Methods for producing acetylated and non-acetylated glycolipid amphiphiles - Patents.com

Abstract

The present invention relates to the use of a known enzyme called "Starmelella bombycola lactone esterase (Sble)" to carry out transesterification and / or hydrolysis reactions. More specifically, the Sble enzyme carries out transesterification and / or hydrolysis reactions on bolaamphiphilic glycolipids. The present invention indeed discloses that said Sble is capable of converting bolaamphiphilic sophorolipids into lactone and / or acidic sophorolipids and sugars, and that yeast strains containing non-functional or dysfunctional Sble enzymes and / or dysfunctional sble genes and / or no sble genes produce (acetylated) bolaamphiphilic glycolipids. Additionally, the present invention further discloses a method for producing non-acetylated (bola-type) amphipathic glycolipids in the latter yeast strains by making the acetyltransferase enzymes At1, At2, and At3 non-functional or incompetent and / or by modifying the strains so as not to contain the acetyltransferase αt1, αt2, and αt3 gene(s) and / or by strains not containing the αt1, αt2, and αt3 gene(s).Furthermore, in the above mentioned strains, when glucosyltransferase B (UgtB1) is made non-functional or incompetent and / or when the ugtB1 gene is removed and / or incompetent and / or by strains not containing the ugtB1 gene, they produce acetylated and / or non-acetylated bola-type amphipathic glycolipids. The present invention further discloses methods of producing non-acetylated glycolipids by rendering the acetyltransferase enzymes At1, At2, and / or At3 non-functional or disabled, and / or by removing and / or disabling glycolipid acetyltransferase genes in the glycolipid-making yeast strain, and / or by strains that do not contain the αt1, αt2, and αt3 gene(s).

Description

[Technical field]

[0001] The present invention relates to the use of a known enzyme called "Starmerella bombicola lactone esterase (Sble)" to carry out transesterification and / or hydrolysis reactions. More specifically, the Sble enzyme carries out transesterification and / or hydrolysis reactions on bolaamphiphilic glycolipids. Said Sble is capable of converting bolaamphiphilic sophorolipids into lactone form (transesterification) and / or acidic sophorolipids and sugars (hydrolysis), and the present invention indeed discloses that yeast strains containing non-functional or dysfunctional Sble enzymes and / or dysfunctional sble genes and / or with the sble gene removed produce (acetylated) bolaamphiphilic glycolipids. Additionally, the present invention further discloses a method for producing non-acetylated (bolaamphiphilic) glycolipids in the latter yeast strains by making the acetyltransferase enzymes At1, At2, and At3 non-functional or incompetent and / or by modifying the strains such that the acetyltransferase at1, at2, and at3 gene(s) are incompetent and / or deleted. Furthermore, in the above-mentioned strains, when glucosyltransferase B (UgtB1) is made non-functional or incompetent and / or when the ugtB1 gene is deleted and / or deleted, they produce acetylated and / or non-acetylated bolaamphiphilic glucolipids. The present invention further discloses a method for producing non-acetylated glycolipids by making the acetyltransferase enzymes At1, At2, and At3 non-functional or incompetent and / or by deleting and / or deleting glycolipid acetyltransferase genes in the glycolipid-producing yeast strains. [Background technology]

[0002] In particular, the yeast S. bombicola is known in the art to produce large amounts of sophorolipids (SLs). SLs are composed of the disaccharide sophorose linked to hydroxylated fatty acids. Wild-type S. bombicola strains produce a mixture of acidic and lactone-type SLs, which can be non-acetylated, mono-acetylated, or diacetylated, and contain mainly C18:1 fatty acids. The biosynthetic pathway for SLs has been previously elucidated, and the proposed pathway is shown in FIG. 1.

[0003] All but one of the genes involved in the biosynthesis of SLs are found within one large subtelomeric gene cluster (see FIG. 1). The current understanding of the SL biosynthetic pathway involves five steps that result in the final product being a diacetylated lactone-type SL: the first step consists of the terminal (near) hydroxylation of fatty acids by the action of a cytochrome P450 monooxygenase (Cyp52M1) (Non-Patent Document 1) (step / gene (1) in FIG. 1). Subsequent glycosylation of the hydroxy fatty acid involves two glucosyltransferases. The first glucosyltransferase (Ugta1) (Non-Patent Document 2) is responsible for the transfer of a glucose molecule from UDP-glucose to a hydroxylated fatty acid, resulting in a glucolipid and UDP (step / gene (2) in FIG. 1), while the second glucosyltransferase (Ugtb1) (Non-Patent Document 3) specifically transfers a second glucose molecule from UDP-glucose to the formed glucolipid (not to the hydroxylated fatty acid) (step / gene (3) in FIG. 1). The SL is then acetylated by the action of an acetyltransferase (At1) (Non-Patent Document 4) (step / gene 4 in FIG. 1) and, after secretion by a specific SL transporter (Mdr) (step / gene 5 in FIG. 1), can be further lactonized by the action of a secreted lactone esterase (Sble) (step / gene 6 in FIG. 1) (Non-Patent Document 5, Patent Document 1). In contrast to all other genes involved in the biosynthesis of SLs by S. bombicola, the sble gene is not located in the biosynthetic gene cluster and appears to be differentially regulated. This last step also takes place (mainly) in the extracellular space, since the Sble protein has a secretion signal, is actively secreted and is well found in the extracellular space (Non-Patent Document 5). The SL transporter (Mdr) is also encoded within the biosynthetic gene cluster. Deletion of this gene leads to at least a 90% reduction in SL production (Non-Patent Document 6).

[0004] A single S bombicola at1 deletion strain, S bombicola Δat1, was described by 4 and was reported to produce non-acetylated acidic and non-acetylated lactone-type sophorolipids (SLs). In addition to trace amounts of ring-open / acidic SLs, non-acetylated lactone-type SLs were reported to be the most predominant structure in the mixture. A single S bombicola sble deletion strain, S bombicola Δsble, was subsequently described by 5 and was reported to exclusively produce acidic SLs. The authors also suggested that SLs are secreted by yeast in an acidic form and then lactonized by the extracellularly secreted Sble enzyme. 7 also reported production experiments with this strain, again reporting exclusive production of acidic SLs in a mixture of acetylated and non-acetylated congeners. (2003) subsequently investigated the mechanism of action of the Sble enzyme by in vitro enzyme assays and reported that the enzyme catalyzes the intramolecular esterification (lactonization) of acetylated acidic sophorolipids to acetylated lactone-type sophorolipids in an aqueous environment. Since no lactonization activity was observed by the authors for non-acetylated acidic SLs, acetylation was deemed essential for this esterification reaction by Sble. The acetylated acidic SLs used in the in vitro Sble enzyme assays described in the above study were obtained from a Δsble S Bombicola strain (Non-Patent Document 5), and HPLC and LC-MS analysis suggested that the extracted SLs used in these in vitro enzyme assays consisted of a mixture composed of 50% non-acetylated acidic SLs, 17% monoacetylated acidic SLs, 31% diacetylated acidic SLs, and 2% contaminants, as described by authors in the art.

[0005] It was subsequently found that the combination of these two deletions in one strain (i.e., S. bombicola Δat1 Δsble) unexpectedly resulted in the biosynthesis of bolamorph / bolatype amphipathic glycolipids (the general formula of which is shown in Figures 2A and 2B). More specifically, the authors reported the biosynthesis of non-acetylated bolatype sophorolipids (Figure 2C) (Non-Patent Document 9 and Patent Document 2), which was also explained in subsequent studies (Non-Patent Document 10, Patent Document 3). Based on previous findings and the proposed biosynthetic pathway shown in Figure 1, this strain logically produces non-acetylated acidic sophorolipids. Surprisingly, in addition to these predicted non-acetylated acidic SLs, bolatype sophorolipids (74% of the produced SLs) were also obtained. These bolatype sophorolipids contain an additional sophorose molecule attached to the carboxyl functional group of the acidic sophorolipids, confirmed by LC-MS and NMR analysis. The biosynthesis of bola-type sophorolipids was proved to be due to the promiscuous activity of both UDP-glucosyltransferases UgtA1 and UgtB1 from the sophorolipid biosynthesis pathway, which were found to be active even on the carboxyl group of the non-acetylated intermediate. Since bola-type glycolipid compounds were found to be produced by the Δat1 Δsble strain but not by the Δsble strain, it was hypothesized that the absence of an acetyl group triggers the formation of bola-type glycolipid compounds starting from acidic sophorolipids. The authors also speculated that the presence of the At1 enzyme in the wild-type strain would result in acetylated sophorolipids, which would prevent the formation of bola-type sophorolipids, as a potential reason why these bola-type sophorolipids were detected in only trace amounts (less than 0.1% of the sophorolipids produced) in the culture medium of the wild-type Starmerella bombicola strain (Non-Patent Document 11). LC-MS analysis of the sophorolipid mixture produced by the Δat1 Δsble strain revealed the production of non-acetylated bolasophorolipids with varying fatty acid chain length and position of the fatty acid hydroxyl group (as in wild-type sophorolipids). The sophorolipid mixture produced by this new strain was fractionated, and NMR analysis confirmed the structure of the non-acetylated bolasophorolipid shown in Figure 2.As this was an unexpected finding, the authors again investigated the glycolipid mixtures produced by both of the single deletion strains mentioned above. The Δsble S Bombicola strain was confirmed by the authors to produce only acidic sophorolipids (Non-Patent Document 9), whereas the Δat1 S Bombicola strain, upon reanalysis using a glycolipid extraction procedure adapted for more hydrophilic compounds, was found to produce non-acetylated bora-type sophorolipids in addition to the previously reported non-acetylated acidic and lactone-type sophorolipids reported to be produced by this strain (Non-Patent Document 9). Thus, the authors again suggested that the absence of acetylation, as mentioned above, seems to be the key factor triggering bora-type sophorolipid synthesis, and since a higher production efficiency of bora-type sophorolipids seems to be obtained using the double deletion strain, this effect is enhanced by the absence of lactone-type sophorolipid forms (which do not have any more carboxyl groups freely available). It was hypothesized that non-acetylated glycolipid compounds allow the UgtA1 and UgtB1 enzymes to adopt certain conformational orientations that are not possible for their acetylated equivalents, thus resulting in further glycosylation of non-acetylated acidic sophorolipids leading to non-acetylated bolasophorolipids.

[0006] However, at present, it is not known whether yeast strains containing non-functional or dysfunctional lactone esterase enzyme (Sble) and functional acetyltransferase enzyme 1 (At1) are capable of producing bola-type sophorolipids. It is also not known whether they are capable of producing acetylated bola-type sophorolipids. It is also not known whether yeast strains containing non-functional or dysfunctional acetyltransferase enzyme (At1), encoded by the SL biosynthetic gene cluster and responsible for sophorolipid acetylation, are still capable of producing acetylated (bora-type) sophorolipids. Furthermore, it is not known at all whether the Sble enzyme can carry out transesterification, and more specifically, it is not known at all that the Sble enzyme has the transesterification activity of converting bola-type sophorolipids / bora-type glucolipids into lactone-type sophorolipids / glucolipids, respectively. It is also not known that the Sble enzyme has the hydrolysis activity of converting bola-type sophorolipids / glucolipids into acidic sophorolipids / glucolipids. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2013 / 092421 [Patent Document 2] International Publication No. 2015 / 028278 [Patent Document 3] International Publication No. 2021 / 229017 [Non-patent literature]

[0008] [Non-Patent Document 1] Van Bogaert et al., 2009a [Non-Patent Document 2] Saerens et al., 2011a [Non-Patent Document 3] Saerens et al., 2011c [Non-Patent Document 4] Saerens et al., 2011b [Non-Patent Document 5] Ciesielska et al. 2014 [Non-Patent Document 6] Van Bogaert et al., 2013 [Non-Patent Document 7] Roelants et al. (2016) [Non-Patent Document 8] Ciesielska et al. (2016) [Non-Patent Document 9] Van Bogaert et al., 2016 [Non-Patent Document 10] Van Renterghem et al., 2019 [Non-Patent Document 11] Price et al., 2012 Summary of the Invention

[0009] The present invention relates to:

[0010] Use of a modified yeast strain comprising a non-functional or dysfunctional transesterification enzyme Sble and / or not comprising a functional sble gene and / or having reduced expression of sble compared to unmodified yeast for producing bolaamphiphilic glycolipids.

[0011] The use of the modified yeast strain as described above, wherein the bolaamphiphilic glycolipid is an acetylated bolaamphiphilic glycolipid.

[0012] Use of the modified yeast strain as described above, wherein the bolaamphiphilic glycolipid is a bolasophorolipid.

[0013] Use of the modified yeast strain described above, in which the acetylated bolasophorolipid has an acetylation degree of 4.

