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Biotechnological opportunities in biosurfactant

production
Robin Geys, Wim Soetaert1 and Inge Van Bogaert1

In the recent years, biosurfactants proved to be an interesting for the classic petrochemical surfactants. Because of their
alternative to petrochemically derived surfactants. Two classes interesting properties like low ecotoxicity [2], easy bio-
of biosurfactants, namely glycolipids and lipopeptides, have degradability [3] and mild production conditions, biosur-
attracted significant commercial interest. Despite their factants have numerous potential applications in a wide
environmental advantages and equal performance, variety of industrial sectors [4].
commercialization of these molecules remains a challenge due
to missing acquaintance of the applicants, higher price and Still, some obstacles remain. Often issues like low titers
lack of structural variation. The latter two issues can partially be combined with high production costs hamper market
tackled by screening for novel and better wild-type producers penetration and limited structural variation hinders integ-
and optimizing the fermentation process. Yet, these traditional ration in all application domains. To counter these pro-
approaches cannot overcome all hurdles. In this review, an blems, a lot of effort has been put into engineering the
overview is given on how biotechnology offers opportunities for production process using either waste streams [5], opti-
increased biosurfactant production and the creation of new mizing the fermentation parameters and product recovery
types of molecules, in this way enhancing their commercial [6]. With the rise of biotechnical engineering, new ways
potential. for improving the production of biosurfactants became
Addresses available. Both the engineering of wild-type producers as
Centre of Expertise for Industrial Biotechnology and Biocatalysis, the development of heterologous production systems are
Faculty of Bioscience Engineering, Ghent University, Coupure links 653, viable approaches towards increased titers and enlarged
9000 Gent, Belgium
molecular diversity.
Corresponding author: Van Bogaert, Inge ([email protected])
1
Shared senior authorship. Two classes of biosurfactants are currently considered
of industrial and economical relevance: glycolipids and
lipopeptides, both low-molecular weight biosurfactants.
Current Opinion in Biotechnology 2014, 30:66–72
The ones discussed in this review are the rhamnolipids,
This review comes from a themed issue on Chemical biotechnology
sophorolipids, cellobiose lipids and mannosylerythritol
Edited by Curt R Fischer and Steffen Schaffer lipids for the glycolipids. Lipopeptides will be discussed
in a more general way.

http://dx.doi.org/10.1016/j.copbio.2014.06.002 Rhamnolipids
0958-1669 # 2014 Published by Elsevier Ltd. All right reserved. Rhamnolipids, perhaps the most studied glycolipids to
date, are composed of one or two L-rhamnose molecules
coupled to a mono or dimer of b-hydroxy fatty acids [7]
(Figure 1). They have interesting applications in, for
example, soil remediation [8] or pest control [9]. Rham-
nolipids are produced by several species from the Pseu-
Introduction domonas [10] and Burkholderia [11] genera with
Surfactants are known since ancient times. The earliest Pseudomonas aeruginosa being the top producer with titers
evidence of soap-making dates back to the Babylonians over 100 g/L. Rhamnolipids are commercialized by a
2800 years BC. Throughout history, more applications, couple of companies like Jeneil Biotech Inc. and Rham-
molecule types and resources were discovered. In 2012, nolipid Companies Inc., but the high price still hampers
the global surfactant market generated a revenue of further market penetration.
27 billion dollars and is expected to rise. Most of these
surfactants are obtained by chemical processes from pet- The biosynthesis of rhamnolipids depends on two path-
rochemical and oleochemical resources. During recent ways delivering the necessary molecules. The first one is
years, a shift towards more environmentally friendly the production of dTDP-L-rhamnose by the rmlABCD
surfactants is observed, caused by a growing awareness operon; glucose-1-phosphate is converted to the activated
of both consumers and companies for the adverse rhamnose that serves as a donor for the hydrophilic part
effects that surfactants can have on the environment of the rhamnolipids. The hydrophobic part originates
[1]. Biosurfactants, surfactants produced by micro-organ- from b-hydroxy fatty acids coupled to an acyl carrier
isms from renewable resources, are a worthy alternative protein. Two of these molecules are coupled by RhlA

Current Opinion in Biotechnology 2014, 30:66–72 www.sciencedirect.com

 Biotechnological opportunities for biosurfactants Geys, Soetaert and Van Bogaert 67

Figure 1 the Vitreoscilla hemoglobin gene vgb. Such strains show a

7-fold increase compared to the wild-type [21]. This
H
O might be correlated to the higher oxygen uptake which
O enhances cell density and production.
HO O O
CH3 O
H H OH Production of rhamnolipids results in a mixture of both
H H monorhamnolipids and dirhamnolipids. By knocking-out
OH
rhlC, encoding a rhamnosyltransferase, only monorham-
H
O
nolipids are produced without a big loss in production
O
HO titers [22].
CH3
H H

