# Patent

## Title
Method for the production of medium-chain sophorolipids

## Abstract

The MFE2 gene of a microorganism capable of producing glycolipids, such as, but not limited to C. bombicola is disrupted with the purpose of blocking the beta-oxidation pathway in the strain. Fermentation of a such strain having such disrupted genes on primary or secondary alcohols or diols, preferably on primary alcohols produces short chained glycolipids in high yield and purity.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a §371 national stage entry of International Application No. PCT/EP2009/056190, filed May 20, 2009, which claims priority to European Patent Application 08156696.0, filed May 21, 2008, the entire contents of which are incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to a method for producing glycolipids, in particular sophorolipids with medium length hydrocarbon chains.
The present invention also relates to a process for the site-specific modification of the genome of a host strain of a glycolipids producing micro-organism with the purpose of optimising the strain for the production of medium length hydrocarbon chain glycolipids, preferably sophorolipids.
BACKGROUND OF THE INVENTION
The use of glycolipid biosurfactants in detergents is well known in the art. Glycolipid biosurfactants include rhamnolipids, sophorolipids, cellobioselipids, trehaloselipids, mannosyl erythritol lipids and (bio)chemical modifications thereof. Glycolipid based biosurfactants are understood to comprise those surfactants that have been obtained through microbial cultivation and consist of carbohydrates bound to aliphatic acids or aliphatic hydroxyl acids through glycosidation or acylation. They offer the advantage of being naturally produced molecules that can be produced through microbial cultivation by feeding renewable raw materials and of being fully degradable after use.
Sophorolipids are one of the most promising glycolipids known, one reason being their high production yield and ease of recovery from the microbial cultivation. Several Candida species, a.o. Candida (Starmerela) bombicola (formerly Torulopsis bombicola), Candida apicola (formerly Torulopsis magnoliae and Torulopsis apicola) and Rhodotorula bogoriensis, Wickerhamiella domericqiae are known to produce sophorolipids in large amounts from various substrates such as carbohydrates, vegetable oils, animal fats and n-alkanes. Sophorolipid production mainly takes place during the stationary phase because of nitrogen limitation. It appears to be enhanced when providing simultaneously hydrophilic (e.g. glucose) and hydrophobic (e.g. fatty acids) substrate to Candida species. Candida bombicola for example produces a complex mixture of 22 structurally different sophorolipids from either glucose and or an oily substrate, preferably C16 to C18 alkanes, fatty acids or their esters. The main compounds produced are the lactonic and the acidic form typically in the ratios of 75 and 25 w/w. A typical hydroxyl fatty acid distribution incorporated in non, mono- and diacetylated sophorolipids either in their free acid or lactonic form is for example disclosed in FR2779057:











TABLE 1







Type
Hydroxy fatty acid
Wt. %




















C16:0
15-OH hexadecanoic
1.5



C16:0
16-OH hexadecanoic
2



C18:0
17-OH octadecanoic
3.5



C18:1
17-OH octadecenoic
60



C18:1
18-OH octadecenoic
12



C18:2
17-OH octadecadienoic
7



C18:2
18-OH octadecadienoic
14










Sophorolipids find numerous applications for example in preventing and curing dandruff and body odor attributed to bacteriostatic properties, as disclosed by EP-A-1.082.097; as therapeutically active substances or cosmetic products, in particular skin treatment as disclosed by EP-A-835.118; and for their antifungal properties disclosed by US-A-2005/0164955. EP-A-820.273 discloses that sophorolipids are non-irritant to the skin, weakly irritant to the eyes only and that they show anti-inflammatory as well as elastase inhibiting activity. Other uses of sophorolipids include their use as the sole surfactant in washing and cleaning applications such as disclosed in U.S. Pat. No. 6,433,152. According to EP-A-1.445.302 mixtures of sophorolipids show synergistic activity in laundry and hard surface cleaning applications. EP-A-499.434 discloses to combine sophorolipids with lamellar, usually ethoxylated non-ionic surfactants in washing and cleaning applications. DE-A-19600743 discloses manual dishwashing formulations based on sophorolipids and other glycolipids combined with high foaming surfactants.
However, the physicochemical properties of sophorolipids form an important limitation to their applicability. In this respect it is mentioned that the lactonic form of the sophorolipids is hardly water soluble, it is insoluble in acidic environment, it is spontaneously deacetylated in alkaline pH and upon deacetylation the pH reduces to approximately 6. Fully de-acetylated acidic sophorolipids appear to lose a substantial part of their surface activity with respect to the di-acetylated lactonic form of crude sophorolipids. Shorter-chained sophorolipids are presumed to decrease surface tension more effectively than the known long chain C16-C22 sophorolipids. Therefore several attempts have been made to find an efficient process for their production. Whereas, Candida bombicola and its most commonly used strain (ATCC 22214) have been found capable of readily fermenting C16 and C18 chains and of incorporating them into the glycolipids, shorter hydrocarbon chain precursors C10-C14 were found to be hardly incorporated by the micro-organism: they were assimilated instead. C15-sophorolipids could only be obtained in unsatisfactory yields of below 40% by Jones and Howe (1968), by offering a wide variety of expensive C15 substrates to Torulopsis gropengiesseri. Pentadecanoic acid proved to be too short for appreciable hydroxylation, which is an essential step in sophorolipid production by the micro-organism, and 12-hydroxyoctadecanoic acid did not react because the hydroxyl group is too close to the reaction site.
Traces of a C14 component could only be detected when a glucose/tetradecane mixture was used as hydrocarbon source. According to the teaching of U.S. Pat. No. 6,433,152 short chain 3-alkanols, 4-alkanols and 2, 3 and 4-alkanols could be converted into short chain sophorolipids with the envisaged chain length, provided the conversion was carried out under reduced oxygen concentrations and with an exceptionally high glucose feed. However, the yield was only between 3-17 g/l, of which only 41-85% had the envisaged short chain length. Although U.S. Pat. No. 6,433,152 claims incorporation of the easily obtainable and cost-effective 1-alcohols, no examples are given or yields specified. It therefore seems likely that 1-alcohols do not form a useful source for the production of short-chain sophorolipids, as these are first converted into the corresponding acids before they are transformed into glycolipids rather than being glycosidically bound at the hydroxyl group (Jones et al, 1968). According to the disclosure of DE19518982.5 Candida species are capable of converting secondary alcohols or ketones to non-cyclic sophorose lipids with surface active properties. The yields amount to approximately 20 g/l of C12-C14 sophorolipids, 75-85% of which has the desired chain length. 7% was converted into a fatty diol. However, the low yield, combined with the impurity of the end product which is a mixture containing 10% of standard C18 sophorolipids and the fact that secondary alcohols are expensive, render this process unattractive.
In most Candida species, substrates having a hydrocarbon chain of more than 18 carbon atoms, are first reduced in chain length by one or more units of two carbon atoms and only thereafter hydroxylated, thus yielding C16 or C18 hydroxy fatty acids with the OH group on the penultimate carbon atom (Tulloch, Spencer and Gorin 1962).
Van Bogaert et al (2007) describe the cloning, characterisation and functionality of the orotidine-5′-phosphate decarboxylase gene (URA3) in Candida bombicola. 