[0014] Use of the modified yeast strain as described above, further comprising a non-functional or dysfunctional glucosyltransferase enzyme UgtB1 and / or not comprising a functional ugtB1 gene and / or having reduced expression of ugtB1 compared to an unmodified yeast strain, wherein the bolaamphipathic glycolipid is a bolaamphipathic glucolipid.

[0015] Use of the above modified yeast strain, which further contains a non-functional or dysfunctional acetyltransferase enzyme (At1) and / or does not contain a functional at1 gene from the SL biosynthetic cluster and / or has reduced expression of at1 compared to an unmodified yeast strain, wherein the acetylated bolaamphiphilic glycolipid has a degree of acetylation of 0, 1, or 2.

[0016] Use of the modified yeast strain as described above, further comprising a second (At2) or third (At3) non-functional or dysfunctional glycolipid acetylase and / or not comprising a functional at2 or at3 gene and / or having reduced expression of at2 or at3 compared to an unmodified yeast strain, wherein the acetylated bolaamphiphilic glycolipid has a degree of acetylation of 0, 1 or 2.

[0017] Use of the modified yeast strain as described above, further comprising a second (At2) and a third (At3) non-functional or dysfunctional glycolipid acetylase and / or not comprising functional at2 and at3 genes and / or having reduced expression of at2 and at3 compared to an unmodified yeast strain, for producing a non-acetylated bolaamphiphilic glycolipid, wherein the bolaamphiphilic glycolipid is a non-acetylated bolasophorolipid and / or a non-acetylated bolaglucolipid.

[0018] An isolated acetyltransferase having the amino acid sequence given by SEQ ID NO:6 or SEQ ID NO:8.

[0019] Use of a modified yeast strain comprising non-functional or dysfunctional At1, At2, and At3 enzymes and / or not containing the at1, at2, and at3 genes and / or in which the genes encoding the At1, At2, and At3 enzymes have been completely disabled or removed, for producing non-acetylated glycolipids.

[0020] Use of the modified yeast strain as described above, which comprises non-functional or dysfunctional Sble, UgtB1, At1, At2 and / or At3 enzymes and / or does not contain the sble, ugtB1, at1, at2 and / or at3 genes.

[0021] The yeast strains were: Starmerella (Candida) bombicola, Starmerella (Candida) apicola, Starmerella (Candida) batistae, Starmerella (Candida) magnolia, Candida gropengiesseri, Starmerella (Candida) floricola, Candida tropicalis, Candida riodocensis, Starmerella (Candida) stellata, Starmerella (Candida) quoi, Use of the modified yeast strain as described above, wherein the yeast strain is selected from the group consisting of Candida kuoi, Candida tropicalis, Candida species NRRL Y-27208, Pseudohyphozyma (Rhodotorula, Candida) bogoriensis species, Wickerharmiella domericqiae, Candida antarctica, Pseudohyphozyma antarctica, Pseudohyphozyma bogoriensis, Candida lipolytica, and a strain selected from sophorolipid producing strains of the Starmerella clade.

[0022] Use of a Sble enzyme to carry out transesterification and / or hydrolysis reactions.

[0023] Use of Sble enzymes to carry out transesterification and / or hydrolysis reactions in bolaamphiphilic glycolipids.

[0024] Use of the Sble enzyme as described above for converting a bolaamphiphilic glycolipid into a lactone glycolipid, wherein the bolaamphiphilic glycolipid is a bolasophorolipid and the lactone glycolipid is a lactone sophorolipid, or the bolaamphiphilic glycolipid is a bolaglucolipid and the lactone glycolipid is a lactone glucolipid, and non-acetylated and / or acetylated glucose and / or sophorose are released.

[0025] Use of the Sble enzyme as described above for converting a bolaamphiphilic glycolipid into an acidic glycolipid, wherein the bolaamphiphilic glycolipid is a bolasophorolipid and the acidic glycolipid is an acidic sophorolipid, or the bolaamphiphilic glycolipid is a bolaglucolipid and the acidic glycolipid is an acidic glucolipid, and non-acetylated and / or acetylated glucose and / or sophorose are released.

[0026] The use of the above Sble enzyme, wherein the bola-type sophorolipid is a tetraacetylated bola-type sophorolipid and the lactone-type sophorolipid is a diacetylated lactone-type sophorolipid, and non-acetylated and / or acetylated glucose and / or sophorose are released.

[0027] The use of the above Sble enzyme, wherein the bola-type sophorolipid is a non-acetylated, mono-acetylated, di-acetylated, and / or tri-acetylated bola-type sophorolipid, and the lactone-type sophorolipid is a non-acetylated, mono-acetylated, and / or di-acetylated lactone-type sophorolipid, and non-acetylated and / or acetylated glucose and / or sophorose is released.

[0028] The use of the above Sble enzyme, wherein the bolasophorolipid is a tetraacetylated bolasophorolipid and the acidic sophorolipid is a diacetylated acidic sophorolipid, and non-acetylated and / or acetylated glucose and / or sophorose are released.

[0029] The use of the above Sble enzyme, wherein the bola-type sophorolipid is non-acetylated, mono-acetylated, di-acetylated, and / or tri-acetylated bola-type sophorolipid, and the acidic sophorolipid is non-acetylated, mono-acetylated, and / or di-acetylated acidic sophorolipid, and non-acetylated and / or acetylated glucose and / or sophorose is released. [Brief description of the drawings]

[0030] [Figure 1] (a) Chromosome II of S. bombicola containing the sophorolipid biosynthesis gene cluster (±11 kb) and the gene responsible for lactonization (sble) on the opposite side of the chromosome. (b) The complete sophorolipid biosynthesis pathway consisting of (1) hydroxylation of fatty acids (mainly C16 or C18) by Cyp52M1 monooxygenase, (2) glucosylation of FA-OH by the first glucosyltransferase UgtA1, (3) and a second glucosylation step by the second glucosyltransferase UgtB1 to make the formed glucolipid into an acidic sophorolipid, (4) an acidic sophorolipid that can be acetylated by the action of acetyltransferase At1. The various sophorolipids are transported into the extracellular space by the multidrug transporter protein (Mdr) (5). Lactonization (6) mainly occurs extracellularly when the responsible enzyme (Sble) is secreted. [Diagram 2](A and B) Bolamorph / Bola-type amphiphilic glycolipids, where R1=H or CO-CH3, R2=H or CO-CH3, R3=H or CO-CH3, R4=H or CO-CH3, R5=unsubstituted or hydroxy-substituted, unbranched, divalent organic moiety of 6-32 carbon atoms, which may contain 1-3 double or triple bonds, R6=H, CH3, or unsubstituted or hydroxy-substituted, unbranched, organic moiety of 2-10 carbon atoms, which may contain 1-3 double or triple bonds, n=1 or 0, m=1 or 0. (C) Non-acetylated bola-type sophorolipids, which have been reported to be produced by the S bombicola Δat1 Δsble strain and also by the S bombicola Δat1 strain. Non-patent literature 9 reported that only non-acetylated variants were produced by these strains. [Diagram 3] Figure 1 shows the deletion cassette 1 for the complete deletion of the sble gene of S. bombicola by the ura3 marker. HR: homology region. [Figure 4] Figure 2 shows the deletion cassette 2 for the deletion of the at1 gene of S. bombicola by the ura3 marker. HR: homology region. [Diagram 5] Deletion cassette 3 for deletion of the at1 gene of S. bombicola by the hygromycin resistance (HygroR) marker. HR: homology region. [Figure 6] FIG. 1 shows an acetylated borasophorolipid with a degree of acetylation of 4, i.e., acetyl groups are present at the 6′ and 6″ positions of all incorporated glucose moieties. [Figure 7] HPLC-UV chromatogram of sample (1) from an activity assay of rSble with terminally hydroxylated (sophorose attached to the terminal position of the fatty acid, CORD) acetylated acidic sophorolipid after 1 hour incubation at 30° C., 1400 rpm, and pH 3.5. rSble was added at a concentration of 4 μg / ml and acetylated acidic sophorolipid was at a concentration of 5 mM. The black arrow indicates the primary substrate. (2) Negative control (no enzyme added). [Figure 8] HPLC-UV chromatogram of sample (1) from an activity assay of rSble using acetylated acidic sophorolipids with glycosyl groups attached primarily at subterminal positions after incubation at 30° C., 1400 rpm, and pH 3.5 for 1 hour. rSble was added at a concentration of 4 μg / ml and acetylated acidic sophorolipid was at a concentration of 5 mM. The black arrow indicates the primary substrate. (2) Negative control for this experiment (no enzyme added). [Figure 9] HPLC-UV chromatogram of sample (1) from an activity assay of rSble using non-acetylated acidic sophorolipids with glycosyl groups attached primarily at subterminal positions after incubation at 30° C., 1400 rpm, and pH 3.5 for 1 hour. rSble was added at a concentration of 4 μg / ml and non-acetylated acidic sophorolipid was at a concentration of 5 mM. The black arrow indicates the primary substrate. (2) Negative control for this sample (no enzyme added). [Figure 10] HPLC-UV chromatograms of (1) a negative control of the sophorolipid mixture obtained from the Δsble strain used in the activity assay, and (2) a sample from an activity assay of rSble using the acidic sophorolipid mixture shown in panel 1 as substrate. The five-pointed star in panel 2 indicates the lactone sophorolipid produced after incubation. The arrows in panel 1 indicate three peaks that are significantly reduced following the reaction with the addition of rSble enzyme (shown in panel 2), and the black four-pointed star (panels 1 and 2) indicates a peak corresponding to a diacetylated acidic sophorolipid (C18:1) with a near-terminal (ω-1) bond, which is not reduced upon addition of rSble. [Figure 11]LC-MS TIC chromatogram from an activity assay of rSble, containing mainly acetylated bolasophorolipids (code: INV-113). The top chromatogram is the mixture without added enzyme. The peaks indicated by the arrows are all mono- and diacetylated bolasophorolipids (see Table 5) that are reduced in intensity after incubation with enzyme. The bottom chromatogram is obtained from the same sophorolipid mixture, but after incubation with rSble. All peaks shown are lactone sophorolipids (see Table 5). [Figure 12] MS TIC chromatogram from an activity assay of rSble, containing mainly non-acetylated bolaform sophorolipids (code: INV-22). The top chromatogram is the mixture to which no enzyme was added. The peaks indicated by the arrows are mainly non-bolaform sophorolipids (see Table 6) that are reduced in intensity after incubation with enzyme. The bottom chromatogram is obtained from the same sophorolipid mixture, but after incubation with rSble. The peaks shown correspond to non-acetylated and mono-acetylated lactone-type sophorolipids (see Table 6). [Figure 13] Figure 1 shows the adapted sophorolipid biosynthetic pathway in S. bombicola. (1) Cyp52M1, (2) UgtA1, (3) UgtB1, (4) At1, (5) Sble. UDP: uridine diphosphate, CoA: coenzyme A, nA: non-acetylated, DiAC: diacetylated, tetraAc: tetraacetylated. The figure shows the formation of tetraacetylated bolasophorolipids and their conversion to diacetylated lactone sophorolipids and the release of acetylated sophorose. Less acetylated bolasophorolipids are also converted to non-acetylated and monoacetylated lactone sophorolipids and non-acetylated and / or monoacetylated sophorose. [Figure 14] Figure 1 shows the deletion cassette for the deletion of the at2 gene in S. bombicola by the URA3 marker. HR: homology region. [Figure 15]FIG. 1 shows a deletion cassette for deletion of the at3 gene in S. bombicola by the URA3 marker. HR: homologous region. The 5' homologous region corresponds to the last 500 base pairs of the at3 coding sequence and is indicated by the hatched bar. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] The present invention relates to the surprising discovery that Sble enzymes can carry out transesterification and / or hydrolysis reactions on bolaamphiphilic glycolipids. Such transesterification refers to the replacement of an alcohol of one ester with an alcohol of another ester in a process similar to hydrolysis, but using alcohol instead of water. Hydrolysis is therefore the process of replacing an alcohol of an ester with water.

[0032] In the present invention, the term "bolaamphiphilic glycolipid" refers to the molecules described by WO 2005 / 023363 and is generally a compound having the general formula shown in Figure 2 (A and B). The present invention more specifically relates to the fact that Sble enzymes are capable of carrying out transesterification reactions on (acetylated) bolaamphiphilic glycolipids. More specifically, Sble enzymes contain transesterification activity on (acetylated) bolaamphiphilic sophorolipids and bolaamphiphilic glucolipids, converting them to (acetylated) lactone-type sophorolipids and lactone-type glucolipids, respectively, while releasing the (acetylated) sugar. As well as transesterification, hydrolysis of bolaamphiphilic glycolipids is also an activity found in Sble enzymes. Sble enzymes are capable of converting (acetylated) bolaamphiphilic glycolipids to (acetylated) acidic sophorolipids and (acetylated) acidic glucolipids, while releasing the (acetylated) sugar from the reaction.