H H Engineering of species other than P. aeruginosa has been

CH3
CH3 reported as well. Using this strategy, the opportunistic
OH HO
pathogenic nature of this micro-organism, which is a
Current Opinion in Biotechnology
serious burden both on the production and application,
can be circumvented. Usually, the P. aeruginosa rhlAB
Structure of the dirhamnolipid a-L-rhamnopyranosyl-a-L-
rhanopyranosyl-b-hydroxydecanoyl-b-hydroxydecanoate. Further operon is expressed in a strain that is resistant towards
variation is possible in the hydrophobic part by incorporation of b- high biosurfactant concentrations [23]. Using several
hydroxy fatty acids ranging in length from 8 to 16 carbon atoms and Pseudomonas putida strains like KT2440 [23], KT2442
potential unsaturations. [24] and KCTC1067 [25], titers ranging from 0.6 g/L
[24] to 7 g/L [25] were obtained. This lower production
compared to P. aeruginosa might be explained by insuffi-
to form 3-(3-hydroxyalkanoyloxy)alkanoic acids. Finally, cient production of the dTDP-L-rhamnose precursor, a
RhlB and RhlC will link the hydrophilic and hydrophobic necessary substrate for the rhamnosyltransferases [25].
part to form respectively monorhamnolipids and dirham-
nolipids [7]. Other production hosts were evaluated as well, such as
Escherichia coli strains W2190 [26] and BL21 [27]. When
In P. aeruginosa, direct and indirect factors like quorum using the same strategy as described above, maximum
sensing, nutritional status or stress response influence production titers of 0.18 g/L are feasible. Again, it is
rhamnolipid production [12]. Various attempts have been suggested that the flux through the dTDP-L-rhamnose
made to interfere with these regulatory mechanisms. synthesis pathway is too low to guarantee higher
AlgR, a regulator involved in several pathways like algi- production titers [26]. By introducing the rhlAB operon
nate production [13], has an impact on rhamnolipid from P. aeruginosa PAO1 in Burkholderia kururiensis KP23,
production. A recent paper proves the direct involvement a 6-fold increase from 0.78 g/L to 5.67 g/L could be
of AlgR in the expression of the rhlAB operon by binding detected [28].
to the promotor [14]. Deletion of this regulator shows an
increased rhamnolipid production in biofilm compared to Sophorolipids
the wild-type PAO1-strain [15]. Unfortunately, no differ- Sophorolipids, another well-studied type of glycolipids,
ences could be observed when the strains were grown in are produced by certain yeast strains like Starmerella
liquid medium. RhlR, a regulatory protein binding to the bombicola [29], Wickerhamiella domercqiae [30] and Candida
promoter of the rhlAB operon, has a more subtle role. batistae [31]. The molecules are constituted out of a
Depending on the butanoyl-homoserine lactone (C4- sophorose molecule (2-O-b-D-glucopyranosyl-D-gluco-
HSL) levels, an autoinducer that binds directly to RhlR, pyranose) linked to a terminal or subterminal hydroxyl-
this regulator works either as an activator or as an inhibitor ated fatty acid (Figure 2). The first step in the
for the transcription of the operon [16]. When no C4-HSL biosynthesis of sophorolipids is the terminal or subterm-
is present, RhlR acts as an inhibitor. Mutant strains inal hydroxylation of a fatty acid by the cytochrome P450
deficient in lasR, an essential gene for the production monooxygenase enzyme CYP52M1. In a stepwise man-
of C4-HSL, show no production of rhamnolipids [17]. ner, the UDP-glucose dependent glucosyltransferases
Still, even when RhlR is present with its autoinducer C4- UgtA1 and UgtB1 add the two glucose units to form a
HSL, production is not always guaranteed [18]. Other non-acetylated acidic sophorolipids. These can undergo
regulators like PtxR, RsaL, RsmA, DksA and several further modifications like acetylation by an specific
genes encoding sigma factors like RpoN and RpoS also acetyltransferase and lactonization between the free
influence the expression levels of the rhlAB operon carboxylic end and the C400 of the sophorose unit, which
[12,19,20]. Great potential lies in uncoupling the pro- is governed by an extracellular lactone esterase [32,33,34].
duction of rhamnolipids from this complex regulatory Sophorolipids are used in several products like hard sur-
system. Another strategy for enhancing rhamnolipid pro- face cleaning products from companies like Ecover and
duction is the creation of a P. aeruginosa strain harboring Saraya. S. bombicola, known for the efficient production of

