Biotechnology Advances 31 (2013) 338–345

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Research review paper

Lipopeptides in microbial infection control: Scope and reality for industry

Santi M. Mandal a, Aulus E.A.D. Barbosa b, Octavio L. Franco b,⁎a Central Research Facility, Indian Institute of Technology Kharagpur, Kharagpur 721302, W B, Indiab Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises, Proteômicas e Bioquímicas, Universidade Católica de Brasília Brasília, Brazil

⁎ Corresponding author at: Universidade Catolica de Bra219, Brasilia, DF, CEP 70990-160, Brazil. Tel.: +55 61 3448

E-mail addresses: ocfranco@gmail.com, ocfranco@po

0734-9750/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.biotechadv.2013.01.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 August 2012Received in revised form 4 January 2013Accepted 4 January 2013Available online 12 January 2013

Keywords:LipopeptidesBiotechnologyAntimicrobialsBacterial infections

Lipopeptides are compounds that are formed by cyclic or short linear peptides linked with a lipid tail or otherlipophilic molecules. Recently, several lipopeptides were characterized, showing surfactant, antimicrobial andcytotoxic activities. The properties of lipopeptides may lead to applications in diverse industrial fields includingthe pharmaceutical industry as conventional antibiotics; the cosmetic industry for dermatological product devel-opment due to surfactant and anti-wrinkle properties; in food production acting as emulsifiers in various food-stuffs; and also in the field of biotechnology as biosurfactants. Some lipopeptides have reached a commercialantibiotic status, such as daptomycin, caspofungin, micafungin, and anidulafungin. This will be the focus of thisreview. Moreover, the review presented here will focus on the biotechnological utilization of lipopeptidesin different fields as well as the functional–structure relation, connecting recent aspects of synthesis andstructure diversity.

© 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3382. Lipopeptides' mechanism of action and resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3393. Origin and structural diversity of lipopeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3394. Lipopeptides in the pharmaceutical industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3405. Lipopeptides in the cosmetic industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3426. Lipopeptides in the food industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3437. Lipopeptides in the biotech industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3438. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

1. Introduction

Antimicrobial peptides are multifunctional compounds withmultiple utilities (Franco, 2011). Among them, lipopeptides are smallmolecules that are formed by cyclic or short linear peptides linkedwith a lipid tail or other lipophilic molecules (Arnusch et al., 2012;Raaijmakers et al., 2010). The first lipopeptide discovered with anantimicrobial function, polymyxin A, was isolated in 1949 from thesoil bacterium Bacillus polymyxa (Jones, 1949), but the biosynthesis ofthese molecules has been detected in several bacterial genera, mainly

silia, SGAN 916N, Modulo C, Sala7167; fax: +55 61 3347 4797.s.ucb.br (O.L. Franco).

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Bacillus, Pseudomonas and Streptomyces, as well as in fungi. Recently,several lipopeptides have been characterized, showingdiverse activitieslike surfactant, antimicrobial and cytotoxic (Raaijmakers et al., 2010).Some of them have even reached a commercial antibiotic status, likedaptomycin (Robbel and Marahiel, 2010), caspofungin (Ngai et al.,2011), micafungin (Emiroglu, 2011) and anidulafungin (George andReboli, 2012).

Currently, lipopeptide propertiesmay lead to applications in diverseareas of industry. In the pharmaceutical industry, lipopeptides havebeen used when conventional antibiotics were no longer workingagainst resistant bacteria or fungi. In the cosmetic industry, surfactantand anti-wrinkle characteristics of lipopeptides are applied in dermato-logical products, since lipopeptides present low cytotoxicity againsthuman cells. In food production, lipopeptides are used as emulsifiers invarious foodstuffs. Finally, lipopeptides are applied in biotechnology asbiosurfactants, giving rise to several industrial and medical applications.

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With this in mind, the function–structure relation of lipopeptides,reporting recent aspects in synthesis and structure diversity, as well asseveral applications of these antimicrobialmolecules in pharmaceutical,cosmetic, food and biotech industries will be discussed here.

