ml and hygromycin 200 pg / ml.

[0384] Selected transformants were then grown in buffered glycerol-complex medium (BMGY) in 3 L baffled shake flasks containing 500 ml medium for 48 h at 28 °C, 250 rpm. Then, the induction of gene expression was performed in buffered-methanol complex (BMMY) medium for 48 h at 16 °C, 250 rpm. Every 12 h, 1 % methanol was added for continuous stimulation of protein production. Both BMGY and BMMY consist of 1% (w / v) yeast extract (Lab M), 2% (w / v) peptone (BD), 100 mM phosphate buffer at pH 6.0 and 1.34% (w / v) yeast nitrogen base (YNB, For medium) with 1% (v / v) glycerol or 1% (v / v) methanol as sole carbon source respectively. Finally, the culture containing the produced rSBLE was centrifuged (5000g, 10 min) to collect the supernatant for protein purification.

[0385] Purification of recombinant SBLE mutants

[0386] For purification of mutant SBLE, a two-step purification strategy was utilized by following the protocol described in De Waele et al. (2018). In brief, for the first step, purification was done on an AKTA Purifier system (GE Healthcare). Before sample loading, 0.01 % (w / v) reduced glutathione and 2 mM (final concentration) of magnesium sulfate were added in the supernatant, after which the pH was adjusted to 7.5. After filtering the sample through a Steritop® Filter Unit (EMD Millipore) or VacuCap® (VWR) with a pore size of 0.22 μm, the filtrate was subsequently loaded on a HisTrap™ HP column (5ml, Cytiva) previously equilibrated with binding buffer of 50 mM Na2HPO4, pH 7.5, 500 mM NaCI at a flow rate of 5 ml / min. Following sample loading, the column was washed with binding buffer until the UV (280nm) absorbance reached a steady baseline. Then, a step-wise elution was performed using 20- and 200mM, respectively, imidazole in binding buffer. The 2 eluted fractions were mixed and immediately desalted via a buffer exchange using 25 mM Tris-HCI, pH 7.5, 150 mM NaCI and Amicon® Ultra-15 centrifugal filter devices (Merck) with a 10 kDa cut-off, and concentrated to 1ml eventually. The fractions containing mutant SBLE were concentrated to 1.0 ml using Amicon® Ultra-15 centrifugal filter devices (Merck) with a 10 kDa cut-off. The concentration of protein was determined using the Thermo Scientific™ Coomassie (Bradford) Protein Assay Kit and using the Bio-Rad Microplate Reader model 680 (595nm). The protein was stored at -80°C for further experiments.

[0387] HPLC based assay for activity testing

[0388] An HPLC-based activity assay was followed as described by De Waele et al. (2018) with some adaptations. In brief, 10 pg of purified rSBLE was added to 500pl of reaction buffer, containing 5 mM of substrate and 50 mM sodium citrate at pH 3.5. The mixture was incubated for 2 h at 30°C and 1400 rpm after which the reaction was stopped using 1500pl 100% (v / v) ethanol. The reaction in which rSBLE was replaced by the same amount of buffer (25mM Tris, 150mM NaCI, pH7.5) used for protein purification was prepared as negative control in the assay. After concentrating the sample using a SpeedVac vacuum centrifuge (Thermo Savant, Holbrook, NY) to 250pl, lOOpI of the samples were analyzed by reverse phase HPLC using an Ettan™ LC system (GE Healthcare) implementing a Epic C8 Cartridge Column-220 mm x 2.1 mm (ES Industries), using a gradient separation with UV absorption detection (207nm). The gradient started at 30% acetonitrile (ACN) and linearly increased to 50% in 15 min, after which the gradient increased linearly from 50% ACN until 80% in 30min. The elution solvent was kept at 80 % ACN for 5 min and was then returned to 30% ACN in 1 min. A flow rate of 0.15 mL / min was applied.

[0389] Alternatively, HPLC analysis with CAD detection was performed as described in Example 1.