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Sophorolipids from wild type Candida grown on rapeseed oil (wild type Sophorolipids at 29.9 min).
FIG. 2: Sophorolipids from wild type Candida grown on 1-dodecanol (medium chain Sophorolipids at 30.2 min, no wild type Sophorolipids at 29.9 min).
FIG. 3: Sophorolipids from Candida mutant strain M18 grown on 1-dodecanol (medium chain Sophorolipids at 30.2 min, wild type Sophorolipids at 29.9 min).
FIG. 4: Sophorolipids from Candida mutant strain M33 grown on 1-dodecanol (medium chain Sophorolipids at 30.2 min, wild type Sophorolipids at 29.9 min).
FIG. 5: Creation of the MFE-2 knock-out cassette with the URA3 selection marker for use in C. Bombicola. 



DESCRIPTION OF THE INVENTION
There is thus a need to a process with which shorter-chained glycolipids, in particular sophorolipids may be produced at higher yields and selectivity than could be achieved up to now, as those are presumed to decrease surface tension more effectively than the known long chain C16-C22 glycolipids.
In particular, there is a need for a process with which medium-chain glycolipids or an acetylated derivative thereof, can be produced at an acceptable yield, wherein the process shows a sufficient selectivity towards the desired glycolipid. More particularly, there is a need for a process with which glycolipids or an acetylated derivative thereof, more in particular glycolipids with a hydrocarbon chain length of C14 or less, preferably a hydrocarbon chain length of between 8-14 carbon atoms, can be produced at an acceptable yield, and a sufficient selectivity towards the desired glycolipid, in particular sophorolipid.
It is therefore the object of the present invention to provide a process for producing medium-chain glycolipids, in particular glycolipids with a hydrocarbon chain length of 14 or less, more particularly with a hydrocarbon chain length of between 8 and 14 carbon atoms, at an acceptable yield, and a selectivity towards the desired glycolipid which is sufficiently high.
It is also an object of the present invention to provide a micro-organism which is capable of producing such glycolipids at an acceptable yield and selectivity, and a process for producing such a micro-organism.
This object is achieved according to the present invention when with the technical features of the characterizing portion of the first claim.
Thereto, the present invention provides a process for the site-specific modification of the genome of a host strain of a glycolipids producing micro-organism, which process comprises the steps of:

    
    
        (1) transforming the host by disrupting through homologous recombination, at least one target gene encoding for at least one enzyme in the β-oxidation pathway
        (2) selecting the thus produced transformants by the ability of the transformants to grow in a selective medium.
    
    


Preferably the process of the present invention for the site-specific modification of the genome of a host strain of a glycolipids producing micro-organism, comprises the step of transforming the host by disrupting through homologous recombination at least one target gene encoding for enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase in the β-oxidation pathway.
More preferably the process of this invention is characterised in that the process comprises the step of transforming the host by disrupting at least the MFE2 gene.
A further preferred embodiment of the process for the site-specific modification of the genome of a glycolipids producing micro-organism the present invention is characterised in that it comprises the steps of:

    
    
        (1) transforming the host by disrupting through homologous recombination a target gene selected from the group consisting of the MFE2 and combinations thereof with a DNA fragment comprised of a selectable marker gene;
        (2) selecting the thus produced transformants by the ability of the transformants to grow in a selective medium.
    
    