www.sciencedirect.com Current Opinion in Biotechnology 2014, 30:66–72

68 Chemical biotechnology

Figure 2 form in concentrations comparable to the wild-type

strains [34]. Overexpression of the lactonase results in
OR1 CH3 producing lactonic sophorolipids at relative higher pro-
O
duct titers compared to the wild-type. These engineering
H O
H attempts show that it is possible to steer the production of
OH H very specific types of molecules with this yeast.
HO H
OR2 H Cellobiose lipids
H O Cellobiose lipids are produced by several fungi like
H Ustilago maydis and Pseudozyma flocculosa. They are com-
OH H O
posed of cellobiose (4-O-b-D-glucopyranosyl-D-glucopyr-
HO H
anose) attached to a hydroxylated palmitic acid
H OH (Figure 3a). The cellobiose lipids from P. flocculosa have
been used as a biological fungicide under the name
Sporodex L [38]. In U. maydis, the production is initiated
under nitrogen starvation. It has to be said that U. maydis
R1 = H or COCH3
produces two classes of glycolipids under these con-
R2 = H or COCH3 ditions, cellobiose lipids and mannosyl erythritol lipids
(MEL’s), with titers of glycolipids up to 30 g/L with
varying amounts of MEL’s and cellobiose lipids [39].

O Deletion of either the mannosyltransferase emt1 or the

cytochrome P450 monooxygenase cyp1 genes results in
OH total blockage of respectively the MEL’s or cellobiose
Current Opinion in Biotechnology
lipids biosynthesis, allowing recovery of one type of
glycolipid. Biosynthesis of cellobiose lipids in U. maydis
Structure of an acidic sophorolipid with oleic acid as hydrophobic group. is carried out by a gene cluster containing al the genes
necessary. The synthesis is initiated by Cyp1, a
cytochrome P450 enzyme necessary for terminal
hydroxylation of palmitic acid. A second hydroxyl group
sophorolipids up to 400 g/L [29], is the best studied is introduced by Cyp2 at the v-1 position. The cello-
producer so far. biose moiety is coupled to this 15,16-dihydroxy palmi-
tic acid by Ugt1, a glucosyltransferase capable of
Several knock-out strains were created for the production sequential glucosylation. Further modifications are
of modified sophorolipids. Production of medium-chain carried out by Uat1, an acetyl/acyl transferase, and
sophorolipids is challenging as the required special sub- Ahd1, responsible for the partial a-hydroxylation of
strates such as primary alcohols and diols are readily the palmitic acid [40].
metabolized in b-oxidation. Knocking out the multifunc-
tional enzyme type 2 (MFE-2), catalyzing the second and Regulation of cellobiose lipids production in U. maydis is
third step of the b-oxidation, enhances the production of governed by Rua1, a transcription factor interacting with
sophorolipids with shorter chain lengths up to 3 times the cluster genes and located herein [41]. Overexpression
compared to the wild-type strain [35]. Still, the depen- of this regulator promoted biosurfactant production
dence on special substrates instead of vegetable oil already in the exponential growth phase.
greatly affecting the production cost.
Other engineering possibilities have been reported as
Another example involves the acetyltransferase, necessary well. Deletion mutants of the cyp2, ahd1, uat1 and uhd1
for acetylation of the sophorose group. A knock-out strain genes in U. maydis produce altered cellobiose lipids [42].
of this gene produces purely unacetylated sophorolipids These genes are respectively responsible for the v-1
[36]. Yet another example of a successful knock-out hydroxylation (cyp2) and a-hydroxylation (ahd1) of the
involves ugtB1, the gene responsible for the addition of fatty acid tail, the acylation and acetylation of the cello-
a second molecule of glucose in the sophorolipid biosyn- biose group (uat1) and the hydroxylation of the short-
thesis. After a prolonged fermentation, a mixture of acetyl- chain fatty acid coupled to the cellobiose group (uhd1).
ated and non-acetylated glucolipids was obtained [37]. Unfortunately no data are available on the cellobiose
lipids titers of these knock-out strains.
A final engineering example is that of the lacton esterase,
the enzyme catalyzing lactonization of the sophorolipids. Interestingly, only one glucosyltransferase is present in
Knock-out strains are capable of producing the acidic the cellobiose lipid gene cluster [42] and it is suggested

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 Biotechnological opportunities for biosurfactants Geys, Soetaert and Van Bogaert 69