[0390] Results:

[0391] Docking of substrate into rSBLE active site

[0392] The model substrate, tetra acetylated bolasophorolipid, was used for in silico docking of the substrate in the active site of the rSBLE structure (model 1 in Example 17). Furthermore, we used OpenMM program to run MD simulations of SBLE or its mutants with acetylated or non-acetylated sophorolipid to investigate the binding of the substrate. From the different solutions provided by the molecular dynamics solutions, the one with best energy fit combined with correct positioning of the carboxylester of the substrate in view of the nucleophilic attack of the active site serine in the first step of the mechanism was selected. Figure 12 displays how the substrate fits in a cleft. The coordinates of the atomic structure of the protein substrate complex are listed in Table 11. The rSBLE protein contains two flexible loops I235-K243 and G344-L351 that are proposed to move out to allow entry of the substrate. In this model, two sophorose head groups enter the active site pocket wherease the lipid tail is proposed to interact with a number of hydrophobic residues in this packet, containing, amongst others Tyr237, Tyr241 and Ile246. The model exposes the sophorosyl ester bond to the active site residues allowing a nucleophilic attack of Serl81 to form an acyl intermediate. The consequent release of sophorose seems to leave room for the consequent attack from the second sophorose group. The open structure of the acyl intermediate is different from classical lipases where the acyl intermediate recruits a water molecule that is activated in a hydrophobic environment and the presence of the acyl side chain of a Glu or Asp residue. The activated water molecule would then induce a hydrolysis of the acyl intermediate to release an acid. Amino acid residues around positions 132, 159, 160, 225, 348 and 382 in SBLE could be alternative positions to act as water activating residues if they would be acidic in nature, but are hydrophobic instead.

[0393] From this docking experiment several residues can be defined that play an important role in binding the substrate taking into account some of these residues are positioned on a flexible loop that is proposed to move upon substrate binding. These can be divided in residues that form hydrogen bonds to sugar alcohols or those that bind to the acetyl groups via hydrogen bonds. The following residues in SBLE are proposed to make contacts with the sophorose heads : Phel04, Glul05, Aspl06, Serl25, Asnl26, Leul28, Prol29, Tyrl93, Serl94, Met249, Cys286, Asn287, Gln288, Glu347, Leu348, His378, Asp379, Ala382 and Tyr383.

[0394] Results of assays on purified SBLE mutants.

[0395] For the mutant enzyme S125P_P129G a clear shift in the chromatographic pattern compared to the wild type was observed when either acetylated or non-acetylated bolaform sophorolipids were used as the substrate (Fig 13). Compared to the wild type, almost no lactonic sophorolipids were obtained, whereas the peak corresponding to acidic sophorolipids increased dramatically, as predicted from the structural model. Integration of the peaks revealed that using the mutant enzyme 94.3% of the substrate was converted into acidic SLs, compared to 75.1 % when the WT enzyme is used. Additionally, an A382E and L348D mutant enzyme showed improvement in hydrolytic activity compared to the pure WT enzyme when acetylated and non-acetylated SLs, respectively, were used as the substrates (Table 12). Table 12.

[0396] Example 19

[0397] Use of the SBLE enzyme to synthesize SL oligomers / polymers / esters from triglycerides and bola sophorolipids

[0398] Material and methods

[0399] The capacity of SBLE enzyme to catalyze transesterification reaction on bola SLs and triglycerides to synthesize sophorolipid oligomers / polymers / esters (SL oligomers / polymers / esters) reported as the third-most abundant component in the natural S. bombicola sophorolipid products (Kobayashi et al., 2023) was evaluated. 500ug of purified recombinant SBLE (produced according to Example 17, dissolved in (25mM Tris, 150mM NaCI, pH7.5)) was added to 500pl of reaction mixture containing bola SLs (code: INV-113, containing mainly acetylated bola SLs) and a commercial food grade rapeseed oil (Brassica, dissolved in n-Hexane) diluted in a 1:1 ratio (final concentration 5mM) in 50 mM sodium citrate (Merck) at pH 3.5. The reaction mixture was incubated at 30°C under agitation at 1500rpm for lh, 2h, 4h, 8h, 12h and 24h, followed by the addition of 1500pl 100% (v / v) ethanol (Chem-Lab) to terminate the reaction. Afterwards, the samples were concentrated to 250pl using a SpeedVac vacuum centrifuge (Thermo Savant, Holbrook, NY). Blank reaction mixtures contained the same substrates, but instead of enzyme solution, the same amount of a buffer solution (25mM Tris, 150mM NaCI, pH7.5) was used in the assay and incubated at 30°C, 1500rpm for 4h, 12h and 24h.