[0033] The present invention further relates to the use of Sble enzymes to convert (acetylated) bola-type sophorolipids to (acetylated) lactone-type sophorolipids while releasing (acetylated) sugars. The present invention also relates to the use of Sble enzymes to convert (acetylated) bola-type sophorolipids to (acetylated) acidic sophorolipids while releasing (acetylated) sugars.

[0034] Furthermore, the present invention relates to the use of Sble enzymes to convert (acetylated) bolasophorolipids to (acetylated) lactone sophorolipids while releasing (acetylated) sophorose and / or (acetylated) glucose. The present invention also relates to the use of Sble enzymes to convert (acetylated) bolasophorolipids to acidic sophorolipids and (acetylated) sophorose and / or glucose.

[0035] The present invention further relates to the surprising discovery that yeast strains that contain a non-functional and / or dysfunctional Sble enzyme and / or that do not contain a (functional) sble gene are capable of producing acetylated bolaamphiphilic glycolipids.

[0036] The present invention further relates to the surprising discovery that yeast strains which comprise a non-functional and / or dysfunctional At1 enzyme and / or in which the at1 gene is absent and / or impaired are capable of producing acetylated (bolaamphiphilic) glycolipids and, surprisingly, that yeast strains which comprise, in addition to the At1 acetyltransferase enzyme / gene, additional non-functional and / or dysfunctional acetyltransferase enzymes At2 and At3 and / or in which the at2 and at3 genes are absent and / or impaired produce non-acetylated (bolaamphiphilic) glycolipids.

[0037] The present invention further relates to yeast strains which, in addition to containing a non-functional and / or dysfunctional Sble enzyme, further contain a non-functional and / or dysfunctional UgtB1 enzyme and / or the ugtB1 gene has been deleted and / or rendered defective, such that the strain produces (acetylated) bora-type glucolipids.

[0038] The term "non-functional or dysfunctional" generally refers to an enzyme as described above, or a fragment or variant thereof, that does not function "normally" and / or has no activity (non-functional) or has defective activity (dysfunctional). Thus, the term refers to an enzyme that is a) absent and therefore not functional, b) still present but non-functional, or c) still present but with weakened or reduced activity, where weakened or reduced activity is significantly less than 90%, 80%, 70%, 60%, or 50%, 40%, or 30% (p<0.05) of the activity of the corresponding wild-type enzyme, preferably less than 20%, more preferably less than 10%, even more preferably less than 5%, e.g., less than 4%, 3%, 2%, or 1%.

[0039] A situation in which the enzyme, or a fragment or variant thereof, is a) absent and therefore not functional, b) still present but non-functional, or c) still present but with weakened or reduced activity, can be obtained by any known means of avoiding, reducing and / or silencing the transcription and / or translation of the nucleic acid sequence encoding the enzyme, or by any known means of impairing the enzyme activity, such as, but not limited to, knockout; insertion of a nucleic acid fragment containing a marker gene or any other nucleotide fragment in a target gene resulting in impairing the transcription or translation of the nucleic acid sequence encoding the enzyme; use of CRISPR; homologous recombination; siRNA; CRISPRi; use of riboswitches; recombineering; ssDNA mutagenesis; RNAi, miRNA or asRNA; mutation of the enzyme or of the nucleic acid sequence encoding the enzyme; transposon mutagenesis; disruption of (the function of) a necessary regulator / activator protein; of the target enzyme or of the activator / regulator. interference with cellular synthesis; use of one or more aptamers; use of one or more ribozymes; use of antibodies, amino acids, peptides, or any small molecules that interfere with transcription, translation, synthesis, or enzymatic activity of an active enzyme; use of oligoribonucleotide sequences, such as dsRNA or antisense nucleic acids used to initiate RNA interference (RNAi); introduction of point mutations; use of truncated, modified, or mutated enzymes; use of inhibitors or antibodies; mutations (spontaneous, induced and / or directed, point mutations, deletions, frameshifts, insertions, or any other type of mutation); or by any other means known to those skilled in the art.

[0040] The term "aborted" in the context of a gene generally refers to a gene, or a fragment or variant thereof, that does not function "normally" and / or has no activity or has defective activity. Thus, the term refers to a gene that is a) absent and therefore not functional, b) still present but not functional, or c) still present but with weakened, reduced or altered activity. The situation in which the gene, or a fragment or variant thereof, is a) absent and therefore not functional, b) still present but not functional, or c) still present but with weakened or reduced activity, can be obtained by any known means of avoiding, reducing, altering and / or silencing the transcription and / or translation of the nucleic acid sequence encoding the enzyme.For example, but not limited to, knockout; insertion of a nucleic acid fragment containing a marker gene or any other nucleotide fragment into a target gene resulting in transcription or translation failure of the nucleic acid sequence encoding the enzyme; promoter recombination, promoter removal, promoter switching, Kozak sequence recombination, Kozak sequence removal, Kozak sequence switching, RBS (ribosome binding site) recombination, RBS removal, RBS sequence switching, UTR (untranslated region) recombination, UTR removal, UTR sequence switching, use of CRISPR; homologous recombination; siRNA; CRISPRi; use of riboswitches; recombination; ssDNA mutagenesis; RNAi, miRNA, or asRNA; mutation of the nucleic acid sequence encoding the enzyme; transfection. poson mutagenesis; disruption of (the function of) a necessary regulator / activator protein; interference with the cellular synthesis of a target enzyme or of an activator / regulator; use of one or more aptamers; use of one or more ribozymes; use of antibodies, amino acids, peptides, or any small molecules that interfere with the transcription, translation, or synthesis of an active enzyme; use of oligoribonucleotide sequences, such as dsRNA or antisense nucleic acids, used to initiate RNA interference (RNAi); introduction of point mutations; use of truncated, modified, or mutated enzymes; use of inhibitors or antibodies; mutations (naturally occurring, induced and / or directed, point mutations, deletions, frameshifts, insertions, or any other type of mutation); or by any other means known to those skilled in the art. The term "make defective" refers to the act of conferring a defective gene.

[0041] The term "removed" in the context of a gene generally refers to a gene or a fragment or variant thereof that has been removed, in whole or in part, from genomic DNA. Such removal can be obtained by any known means, including but not limited to knocking out coding sequences by homologous recombination, knocking out genes by homologous recombination; knocking out coding sequences by using CRISPR technology, knocking out genes by using CRISPR technology; or any other means known to those skilled in the art. The term "removing" refers to the act of providing a removed gene.

[0042] The term "reduced expression" refers to an expression that is significantly less than 90%, 80%, 70%, 60%, or 50%, 40%, or 30% (p<0.05), 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 expression of the corresponding wild-type gene. Such reduced expression can be obtained by any known means that avoids, reduces, alters, and / or silences the transcription and / or translation of the nucleic acid sequence encoding said enzyme. For example, but not limited to, knockout; knockdown; insertion of a nucleic acid fragment containing a marker gene or any other nucleotide fragment into a target gene resulting in transcription or translation failure of the nucleic acid sequence encoding the enzyme; promoter recombination, promoter removal, promoter switching, Kozak sequence recombination, Kozak sequence removal, Kozak sequence switching, RBS (ribosome binding site) recombination, RBS removal, RBS sequence switching, UTR (untranslated region) recombination, UTR removal, UTR sequence switching, use of CRISPR; homologous recombination; siRNA; CRISPRi; use of riboswitches; recombination; ssDNA mutagenesis; RNAi, miRNA, or asRNA; mutation of the nucleic acid sequence encoding the enzyme; transcription. transposon mutagenesis; disruption of (the function of) a necessary regulator / activator protein; interference with the cellular synthesis of a target enzyme or of an activator / regulator; use of one or more aptamers; use of one or more ribozymes; use of antibodies, amino acids, peptides, or any small molecules that interfere with the transcription, translation, or synthesis of an active enzyme; use of oligoribonucleotide sequences, such as dsRNA or antisense nucleic acids used to initiate RNA interference (RNAi); introduction of point mutations; use of truncated, modified, or mutated enzymes; use of inhibitors or antibodies; mutations (spontaneous, induced and / or directed, point mutations, deletions, frameshifts, insertions, or any other type of mutation); or by any other means known to the skilled artisan.

[0043] The term "variant" refers to a protein or peptide or polypeptide as described by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and / or SEQ ID NO:62 having at least 34% sequence identity, preferably at least 51%-70% sequence identity, more preferably at least 71%-90% sequence identity, or most preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2, or a fragment thereof, and which retains the enzymatic activity as described above.

[0044] The percentage of amino acid sequence identity is determined by aligning the two sequences and identifying the number of identical amino acid positions / number of amino acids in the shorter sequence×100.

[0045] The latter "variants" may differ from the proteins described by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and / or SEQ ID NO:62 only by conservative substitutions and / or modifications, resulting in retention of activity capability. A "conservative substitution" is one in which an amino acid is replaced with another amino acid of similar properties, such that the properties of the protein are substantially unchanged, as would be expected by one of skill in the art of protein chemistry. In general, the following groups of amino acids represent conservative changes: (1) Ala, Pro, Gly, Glu, Asp, Gln, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) Val, Ile, Leu, Met, Ala, Phe; (4) Lys, Arg, His; and (5) Phe, Tyr, Trp, His.

[0046] Variants may also (or alternatively) be proteins as described herein that have been modified, for example by deletion or addition of amino acids that have minimal effect on the enzymatic activity, secondary structure and hydropathic nature of the enzyme as defined below.

[0047] Furthermore, the term variant also refers to any glycosylated protein or any protein modified in any other manner described by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, and / or SEQ ID NO:62, or fragments thereof. A non-limiting list of such protein modifications includes acetylation, acylation, ADP-ribosylation, amidation, covalent binding, cross-linking, cyclization, disulfide bond formation, demethylation, covalent cross-link formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, lipid attachment, sulfation, selenoylation, transcription-RNA mediated addition of amino acids to proteins (such as arginylation), and ubiquitination.

[0048] Thus, orthologs and paralogs, or any genes in genera and species (other than the strain Starmelella bombicola ATCC 22214 from which SEQ ID NOs: 1 to 8, and SEQ ID NOs: 61 and 62 are derived) that code for a polypeptide having the described activity are part of the present invention.

[0049] The term "Sble enzyme" relates to an enzyme previously called "lactonase" or "Starmelella bombycola lactone esterase" and described in detail in WO 02 / 04396. The Sble enzyme of the present invention therefore relates to a polypeptide comprising the amino acid sequence given by SEQ ID NO:2 or a fragment thereof, retaining the above mentioned enzymatic activity (i.e. transesterification and / or hydrolysis activity on (acetylated) bolaamphiphilic glycolipid compounds, more specifically the conversion of (acetylated) bolaform sophorolipids / (acetylated) bolaform glucolipids to (acetylated) lactone type sophorolipids / (acetylated) lactone type glucolipids, respectively, while releasing (acetylated) sugars such as (acetylated) sophorose and / or (acetylated) glucose, and / or the conversion of (acetylated) bolaform sophorolipids / (acetylated) bolaform glucolipids to (acetylated) acidic sophorolipids / (acetylated) acidic glucolipids, while releasing (acetylated) sugars such as (acetylated) sophorose and / or (acetylated) glucose), or a variant thereof having at least 34% sequence identity with SEQ ID NO:2 and having the above mentioned enzymatic activity.

[0050] The nucleic acid sequence set forth in SEQ ID NO:1 corresponds to a 1233 base pair open reading frame that encodes the polypeptide sequence of the Sble enzyme of the invention, as set forth by the 410 amino acid sequence of SEQ ID NO:2.

[0051] SEQ ID NO:1:

[0052] SEQ ID NO:2: MLALFFSLAPLLSQALPLGYTAAPAESFYFWPENISSLQAGEIFRKRELLTLPDIFDFGPNLEKVVQVAYKTRLTDGNDSFSIASIFIPKNPSPELKLYSYQTFEDAVQLDCAPSYALEVGNKSSNYLPVTSNLSAISRELEKGRHCIIIPDHEGYISGFFAGRQEGYAGLDGIRAARNYLNGTNETPIGIFGYSGGAQATAWIVD LHDEYAPDLNFVGTVSGGTLVDAWGTFQYIDYPKVYLKGSILIMYTGLFSGYPAQFEVIWPYIEPVIQENMLLLRLAPNDCNQSPILQGYNNSIMAGIHVDL PEFPASKYIFQHESLLANYSVVPVSTPKFPRYMYHGGSDELAKLSLVEQYVDQQWNTGANLTFVVYPGLLHDETAYRGFDAAMDWLDAQLDSGYLPPVNSTHT

[0053] The term "fragment" further refers to a protein or peptide or polypeptide that contains fewer amino acids than the amino acid sequence described by SEQ ID NO: 2 and retains the above enzymatic activity, i.e., "transesterification and / or hydrolysis reactions in bolaamphiphilic glycolipid compounds." Such a fragment can be, for example, a protein in which 10% or less of the total number of amino acids have been deleted at the C-terminus and / or N-terminus.