2. Lipopeptides' mechanism of action and resistance

Several questions have been raised over lipopetides' mechanisms ofaction. Data have shown that pore formation inmembranes occurs afterlipopeptide oligomer binding, some of which are Ca2+ dependentmultimers (Scott et al., 2007). These pores may cause transmembraneion influxes, including Na+ and K+, which result in membrane disrup-tion and cell death (Mangoni and Shai, 2011; Ostroumova et al., 2010;Scott et al., 2007). In the case of daptomycin, oligomer formation prob-ably occurs before membrane contact. This process seems to be relatedto ion binding, as after calcium binding the lipopeptide undergoes aconformational modification that leads to oligomerization, even inhigh concentrations. Daptomycin/calcium complex interacts with thenegatively charged membrane phosphatidylglycerol head groups andundertakes a second conformational alteration that induces oligomeri-zation of the membrane, leading to membrane penetration (Muraih etal., 2011).

In addition, some research has showed that lipopeptides can inhibitfungi cell wall formation (Schneider and Sahl, 2010a, b). Echinocardinsact by specific and non-competitive inhibition of enzyme β-(1,3)-D-glucan synthase (Yao et al., 2012). This carbohydrate is an essentialcomponent for the fungal cell wall to keep its structural integrity. Thelack of β-(1,3)- D-glucan leads to cell wall deterioration and consequen-tial cell death (Yao et al., 2012). The relationship between structure andactivity has also recently been verified for the echinocardins (Fig. 2F).The authors suggested that chemical modifications in some parts ofthe molecule, like alterations in non-proteinogenic amino acids or inthe fatty acid chain, may increase its activity or modify the specificity(Yao et al., 2012). However, it is not only the membrane that could bethe main target of lipopeptides. For example, at lower concentrations,the antifungal lipopeptide from Bacillus amyloliquefaciens can causeapoptosis by binding to ATPase on the mitochondrial membrane(Qi et al., 2010).

These modes of action may confer upon lipopeptides' high activityagainst multidrug-resistant bacteria (Mangoni and Shai, 2011), andthe emergence of resistance against lipopeptides is extremely rare(Sader et al., 2011). One example occurs in the bacterial resistanceprocess of lipopeptide daptomycin in Bacillus subtilis after artificial selec-tion (Hachmann et al., 2011). Changes in phosphatidylglycerol content inB. subtilis mutants explain this resistance, probably due to a decrease inthe net negative charge of the membrane which reduces the interactionwith daptomycin-Ca2+ complex (Hachmann et al., 2011). Otherwise,the emergence of lipopeptide resistance that inhibits peptidoglycansynthesis, like MX-2401, seems to be more unlikely (Rubinchik et al.,2011). MX-2401 binds to undecaprenylphosphate, a carbohydratecarrier involved in several biosynthetic pathways, and this linkageresults in several cell wall precursors in biosynthesis inhibition(Rubinchik et al., 2011).

3. Origin and structural diversity of lipopeptides

Lipopeptides with antimicrobial activity have been found in a widenumber of microorganisms, and have been purified from the followingbacterial genera: Actinoplanes (Schneider et al., 2009), Bacillus (Velho etal., 2011; Yuan et al., 2011), Brevibacillus(Desjardine et al., 2007),Lyngbya (Balunas et al., 2010), Paenibacillus(Guo et al., 2012; Qian etal., 2012), Pseudomonas (de Bruijn and Raaijmakers, 2009; de Bruijn etal., 2008), Streptomyces (Alexander et al., 2011; Gu et al., 2011),Tolypothrix (Neuhof et al., 2005; Neuhof et al., 2006), and in the fungiAspergillus nidulans (Cortes and Russi, 2011; De Lucca and Walsh,1999). All of these natural lipopeptide sources provide high levels of

structure diversity reflecting several modes of action and targets(Tally et al.,1999). There are variations in length, configuration, number,and composition of lipids and amino acids in the structure.