Preferably the marker gene is selected from the group of an auxotrophic marker, antibiotic marker.
Preferably, the selectable marker gene is flanked on both ends by DNA sequences having homology to the target gene or having homology to DNA sequences flanking a chromosomal target gene. One aspect of the present invention relates to a process for the site-specific modification of the C. bombicola genome, comprising transforming a C. bombicola host cell with a linear DNA fragment comprised of a selectable marker gene, wherein said selectable marker gene is flanked on both ends by DNA sequences having homology to a chromosomal target gene or having homology to DNA sequences flanking a chromosomal target gene.
Preferably the DNA fragment is a selectable marker gene flanked on both ends by a MFE2 sequence of at least 100 base pairs, preferably at least 500 base pairs, more preferably at least 1000 base pairs. The inventors have namely found that the amount of transformants that may be obtained, significantly increases when using a fragment which on both ends is flanked with at least 1000 base pairs.
According to the teaching of Brachmann et al, 1998 knocking out genes in S. cerevisiae is relatively simple. Since S. cerevisiae laboratory strains are efficient in homologues recombination, events of double cross-over or gene replacement occur frequently enough to yield the right transformants. However, this strategy failed for C. Bombicola and did not yield proper transformants. The inventors have now found that a sufficient amount of transformants could only be obtained provided the selectable marker gene is flanked on both ends by a MFE2 sequence of at least 100 base pairs.
Preferably the selectable marker gene is the URA3A gene.
Preferred host micro-organisms include Candida (Starmerela) bombicola (formerly Torulopsis bombicola), Candida apicola (formerly Torulopsis magnoliae and Torulopsis apicola) and Rhodotorula bogoriensis, Wickerhamiella domericqiae, the more preferred host micro-organism being Candida Bombicola ATCC22214.
When analysing sophorolipid production by existing C. bombicola cultures, the inventors have observed that when C. bombicola is cultivated with a less favourable substrate for sophorolipid production such as shorter primary alcohols, part of the substrate is not converted into sophorolipids but is metabolized in the β-oxidation pathway instead. The analysis by the inventors also learned a feed of fatty acids is converted to acyl-CoA by acyl-CoA-synthetase before it enters the actual β-oxidation pathway, and is then transported into the peroxisomes. The analysis by the inventors further learned that the β-oxidation is a cyclic pathway, which comprises four reactions. After every cycle an acetyl-CoA molecule and an acyl-CoA lacking two carbon atoms are obtained, this until in the final cycle two acetyl-CoA molecules are released.
The first reaction of the β-oxidation pathway is performed by acyl-CoA dehydrogenase. In some yeast species, multiple acyl-CoA dehydrogenase genes or PDX genes occur, which are all functionally translated into isozymes. C. maltosa for example has two such genes (and C. tropicalis three. In the genome of Yarrowia lipolytica, even five PDX genes were found some of them show activity against short-chain fatty acids (with a chain length of less than 6 carbon atoms), others against long-chain fatty acids (with a chain length of more than 18 carbon atoms) and some are active against molecules of all chain lengths. According to the teaching of U.S. Pat. No. 5,254,466, in order to completely shut down the β-oxidation pathway, a quadruple mutant has to be created. From this the inventors conclude that the number of PDX genes is variable, but that there is a bigger chance to the occurrence of isozymes for yeasts that readily metabolize alkanes or fatty acids. C. bombicola belongs to this latter group, and therefore it can be expected that in this species multiple PDX genes are active as well. From this, the inventors conclude that creation of a mutant blocked in its β-oxidation by knocking out PDX genes is not very convenient, since all PDX genes must be isolated and possibly multiple deletions must be made.
The inventors have further observed that the second step of the β-oxidation pathway which involves a hydration step and the third step of the β-oxidation pathway which involves a second dehydrogenation, are performed by one and the same enzyme in eukaryotic organisms: the so called multifunctional enzyme. Contrary to mammals, yeasts only possess MFE-2 and until now there is no evidence for the presence of isozymes in any yeast or fungal species. Therefore, the MFE-2 from C. bombicola is considered as an attractive target for gene disruption. It is expected that disabling of the gene would permit simultaneous inhibition of two biochemical reactions.
The last reaction of the β-oxidation cycle is the thiolytical cleavage. As in the genome of C. tropicalis four isozymes with different activities and specificities were found, only with double mutants β-oxidation could be inhibited (Kanayama et al, 1998). As it is likely that also in C. bombicola several isozymes are present, targeting of the thiolytical cleavage for blocking the β-oxidation pathway, seems less favourable.
Based on this analysis, the inventors came to the conclusion that an optimum improvement of the glycolipid production based on specialty substrates could be achieved by eliminating the competing β-oxidation pathway either by chemical or by genetic inhibition.
Experiments with chemical inhibitors of the β-oxidation added to the medium (e.g. bromobutyric acid) did not result in better use of the substrate. Either the chemicals were not active against the yeast pathway or could not perform properly due to the long fermentation period at low pH.
The present invention also relates to a process for restoring auxotrophy, preferably uracil auxotrophy to cells of a strain of glycolipid producing micro-organisms previously transformed to prototrophy with a selectable marker, preferably a URA 3 selectable marker, comprising the steps of:
(a) selecting or screening for spontaneous mutations which inactivate the selectable marker and isolate auxotrophic mutants derived from said previously transformed strain,
(b) confirming the selectable marker auxotrophy of the mutants,
(c) confirming the parental genotype of the auxotrophs by Southern hybridization to appropriate gene probes.
Preferably the process for restoring auxotrophy, preferably uracil auxotrophy to cells of a strain of glycolipid producing micro-organisms previously transformed to prototrophy with a selectable marker, preferably a URA 3 selectable marker, comprises the steps of:
(a) transforming prototrophic host cells with a non-functional selectable marker gene which has been made non-functional by an in-vitro deletion of a portion of the central coding sequence of the gene to produce auxotrophic mutants,
(b) confirming the selectable marker auxotrophy of the mutants,
(c) confirming the parental geneotype of the auxotrophs.
Yet another aspect of the present invention relates to a process for blocking the beta-oxidation pathway in a glycolipid producing host strain.
This process comprises the steps of disrupting in the host through homologous recombination at least one gene encoding for enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase. A preferred embodiment of this process comprises the step of disrupting through homologous recombination at least the MFE2 gene or at least one combination thereof with a linear DNA fragment comprised of a selectable marker gene. Herein, the selectable marker gene is preferably flanked on both ends by at least one DNA sequence having homology to the target genes of the host strain, preferably a C. bombicola host strain. Preferably, the selectable marker gene is a URA3A marker gene.
A further aspect of the invention relates to a cell of a glycolipid producing micro-organism strain, having a chromosomal gene which has been disrupted through homologous recombination with a linear DNA fragment comprised of a selectable marker gene which is flanked on both ends by DNA sequences having homology to the chromosomal gene of the corresponding host strain. Herein, preferably the chromosomal gene is a chromosomal MFE2 gene. Herein preferably the selectable marker gene is a URA3A marker gene. The preferred micro-organism is Candida bombicola. 
The invention further relates to a method for producing glycolipids, using the mutated micro-organism obtained with the process as described above or using the micro-organism as described above.
The process for producing glycolipids of this invention comprises of the following steps:

    
    
        1. culturing a mutant strain of a microorganism producing glycolipids obtained by mutagenesis as described above, in a medium consisting essentially of an energetic substrate including at least one carbon source and
        2. subjecting said strain to a bioconversion substrate, which contains at least one primary or secondary alcohol or diol, at least one fatty acid or fatty acid ester, in which the hydrocarbon chain which determines the chain length contains about 4 to about 24 carbon atoms or a mixture of two or more of those.
    