[0054] Furthermore, the present invention relates to the use of Sble enzyme to convert tetraacetylated bolasophorolipids to diacetylated lactone sophorolipids while releasing (acetylated) sugars such as (acetylated) glucose and / or (acetylated) sophorose.

[0055] Furthermore, the present invention relates to the use of Sble enzyme to convert non-acetylated, mono-acetylated, diacetylated, and / or triacetylated bolasophorolipids to non-acetylated, mono-acetylated, and / or diacetylated lactone-type sophorolipids while releasing non-acetylated, mono-acetylated, or diacetylated sophorose and / or glucose.

[0056] Furthermore, the present invention relates to the use of Sble enzyme to convert tetraacetylated bolasophorolipids into diacetylated acidic sophorolipids while releasing (acetylated) sugars such as (acetylated) glucose and / or (acetylated) sophorose.

[0057] Furthermore, the present invention relates to the use of Sble enzyme to convert non-acetylated, mono-acetylated, di-acetylated and / or tri-acetylated bolasophorolipids into non-acetylated, mono-acetylated and / or di-acetylated acidic sophorolipids while releasing (acetylated) sugars, such as non-acetylated and / or mono-acetylated glucose and / or non-acetylated, mono-acetylated and / or di-acetylated sophorose.

[0058] The present invention further relates to the use of modified yeast strains which comprise a non-functional and / or dysfunctional Sble enzyme and / or in which the sble gene has been deleted and / or rendered abortive, and which strains are capable of producing (acetylated) bolaamphiphilic glycolipids, such as bolasophorolipids and / or bolaglycolipids.

[0059] The term "modified yeast strain" relates to a yeast strain which has been modified in any way such that the Sble enzyme is non-functional or disabled and / or the sble gene has been deleted and / or disabled.

[0060] More specifically, the present invention relates to the use of the modified yeast strain described above, wherein the bolasophorolipid has an acetylation degree of 0, 1, 2, 3, or 4.

[0061] The term "degree of acetylation" of 4 means that all four glucose moieties present in the bola-SL are acetylated.

[0062] Furthermore, the present invention relates to the use of modified yeast strains comprising a non-functional and / or dysfunctional At1 enzyme and / or not containing a (functional) at1 gene encoded in the SL biosynthetic gene cluster, wherein the strains are surprisingly capable of producing acetylated (boratype) amphipathic glycolipids, more specifically acetylated (boratype) sophorolipids and / or acetylated (boratype) glucolipids, said acetylated bolatype sophorolipids and / or glucolipids having a degree of acetylation of 0, 1 or 2. The terms "degree of acetylation" of 0, 1 or 2 means that 0, 1 or 2 glucose moieties, respectively, present in the bolatype amphipathic glycolipid are acetylated.

[0063] The term "modified yeast strain" relates to a yeast strain which has been modified in any way as already described above for the Sble enzyme, such that the At1 enzyme is non-functional or impaired as described above and / or the at1 gene is deleted and / or impaired.

[0064] The term "acetyltransferase (At1) enzyme 1" relates to an enzyme previously described in detail in WO 2012 / 080116, as well as in Fischer et al. (2015). This At1 enzyme is referred to as "At1" in the present invention and is therefore responsible for the acetylation of glycolipids produced by S. bombicola (Saerens et al. (2015)). The At1 enzyme of the present invention therefore relates to the nucleic acid sequence as set forth in SEQ ID NO: 3, corresponding to an open reading frame of 780 base pairs encoding a polypeptide comprising the amino acid sequence given by SEQ ID NO: 4, or a fragment thereof that retains the above-mentioned enzymatic activity (i.e. "acetylation of (bora-type) amphipathic glycolipid compounds, more specifically acetylation of (bora-type) sophorolipids and / or (bora-type) glucolipids") and thus relates to the nucleic acid sequence as set forth in SEQ ID NO: 3, corresponding to an open reading frame of 780 base pairs encoding a polypeptide comprising the amino acid sequence given by SEQ ID NO: 4, or a fragment thereof that retains the enzymatic activity, or a variant thereof having at least 34% sequence identity with SEQ ID NO: 4 and having the above-mentioned enzymatic activity.

[0065] SEQ ID NO:3: atggttgtaaactctcgaaggaccctcaaaacaaaggaatgactcctagaaagaaattgaccaggaaatggtctcttgggccaaaaaaaacctcaaaaacacccctggcaatgaaaactatgagaagatggtctcaggagttccttacaatccatacgatccagatcttatgtttagagccctggctactagt gagaaagttagggagttcaataccattgcaagtgaaagtcgtacttttgagtcaaatcacgctgcttatatcaagaaggtcgagattctcaaagacacttttggtcaaacaaaggatattgtctggctgaccgctccattctcagttgattttggattcaacatcagcgtaggcgagcacttttacgccaacttc aacgtttgcttcttggactcggctccaataatctttggtgatgaggtgattgtagggcccaatacaacgttcgtgactgcgactcatcctattagccccgagaaacgtgcgaggagaattgtgtatgctcttcctatcaaggtggggaataatgtatggattggtgcgaatgtgactgtcctgccgggtgttacg attggagatggctcaacaattgcggctggtgctctcgttcgagaagatgttcctctcctactgtggtgggaggatccctgcgcgaatcctcaagcatattccagaggaggatcccgacgaggctgaaggagagactggaattccttcttccaggtgaaatgaacgtcaataccgctaaccagaaggtctag

[0066] sequence number 4: MVVNSSKDPQNKGMTPRKEIDQEMVSWAKKNLKNTPGNENYEKMVSGVPYNPYDPDLMFRALATSEKVREFNTIASESRTFSNHAAYIKKVEILKDTFGQTKDIVWLTAPFSVDFGFNISVGEHFYAN FNVCFLDSAPIIFGDEVIVGPNTTFVTATHPISPEKRARRIYALPIKVGNNVWIGANVTVLPGVTIGDGSTIAAGAVVREDVPPRTVVGGVPARILKHIPEEDPDEAEGEELEFLLPVEMNVNTANQKV

[0067] The term "fragment" further refers to a protein or peptide or polypeptide that contains fewer amino acids than the amino acid sequence described by SEQ ID NO: 4 and retains the above enzymatic activity, i.e., "acetylation of (bora) amphipathic glycolipid compounds, more specifically, acetylation of (bora) sophorolipids and / or (bora) glucolipids." Such a fragment can be, for example, a protein in which 10% or less of the total number of amino acids has been deleted at the C-terminus and / or N-terminus.

[0068] The present invention further relates to the use of the above-mentioned modified yeast strains further comprising, besides the above-mentioned dysfunctional At1 enzyme, two additional non-functional or dysfunctional glycoside O-acetyltransferase enzymes (At2 and At3) and / or a combination of both enzymes, non-functional or dysfunctional, for producing fully non-acetylated (bora-type) amphipathic glycolipids, such as (bora-type) sophorolipids / glucolipids. The term "glycoside O-acetyltransferase enzymes" relates to the enzymes, referred to in the present invention as At2 and At3, responsible for the acetylation of (bora-type) amphipathic glycolipids.

[0069] The term "modified yeast strain" relates to a yeast strain which has been modified in any way so that the At2 and / or At3 enzymes are non-functional or impaired, as described above, and / or in which the at2 and / or at3 genes have been deleted and / or impaired.

[0070] The present invention further relates to the use of modified yeast strains comprising one of the three non-functional or dysfunctional acetyltransferase enzymes (At1, At2, At3) and / or a combination of the three enzymes rendered non-functional or dysfunctional, for the production of non-acetylated glycolipids, such as non-acetylated lactone-type SLs, non-acetylated acidic glucolipids, non-acetylated acidic sophorolipids, non-acetylated bola-type sophorolipids, non-acetylated bola-type sophorolipids, non-acetylated bola-type glucolipids, etc. The term "modified yeast strain" relates to a yeast strain modified in any manner as described above for the Sble enzyme such that the At1, At2, and / or At3 enzymes are non-functional or dysfunctional.

[0071] The At2 enzyme of the present invention therefore relates to the nucleic acid sequence set forth by SEQ ID NO:5, which corresponds to an open reading frame of 663 base pairs encoding a polypeptide comprising 220 amino acids having the sequence given by SEQ ID NO:6, or a fragment thereof that retains the above-mentioned enzymatic activity (i.e. "acetylation of (bola-) amphipathic glycolipid compounds, more specifically acetylation of (bola-) sophorolipids and / or (bola-) glucolipids"), or a fragment thereof that retains the enzymatic activity, or a variant thereof having at least 34% sequence identity with SEQ ID NO:6 and having the above-mentioned enzymatic activity.

[0072] SEQ ID NO:5: ATGCCTAGCGGAGCCCCAAGAATCGAGTACAATTGGGACCTGATCAAGTGGGCTCGCGAAAATTGTCCCATTTGCCGGTAGATGATGACAACTATCACCGGATGATTAGTGGGTTGCCATATGAGGCAACCCGCACAGATTATTCGCGCCATCGAATAGAGTCCCATGAATTACTTCTAGAATACTTGAATATGAAACTGAAGGACTTCGCTACGTTGGAAAAATAATCAGGCGCGAGCAGATTTGCTTTCAAAGGTTGTTGGCTCCATGGGCACCAAACTGCTTCATTGAGCAACACTATTGTAGATTATGGTTGCAACATTAAAG TCGGCAATAACTTTTATGCGAACAACAACCTTACAATGCTCGATTGCTCTGTCATTGAGATTGGCGACAATGTGTTTTTTGGACCTAATGTAACAATCACTACGGCATCTCACCCGTTGGAATCGAAGCCTAGGGCCGAAGGGGTCGAATTCGCTTTCAATGTCAA AATCGGAAACAACGTCTGGATAGGTTCCAACGCTGTGGTCTTGCCGGGAGTTACCATTGGAGATGACGTAGTCGTTGCAGCTGGCGCAGTGGTCAACAAGGATGTGCCCCTTCAGTCGTAGTGGGCGGTGTCCCGGCGAAAATTCTTAAGCAAATCCAGAATTGA

[0073] sequence number 6: MPSGAPRIEYNWDLIKWARENLSHLPVDDDNYHRMISGLPYEATRTDYSRHRIESHELLLEYLNMKLKDFATLEKYNQARADLLSKVFGSMGTNCFIEQHLFWVDYGCNIKVGNNFYANNNLTMLDCSVIEIGDNVFFGPNVTITTASHPLESKPRAEGVEFAFNVKIGNNVWIGSNAVVLPGVTIGDDVVVAAGAVVNKDVPPSVVVGGVPAKILKQIQN

[0074] The term "fragment" further refers to a protein or peptide or polypeptide that contains fewer amino acids than the amino acid sequence described by SEQ ID NO: 6 and retains the above enzymatic activity, i.e., "acetylation of (bora-) amphiphilic glycolipid compounds, more specifically, acetylation of (bora-) sophorolipids and / or (bora-) glucolipids." Such a fragment can be, for example, a protein in which 10% or less of the total number of amino acids has been deleted at the C-terminus and / or N-terminus.

[0075] The At3 enzyme of the present invention therefore relates to the nucleic acid sequence set forth by SEQ ID NO:7, which corresponds to an open reading frame of 747 base pairs encoding a polypeptide comprising the amino acid sequence given by SEQ ID NO:8 of 248 amino acids, or a fragment thereof that retains the above-mentioned enzymatic activity (i.e. "acetylation of (bora-type) amphipathic glycolipid compounds, more specifically acetylation of (bora-type) sophorolipids and / or (bora-type) glucolipids"), or a variant thereof having at least 34% sequence identity with SEQ ID NO:8 and having the above-mentioned enzymatic activity.