Figure 3

(a) O OH O
CH3

O OH
O R1
H O
H R1 = H or OH
OH H
HO
H n = 2 or 4
H O H OH
H
OH H O
HO H
(b) HO
H O
O
HO H
R1 O
HO H
OH
O
( (n H O
H
CH3 OR3 OR4
R1 , R2 = H or COCH3
R2O H
R3 , R4 = H or C2-C18 fatty acid
H H
Current Opinion in Biotechnology

Structures of the glycolipids produced in U. maydis (a) cellobiose lipids; (b) mannosylerythritol lipids.

that this transferase performs both glucosylation reactions. higher production. The discovery of Mat1, the acetyl-
The codon optimized ugt1 gene from U. maydis was used transferase responsible for both acetylation reactions,
for the heterologous production of cellobiose lipids in S. showed that it is possible to produce deacetylated MEL’s
bombicola. By replacing the second glucosyltransferase without highly influencing the production capacity [48].
ugtB1 from S. bombicola, a new strain capable of synthesiz-
ing cellobiose lipids [43] was created. Even though 70% Lipopeptides
of the molecules are mainly glucolipids, a diverse popu- Several species like Aspergillus, Bacillus or Pseudomonas are
lation of cellobiose lipids was observed as well. Most of capable of synthesizing lipopeptides. These molecules
these cellobiose lipids are new-to-nature and consist of a are characterized by a small oligopeptide, either linear or
C18 hydroxy fatty acid tail and no acylations of the circular, coupled to a b-hydroxy fatty acid. The oligopep-
cellobiose group. Also no lactonization is observed. Still, tide varies both in number and types of amino acids [49].
optimization is required because of the glucolipid con- Unusual or non-proteiogenic amino acids are incorporated
tamination and less than optimal product concentrations. as well, for example in surfactin (Figure 4), synthesized by
Bacillus subtilis, two D-leucine molecules are present [50].
Mannosylerythritol lipids In the case of surfactin, titers of 3.6 g/L are possible [51].
Mannosylerythritol lipids (MEL’s) are interesting com-
pounds consisting of a 4-O-b-D-mannopyranosyl-meso- Figure 4
erythritol with various acylation and acetylation patterns,
depending on the production organism and substrates
Asp
used (Figure 3b). The titers of these molecules can easily Leu Val
reach 100 g/L or more [44]. Special interest goes out to Leu
these molecules because of their self-assembling proper- Leu
ties [45], potential pharmaceutical applications [46] and O
H3C Leu
their usage in cosmetics [47]. The Japanese companies
Toyobo and Kanebo Cosmetics commercialize MEL’s. Glu
( )9
As mentioned in ‘‘Cellobiose lipids’’ section, some engin- H3C
eering is done to optimize production in U. maydis. O
Deletion of emt1, the gene encoding the erythritol/ Current Opinion in Biotechnology

mannose transferase, resulted in total loss of the pro-

duction while overexpression did not necessarily lead to Structure of surfactin, a lipopeptide from B. subtilis.

www.sciencedirect.com Current Opinion in Biotechnology 2014, 30:66–72

70 Chemical biotechnology

Lipopeptides have drawn significant interest because of new possibilities to fully capture the metabolic potential
their potential as antimicrobial agents [52]. of micro-organisms have risen. The development of heter-
ologous production systems with non-pathogenic species,
Significant effort has been put into engineering of the the progress towards entire production platforms for a
hydrophilic oligopeptide structure. This peptide is syn- range of molecules and utilizing previously unsuitable
thesized by a nonribosomal peptide synthetase (NRPS). waste streams as cheap substrates are just a few examples
This is a multienzyme complex which can further be of what is possible thanks to biotechnology. Eventually,
divided in subunits, modules and domains. Each module the combination of each success in the creation of cheaper
is responsible for the incorporation of one amino acid and and more diversified biosurfactants will result in further
they contain the necessary domains for the elongation of commercialization of these molecules.
the peptidyl chain. Several modules can be grouped
together in a subunit, a monomer being part of the Acknowledgements
multienzyme complex. A first strategy is the exchange This work was made possible by the Flemish Agency for Innovation by
Science and Technology (IWT; Grant 121129) and the European FP7
of subunits of the NRPS. In the case of daptomycin, project Biosurfing (Grant 289219).
a lipopeptide produced by Streptomyces roseosporus,
replacing the final subunit DptD results in compounds References and recommended reading
differing in the last two amino acid of the peptide core Papers of particular interest, published within the period of review,
[53]. The second option is the exchange or deletion of have been highlighted as:

modules [54] and of domains within these modules [55].

 of special interest
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