Lipopeptides are synthesized in microorganisms by nonribosomalpeptide synthetases (NRPSs) (Mitchell et al., 2012). These enzymespossess a modular structure formed by multiple catalytic domainsthat act like a multidomain protein (Mitchell et al., 2012). There arethree domains in the NRPSs, each one with a different function thatincludes adenylation (domain A), condensation (domain C) andthioesterase (TE) (Fig. 1). At the beginning of the synthesis process,amino acids and peptides are adenylated and then covalently linkedto the peptidyl carrier protein (PCP). The variation of selected aminoacid residues in NRPSs can explain the variation in the lipopeptideamino acid sequence. Following PCP linkage, the NRPS condensationdomain catalyzes the peptide bond formation between two aminoacids. Ending the reaction, peptide release is performed after thioesterhydrolysis (Mitchell et al., 2012). This final step, inmost cases, catalyzesthe cyclization ofmature lipopeptides (Samel et al., 2006). The fatty acidmoiety is linked at the peptide N-terminus by the action of severalenzymes (Hansen et al., 2007; Kraas et al., 2010, 2012), and this variablemode of synthesis produces several kinds of fatty acid chains inlipopeptides.

In nature, variations in selected amino acids and fatty acids duringlipopeptide biosynthesis by microorganisms can yield a vast anddifferent number of molecules. They could present differences in aminoacid composition, in structure, being linear (Desjardine et al., 2007) orcyclic (Schneider et al., 2009), and in composition and also fatty acidchain length (Fig. 2). In counterpart, the artificial chemical synthesisof lipopeptides has been performed using solution phase synthesis[3+3] (Yao et al., 2012), solid-phase synthesis (Brunsveld et al., 2006)and by the chemoenzymatic approach (Grunewald and Marahiel,2006). However, solid-phase chemistry produces purified peptides inless time and with superior overall yields (Brunsveld et al., 2006).Modifications in these chemical synthesis methods can be appliedto simplify and optimize the lipopeptide structure (Yao et al.,2012) and even by reduction of the peptide length or lipid tail, theartificial lipopeptides show similar activities and mode of action.Makovitzki et al. (2006) show that short artificial lipopeptides, withonly 4 amino acid residues and a 16 carbons fatty acid chain, are activeagainst Gram-positive bacteria and fungi. Likewise, it has been demon-strated that modifications in lipid tail length may change lipopeptides'activities and specificities; for example, lipopeptides synthesized with10 or 12 carbon atoms at the lipid tail seem to be non-hemolytic and ac-tive towards both bacteria and fungi, whereas larger lipopeptides, with14 or 16 carbon atoms, showed enhanced efficiency in fungal control(Malina and Shai, 2005). Further modifications, like the replacementof lipid tail by lipophilic biomolecules, such as vitamin E and cholesterol,increased the activity against fungi and simultaneously reduced thehemolytic activity; Nevertheless, the motives for this are still notknown (Arnusch et al., 2012). In addition, the cyclization of chemicallysynthetized lipopeptides can be aided using NRPS TE excised domains(Grunewald and Marahiel, 2006).

Among linear lipopeptides from natural sources, tauramadine anddragomide E could be cited. Tauramadine (Fig. 2A) is a linearlipopeptide, isolated from Brevibacillus laterosporus, with five aminoacids and a fatty acid tail with six carbons (Desjardine et al., 2007).Dragomide E (Fig. 2B) is a linear lipopeptide from Lyngbya majusculethat shows five amino acids chain-linked in their structure with afive-carbon fatty acid tail (Balunas et al., 2010). Otherwise, cycliclipopeptides were also widely found. The lipopeptide friulimicin(Fig. 2C), isolated from Actinoplanes friuliensis, is formed by a cyclicpeptide with 10 amino acid residues and a branched fatty acid sidechain with 11 carbons (Schneider et al., 2009). Another pair of cycliclipopeptides was isolated from the Paenibacillus bacterial genus. Thefirst consists of a cyclic lipopeptide with a 15 fatty acid chain and 13amino acid residues (FA-Orn-Val-Thr-Orn-Ser-Val-Lys-Ser-Ile-Pro-

Fig. 1. NRPS 3-D Structure. (A) SrfA-C, a NRPS from Bacillus subtilis (Tanovic et al., 2008) and the related catalytic domains. (B) Domain A catalytic amino acids are orange,Leu943–Thr944–Thr945–Asm946-Gly947. (C) PCP domain and catalytic Ala1003are orange. (D) Domain C and the catalytic His147 are orange. (E) TE domain and the catalytic Ser1120. All structuralrepresentations were constructed by using Pymol.