[0400] HPLC and MALDI-TOF MS analysis of sophorolipids and sophorolipids glycerides were performed as follows: 100 microliter of the reaction products were fractionated by HPLC on an Ettan™ LC system (GE Healthcare) using an Epic C8 column-150 x 2.1mm, 3pm (Perkin Elmer®) and UV absorption detection (207nm, GE Healthcare) using a gradient separation. The gradient started at 30% ACN and linearly increased to 50% in 15 min, after which the gradient increased linearly from 50% ACN till 80% in 30min. The mixture was kept in this way for 5 min and was then brought back to 30% ACN in 1 min. A flow rate of 0.15 mL / min was applied.

[0401] The chromatograms from the enzyme reactions were compared with those from the blank control. Fractions collected from significantly increased peaks after reaction where submitted to Matrix- Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The collected fractions were first dried under SpeedVac vacuum centrifuge (Thermo Savant, Holbrook, NY) and the dried compounds were then resuspended in 5 pl of 50% ACN (BioSolve) / 0.1% trifluoroacetic acid (TFA, Sigma-Aldrich) solution. 1 pl of resuspended compound, mixed with a saturated a-cyano-4- hydroxycinnamic acid solution in a 1:1 ratio was spotted onto an Opti-TOF 384 Well MALDI Plate Insert for MALDI-TOF MS analysis with the MALDI TOF / TOF 4800 Plus (ABSciex).

[0402] Results

[0403] Activity assay of rSble using triglycerides and bola sophorolipids

[0404] To evaluate intermolecular transesterification activity of SBLE enzyme, rSBLE enzyme was incubated with bola SLs containing acetylated bola SLs (code: INV-113) and triglycerides (rapeseed oil was used in the test) as substrates in an approximately 1:1 ratio. Triglycerides were used as acyl acceptor, while bola SLs were used as the acyl donors in the reaction. The reaction products were analyzed using HPLC and MALDI-TOF MS.

[0405] As a result, after 12h of reaction, 3 new compounds emerged in the HPLC chromatograms (Fig. 14) that eluted later than di-acetylated lactonic SLs (Ci8:i, MW= 688, retention times (RTs) at 37.44min and 38.10min). Among them, two novel peaks at RTs of 39.4min and 39.8min, respectively, were identified as an SL oligomer (SL-di-glyceride) with MW of 1468 as described by Kobayashi et al. (2023), whereas the third novel peak, at RT of 54.45min, corresponded to an SL oligomer (SL-tri-glyceride) with MW of 2157. Furthermore, our results also showed that both synthesized SL-di-glycerides (RT at 39.36min and 39.81min) disappeared after 24h of reaction, in contrast, SL-tri-glyceride (RT at 54.45min) increased (Fig. 14), indicating that SL-di-glycerides are intermediates of reaction and are converted to SL-tri-glyceride after the addition of a 3rdSL moiety after prolonged reaction time.

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Claims

Claims1. A mutant of a lactone esterase (LE) enzyme, said mutant comprising at least one mutation in a substrate binding pocket of the LE enzyme.

2. The mutant according to claim 1, wherein said substrate binding pocket consists of regions corresponding to the loop forming region T103-A113, the helix forming region S125-T131, the loop forming region S132-A136, the loop forming region D151-G162, the loop forming region G192-S194, the helix forming region G195-D208, the loop forming region G223-A228, the helix forming region W229-I235, the loop forming region D236-L242, the helix forming region K243- G256, the loop forming region A282-S298, the loop forming region G344-K349, the loop forming region P374-H378 and the helix forming region D379-D398 of SEQ ID NO:2.

3. The mutant according to claim 1 or 2, wherein said mutant comprises at least one mutation in at least one region corresponding to a region selected from the group consisting of: the helix forming region S125-T131, the helix forming region K243-G256, the loop forming region A282-S298, the loop forming region G344-K349 and the helix forming region D379-D398 of the amino acid sequence of SEQ ID NO:2.

4. The mutant according to any one of claims 1 to 3 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E;N126G;S125P and P129G;G244S, G244T, G244C, G244N, G244Q, G244W or G244Y;P290S, P290T, P290C, P290N, P290Q, P290F or P290Y;L128T or L128Y;Q288Y or Q288V;N287F; l248A or I248G; andL348D.

5. The mutant according to any one of claims 1 to 4 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E;N126G;S125P and P129G;G244S, G244T, G244C, G244N, G244Q, G244W or G244Y;P290S, P290T, P290C, P290N, P290Q, P290F or P290Y;L128T;Q288Y or Q288V;N287F; l248A or I248G.; andL348D.