    


A preferred embodiment of this invention relates to a method for producing sophorolipids with medium length hydrocarbon chains, i.e. hydrocarbons having 6-14 carbon atoms in the length determining chain.
Within the scope of this invention glycolipid is understood to comprise those surfactants that have been obtained through microbial cultivation and consist of carbohydrates bound to aliphatic acids or aliphatic hydroxyl acids through glycosidation or acylation. The glycolipid, may be any glycolipid known to the person skilled in the art, in particular it may be a rhamnolipid, sophorolipid, cellobioselipid, trehaloselipid, mannosyl erythritol lipid and (bio)chemical modifications thereof. The person skilled in the art will be capable of selecting the appropriate micro-organism taking into account the nature of the envisaged glycolipid.
Preferably, the glycolipid is a sophorolipid corresponding to the formula:




    
    
        wherein
        R3=—(CH2)mH
        R4=—(CH2)pH
        In which (m+n+p)≧2 and ≦22 and m, n, and p are independent of one another and ≧0 and ≦22,
        R7=—COON or —C(R4)(R5)H
        R5=—H, —OH or R6
    
    






    
    
        R1 and R2 are independent of one another and —H or —C(O)CH3.
    
    


The process of this invention for producing glycolipids, in particular sophorolipids described above preferably comprises culturing C. bombicola mutant strain M18, M30 or M33 obtained according to claims 1-10 in a culture medium containing a nitrogen source, an organic substrate and a co-substrate, the M30 strain being most preferred.
The pH of the culture medium after maximum cell density is reached is preferably maintained between 2 and 7, preferably between 3 and 5, more preferably about 3.5.
It is preferred to add the substrate continuously to the culture medium, more preferably at a rate of between 0.1 and 1 gram per litre per hour. The residual concentration of the substrate in the culture medium is preferably kept below 10 g/l, preferably 8 g/l, more preferably 2 g/l.
As a co-substrate preferably use is made of glucose at an initial concentration of at least 100 gram glucose per litre, preferably at least 120 g/l, and in that an excess glucose concentration is maintained above an excess of about 20 g/l. Thereto, glucose may be added in a discontinuous or continuous manner. The oxygen partial pressure of the culture solution is preferably kept ≦40% of the saturation value, preferably ≦15% of the saturation value. The culture medium may contain a nitrogen source, however in that case the concentration corresponds to 0.5-5 g/l of ammonium chloride, preferably about 1.5 g/l ammonium chloride.
Optimum yield is achieved with a culture medium containing between 0.5 and 25 g/l of yeast extract, preferably between 1 and 15, more preferably between 1 and 10, most preferably about 4 g/l yeast extract.
Within the scope of the present invention, all substrates considered suitable by the person skilled in the art may be used. Suitable examples include compounds responding to the formula

R10—R1—X—Y—Z—R2—R20  (formula I)

which contains at least one cleavable bond,

wherein

    
    
        —Y is —O—, —S—, —NH—, a mono or di unsaturated bond
        X and Z may be the same or different and are chosen from the group of a —CO— group or a —CHn(CH2)m— group which may be straight or branched, with m having a value between 0 and 4 and n between 0 and 2
        R1 and R2 each are aliphatic hydrocarbon chains which may be the same or different chain, which may be branched or unbranched, may contain one of more unsaturated bonds and may contain one or more substituents
        —R10 and R20 may be the same or different and are chosen from the group of —H, —CH3, —CHO, —CH2OH, —COOH, —CHS, —CH2SH, —CN or —CH2NR3p, R3 being an aliphatic hydrocarbon chain or H, p being 1 or 2

or a salt or a methyl, ethyl or glycerol ester thereof.

    
    


The preferred substrate however is a primary or secondary alcohol or diol having from about 4 to about 24 carbon atoms, preferably from about 8 to about 14 carbon atoms, primary alcohols being particularly preferred. Preferred primary alcohols include 1-dodecanol or 1-tetradecanol.
For the production of medium chained sophorolipids at high yield and selectivity, preferably use is made of a C. bombicola strain M30 which has been cultured in a culture medium containing a nitrogen source, an organic substrate and a co-substrate, the substrate being selected from primary or secondary alcohols or diols, preferably a primary alcohol.
The invention is further elucidated in the example given below.
Example 1
Strains, Plasmids and Culture Conditions—DNA Isolation and Sequencing
C. bombicola ATCC 22214 was used for the preparation of genomic DNA. Escherichia coli DH5α was used in all cloning experiments. C. bombicola was maintained on agar plates containing 10% glucose, 1% yeast extract and 0.1% urea. Yeast strains presumed to lack β-oxidation activity were tested for growth on Yeast Nitrogen base with Oleic acid (YNO) plates (0.67% yeast nitrogen base without amino acids, 0.02% Tween 40 and 0.1% oleic acid). Sophorolipid synthesis of yeast strains with a blocked β-oxidation activity was tested on medium as described by Lang et al. (2000) with either 1-dodecanol (Sigma) used as hydrophobic carbon source in a concentration of 20 or 5 g/L or rapeseed oil (Sigma) used at 37.5 g/L. The hydrophobic carbon source was added two days after inoculation and the cultures were incubated for another eight days, unless specified otherwise.
Liquid yeast cultures were incubated at 30° C. and 200 rpm. E. coli was grown in Luria Bertani (LB) medium (1% trypton, 0.5% yeast extract and 0.5% sodium chloride) supplemented with 100 mg/l ampicillin and 40 mg/l X-gal if necessary. Liquid E. coli cultures were incubated at 37° C. and 200 rpm.
All PCR products were cloned into the pGEM-T® vector (Promega).
Yeast genomic DNA was isolated with the GenEluteTM Bacterial Genomic DNA Kit (Sigma). Cell lysis was performed by incubation at 30° C. during 90 minutes with zymolyase (Sigma). Plasmid DNA was isolated with the QIAprep Spin Miniprep Kit (Qiagen). All DNA sequences were determined at the VIB Genetic Service Facility (Belgium).
Example 2
Transformation
C. bombicola cells were transformed with the lithium acetate method (Gietz and Schiestl, 1995), but 50 mM LiAc was used instead of 100. Transformants were selected on synthetic dextrose (SD) plates (0.67% yeast nitrogen base without amino acids (DIFCO) and 2% glucose). E. coli cells were transformed as described by Sambrook and Russell (2001).
MFE-2 Sequence.
The nucleotide sequence of the MFE-2 gene of C. bombicola has been deposited at the GenBank nucleotide database under the accession number EU315245 and is given in enclosure 1.