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Val-Lys-Ile) (Guo et al., 2012). On the other hand, the second fromPaenibacillus tianmuensis, named battacin, shows 8 amino acid residues(Qian et al., 2012). Two lipopeptides from cyanobacteria of Tolypothrixgenus were found and named hassallidin A and B. Hassallidin A(Fig. 2D) presents an eight amino acid cyclic peptide linked withmannose and a fatty acid residue (α,β-dihydroxytetradecanoic acid)(Neuhof et al., 2005), and hassallidin B (Fig. 2E) presents a very similarstructure, yet the single difference is a rhamnose linked to the3-hydroxyl group of the C14-acyl side chain (Neuhof et al., 2006).These different attached carbohydrates seem to have no influence onhassallidin A and B antifungal activities (Neuhof et al., 2006). Today, an-timicrobial lipopeptides have been found in Aspergillus fungi genera,named eichnocardins (De Lucca and Walsh, 1999). These lipopeptidespossess a similar cyclic peptide with 7 amino acids and a variable fattyacid tail (Fig. 2F) (De Lucca and Walsh, 1999). Variations of aminoacid residues in lipopeptides have received considerable attention toalter their activity against pathogenic microorganisms, which have asignificant impact on antibacterial activity. For example, massetolide Aand viscosin showed a two-fold difference in their activity againstMycobacterium tuberculosis and Mycobacterium avium-intercellulareby the difference of only one amino acid residue in their peptidemoiety(Gerard et al., 1997). However, recently several variants and modifica-tions of lipopeptide architecture have been reported. Remarkably,sometimes the cyclic peptide is also formed by non-proteinogenicamino acids, for example D-Ala, D-Asn, kynurenine, methylglutamate,ornithine, D-Ser (Robbel and Marahiel, 2010; Strieker and Marahiel,2009).

All these variations in length and branching of the fatty acid chainsand amino acid substitutions lead to remarkable lipopeptide diversityand activities. This dual composition of lipopeptides, formed byamino acids and lipids, causes the rearranged molecules to form mi-celles (Horn et al., 2012) or oligomers (Muraih et al., 2011) in solu-tion. After binding to the bacterial surface bilayer, lipopeptides alter

the local lipid organizational linking on negatively charged fattyacids. This causes a dramatic reorganization in lipid bilayer, alteringdiverse cellular processes that depend on specific bilayer composition(Horn et al., 2012). Single alterations in the amino acids and fatty acidsof a lipopeptide can alter its specificity for other bacteria or fungi(Jenssen et al., 2006).

4. Lipopeptides in the pharmaceutical industry

The rapid increase in the incidence of bacterial infections, particularlybacteria that are resistant to several antibiotics, is of renewed interest inthe development of novel antibiotics to infection control (Franco, 2011;Maria-Neto et al., 2012). Despite the development of several newantimi-crobial agents, and in some cases their limited utility, the need for novelantibiotics continues. Antibiotics generally lose potency and effective-ness because of repetitive exposure to bacteria, leading to the resistantvariants which are currently life-threatening. One commonly usedlipopeptide-based antibiotic, daptomycin, has potent antibacterialactivity against clinically-relevant resistant pathogens, such asmethicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), glycopeptide-intermediate-susceptibleS. aureus (GISA), coagulase-negative Staphylococci (CNS), and penicillin-resistant Streptococcus pneumoniae (PRSP) (Tally et al., 1999). A seriesof identified lipopeptides and their possible activity against microbialinfection control is shown in Tables 1 and 2.