[0076] SEQ ID NO:7: ATGTTGCCTGCAACAGAAATCGATAGAGAACTCGTGCAATGGGCTCGCGAAAATCTTCCAAACCTCCCTCAAAGCACACATTATGACAAGCAGATTAGTGGCATGCTGATCAAGCCCAAATGGTCCTCAATGGTTCACGAGACAAAGATGAAACAGCTCACAAGGGACTATGACAGCATCAATCTC AACCATTTCAGCTCTGTGGCGAAATACTTTGAGGCCAGGACAAGCTTTATCCAGAAGCATCTCCTCGGCAAACAGGAAAGAGAGTCTACCTCGAATCCCCAGTTCACATCAATCACGGATACAATATATCGGTAGGCGAAAACTTCTATTGCAACTTTAATTGCATATTTCTCGACTGGTCCATAA TCAGAATTGGCGACAACGTTGCGATTGGCCCCAACTGTACCTTAAGTTGCATTAATCATCCCTTGAGTGGCGATGATCGCAAAAATGGTGCGGGTTATACGCTTTCCCTATCTTTATCGATGACAATGTCTGGATAGGGGCGAACTGTGTGATTCTTTCAGGGATTCATGTTGCTGAAGGGTCGGT TGTCGCCGCAGGATCGGTAGTGCAAAAAAGTGTGCCCCCTCATGTTATCGTGGCTGGCAATCCTGCGAAGATCATTGCGAAGGCAACAGACCGACGACTTCGGGGCTGCCGCAGAGGATTCATCCTCCCCAGAATCTTCGGACGCCGAAGAGAGCTACATGTTCATTACCAAGACTGCGGATCCCTGA

[0077] sequence number 8: MLPATEIDRELVQWARENLPNLPQSTHYDKQISGMLIKPKWSSMVHETKMKQLTRDYDSINLNHFSSVAKYFEARTSFIQKHLLGKTGKRVYLESPVHINHGYNISVGENFYCNFNCIFLDWSI IRIGDNVAIGPNCTLSCINHPLSGDDRKNGAGLYAFPIFIDDNVWIGANCVILSGIHVAEGSVVAAGSVVTKSVPPHVIVAGNPAKIIAKATDRRLRAAAEDSSSPESSDAEESYMFITKTADP

[0078] The term "fragment" further refers to a protein or peptide or polypeptide that contains fewer amino acids than the amino acid sequence described by SEQ ID NO: 8 and retains the above enzymatic activity, i.e., "acetylation of (bora) amphipathic glycolipid compounds, more specifically, acetylation of (bora) sophorolipids and / or (bora) glucolipids." Such a fragment can be, for example, a protein in which 10% or less of the total number of amino acids has been deleted at the C-terminus and / or N-terminus.

[0079] Thus, the present invention also relates to isolated acetyltransferases having the amino acid sequence given by SEQ ID NO:6 or SEQ ID NO:8, designated acetyltransferase 2 (At2) and acetyltransferase 3 (At3), respectively.

[0080] Furthermore, the present invention relates to the use of the modified yeast strains described above, which further comprise a non-functional or dysfunctional glucosyltransferase UgtB1 enzyme or in which the ugtB1 gene has been deleted and / or disabled, and acetylated and / or non-acetylated bora-type glucolipids are produced instead of bora-type sophorolipids. The term "UgtB1 enzyme" refers to the enzyme described in detail by Friedrichs et al. (1999) and Saerens et al. (1995) with activity of glycosylating (bora-type) glucolipids to (bora-type) sophorolipids. The UgtB1 enzyme of the present invention therefore relates to the nucleic acid sequence described by SEQ ID NO: 61, which corresponds to an open reading frame of 1299 base pairs encoding the polypeptide sequence of the UgtB1 enzyme described by the 432 amino acid sequence of SEQ ID NO: 62, or a fragment thereof that retains the enzymatic activity, or a variant thereof that has at least 34% sequence identity with SEQ ID NO: 62 and has the above enzymatic activity. The term "modified yeast strain" relates to a yeast strain in which the UgtB1 enzyme encoded in the SL biosynthetic gene cluster (Non-Patent Document 3) is modified in any way so that it is non-functional or dysfunctional, as described above.

[0081] SEQ ID NO:61:

[0082] SEQ ID NO:62: MAIEKPVIVACACPLAGHVGPVLSLVRGLLNRGYEVTFVTGNAFKEKVIEAGCTFVPLQGRADYHEYNLPEIAPGLLTIPPGLEQTGYSMNEIFVKAIPEQYDALQTA LKQVEAENKSAVVIGETMFLGVHPISLGAPGLKPQGVITLGTIPCMLKAEKAPGVPSLEPMIDTLVRQQVFQPGTDSEKEIMKTLGATKEPEFLLENIYSSPDRFLQL CPPSLEFHLTSPPPGFSFAGSAPHVKSAGLATPPHLPSWWPDVLSAKRLIVVTQGTAAINYEDLLIPALQAFADEEDTLVVGILGVKGASLPDSVKVPANARIVDYFP YDELLPHASVFIYNGGYGGLQHSLSHGVPVIIGGGMLVDKPAVASRAVWAGVGYDLQTLQATSELVSTAVKEVLATPSYHEKAMAVKKELEKYKSLDILESAISELAS

[0083] The term "fragment" further refers to a protein or peptide or polypeptide that contains fewer amino acids than the amino acid sequence described by SEQ ID NO: 62 and retains the above enzymatic activity, i.e., "glycosylation of (bola-)amphipathic glucolipid compounds, more specifically, glycosylation of (bola-)glucolipids." Such a fragment can be, for example, a protein in which 10% or less of the total number of amino acids has been deleted at the C-terminus and / or N-terminus.

[0084] More specifically, and as already mentioned above, the present invention further relates to the use of the modified yeast strains mentioned above, said yeast strains being selected from the group consisting of Starmerella bombicola (formerly Candida) (Spencer et al., 1970), Starmerella apicola (Gorin et al., 1961) (formerly Candida) (originally identified as T. magnolia), Wickelhamieradomerichiae (Chen et al., 2006), Pseudohyphosima bogoriensis species (formerly Rhodotorula or Candida) (Tulloch et al., 1968), Starmerella batistae (Konishi et al., 2008) (formerly Candida), Starmerella (formerly Candida) phylloricola (Imura et al., 2010), Starmerella (formerly Candida) batistae, Candida iodocensis, Candida tropicalis, Starmerella stellata (formerly Candida) and Candida species NRRL Y-27208 (Kurtzman et al., 2010), Starmerella koi (Kurtzman, 2012) (formerly Candida), Candida glopenghiesseri, Candida magnoliae, Candida antarctica, Pseudozyma antarctica, Candida tropicalis, Candida lipolytica, and any other SL-producing strain (of the Starmerella clade).

[0085] Furthermore, the present invention relates to the use of the modified yeast strains described above, which are defective in the activity of the Sble enzyme, the UgtB1 enzyme, and / or the acetyltransferase enzymes At1, AT2, and At3, and / or the genes encoding them. EXAMPLES

[0086] Example 1: Production of acetylated bolaglycolipids.

[0087] Materials and Methods Strains and culture methods Cloning experiments and plasmid maintenance were performed in top 10 cells of Escherichia coli. E. coli cells were grown in Luria-Broth medium (37°C, 10 g / l tryptone, 5 g / l yeast extract, 5 g / l sodium chloride, and 15 g / l agar when required; Sigma-Aldrich) supplemented with 100 mg / L ampicillin (LB-amp; MP Biomedicals) when applicable. Wild-type S. bombicola (WT; ATCC 22214) and a URA3 auxotrophic mutant strain (PT36) were used during this study (Lodens et al., 2018). Two existing S. bombicola strains developed in the past were also included: a single deletion strain Δsble (Non-Patent Document 5) and a double deletion strain Δat1 Δsble (Non-Patent Document 6 and Patent Document 2). Uracil-free complete supplement mix (6.7 g / L of amino acid-free yeast nitrogen base (Sigma-Aldrich), 20 g / L of glucose (Cargill), 20 g / L of agar Noble (Difco), 0.77 g / L of uracil-free complete supplement mix (MP biomedicals)) and yeast extract peptone dextrose supplemented with hygromycin (solid synthetic dextrose containing 20 g / L glucose (Cargill), yeast extract (DSM), 20 g / L bactopeptone (BD biosciences), agar (Biokar Diagnostics), 1 g / L hygromycin B (Sigma-Aldrich) were used for selection of positive deletion mutants after transformation with the URA3 auxotrophic or hygromycin resistance markers, respectively.

[0088] For glycolipid production experiments, the production medium described by (Lang et al., 2000) was used. Precultures (5 mL) were inoculated from cryovials (1%) and incubated (30°C, 200 rpm) for 48 h. Then, shake flasks (n=3) containing 100 mL of production medium were inoculated from the precultures (1%). The shake flasks were incubated (30°C, 200 rpm) for 240 h. After 48 h of cultivation, 37.5 g / L oleic acid (Sigma-Aldrich) was added.

[0089] analysis technology The cell dry weight (CDW) was determined by a centrifugation step (5 min, 14000 rpm) until the shake flask broth was reduced to 1 mL, after which the supernatant was discarded. The biomass pellet was resuspended in 0.9% (w / v) NaCl solution and centrifuged once more at 14000 rpm for 5 min. After this washing step, the supernatant was discarded and the biomass was placed in a 60°C oven for at least 50 h to remove residual moisture. Finally, the net dry biomass was determined gravimetrically once more and the total CDW was measured and expressed in g / L dry biomass.

[0090] The pH of the sF broth samples was measured with a two-point calibrated Five easy F20 Mettler Toledo pH / mV meter.

[0091] Production samples were analyzed by UPLC-HRMS (Thermo Scientific™ Exactive™ Plus Orbitrap Mass Spectrometer). Products were separated by UPLC according to (Van Renterghem et al., 2018). Sample preparation was performed on SF broth samples. First, 70% EtOH (3:1, v / v) was added to the sample and vortexed vigorously for 5 min. This was followed by a centrifugation step (5 min, 14000 rpm) where the supernatant was filtered through a PES filter (0.2 μm, sartorius). Four in-house SL standards were analyzed alongside the production samples.

[0092] molecular method Circular polymerase extension cloning (CPEC) pieces and linear deletion cassettes were amplified by Primestar™ GXL according to the manufacturer's instructions. Colony PCR was performed in E. coli and S. bombicola according to (De Graeve et al., 2019). S. bombicola colony PCR was performed to analyze the 5', 3', and complete overlap of the integrated genomic deletion cassettes. CPEC was performed with Q5™ Hifi DNA polymerase according to the manufacturer's instructions and as described in (Quan and Tian, ​​2009). CPEC assembly products and linear deletion cassettes were transformed into E. coli and S. bombicola, respectively, by electroporation according to (De Graeve et al., 2019). Plasmids assembled by CPEC were sequenced by Macrogen inc.

[0093] For subsequent gene deletion in S. bombicola, three different deletion cassettes were constructed (Figures 3–5).

[0094] Genetic elements were derived from the S. bombicola genome, except for the hygromycin B selection marker (HygroR) and the herpes simplex virus tyrosine kinase (tTK) terminator, which were used as described by (Van Bogaert et al., 2008). To construct the deletion cassettes, fragments were first amplified and assembled in a pJET vector backbone (pJET; Thermo scientific) using circular polymerase extension cloning (CPEC) plasmid assembly (Quan and Tian, ​​2009). These plasmids were transformed into top 10 cells of E. coli, and positive colonies were selected from LB-amp and verified by colony PCR and subsequent DNA sequencing. The primers used for amplification of the fragments, the origin of amplification of the fragments, and the primers used for E. coli colony PCR are listed in Table 1. Disruption of the ugtB1 gene was achieved as described by Lodens et al. (2020).

[0095] result Evaluation of existing S. bombicola strains Recently performed biosurfactant production experiments described under Materials and Methods using three previously developed and described S. bombicola strains: Δsble (Non-Patent Document 5 and Patent Document 1), Δat1 Δsble (Non-Patent Document 9 and Patent Document 2) and Δat1 (Non-Patent Document 4) have led to two unexpected observations that are in contradiction with the state of the art. The first observation concerns the surprising detection of masses corresponding to (acetylated) bola-type sophorolipids with a degree of acetylation up to 4 in samples derived from experiments using Δsble strains described by (Non-Patent Document 5, Non-Patent Document 7 and Patent Document 1), thus in contrast to previous observations and reports in which it was described that only acidic SLs were produced. The second observation also concerns the surprising detection of acetylated (bola-type) sophorolipids with a degree of acetylation of up to 2 (mainly 1) in samples from experiments with the Δat1 Δsble strain (Non-Patent Document 9 and Patent Document 2) and with the Δat1 strain (Non-Patent Document 4), thus in contrast to previous observations and reports. Both observations are in contradiction with the art and unexpected, since the Δsble strain has been described as exclusively producing (acetylated) acidic SLs. Diacetylated acidic SLs have been described as substrates for the Sble enzyme, which converts them into diacetylated lactone-type SLs (Non-Patent Document 5 and Non-Patent Document 8). The Δat1 Δsble strain and Δat1 have been described as not producing any acetylated (bola-type) SLs due to a mutation in the at1 gene, which has been described as the (only) enzyme responsible for the acetylation of sophorolipids. Therefore, the production of acetylated bola-type sophorolipids has not been described. These findings were therefore highly unexpected and further experiments were performed.

[0096] Since the existing strains were developed using restriction enzyme-mediated methods, some of the ORFs of sble and at1 genes were still present in the above-mentioned modified strains, which may result in residual enzyme activity. Therefore, new S. bombicola strains with completely deleted ORFs / coding sequences described under Materials and Methods were generated as described below, and their glycolipid production profiles were evaluated after production experiments.