TABLE 2





SEQ 





ID NO
Name
Feature
Sequence







SEQ ID
MFE2_3545For
cloning
5′-CTCCTAAGCATTTAACTGCCTTGAG-3′


NO 4

MFE2



 


SEQ ID
MFE2_3545Rev
cloning
5′-AACCGAGATACGCCTAATCAGTC-3′


NO 5

MFE2



 


SEQ ID
URA3infMFE2For
ligating
5′-TGCGTTGCCCCTACACTGACGGGCGGATAGTACA-3′


NO 6

Ura3 into





MFE2



 


SEQ ID
URA3infMFE2Rev
ligating
5′-TGGTCTTCGCCCTCACATCATCGTCACTATACAC-3′


NO 7

Ura3 into





MFE2



 


SEQ ID
MFE2knock1000For
4095 bp
5′-GGCAACTTTGGCCAGGCCAATTA-3′


NO 8

knock-out





fragment



 


SEQ ID
MFE2knock1000Rev
4095 by
5′-GTTTAGATGTGGCTCAAGTA-3′


NO 9

knock-out





fragment



 


SEQ ID
MFE2knock500For
3070 by
5′-TACTGGTGCGGGAGGCGGAATTG-3′


NO 10

knock-out





fragment



 


SEQ ID
MFE2knock500Rev
3070 by
5′-GGTGGCTTGTTGGCTGCTGTGAT-3′


NO 11

knock-out





fragment









Creation of the Knock-Out Fragments

The 2655 bp coding fragment and 493 and 397 bp upstream and downstream of the MFE-2 gene were amplified with the primers MFE2—3545For and MFE2—3545Rev, yielding a fragment of 3545 bp which was cloned into the pGEM-T® vector (Promega). The created vector was digested with EcoNl and BbvCl, which both cut the coding sequence of MFE-2 once, thus deleting 309 bp of the MFE-2 sequence. The URA3 gene of C. bombicola was inserted by means of the In-FusionTM 2.0 Dry-Down PCR Cloning Kit (Clontech). The primers URA3infMFE2For and URA3infMFE2Rev were designed according to the guidelines of the manual and used for integration of the functional C. bombicola URA3 sequence (2055 bp) into MFE-2. The primerpair MFE2knock1000For and MFE2knock1000Rev were used for the amplification of a 4095 bp fragment containing the URA3 sequence with approximately 1000 bp of the MFE-2 sequence on each site. In the same way the primers MFE2knock500For and MFE2knock500Rev were used to create a construct of 3070 bp with 500 bp flanking regions. Finally, a larger fragment (5287 bp) with 1962 bp and 1272 bp flanking respectively the 5′ and 3′ site of the URA3 sequence was created with the MFE2—3545For and MFE2—3545Rev primerpair. The creation of the knock-out cassettes is illustrated in FIG. 5.
Cell Dry Weight.
Cell dry weight was measured by centrifugation of 2 mL culture broth for 5 min at 9 000 g. Pellets were washed two times with ethanol to remove sophorolipids and lipophilic substrate and finally dissolved in distilled water. The suspension was transferred to a cellulose nitrate filter with a pore diameter of 0.45 μm (Sartorius) and the dry weight was determined in the XM60 automatic oven from Precisa Instruments Ltd.
Sophorolipid Production.
Analytical sophorolipid samples were prepared as follows: 440 μL ethyl acetate and 11 μL acetic acid were added to 1 mL culture broth and shaken vigorously for 5 min. After centrifugation at 9 000 g for 5 min, the upper solvent layer was removed and put into a fresh eppendrof tube with 600 μL ethanol. Samples were analysed as described below.
Final Sophorolipid Extraction from Culture Broth.
This protocol is described in Fleurackers (2006).
HPLC-Analysis.
Sophorolipid samples were analysed by High Performance Liquid Chromatography (HPLC) on a Varian Prostar HPLC system using a Chromolith® Performance RP-18e 100-4.6 mm column from Merck KGaA at 30° C. and Evaporative Light Scattering Detection (ELSD, Alltech). A gradient of two eluents, a 0.5% acetic acid aqueous solution and acetonitrile, had to be used to separate the components. The gradient started at 5% acetonitrile and linearly increased till 95% in 40 min. The mixture was kept this way for 10 min and was then brought back to 5% acetonitrile in 5 min. A flow rate of 1 mL/min was applied.
According to the teaching of Brachmann et al, 1998, knocking out genes in S. cerevisiae is relatively simple: by means of PCR one can construct a linear fragment containing a marker flanked on each site by only 40 bp of the target gene and transform the yeast cells with this construct. Since S. cerevisiae laboratory strains are very efficient in homologues recombination, events of double cross-over or gene replacement occur frequently enough to yield the right transformants. This strategy was also tested for C. bombicola; the functional URA3 sequence was flanked with 60 bp of the C. bombicola MFE-2 gene and used to transform the ura3-auxotrophic G9 strain described by Van Bogaert et al, (2008). This did however not yield proper transformants. 60 bp homology is probably too short for efficient recombination with the C. bombicola genome. Therefore, a disruption vector was created as described in the Materials and Methods. It contains the 2055 bp of the URA3 coding sequence with promoter inserted into the MFE2 sequence. The vector was then used as a template in PCR reactions with the primers listed in Table V-1 to create a linear marker sequence flanked with MFE-2 sequences of a variable length. In this way fragments with about 500, 1000 or more bp of the MFE-2 gene on both ends were created with a total length of respectively 3070, 4095 and 5287 bp. 1.5 μg of each of the three fragments were used to transform C. bombicola. The fragment with 1000 bp flanking regions yielded about threefold more transformants compared to the other two.
It was found that when transforming the ura3-auxotrophic G9 C. bombicola strain with a functional URA3 gene, double cross-over with a consequent gene replacement takes place at a rate of 19%.
All other transformants arise owing to events of single cross over or illegitimate recombination. For this reason, the MFE2-negative candidates obtained were further tested. The correct phenotype was tested by plating the mutants on medium with oleic acid as the sole carbon source (YNO); mutants with a deficient β-oxidation will not succeed in growing on this medium. 9 transformants were not able to grow on this medium, while the growth on general YPD medium was good: M9, M15, M16, M18, M24, M27, M30, M32 and M33.
The genotype of the obtained mutants was confirmed by a yeast colony PCR targeting the MFE-2 sequence with the primers MFE2—3545For and MFE2—3545Rev (Table 2). Presence of the intact wild type MFE-2 gene should yield a fragment of 3545 bp, whereas for yeasts with the knocked-out MFE-2, this fragment must be either absent or appear as a larger band of about 5000 bp. All 9 strains selected by their non-growth on YNO-plates displayed the right genotype.
Evaluation of Sophorolipid Synthesis by the MFE-2 Negative Strains.
The inventor has observed that by knocking out the β-oxidation, the hydroxylated medium-chain substrates could be re-directed towards sophorolipid biosynthesis.
Thereto, use was made of 1-dodecanol as a substrate because of its good solubility at 30° C. which facilitates sophorolipid synthesis and sampling. The results of the fermentations run on 1-dodecanol are illustrated in HPLC chromatograms given in FIG. 1 for the wild type Candida bombicola ATCC 22214 grown on rapeseed oil (FIG. 1) and grown on 1-dodecanol (FIG. 2) and for two mutant strains M18 and M30 grown on 1-dodecanol (FIGS. 3 and 4). Wild type sophorolipids from rapeseedoil, which mainly contains monounsaturated C18 sophorolipids, elute at about 29.2 minutes (see FIG. 1) medium C12 chain sophorolipids at about 30.1-30.2 minutes. FIG. 2 shows that the wildtype Candida strain hardly produces any of these medium chain sophorolipids, whereas M18 and M30 yield a very pronounced C12 peak and virtually no C18 sophorolipids, so with highly selectively transforming the alcohol substrate to the envisaged medium chain sophorolipids. The medium chain sophorolipid yields are listed in Table 3 for all obtained mutant strains.