6. The mutant according to any one of claims 1 to 5 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E;N126G;S125P and P129G;G244S, G244T, G244C, G244N, G244Q, G244W or G244Y;P290S, P290T, P290C, P290N, P290Q, P290F or P290Y;L128T;Q288Y or Q288V;N287F; and l248A or I248G.

7. The mutant according to any one of claims 1 to 6 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E;N126G;S125P and P129G;G244S, G244T, G244C, G244N, G244Q, G244W or G244Y;P290S, P290T, P290C, P290N, P290Q, P290F or P290Y;L128T;Q288Y;N287F; and l248A or I248G.

8. The mutant according to any one of claims 1 to 6 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: A382E;S125P and P129G; andQ288V.

9. The mutant according to any one of claim 1 to 4 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2:G244S, G244C or G244Q;P290S, P290T, P290C, P290N or P290Q;L128Y;Q288Y; and l248A or I248G.

10. The mutant according to any one of claims 1 to 5 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: S125P and P129G;G244S or G244C;P290S, P290T, P290C, P290N or P290Q;Q288Y or Q288V; l248A or I248G; andL348D.

11. The mutant according to any one of claims 1 to 5, 9 or 10 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244S or G244C;P290S, P290T, P290C, P290N or P290Q;Q288Y; and l248A or I248G.

12. The mutant according claim 11 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2:G244C;P290S, P290T, P290C, P290N or P290Q; and l248A or I248G.

13. The mutant according to any one of claims 1 to 5, or 10 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: S125P and P129G;Q288V; andL348D.

14. The mutant according to any one of claims 1 to 5 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: S125P and P129G;G244S or G244C;P290S, P290T, P290C, P290N or P290Q;Q288Y or Q288V; and l248A or I248G.

15. The mutant according to any one of claims 1 to 4, or 9 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2: G244S or G244Q;L128Y; andQ288Y.

16. The mutant according to claim 15 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2:G244Q; andL128Y.

17. The mutant according to claim 11 or 15 comprising at least one mutation corresponding to at least one of the following mutations of the SBLE enzyme of SEQ ID NO:2:G244S; andQ288Y.

18. The mutant LE enzyme according to any one of claims 1 to 17, wherein said LE enzyme is a Starmerella bombicola SBLE enzyme, such as the S. bombicola enzyme having the amino acid sequence set forth in SEQ ID NO:2 or 13, or a functional variant or fragment thereof.

19. The mutant LE enzyme according to any one of claims 1 to 18, wherein said mutant has improved hydrolytic activity compared to the original LE enzyme.

20. The mutant LE enzyme according to any one of claims 5, 9-11 or 14 to 18, wherein said mutant has improved transesterification activity compared to the original LE enzyme.

21. A genetically modified yeast strain comprising a gene encoding a mutant LE enzyme as defined in any one of claims 1 to 20, wherein said yeast strain is a naturally sophorolipid producing yeast strain.

22. The modified yeast strain according to claim 21, wherein said yeast strain is a strain belonging to the Wickerhamiella or the Starmerella genus, preferably a strain belonging to the Starmerella genus, more preferably a Starmerella bombicola strain.

23. The modified yeast strain according to claim 22, wherein said yeast strain has further been modified to have (a) dysfunctional acetyltransferase enzyme(s) Atl, At2 and / or At3, preferably dysfunctional acetyltransferase enzymes Atl, At2 and At3, more preferably nonfunctional acetyltransferase enzymes Atl, At2 and At3.

24. Use of a mutant LE enzyme according to any one of claims 1 to 19 for the production of acidic glycolipids.

25. A method for the production of acidic glycolipids, said method comprising contacting bola glycolipids with a mutant LE enzyme according to any one of claims 1 to 19.

26. Use of a modified yeast strain according to any one of claims 21 to 23 to produce acidic glycolipids.

27. A method to produce acidic glycolipids, said method comprising culturing a modified yeast strain according to any one of claims 21 to 23 to produce a broth comprising acidic glycolipids.

28. The use according to claim 24 or 26, or the method according to claim 25 or 27, wherein said acidic glycolipids are acid sophorolipids.

29. Use of a mutant LE enzyme according to any one of claims 5, 9-11, 14 to 18 or 20 for the production of lactonic sophorolipids.

30. A method for the production of lactonic glycolipids, said method comprising contacting bola glycolipids, optionally non-acetylated bola glycolipids such as non-acetylated bola sophorolipids, with a mutant LE enzyme according to any one of claims 5, 9-11, 14 to 18 or 20.