[0097] Construction and evaluation of novel S. bombicola strains The deletion cassettes described under Materials and Methods and shown in Figures 3-5 were amplified from the corresponding plasmids and used for transformation into S. bombicola strains. Cassettes 1 and 2 were used for the generation of S. bombicola strains containing single gene deletions by homologous recombination, namely Δsble_full and Δat1_full, respectively. After the gene deletion was successfully verified, cassette 3 was used for the deletion of at1 in the new Δsble_full strain. Table 2 lists the strains generated, the corresponding deletion cassettes, the primers used for amplification of the deletion cassettes, the primers used for colony PCR evaluation, the original strains, and the genotypes obtained.

[0098] The production characteristics of the newly developed strains were evaluated in shake flask (SF) experiments together with the S. bombicola wild type (WT) strain. A similar pH drop was observed in all SFs, with an initial pH of approximately 5.8, and a rapid drop to approximately 3 at about 48 hours after inoculation, where it was maintained. A similar growth was observed for all strains, with a maximum cell dry weight (CDW) of 19 g / L at 84 hours after inoculation. Thereafter, the CDW remained constant for all strains except the WT strain. This is mainly due to the fact that solid lactone-type sophorolipids (SLs) remained with the cell pellet for the wild type, causing a distorted CDW measurement. These lactone-type SLs were clearly visible as a separate layer in the centrifuged SF broth samples collected from the WT strain from 84 to 240 hours of production, while no such layer was detected in the similar SF samples from the Δat1, Δsble, and Δat1 Δsble S. bombicola strains.

[0099] Production samples obtained 180 hours after seeding were subjected to UHPLC-HRMS analysis, the results of which are described in the text below and summarized in Table 4.

[0100] As expected, wild-type S. bombicola mainly produces C18:1 diacetylated (diAc) lactone-type SL (L SL).

[0101] The novel Δsble_full strain was evaluated. The SL spectrum is composed mainly of m / z values ​​corresponding to the monoisotopic masses of nAc C18:1 bola-type SL, mAc C18:1 bola-type SL, diAc C18:2 bola-type SL, diAc C18:1 bola-type SL, triacetylated (triAc) C18:1 bola-type SL, tetraacetylated (tetraAc) C18:1 bola-type SL, tetraAc C18:0 bola-type SL, diAc C18:1 acidic SL and diAc C18:10 acidic SL. Indeed, in surprising contrast to previous observations and reports, the novel sble deletion strain (Δsble_full), in which the entire coding sequence is removed for the strain, also produces (acetylated) bola-type SL. Moreover, the fully acetylated bola-type SL (tetraacetylated C18:1 bola-type SL) is produced in extremely large amounts. This was completely unexpected, since Non-Patent Document 9 stated that the absence of an acetyl group triggers the formation of bolasophorolipids starting from acidic sophorolipids.

[0102] During the analysis of the new Δat1_full strain, in which the at1 gene from the SL biosynthetic gene cluster had been completely deleted, acetylated glycolipid compounds were indeed also detected, as described above for the original strain. However, a lower degree of acetylation, as described above for the Δsble strain, was also observed. The product spectrum of the Δat1 strain is mainly composed of m / z values ​​corresponding to the monoisotopic masses of non-acetylated (nAC) C18:1 bola-type SL (bola-type SL), monoacetylated (mAc) C18:1 bola-type SL, nAc C18:1 triglucolipid, nAc C16:0 acidic SL, nAc C18:1 acidic SL, nAc C18:0 acidic SL, nAc C18:1 glucolipid, nAc C18:1 L SL, and monoacetylated C18:1 lactone-type SL. The acetyltransferase from the SL biosynthetic gene cluster (Figure 1, at1) was completely removed in the new Δat1_full strain as shown in Figure 4, and acetylated compounds were still observed, indicating the activity of an unknown acetyltransferase active in SL.

[0103] Finally, the Δat1Δsble_full S Bombicola strain was generated, in which both the sble and at1 genes / enzymes were completely deleted. The new full Δat1Δsble deletion strain was evaluated as described above, and the SL production spectrum was evaluated and found to be composed primarily of m / z values ​​matching the monoisotopic masses of nAc C16:1 bola-type SL, nAc C16:0 bola-type SL, nAc C18:1 bola-type SL, mAc C18:1 bola-type SL, nAc C18:0 bola-type SL, nAc C18:1 acidic SL, nAc acidic C18:0 SL, and nAc C18:1 glucolipid. Table 4 lists all detected m / z values, corresponding retention times, and SL congeners with matching monoisotopic masses.

[0104] In general, it can be seen that when no deletions were made in the sble ORF, only lactone-type SLs were observed. Furthermore, the Δat1 strain mainly produces bola-type SLs with low acetylation (mAc) and lactone-type SLs. On the other hand, the Δsble strain mainly produces bola-type SLs with high acetylation (di-, tri-, and tetra-Ac). The Δat1Δsble strain mainly produces bola-type SLs with low acetylation (mAc). This indicates that the Sble enzyme preferentially transesterifies acetylated bola-type amphiphilic glycolipids.

[0105] These findings are in contrast to what has been described in the art (Non-Patent Document 9, Non-Patent Document 10, Patent Document 1 and Patent Document 3), where it has been described that deletion of the at1 gene is necessary to produce boragosophorolipids, and therefore boragosophorolipids can only be produced as completely non-acetylated molecules. The At1 enzyme has further been described as the only enzyme that acetylates (boragosophorolipids) in S. bombicola (Non-Patent Document 4, Non-Patent Document 9), and therefore the boragosophorolipids described in the art did not contain any acetyl groups.

[0106] Deletion of the ugtB1 gene in the Δsble1 strain produced the Δsble1Δugtb1 strain. The product spectrum of the Δsble1Δugtb1 strain is composed mainly of m / z values ​​corresponding to the monoisotopic masses of nAc C18:1 bola-type GL, nAc C18:1 acidic GL, nAc C18:0 acidic GL, and mAc C18:1 acidic GL. Small amounts of mAc C18:1 bola-type GL, nAc C16:0 acidic GL, mAc C18:0 acidic GL, and mAc C16:0 GL were found to be present. This finding is also in contrast to what has been described in the art, namely, that the Δsble1Δugtb1Δat1 strain is required to produce bola-type glucolipids, and that these bola-type glucolipids are expected to be completely unacetylated. Acetylated bola-type glucolipids were also detected when analyzing this strain, i.e., Δsble1Δugtb1Δat1. The Δsble1Δugtb1Δat1Δat2Δat3 strain can be used to produce the complete non-acetylated bola-glucolipid (also described in Example 3).

[0107] [Table 1]

[0108] [Table 2]

[0109] [Table 3]

[0110] [Table 4]

[0111] Example 2: Use of Sble enzymes to carry out transesterification and / or hydrolysis reactions:

[0112] Materials and Methods Production of recombinant Sble For recombinant Sble (rSble) production, the HAC1 co-expressing strain NRRL-Y-11430 of P. pastoris (syn: Komagataella phaffii) transformed with the pPICZαB_rSbleopt construct with the highest yield of rSble described in De Waele et al. (2018) was utilized in the study. The strain was grown in buffered glycerol complex medium (BMGY) in a 3L baffled shake flask containing 500 ml of medium for 48 hours at 28 °C and 250 rpm. Then, induction was performed in buffered methanol complex (BMMY) medium for 48 hours at 16 °C and 250 rpm. Every 12 hours, 1% methanol was added to continuously stimulate protein production. Both BMGY and BMMY were composed of 1.34% (w / v) yeast nitrogen base (YNB, Formedium) containing 1% (w / v) yeast extract (Lab M), 2% (w / v) peptone (BD), 100 mM phosphate buffer (Chem-Lab) (pH 6.0), and 1% (v / v) glycerol (Chem-Lab) or 1% (v / v) methanol (Chem-Lab) as the sole carbon source, respectively. Finally, the culture containing the produced rSble was centrifuged (5000g, 10 min) and the supernatant was collected for protein purification.

[0113] Purification of recombinant Sble For purification of rSble, a two-step purification method was used according to the procedure described by De Waele et al. (2018). Briefly, in the first step, purification was performed with an AKTA Purifier system (GE Healthcare). Prior to loading the sample, 0.01% (w / v) reduced glutathione (Sigma-Aldrich) and 2 mM (final concentration) magnesium sulfate (Sigma-Aldrich) were added to the supernatant, and then the pH was adjusted to 7.5. After removing the precipitate by 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 diluted with 50 mM NaCl. 2The HisTrap™ HP column (5 ml, Cytiva) pre-equilibrated with binding buffer HPO4 (Chem-Lab) (pH 7.5), 500 mM NaCl (Chem-Lab) was loaded at a flow rate of 5 mL / min. After loading the sample, the column was washed with binding buffer until the UV (280 nm) absorbance reached a stable baseline. Elution was then performed stepwise with 20 mM and 200 mM imidazole (Chem-Lab) in binding buffer. The two eluted fractions were combined and immediately desalted by buffer exchange using 25 mM Tris-Hcl (Sigma-Aldrich) (pH 7.5), 150 mM NaCl, and an Amicon™ Ultra-15 centrifugal filter device (Merck) with a 10 kDa cutoff, and finally concentrated to 1 mL. In the second step, 1 mL of the concentrated IMAC fraction was injected onto a HiLoad™ 16 / 600 Superdex™ 200 pg column (GE Healthcare) equilibrated with desalting buffer (25 mM Tris-HCl (pH 7.5), 150 mM NaCl) and eluted with the same buffer. The fraction containing rSble was concentrated to 1.0 mL using an Amicon™ Ultra-15 centrifugal filter device (Merck) with a 10 kDa cutoff. The concentration of rSble was measured using a Coomassie (Bradford) protein assay kit from Thermo Scientific™ and using a Bio-Rad Microplate Reader model 680. The protein was stored at -80°C for further catalytic experiments.

[0114] Evaluation of the catalytic properties of rSble The HPLC-based activity assay was followed as described by De Waele et al. (2018) with minor adaptations. Briefly, 2 μg of purified rSble was added to 500 μl of reaction buffer containing 5 mM acidic SL or bolaform SL and 50 mM sodium citrate (Merck) at pH 3.5, provided by INBIO. The mixture was incubated for 1 h at 30° C. and 1400 rpm, after which the reaction was stopped with 1500 μl of 100% (v / v) ethanol (Chem-Lab). After concentrating the sample to 250 μl using a SpeedVac vacuum centrifuge (Thermo Savant, Holbrook, NY), 100 μl of the sample was analyzed using HPLC equipped with a UV detector. Reactions in which rSble was exchanged with the same volume of buffer used for protein purification (25 mM Tris, 150 mM NaCl, pH 7.5) were used as negative controls in the assay.

[0115] HPLC and MALDI-TOF MS analysis of sophorolipids Samples of acidic SLs from catalytic assays were analyzed by HPLC on an Ettan™ LC system (GE Healthcare) using a ZORBAX Eclipse Plus C18 Rapid Resolution 4.6 mm×100 mm column (Agilent) and a UV absorbance detector (280 nm, GE Healthcare). To separate the components, a gradient of two eluents, aqueous and acetonitrile (ACN), had to be used. The gradient started with 30% ACN and increased linearly to 50% in 15 min, after which the gradient increased linearly from 50% ACN to 60% in 10 min. The mixture was held in this manner for 10 min, then returned to 30% ACN in 0.1 min. A flow rate of 0.6 mL / min was applied.

[0116] Samples of bolaform SL from the catalytic assay were analyzed by another HPLC analysis method on the same LC system using a Brownlee Spheri-5 RP-18 Cartridge Column-220 mm×2.1 mm (Perkin Elmer™) and a UV absorbance detector (280 nm, GE Healthcare). A gradient of two eluents, aqueous and acetonitrile (ACN), was used to separate the components. The gradient started with 30% ACN and increased linearly to 50% in 15 min, after which the gradient increased linearly from 50% ACN to 80% in 30 min. The mixture was held in this manner for 5 min, then returned to 30% ACN in 1 min. A flow rate of 0.15 mL / min was applied.

[0117] Fractions of the peaks that changed significantly before and after the reaction were collected, and the corresponding compounds were identified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS). The collected fractions were first dried under a SpeedVac vacuum centrifuge (Thermo Savant, Holbrook, NY), and then the dried compounds were resuspended in 50% ACN (BioSolve) / 0.1% trifluoroacetic acid (TFA, Sigma-Aldrich) solution (12 μl). 1 μl of the resuspended compounds mixed with saturated α-cyano-4-hydroxycinnamic acid solution in a 1:1 ratio were spotted onto an Opti-TOF 384 Well MALDI Plate Insert for MALDI-TOF MS analysis by MALDI TOF / TOF 4800 Plus (ABSciex).