TABLE 3







Sophorolipid production on 1-dodecanol of mutant strains


relative to the wild type.











Relative amount
CDW
CDW corrected relative


Strains
of sophorolipids (%)
(g/L)
amount of sophorolipids (%)













Wild type
100.0
7.0
100.0


M9
285.7
9.0
222.2


M15
262.9
9.5
193.7


M16
237.1
9.0
184.4


M18
274.3
8.0
240.0


M24
217.1
7.5
202.7


M27
254.3
9.0
197.8


M30
308.6
7.5
288.0


M32
262.9
11.0
167.3


M33
278.0
8.5
228.9









A total mass of 9.5 g/L sophorolipids was synthesized by the wild type, while for all mutant strains clearly better results were obtained; the overall yield was 2.2 till 3.1 times higher.
Since biomass formation differs among the strains and is slightly lower for the wild type, this factor was taken into account for correct comparison of the obtained sophorolipid values. Still, sophorolipid production of the mutant strains stayed 1.7 to 2.9 times higher as compared to the wild type; it therefore can be concluded that strains with a blocked β-oxidation route perform better when synthesizing sophorolipids on 1-dodecanol.
To produce dodecyl sophoroside, 100 ml of a culture medium of the following composition was introduced into a 500 ml Erlenmeyer flask with baffles:
Glucose.H2O 150.00 g/l (U.S. Pat. No. 5,767,255 slechts 100 g/l)
Sodium citrate.3H2O 5.00 g/l
Yeast extract (granulated, Merck, Darmstadt) 4.00 g/l
Ammonium chloride 1.54 g/l
Potassium dihydrogen phosphate 1.00 g/l
Magnesium sulfate.7H2O 0.70 g/l
Sodium chloride 0.50 g/l
Calcium chloride.2H2O 0.27 g/l
Dipotassium hydrogen phosphate.3H2O 0.16 g/l
The medium was inoculated with the yeast Candida bombicola ATCC 22214 and incubated on a rotary shaker at 100 rpm at a temperature of 30° C. After a cultivation time of 48, 72 and 96 h, in each case 5 g/l 1-dodecanol were added under aseptic conditions to the culture solution. The culture was carried out under unchanged conditions between and after the additions of the alcohol. The pH of the culture suspension decreased over the entire range of cultivation. After a cultivation period of 10 days, the supplied amount of alcohol had been converted; the cultivation was then stopped.
To isolate the products, the culture suspension was neutralized with 1N sodium hydroxide solution and then extracted twice with twice the volume of ethyl acetate. The organic phases were separated off, combined and dried over anhydrous sodium sulphate. After removal of the desiccant on a paper filter, the solvent was removed by distillation under reduced pressure in a rotary evaporator.
Candida bombicola strains M18, M30 and M33 are the optimum yeast strains used in the present invention to produce medium chained sophorolipids and were deposited at the BCCM/MUCL Culture Collection of the Mycothèque de I'Université Catholique de Louvain, Belgium under the provisions of the Budapest Treaty on May 21, 2008, under accession numbers MUCL 51389, MUCL 51390 and MUCL 51391 respectively. The subject culture deposit will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of Micro-organisms.
Comparative Example A
Production of medium chain sophorolipids by the wild-type Candida were carried out as described below. All experiments were carried out using 1-dodecanol as the lipophilic substrate and utilised medium according to Lang et al. (Chem. Today, 2000, 18, p. 76-79). One shakeflask experiment and two bioreactor experiments were carried out. The shakeflask experiment used the wild-type Candida as a reference and was carried out in twofold. Of the bioreactor experiments, one was done under aerobic conditions and the other employed a limited aeration.
Shakeflask Experiments.
For each shakeflask, a test tube containing 10 ml of medium was inoculated with either the wild-type or the M30 strain and left to grow for two days at 30° C. The test tubes were shaken regularly (approx. 7 times a day) to prevent the cells settling on the bottom. Two 500 ml baffled shakeflasks per strain, each containing 200 ml of medium, were inoculated with the contents of one appropriate test tube and were left to grow for two and a half days at 30° C., shaking at 150 rpm. The first portion of 3.45 g 1-dodecanol per flask was added after this period of growth. The second, equally large portion of substrate was added four days after. Four days later the experiment was stopped. Sampling was performed on the 1st, 2nd, 3rd, 4th, 7th and 8th day after the first portion was added. 1 mL of broth was transferred to 3 mL of ethanol, mixed vigorously and filtered over a 0.45 μm membrane to remove the cells. The resulting solution was then analysed by reversed phase HPLC. Chromatograms obtained from the M30 samples of day 8 clearly showed the emergence of new peaks in the area between the fronting peak and the area in which the known C16/C18 sophorolipids occur. This area is concurrent with molecules that are more hydrophilic than these known sophorolipids but more hydrophobic than salts and, for example, wetting agents. The theoretically predicted structure of short chained sophorolipids indicates that they are to be found in this region. No peaks were found in this region for samples taken from the wild-type cultures. Identical peaks and peak patterns were found after cultivation in a bioreactor.