Several lipopeptides are also effective in controlling fungal infectionssuch as echinocandins and their derivatives. Another is the semi-synthetic caspofungin from the fungus Glarea lozoyensis (Letscher-Bruand Herbrecht, 2003). Caspofungin is able to inhibit synthesis of thefungal cell wall by blocking the enzyme of β(l,3)-D-glucan-synthase.In addition to this mechanism of action, pneumocandin (Schwartz etal., 1988), aculeacin (Mizuno et al., 1977) and other related compoundssuch as WF11899s, WF738s, WF14573,WF16616 and WF22210 (Hino

Fig. 2. Examples of lipopeptides' molecular structures. Linear lipopeptides taumaradine A (A) (Desjardine et al., 2007) and dragomide E (B) (Balunas et al., 2010). A cycliclipopeptide, friulimicin (C) (Schneider et al., 2009). Cyclic lipopeptides with sugar residues, hassallidin A (D) and B (E) (Neuhof et al., 2005, 2006). Pneumocadin, a lipopeptidefrom fungi (F) (De Lucca and Walsh, 1999).

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et al., 2001) are known as selective inhibitors of β-1,3-glucan biosyn-thesis. Antifungal lipopeptides have also been used, mainly, to controlpathogens like Candida glabrata (Arendrup et al., 2012), Aspergillusfumigatus (L. Zhao et al., 2012, Y. Zhao et al., 2012), Scedosporiumprolificans (Lackner et al., 2012) and Pseudallescheria polysperma(Lackner et al., 2012).

Lipopeptides have strong antiviral activity and receive immenseattention nowadays. Nybroe and Sorensen (2004) demonstratedthat viscosin has the ability to control enveloped human-pathogenicviruses, including the highly infectious bronchitis virus. Similarly,surfactin was also active against several enveloped viruses, includingherpes viruses and retroviruses (Vollenbroich et al., 1997). Severalstudies revealed that lipopeptides act directly on the lipid envelopeof the virus, and hydrophobicity is also an important factor due toits nonspecific detergent activity which easily disintegrates the virusparticles (Kracht et al., 1999). Another option of HIV control couldbe focused on alternative targets such as glycoproteins gp120 andgp41 (Zhao et al., 2012a, Y. Zhao et al., 2012b). In this view amulti-functional peptide inhibitor for anti-HIV bearing cholesterolgroup (C34-Chol) was constructed (Ingallinella et al., 2009). Thispeptide shows dramatically increased antiviral potency with IC(90)values 15- to 300-fold lower than enfuvirtide and the second-generation inhibitor T1249, designating the lipopetide C34-Chol as a

powerful antiviral tool. Years later, a different version of C34-Chol, L′644, was produced showing increased and sustained antiviral activity(Harman et al., 2012). A few peptides are already marketed as vaccines,and several are in the pipeline with clinical trials, as listed in Table 3.Most of them are applied in HIV immunization, combining an HIV de-rived peptide and a lipid tail (Durier et al., 2006; Rizos et al., 2007).The Monash Institute of Pharmaceutical Sciences has recently devel-oped a novel lipopeptide antibiotic to target Gram-negative multi-drug-resistant bacteria, promising to be less apoptopic than polymyxinsand with a longer half-life (patent no. PCT/AU2010/000568).

Nowadays, a huge amount of resources is being invested in R&D tocontrol the problem of antibiotic resistance (Maria-Neto et al., 2012).The antibioticsmarket is looking to increase the number of newproductswith improved effectiveness. The pharmaceutical industry has realizedthe difficulty of introducing new antimicrobials, and only two novelclasses of antibiotics, the oxazolidinones and the cyclic lipopeptides,have entered the market since the late 1960s (Rodriguez de Castro etal., 2009). Recently, several antibacterial compounds have been undertrial. The global antibiotics market, which was traditionally a growingfield due to long-term success, has been primarily retarded by severalfactors such as antibiotic resistance, the inappropriate usage of antibi-otics, and generic competition. Themarket for antibacterial drugs is high-ly competitive, and many companies are engaged in the development of

Table 1List of identified highly active lipopeptides for infection control.