31. Use of a modified yeast strain according to any one of claims 21 to 23 to produce lactonic glycolipids.

32. A method to produce lactonic glycolipids, said method comprising culturing a modified yeast strain according to any one of claims 21 to 23 to produce a broth comprising lactonic glycolipids.

33. The use according to claim 29 or 31, or the method according to claim 30 or 32, wherein said lactonic glycolipids are lactonic sophorolipids.

34. The use according to claim 29, 31 or 33, or the method according to claim 30, 32 or 33, wherein said lactonic glycolipids are non-acetylated lactonic glycolipids.

35. A functional homologue of the Starmerella bombicola SBLE enzyme, wherein said homologue has improved hydrolytic activity and / or improved transesterification activity compared to the Starmerella bombicola SBLE enzyme.

36. The homologue according to claim 35, wherein said homologue is a Starmerella kuoi LE enzyme having the amino acid sequence set forth in SEQ ID NO:6, or a variant or fragment thereof; a Starmerella batistae LE enzyme having the amino acid sequence set forth in SEQ ID NO:8, or a variant or fragment thereof; a Starmerella riodocensis LE enzyme having the amino acid sequence set forth in SEQ ID NO:114, or a variant or fragment thereof.

37. A modified yeast strain comprising a homologue of the Starmerella bombicola SBLE enzyme according to claim 35 or 36, wherein said yeast strain is a naturally sophorolipid producing yeast strain.

38. The modified yeast strain according to claim 37, wherein said yeast strain is a strain belonging to the Wickerhamiella or the Starmerella genus, preferably a strain belonging to the Starmerella genus, more preferably a Starmerella bombicola strain.

39. The modified yeast strain according to 37 or 38, wherein said yeast strain has further been modified to have (a) dysfunctional acetyltransferase enzyme(s) Atl, At2 and / or At3, preferably (a) nonfunctional acetyltransferase enzyme(s) Atl, At2 and / or At3, more preferably a nonfunctional acetyltransferase enzymes At3, even more preferably a nonfunctional acetyltransferase enzymes Atl, At2 and At3.

40. Use of a homologue of the Starmerella bombicola SBLE enzyme according to claim 35 or 36 for the production of acidic glycolipids.

41. A method for the production of acidic glycolipids, said method comprising contacting bola glycolipids with a homologue of the Starmerella Starmerella bombicola SBLE enzyme according to claim 35 or 36.

42. The use according to claim 40, or the method according to claim 41, wherein said acidic glycolipids are acetylated acidic glycolipids, preferably mono-acetylated or di-acetylated acidic glycolipids.

43. The use according to claim 40, or the method according to claim 41, wherein said acidic glycolipids are non-acetylated acidic glycolipids.

44. The use according to any one of claims 40, 42 or 43, or the method according to any one of claims 41 to 43, wherein said acidic glycolipids are acidic sophorolipids.

45. The use of a homologue of the Starmerella bombicola SBLE enzyme according to claim 35 or 36 for the production of glycolipid oligomers / polymers / esters.

46. A method for the production of glycolipid oligomers / polymers / esters, said method comprising contacting bola glycolipids and another ester, preferably an acyl ester, more preferably an acyl glycerol ester such as a mono acyl glycerol ester, a di acyl glycerol ester and / or a tri acyl glycerol ester, with a homologue of the Starmerella bombicola SBLE enzyme according to claim 35 or 36.

47. The use according to claim 45, or the method according to claim 46, wherein said glycolipid oligomers / polymers / esters are sophorolipid oligomers / polymers / esters.

48. Use of a modified yeast strain according to any one of claims 37 to 39 to produce acidic glycolipids.

49. The use according to claim 48, wherein said acidic glycolipids are acetylated acidic glycolipids, preferably mono-acetylated or di-acetylated acidic glycolipids.

50. The use according to claim 48, wherein said acidic glycolipids are non-acetylated acidic glycolipids.

51. The use according to any one of claims 48 to 50, wherein said acidic glycolipids are acidic sophorolipids.

52. Use of a modified yeast strain according to any one of claims 37 to 39, to produce glycolipid oligomers / polymers / esters.

53. The use according to claim 52, wherein said glycolipid oligomers / polymers / esters are sophorolipid oligomers / polymers / esters.