Bioreactor Experiments.
For each experiment, two test tubes containing 10 ml of medium were inoculated with the M30 strain and grown as indicated above for the shakeflask experiments. These were used to inoculate 500 ml of medium which was left to grow for two days at 30° C. while magnetically stirring. This inoculum was then added to 3 L of medium and grown for two and a half days. The medium was kept at 30° C. and aerated with 1 v/vm which is 1 volume of air per volume medium and per minute of air while stirring at 300 rpm, no pH correction was made. For the fully aerated experiment, the stirring speed was increased to 550 rpm after the growth phase and kept at this value throughout the entire experiment so the pO2 never dropped below 10% and had an average of 40%. For the experiment with limited aeration, the airflow and stirring speed were adjusted after the growth phase in order to keep the pO2 between 1% and 10%. In both experiments, the pH remained between 3 and 4. Six days after the experiments were started, an additional 175 g of glucose was added. In each case, the substrate was added step-wise during the first four days after completion of the growth phase. In the fully aerated experiment, 175.8 g of 1-dodecanol was added; the experiment with limited aeration received a total of 156.5 g 1-dodecanol.
Sampling throughout both experiments was done in a similar fashion as for the shakeflask experiments, except that samples of 3 ml of broth were added to 9 ml of ethanol. Additionally for the experiment with limited aeration, the total dry biomass after the growth phase was determined several times on 12 ml of broth and was found to be stable around 10 g/L. The total duration, from inoculation of the bioreactor to final sampling, of both experiments was ten days.
The final broth of the fully aerated experiment exhibited a top layer of insoluble solids and consisted mostly of dodecanoic acid. As samples taken from the aqueous phase did not contain any appreciable amounts of hydrophobic (i.e. more hydrophobic than C16-C18 sophorolipids) compounds, it is assumed that any 1-dodecanol which was not converted into sophorolipids was in fact oxidised to dodecanoic acid.
Analysis of the Reaction Product Using Chromatography.
Analysis of the samples was done similarly as for the shakeflask samples. The resulting chromatograms where divided into four areas as to correlate retention time to a certain functional class (see table 4). The total peak surface of each area was calculated. As the detection method used returns a quadratic response, the square root of each of the four totaled surface areas was calculated and these values were used to determine the percentage in which components of each functional class were present. The analysis of 100 ml of broth from the experiment with limited aeration yielded 27.4 g/L of non-volatile water soluble components (out of 83.3 g/L dry matter, biomass excluded) out of which 30% was found in the 2nd area; it is therefore concluded that 8.2 g/L of medium-chained sophorolipids were formed. The direct comparison of the chromatograms from equal amounts of either fully or limited aerated broth indicate a that the root of total area 2 is approximately 3.4 times larger in the case of limited compared to full aeration. As such it is concluded that using limited aeration increases production more than threefold.







TABLE 4







Table 1.


Division of chromatogram retention times into functional classes











Retention
Retention




time @ start
time @ end
Description of the functional


Area No
(min)
(min)
class













1
0
6
Salts & components having a HLB





higher than surfactants





(i.e. hydrotropes)


2
6
22
Surfactants with a high HLB value


3
22
34
Surfactants with a low HLB value


4
34
51
Lipophilic components









Analysis of the Reaction Product Using Mass Spectrometry.

Mass spectrometry analysis of the bioreactor experiment extract showed concurrent masses as expected from the direct conversion of dodecanol into sophorolipids. A relatively small amount of de novo sophorolipids were found as well. An overview of the molecular masses that were found, their assigned structure and their origin is given in table 5.







TABLE 5







Molecular masses found in the extract of a bioreactor experiment using dodecanol










MW 





(Da)
Assignation
Structure
Origin





222
Diethyl phthalate




Agent used for the denaturation of ethanol


 


510
SL—C12: 0-0a-Meth




Dodecanol sophorolipid; no acetylation


 


552
SL—C12: 0-1a-Meth




Dodecanol sophorolipid; one acetyl group


 


594
SL—C12: 0-2a-Meth




Dodecanol sophorolipid; two acetyl groups


 


706
SL—C18: 1-2a-Carb




de novo sophorolipid; open form









Evidence for said masses was obtained through both positive and negative electrospray ionisation, except for diethyl phthalate, which was only found when using the positive mode. The retention times of the dodecanol-based sophorolipids confirmed that they are to be found in the part of the chromatogram as indicated in table 4.

Analysis of the Reaction Product Using HPLC.