[0118] Further, LC-MS analysis was performed to verify the conversion of bola-SL to lactone-SL by rSble. SL samples (dissolved in ethanol to 1 mg / ml) were separated at 35°C and a flow rate of 1.5 ml / min on an Agilent 1100 series HPLC equipped with a quaternary pump and DAD detector using a Phenomenex Kinetex C18 150 × 4.6 mm 5μ solid core column. The products were separated using a gradient ranging from 20% to 80% acetonitrile in 30 min with 0.1% formic acid. The HPLC system was coupled to an Agilent G1956B single quadrupole MS detector equipped with an ESI ionization source. The mass spectrometer was set to scan the mass-to-charge range from 600 amu to 1200 amu.

[0119] result Activity assay of rSble using acetylated and non-acetylated acidic SL and crude SL The unexpected discovery of bola-type SLs in the Δsble strain raised the question of the actual substrates of Sble. Therefore, activity tests were performed on different SL samples. First, the activity of Sble against acidic SLs was tested using the recombinantly produced enzyme rSble. The samples used were (1) deacetylated acidic SL (C18:1) ω, (2) deacetylated acidic SL (C18:1) mixed ω and ω-1, and (3) nonacetylated acidic SL (C18:1) mixed ω and ω-1.

[0120] The activity of rSble towards the lactonization of three acidic SLs was analyzed using an HPLC-based activity assay following a procedure adapted from (see Methods). A negative control experiment was prepared by adding buffer without added enzyme. The results showed that after reaction of any of the three acidic SLs, the corresponding lactone-type SL was not detected (Figures 7-9). Indeed, the lactone-type SL was expected to elute after 36 and 41 min for mono- and diacetylated lactone-type SLs, respectively (verified by control experiments, data not shown), and no such peak was observed in any of the chromatograms.

[0121] This was a surprising result, so we increased the enzyme reaction time and the enzyme concentration to investigate whether this was due to a lower E:S ratio or a slower reaction. Instead of 2 μg, 10 μg of purified rSble was added to the reaction mixture, and the mixture was then incubated at 30° C. and 1400 rpm for 2 h. The treatment of the reaction mixture and the sample analysis were the same as described above. Again, no lactone-form SL was detected after the reaction, indicating that reaction time and enzyme concentration are not important parameters in terms of lactonization of acidic SL. Furthermore, all the previously mentioned samples were analyzed using MALDI-TOF / MS to analyze the possibility of diacetylated acidic SL forming polymers. However, the examination of the mass spectra at higher m / Z ratios did not give any indication that polymerization had occurred in the reaction.

[0122] Therefore, rSble was surprisingly unable to convert the three provided acidic SLs to lactone-type SLs. We went back to the activity test using the original crude SL mixture used by (8) and used this during further investigations to test the activity of rSble. This mixture was obtained from the Δsble strain described by (5) and was not purified / extracted. Based on data in the art, this crude SL mixture was expected to always contain only acidic SLs. However, as mentioned above, the Δsble strain was surprisingly found to produce a mixture of bola-type and acidic SLs in both acetylated and non-acetylated forms. Indeed, when comparing the HPLC chromatogram of this old SL mixture with a new sample of more homogeneous acidic SLs, it was revealed that additional peaks / compounds (retention (RT) at 15.5, 16.0, and 17.4 min (indicated by arrows)) were indeed present in this old crude sample used in the original Sble activity assay (Figure 10). MALDI-TOF MS was performed to investigate the identity of these compounds, analyzing fractions collected from three peaks (indicated by arrows in Figure 10 above, RTs of 15.5, 16.0, and 17.4 min). The mass spectrum showed a compound with m / z of 988 (for the peak at RT of 15.5 min), which corresponds to a monoacetylated bola-SL (C18:1). Also, diacetylated (C18:2) and diacetylated (C18:1) bola-SL were detected at RTs of 16.0 and 17.4 min, respectively, with MWs of 1028 and 1030, respectively.

[0123] Indeed, these compounds disappear neatly after incubation of the samples (see panel 2 of Figure 10), and diacetylated lactone-type SLs are formed by rSble (indicated by stars, Figure 10 below), while the peaks corresponding to acidic SLs show almost no loss in intensity.

[0124] Activity assay of rSble using acetylated and non-acetylated bola-type SLs Based on the observation that lactone-type SLs were obtained when Sble was incubated with a mixture of bola-SLs and acidic SLs, in contrast to the assay using only acidic SLs (see above), it was possible to state that bola-amphiphilic glycolipids might in fact be the actual substrates for the Sble enzyme, which then catalyzes the transesterification reaction rather than the lactonization esterification reaction. To confirm this, two types of bola-SLs were tested, the main compounds being (1) a triacetylated bola-SL and a diacetylated bola-SL (code: INV-113) in an approximately 1:1 ratio, and (2) a nonacetylated (and only a small amount of monoacetylated) bola-SL (code: INV_22).

[0125] The activity of rSble to transesterify two bola-SL samples to produce lactone-type SLs was analyzed using HPLC and MALDI-TOF, followed by LC-MS analysis based on negative spray analysis for identification. For the acetylated bola-SLs, our data showed that four lactone-type SL products (Figure 11, panel b, Table 5) were formed, among which the diacetylated lactone-type SL (C18:1) was the most abundant product (RT 21.565 min in Figure 11, panel b), compared to the negative control (Figure 11, panel a). The peak corresponding to the main bola-SL (Figure 11, panel a, Table 5) disappeared or was significantly reduced 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) (Figure 12, Table 6). Therefore, the activity assay results further confirm that Sble converts acetylated bola-SLs (degrees of acetylation of 1, 2, 3, and 4 to form the corresponding lactone-SLs). All peaks that decreased in intensity after reaction with rSble corresponded to bola-SLs, whereas peaks corresponding to acidic SLs remained unchanged after reaction.

[0126] [Table 5]

[0127] For the bola-SL sample, which contained mainly non-acetylated and mono-acetylated bola-SL, after the same process and identification of the significantly altered peaks, only trace amounts of lactone-SL were produced by rSble, while bola-SL was significantly decreased (Table 6). Here, the peaks corresponding to non-acetylated acidic SL showed significant accumulation indicating hydrolysis of the ester bond of (non-acetylated) bola-SL, indicating that the enzyme has additional hydrolytic activity towards substrates with low to no acetylation.

[0128] [Table 6]

[0129] Based on these surprising findings, an adapted sophorolipid biosynthesis illustration is shown in FIG. 13. As shown in FIG. 1, the biosynthesis of lactone SLs by S. bombicola, which has always been described and explained in the art, i.e., by internal esterification of acidic SLs to give lactone SLs, can thus be revisited. It is thus found that the biosynthesis of lactone SLs is instead the result of transesterification of bola-type sophorolipids to lactone sophorolipids. In FIG. 13, the formation and conversion of tetraacetylated bola-type SLs is shown, but as mentioned above, less acetylated bola-type SLs are also converted to non-acetylated, mono-acetylated, and diacetylated lactone and / or non-acetylated, mono-acetylated, and diacetylated acidic SLs.

[0130] Activity assay of rSble with methyl esters and sugars. To confirm that the SBLE enzyme is capable of transesterification with other substrates, the enzyme was tested for its ability to transfer disaccharides with fatty acid methyl esters. Sophorose was used as the acyl acceptor, whereas methyl stearate and methyl laurate were used as acyl donors (in two separate experiments). The components were mixed in a 1:2 ratio (0.006 mmol:0.012 mmol) in a total volume of 1 ml, to which 1 mg / ml of SBLE enzyme was added. The reaction mixture was incubated at 30° C. for 24 hours with stirring. Samples were taken at different time points and analyzed by thin layer chromatography and finally by LC-MS as described above. In both experiments the appearance of new compounds was evident, with retention times in the range of those of glycolipids. In the blanc reactions no appearance of new compounds was observed. LC-MS analysis revealed the appearance of compounds with masses of 524.6 and 608.8, corresponding to sophoryl laurate and sophoryl stearate, respectively, in the reaction mixtures fed with SBLE enzyme, confirming the transesterification activity of Sble enzyme on substrates other than bolaglycolipids.

[0131] Example 3: Production of nonacetylated (bora-type) glycolipids.

[0132] Materials and Methods Strains and culture methods Cloning experiments and plasmid maintenance were performed in top 10 cells of Escherichia coli. E. coli cells were grown in Luria-Broth medium (37°C, 10 g / l tryptone, 5 g / l yeast extract, 5 g / l sodium chloride, and 15 g / l agar when required; Sigma-Aldrich) supplemented with 100 mg / L ampicillin (LB-amp; MP Biomedicals) when applicable. During this study, wild-type S. bombicola (WT; ATCC 22214) and a URA3 auxotrophic mutant strain (PT36) were used, which served as base strains for generating a series of novel strains described below (Lodens et al., 2018). Uracil-free complete supplement mix (6.7 g / L of amino acid-free yeast nitrogen base (Sigma-Aldrich), 20 g / L of glucose (Cargill), 20 g / L of agar Noble (Difco), 0.77 g / L of uracil-free complete supplement mix (MP biomedicals)) and solid synthetic dextrose containing yeast extract peptone dextrose (20 g / L glucose (Cargill), yeast extract (DSM), 20 g / L of bactopeptone (BD biosciences), agar (Biokar Diagnostics) supplemented with hygromycin were used for selection of positive deletion mutants after transformation with the URA3 auxotrophic marker.

[0133] For glycolipid production experiments, the production medium described by (Lang et al., 2000) was used. Precultures (5 mL) were inoculated from cryovials (1%) and incubated (30°C, 200 rpm) for 48 h. Then, shake flasks (n=3) containing 100 mL of production medium were inoculated from the precultures (1%). The shake flasks were incubated (30°C, 200 rpm) for 240 h. After 48 h of cultivation, 37.5 g / L oleic acid (Sigma-Aldrich) was added.

[0134] analysis technology Production samples were analyzed by UPLC-HRMS (Thermo Scientific™ Exactive™ Plus Orbitrap Mass Spectrometer). Products were separated by UPLC according to (Van Renterghem et al., 2018). Sample preparation was performed on SF broth samples. First, 70% EtOH (3:1, v / v) was added to the sample and vortexed vigorously for 5 min. This was followed by a centrifugation step (5 min, 14000 rpm), where the supernatant was filtered through a PES filter (0.2 μm, sartorius).

[0135] molecular method Circular polymerase extension cloning (CPEC) pieces and linear deletion cassettes were amplified by Primestar™ GXL according to the manufacturer's instructions. Colony PCR was performed in E. coli and S. bombicola according to (De Graeve et al., 2019). S. bombicola colony PCR was performed to analyze the 5', 3', and complete overlap of the integrated genomic deletion cassettes. CPEC was performed with Q5™ Hifi DNA polymerase according to the manufacturer's instructions and as described in (Quan and Tian, ​​2009). CPEC assembly products and linear deletion cassettes were transformed into E. coli and S. bombicola, respectively, by electroporation according to (De Graeve et al., 2019). Plasmids assembled by CPEC were sequenced by Macrogen inc.

[0136] Linear deletion cassettes were generated from vector backbones that were cloned and maintained in E. coli, and the basic and cloning steps are described below. Two deletion cassettes were constructed for subsequent gene deletion in S. bombicola (Figures 14 and 15, Table 7). Genetic elements were derived from the S. bombicola genome, except for the Herpes Simplex Virus tyrosine kinase (tTK) terminator terminator, which was used as described by (Van Bogaert et al., 2008). To construct the deletion cassettes, fragments were first amplified and assembled in pGEM-T (Promega) and pJET (Thermo Fisher) vectors using circular polymerase extension cloning (CPEC) plasmid assembly (Quan and Tian, ​​2009). These plasmids were transformed into top 10 cells of E. coli, and positive colonies were selected from LB-amp and verified by colony PCR and subsequent DNA sequencing. The primers used for amplifying the fragments, the origin of fragment amplification, and the primers used for E. coli colony PCR are listed in Table 8.