54. A method for the production of acidic glycolipids, said method comprising culturing a modified yeast strain according to any one of claims 37 to 39 to produce a broth comprising acidic sophorolipids.

55. The method according to claim 54 wherein said acidic glycolipids are mono-acetylated or diacetylated acidic glycolipids.

56. The method according to claim 54 wherein said acidic glycolipids are non-acetylated acidic glycolipids.

57. The method according to any one of claims 54 to 55, wherein said acidic glycolipids are acidic sophorolipids.

58. A method for the production of glycolipid oligomers / polymers / esters, said method comprising culturing a modified yeast strain according to any one of claims 37 to 39 to produce a broth comprising glycolipid oligomers / polymers / esters.

59. The method according to claim 58, wherein said glycolipid oligomers / polymers / esters are sophorolipid oligomers / polymers / esters.

60. A three-dimensional structure of a Starmerella bombicola SBLE enzyme characterized by the atomic coordinates as defined in any one of Tables 9 to 11 or a subset thereof.

61. Use of a three dimensional structure represented by a set of atomic coordinates presented in any one of Tables 9 to 11 or a subset thereof, or atomic coordinates which deviate from those in any one of Tables 9 to 11 or a subset thereof by a root mean square deviation (RMSD) of residue over protein backbone atoms by no more than 3 A, for identifying or designing mutants of an SBLE enzyme with modulated hydrolytic and / or transesterification activity.

62. A computer-implemented method for identifying or designing mutants of an SBLE enzyme with modulated hydrolytic and / or transesterification activity, said method comprising:- employing a three dimensional structure of the SBLE enzyme represented by a set of atomic coordinates presented in any one of Tables 9 to 11 or a subset thereof, or atomic coordinates which deviate from those in any one of Tables 9 to 11 or a subset thereof by a root meansquare deviation (RMSD) of residue over protein backbone atoms by no more than 3 A, for fitting or docking a three dimensional structure or atomic coordinates of a model substrate, in particular a tetra acetylated bolaglycolipid, more particularly a tetra acetylated bolasophorolipid, in said three dimensional structure of the SBLE enzyme to generate a three- dimensional computational representation of an enzyme-substrate complex; and- identifying amino acid residues in the enzyme of said three-dimensional representation of the enzyme-substrate complex with unfavorable interactions with the substrate as candidate positions for enzyme mutagenesis; and- identifying mutations of said candidate positions that form more favorable interactions with said substrate.

63. The use according to claim 61, or the method according to claim 62, wherein said subset is the subset of atomic coordinates of Table 11 defining the loops and helices of the S. bombicola SBLE enzyme that bind the substrate, in particular the subset of atomic coordinates of Table 11 defining the loops and helices corresponding to the loop forming region T103-A113, the helix forming region S125-T131, the loop forming region S132-A136, the loop forming region D151-G162, the loop forming region G192-S194, the helix forming region G195-D208, the loop forming region G223-A228, the helix forming region W229-I235, the loop forming region D236-L242, the helix forming region K243-G256, the loop forming region A282-S298, the loop forming region G344-K349, the loop forming region P374-H378 and the helix forming region D379-D398 of the amino acid sequence of SEQ. ID NO:2.

64. A method for producing mutants of an SBLE enzyme with modulated hydrolysis and / or transesterification activity, , said method comprising:- identifying or designing mutants of the SBLE enzyme by a method according to claim 62 or 63,- generating mutants of the SBLE enzyme that comprise one or more of the identified mutations, and- optionally testing the generated mutants of the SBLE enzyme for modulated hydrolysis and / or transesterification activity to identity mutants of the SBLE enzyme with modulated hydrolysis and / or transesterification activity.

65. A computer program product comprising instructions which when the program is executed by a computer, cause the computer to carry out the steps of the method according to claim 62 or 63.

66. A computer-readable storage medium, comprising a data storage material encoded with computer readable data wherein said data comprises the information needed for and configured to perform the method according to claim 62 or 63.

67. A method for determining a crystal structure of an SBLE enzyme, said method comprising: (a) co-crystallizing the SBLE enzyme and a substrate analog, in particular a bolaform glycoside, more particularly a bolaform sophoroside, to form a crystalline complex,(b) subjecting the crystalline complex to X-ray diffraction, and(c) determining the crystal structure based on said X-ray diffraction data.

68. A crystal of a S. bombicola SBLE enzyme comprising a structure characterized by the atomic coordinates as presented in any one of Tables 9-11 or a subset thereof.