The analytical method used is described in Fleurackers (2003). The gradient used for the analysis was as follows: 0.5% acetic acid to 100% acetonitrile over a time of 40 minutes, 5 minutes of 100% acetonitrile, 1 minute to go to 100% water and 5 minutes of 100% water, always using a flow of 1 ml/min. Ultra pure water was produced in a Synergy 185 ultra pure water system (Millipore) and used in all of the experiments. Additionally the water was kept for approximately 2 hours at 20 mbar to remove any dissolved air. Acetonitrile was Chromesolv® grade (Riedel-de Haën) 100 μl sample was injected twice for analysis. The column was a ChromolithTM Performance RP-18e (Merck; C18; Ø 4.6 mm; length 100 mm), which was protected with a C8 guard column (Supelco PelliguardTM LC-8, 2 cm). A P580 pump (Dionex) and ASI-100 autosampler (Dionex) were coupled to a PL-ELS1000 (Polymer Laboratories) evaporative light scattering detector. Pump and autosampler were controlled by the ChromeleonTM software version 6.40 (Dionex) and the detector by the PL-ELS1000 control software ver4.0 (Polymer Laboratories). For the detector, nitrogen gas was used at a flow of 11/min. Nebulizer temperature was set at 85° C. and evaporator temperature at 90° C. HPLC-data processing was also done using the ChromeleonTM software. The peaks were identified by Intertek using RP-HPLC-MS (Waters ZQ) with electrospray ionisation and quadrupole detection.
From the above given results it appears that no medium chained sophorolipids were synthesized when using the wild-type strain and the use of limiting aeration showed more than a threefold production increase of these sophorolipids when compared to working under fully aerated conditions.
REFERENCES


    Van Bogaert I N A, De Maeseneire S L, De Schamphelaire W, Develter D, Soetaert W, Vandamme E J, 2007 Cloning, characterisation and functionality of the orotidine-5′-phosphate decarboxylase gene (URA3) in Candida bombicola. Yeast 24, 201-208.
    Van Bogaert I N A, De Maeseneire S L, Develter D, Soetaert W, Vandamme E J, 2008. Development of a transformation and selection system for the glycolipid producing yeast Candida bombicola. Yeast 25, 273-278
    U.S. Pat. No. 5,254,466 Picataggio S, Deanda K, Eirich L D, 1993. Site-specific modification of the candida tropicals genome.
    WO9924448 Lang S, Brakemeier A, Wullbrandt D, Seiffert-Stoeriko A, 1999. New sophoroselipids, method for their production and use
    U.S. Pat. No. 5,767,255 Wullbrandt D, Giani C, Brakemeier A, Lang S, Wagner S, 1998. Glucose- and sophorose-lipids, a process for their preparation and their use
    Sambrook J. and Russell D. W. 2001. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbour Laboratory Press, Cold Spring Harbour, New York.
    Gietz, R. D. and R. H. Schiestl. 1995. Transforming yeast with DNA. Methods Mol. Cell. Biol. 5: 255-269.
    Fleurackers S J J. 2006. On the use of waste frying oil in the synthesis of sophorolipids. Eur J Lipid Sci Technol 108, 5-12.
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## Claims

1. A process for producing sophorolipids comprising the steps of:
(1) culturing a mutant strain of a sophorolipids producing Candida bombicola comprising disruption of the MFE2 gene, in a medium comprising an energetic substrate including at least one carbon source; and
(2) contacting said strain with a bioconversion substrate, which contains at least one primary or secondary alcohol or diol, or at least one fatty acid or fatty acid ester, in which the hydrocarbon chain which determines the chain length contains about 4 to about 24 carbon atoms.

2. A process as claimed in claim 1, wherein the sophorolipid corresponds to the formula:



wherein
R3=—(CH2)mH
R4=—(CH2)pH
in which (m+n+p)≧2 and ≦22 and m, n and p are independent of one another and ≧0 and ≦22,
R7=—COOH or —C(R4)(R5)H




R1 and R2 are independent of one another and —H or —C(O)CH3.

3. A process as claimed in claim 1, comprising culturing C. bombicola mutant strain M18, M30 or M33 in a culture medium containing a nitrogen source, an organic substrate and a co-substrate.

4. The process of claim 3, wherein the mutant strain is M30.

5. The process of claim 1, wherein the pH of the culture medium after maximum cell density is reached is maintained between 3 and 5.

6. The process of claim 1, wherein the bioconversion substrate is continuously added to said culture medium at a rate of between 0.1 and 1 gram per liter per hour.

7. The process of claim 1, wherein a residual concentration of the bioconversion substrate in the culture medium is kept below 10 g/l.

8. The process of claim 1, wherein the energetic substrate is glucose at an initial concentration of at least 100 gram glucose per liter, and the excess of glucose concentration is maintained above an excess of about 20 g/l.

9. The process of claim 1, wherein an oxygen partial pressure of the culture solution is ≦40% of the saturation value.

10. The process of claim 1, wherein the culture medium contains a nitrogen source at a concentration which corresponds to 1.5 g/l ammonium chloride.

11. The process of claim 1, wherein the culture medium contains about 4 g/l yeast extract.

12. The process of claim 1, wherein the bioconversion substrate is an aliphatic linear or branched hydrocarbon, which may contain one or more substituents selected from the group consisting of OR, COOH, and an ester with a carbon chain length of about 4 to about 24.

13. The process of claim 12, wherein said bioconversion substrate is a primary or secondary alcohol or diol having from about 4 to about 24 carbon atoms.

14. The process of claim 1, wherein the bioconversion substrate is a primary alcohol.

15. The process of claim 1, wherein the bioconversion substrate is 1-dodecanol or 1-tetradecanol.

16. The process of claim 5, wherein the pH of the culture medium after maximum cell density is reached is maintained at about 3.5.

17. The process of claim 7, wherein the residual concentration of the bioconversion substrate in the culture medium is kept below 8 g/l.

18. The process of claim 7, wherein the residual concentration of the bioconversion substrate in the culture medium is kept below 2 g/l.

19. The process of claim 8, wherein the energetic substrate is glucose at an initial concentration of at least 120 g/l, and the excess of glucose concentration is maintained above an excess of about 20 g/l.

20. The process of claim 9, wherein an oxygen partial pressure of the culture solution is <15% of the saturation value.

21. The process of claim 13, wherein said bioconversion substrate is a primary or secondary alcohol or diol having from about 8 to about 14 carbon atoms.