[0137] [Table 7]

[0138] [Table 8]

[0139] result Evaluation of At2 and At3 enzyme activities in S. bombicola Biosurfactant production experiments described under Materials and Methods using S. bombicola strains described in the art: Δat1 Δsble strain (Non-Patent Document 9 and Patent Document 2) Δat1 (Non-Patent Document 4) led to an unexpected observation that contradicts the art, namely that in samples from experiments with Δat1 and Δat1 Δsble strains, acetylated (bora-type) sophorolipids with an acetylation degree of up to 2 (mainly with an acetylation degree of 1) were surprisingly detected, thus in contrast to previous observations and reports. This observation was in contradiction to the art and unexpected, since it has been described that Δat1 Δsble strains and Δat1 do not produce any acetylated (bora-type) SLs due to a mutation in the at1 gene present in the SL biosynthetic gene cluster, and since the corresponding At1 enzyme (Genbank accession number HQ670751) has been described as the (only) enzyme responsible for the acetylation of sophorolipids. As already mentioned in Example 1, this points to the unexpected activity of other unknown acetyltransferases active on glycolipids in S. bombicola. A BlastP analysis was performed using the acetyltransferase protein sequences of the SL biosynthetic gene cluster (SEQ ID NO: 3 and SEQ ID NO: 4) against all translated ORFs from the S. bombicola genome, resulting in a large number of 71 hits. Two were selected for further investigation and the corresponding protein and gene sequences are shown in SEQ ID NO: 5 to SEQ ID NO: 8. When performing DELTA-BLAST in NCBI, these proteins show the greatest homology with maltose- and galactoside O-acetyltransferases, mostly of bacterial origin.

[0140] Construction and evaluation of novel S. bombicola strains The deletion cassettes described under Materials and Methods and shown in Figures 14 and 15 were amplified from the corresponding plasmids and used for transformation into S. bombicola strains. After successful validation, the deletion cassettes were used to generate the strains listed in Table 9.

[0141] [Table 9]

[0142] The production characteristics of the newly developed strains were evaluated in shake flask (SF) experiments together with S. bombicola wild type (WT) strain. The production samples obtained 180 hours after inoculation were subjected to UHPLC-HRMS analysis. Table 10 lists all detected m / z values, corresponding retention times and SL congeners with monoisotopic mass match.

[0143] [Table 10]

[0144] Wild-type S. bombicola produces mainly C18:1 diacetylated (diAc) lactone-type SL (L SL), as expected. The spectrum of products from the Δat1 strain is mainly composed of nonacetylated (nAC) C18:1 bola-type SL (bola-type SL), monoacetylated (mAc) C18:1 bola-type SL, nAc C18:1 triglucolipid, nAc C16:0 acidic SL, nAc C18:1 acidic SL, nAc C18:0 acidic SL, nAc C18:1 glucolipid, nAc C18:1 L SL, and m / z values ​​corresponding to the monoisotopic mass of the monoacetylated C18:1 lactone-type SL. The SL production spectrum of the Δat1Δsble deletion strain was found to be composed mainly of m / z values ​​corresponding to the monoisotopic masses of nAc C16:1 bola-type SL, nAc C16:0 bola-type SL, nAc C18:1 bola-type SL, mAc C18:1 bola-type SL, nAc C18:0 bola-type SL, nAc C18:1 acidic SL, nAc acidic C18:0 SL, and nAc C18:1 glucolipid. These findings are in contrast to what has been described in the art (Non-Patent Document 9, Non-Patent Document 10, Patent Document 1 and Patent Document 3), where deletion of the at1 gene was described as necessary to produce bola-type sophorolipids, and therefore both sophorolipids could only be produced as completely non-acetylated molecules. The At1 enzyme was further described as the only enzyme that acetylates (bora-type) glycolipids in S. bombicola (Non-Patent Document 4, Non-Patent Document 9), and therefore the bora-type sophorolipids described in the art did not contain any acetyl groups.

[0145] The SL production spectrum of the Δat1Δat2Δat3 strain is mainly composed of nAc C18:1 bola-type SL and nAc C18:1 lactone-type SL, but also nAc C18:1 acidic SL. Upon deletion of the sble gene in this last strain, the Δat1Δat2Δat3Δsble strain was obtained as described in Materials and Methods, and it was found that this strain mainly produces non-acetylated bola-type sophorolipids such as nAc C16:1 bola-type SL, nAc C16:0 bola-type SL, nAc C18:1 bola-type SL, nAc C18:0 bola-type SL, nAc C18:1 acidic SL, and nAc C18:1 glucolipid, but no longer produces acetylated SL / GL or other acetylated (bola-type) amphipathic glycolipids. In these analyses, the clear appearance of nAc C18:1 acidic SL for Δat1Δat2Δat3 compared to trace amounts in strain Δat1Δat2Δat3Δsble is consistent with the in vitro data described above. Sble prefers to carry out transesterification reactions with acetylated bolaamphiphiles, but hydrolysis reactions are present along with transesterification reactions with nonacetylated bolaamphiphiles.

[0146] This was unexpected, since the acetyltransferase gene present in the SL biosynthetic gene cluster (at1) was assumed to be solely responsible for the acetylation of glycolipids in S. bombicola. We have discovered and shown herein that other previously unknown genes / enzymes (at2 / At2 and at3 / At3) present in the S. bombicola genome also have this acetylation activity in (bora-type) sophorolipids and glucolipids, to a lesser extent and with different specificity. The fully non-acetylated glycolipid products are interesting due to the altered nature of the corresponding fully non-acetylated glycolipid compounds, but also have a clear advantage compared to acetylated glycolipids, namely the natural "release" of acetic acid in the aqueous environment upon the natural hydrolysis of the acetyl group. Although this results in an unpleasant odor, this is not the case for glycolipids derived from strains containing the Δat1Δat2Δat3 combination.

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J.; Saey, L. and Soetaert, W. (2011b). One-Step Production of Unacetylated Sophorolipids by an Acetyltransferase Negative Candida bombicola. Biotechnology and Bioengineering, 108 (12): 2923-2931. Saerens, K. M. J.; Zhang, J.; Van Bogaert, I. and Soetaert, W. (2011c). Cloning and functional characterization of the UDP-glucosyltransferase UgtB1 involved in sophorolipid production by Candida bombicola and creation of a glucolipid-producing yeast strain. Yeast, 28: 279-292. Saerens, K.M.J; Van Bogaert, I.N.A.; Soetaert, W. (2015). Characterization of sophorolipid biosynthetic enzymes from Starmerella bombicola. FEMS Yeast Research, 15 (7):. Spencer, J. F. T., Gorin, P. A. J., & Tulloch, A. P. (1970). Torulopsis bombicola sp. n. Antonie van Leeuwenhoek, 36(1), 129-133. https: / / doi.org / 10.1007 / BF02069014 Tulloch, A. P., Spencer, J. F. T., & Deinema, M. H. (1968). A new hydroxy fatty acid sophoroside from Candida bogoriensis. 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Claims

1. Use of a modified yeast strain that contains a non-functional or dysfunctional form of the transesterification enzyme Sble for producing vola-type amphiphilic glycolipids, and / or does not contain the functional sble gene, and / or exhibits reduced sble expression compared to unmodified yeast.

2. The modified yeast strain according to claim 1 is used, wherein the bora-type amphiphilic glycolipid is an acetylated bora-type amphiphilic glycolipid.

3. The use of the modified yeast strain according to claim 1 or 2, wherein the bora-type amphiphilic glycolipid is a bora-type sophorolipid.

4. The use of the modified yeast strain according to claim 3, wherein the bora-type sophorolipid has a degree of acetylation of 4.

5. Use of the modified yeast strain according to Claim 1, wherein the modified yeast strain further comprises a non-functional or dysfunctional glucosyltransferase enzyme UgtB1 and / or does not contain a functional ugtB1 gene and / or exhibits reduced expression of ugtB1 compared to an unmodified yeast, wherein the vola-type amphiphilic glycolipid is a vola-type glycolipid.

6. Use of the modified yeast strain according to Claim 1, wherein the modified yeast strain further comprises a non-functional or dysfunctional acetyltransferase enzyme (At1) and / or does not contain a functional at1 gene derived from the SL biosynthesis cluster and / or has reduced at1 expression compared to an unmodified yeast, wherein the vola-type amphiphilic glycolipid has a degree of acetylation of 0, 1, or 2.

7. Use of the modified yeast strain according to claim 6, wherein the modified yeast strain further comprises a second (At2) or third (At3) non-functional or dysfunctional glycolipid acetyltransferase and / or does not contain a functional at2 or at3 gene and / or has reduced at2 or at3 expression compared to an unmodified yeast, wherein the acetylated vola-type amphiphilic glycolipid has a degree of acetylation of 0, 1, or 2.

8. Use of the modified yeast strain according to claim 6, wherein the modified yeast strain further comprises a second (At2) and a third (At3) non-functional or dysfunctional glycolipid acetyltransferase for producing a non-acetylated boran amphiphilic glycolipid, and / or does not contain functional at2 and at3 genes, and / or has reduced expression of at2 and at3 compared to an unmodified yeast, wherein the boran amphiphilic glycolipid is a non-acetylated boran sophorolipid and / or a non-acetylated boran glucolipid.

9. A composition for producing vola-type amphiphilic glycolipids, comprising a modified yeast strain that contains a non-functional or dysfunctional type of esterification enzyme Sble and / or does not contain a functional sble gene and / or has reduced sble expression compared to an unmodified yeast strain.

10. An isolated acetyltransferase having the amino acid sequence given by SEQ ID NO: 6 or SEQ ID NO:

8.

11. Use of modified yeast strains for producing non-acetylated glycolipids, which contain non-functional or dysfunctional At1, At2, and At3 enzymes and / or do not contain the At1, At2, and At3 genes and / or have completely dysfunctional or removed genes encoding the At1, At2, and At3 enzymes.

12. A composition for producing non-acetylated glycolipids, comprising a modified yeast strain that contains non-functional or dysfunctional At1, At2, and At3 enzymes, and / or does not contain the at1, at2, and at3 genes, and / or the genes encoding the At1, At2, and At3 enzymes are completely dysfunctional or removed.

13. Use of the modified yeast strain according to claim 1 or 11, wherein the modified yeast strain contains non-functional or dysfunctional Sble, UgtB1, At1, At2, and / or At3 enzymes, and / or does not contain sble, ugtB1, at1, at2, and / or at3 genes.

14. The modified yeast strains mentioned above are: Stalmellera (Candida) bombicola, Stalmellera (Candida) apicola, Stalmellera (Candida) batistae, Stalmellera (Candida) magnolia, Candida globengieseri, Stalmellera (Candida) floricola, Candida tropicalis, Candida iodsensis, Stalmellera (Candida) stellata, Stalmellera (Candida) cuoi, Candida tropicalis, and Candida species NRRL. Use of a modified yeast strain according to claim 1 or 11, which is a yeast strain selected from strains selected from Y-27208, Pseudohyphozima (Rhodotorula, Candida) bogoriensis, Wickelhamieradmelichiae, Candida antarctica, Pseudohyphozima antarctica, Pseudohyphozima bogoriensis, Candida lipopolitica, and sophorolipid-producing strains of the Stalmellera clade.

15. Use of Sble enzyme for transesterification and / or hydrolysis reactions.

16. A composition comprising Sble enzyme, used for carrying out transesterification and / or hydrolysis reactions.

17. Use of the Sble enzyme according to claim 15 for carrying out transesterification and / or hydrolysis reactions in boran-type amphiphilic glycolipids.

18. Use of the Sble enzyme according to claim 17 for converting the bora-type amphiphilic glycolipid to a lactone-type glycolipid, wherein the bora-type amphiphilic glycolipid is a bora-type sophorolipid and the lactone-type glycolipid is a lactone-type sophorolipid, or the bora-type amphiphilic glycolipid is a bora-type glucolipid and the lactone-type glycolipid is a lactone-type glucolipid, and non-acetylated and / or acetylated glucose and / or sophorose are released.

19. Use of the Sble enzyme according to claim 17 for converting the bora-type amphiphilic glycolipid to an acidic glycolipid, wherein the bora-type amphiphilic glycolipid is a bora-type sophorolipid and the acidic glycolipid is an acidic sophorolipid, or the bora-type amphiphilic glycolipid is a bora-type glucolipid and the acidic glycolipid is an acidic glucolipid, and non-acetylated and / or acetylated glucose and / or sophorose is released.

20. The bora-type sophorolipid is a tetraacetylated bora-type sophorolipid, and the lactone-type sophorolipid is a diacetylated lactone-type sophorolipid, and non-acetylated and / or acetylated glucose and / or sophorose are released; or The bora-type sophorolipid is a non-acetylated, monoacetylated, diacetylated, and / or triacetylated bora-type sophorolipid, and the lactone-type sophorolipid is a non-acetylated, monoacetylated, and / or diacetylated lactone-type sophorolipid, and non-acetylated and / or acetylated glucose and / or sophorose are released. Use of the Sble enzyme according to claim 18.

21. The bora-type sophorolipid is a tetraacetylated bora-type sophorolipid, and the acidic sophorolipid is a diacetylated acidic sophorolipid, and non-acetylated / or acetylated glucose and / or sophorose are released; or The bora-type sophorolipid is a non-acetylated, monoacetylated, diacetylated, and / or triacetylated bora-type sophorolipid, and the acidic sophorolipid is a non-acetylated, monoacetylated, and / or diacetylated acidic sophorolipid, and non-acetylated and / or acetylated glucose and / or sophorose are released. Use of the Sble enzyme as described in claim 19.

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