Lipopeptides Source Activity Ref.

Viscosin Pseudomonas sp. Mycobacterium avium, Mycobacterium tuberculosis (Gross and Loper, 2009)Amphisin Pseudomonas sp. Bacillus sp. (Bassarello et al., 2004)Tolaasin Pseudomonas sp. Gram positive bacteria (Bassarello et al., 2004)Syringomycin Pseudomonas sp. Geotrichum candidum, Rhodotorula pilimanae (Ramos, 2004)Putisolvin Pseudomonas sp. Inhibit biofilm formation of Pseudomonas sp. (Kuiper et al., 2004)Massetolide Pseudomonas sp Mycobacterium avium, Mycobacterium tuberculosis (de Bruijn et al., 2008)Entolysin Pseudomonas sp Staphylococcus aureus SG 511, Arthrobacter crystallopoietes,

Bacillus subtilis 168(Reder-Christ et al., 2012)

Surfactin Bacillus subtilis Enterococcus faecalis ATCC 2912, Lactococcus garviae KCCM 40698,Streptococcus parauberis DSM 6631, Flexibacter tractuosus ATCC23168,Vibrio harveyi ATCC 14126

(Kim et al., 2009)

Mycosubtilin Bacillus subtilis Micrococcus luteus (Peypoux et al., 1979)Fusaricidin A Bacillus subtilis MRSA S. aureus and S. epidermidis, Vancomycin-resistant E. faecium (Stawikowski and Cudic, 2009)Lichenycin Bacillus subtilis Corynebacterium variabiliss and Acinetobacterr sp. (Nerurkar, 2010)Bacillomycin D Bacillus subtilis Antifungal (Sclerotinia sclerotiorum) (Kumar et al., 2012)Friulimicin B Actinoplanes friuliensis Gram-positive pathogens (Schneider et al., 2009)LY146032 Streptomyces roseosporus Broad spectrum activity against staphylococci (MRSA), beta-hemolytic Streptococcus spp.,

Pneumococci, Clostridium spp., and Enterococci sp.(Eliopoulos et al., 1986)

Iturin A Bacillus subtilis Antifungal (Fusatium oxysporum) (Thimon et al., 1995; Yuan et al., 2012)Daptomycin Streptomyces roseosporus MRSA-Staphylococcus aureus (Miro et al., 2012)Echinocandin Papularia sphaerosperma Yeast, most species of Candida (Cortes and Russi, 2011)MSI-843 Synthetic E. coli, S.aureus, P. aeruginosa, C. albicans (Thennarasu et al., 2005)MX-2401 Semi-synthetic S. aureus, E. faecalis (Dugourd et al., 2011)

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anti-MRSA or multi-drug-resistant treatments, where lipopeptides arethe target compound of most companies. The US has the largest marketfor antibiotics, followed by Europe and Asia-Pacific (mostly India andChina). Scientists at the Monash Institute of Pharmaceutical Sciences es-timated that in theUSA, twomillion people are infected by bacterial path-ogens in hospitals each year, and 90,000 die as a result. About 70% ofthose infections are resistant to at least one antibiotic. Resistant patho-gens lead to higher health care costs due to requirements for more ex-pensive drugs and extended hospital stays; in the USA, the total cost isaround 5 billion annually. In Europe each year, over 25,000 people dieof bacterial infections. TheWHO reported thatmillions of people are in-capacitated by infectious diseases which cause disorders includingblindness. An estimated 154 million are infected each year inAfrica and Asia. The situation also demands that small biotech firmstake advantage of government incentives and encourage antibioticdevelopment.

Table 2Recent patents on lipopeptide as infection control.

Patent no. Date of patent Name of the patent

US 2011/0224129 A1 Sep. 15, 2011 Lipopeptide compounds and their useUS 2011/0030103 A1 Feb. 3, 2011 Lipopeptides and lipopeptide synthetasesUS 7,868,135 B2 Jan. 11, 2011 Composition of lipopeptide antibiotic deri

and methods of use thereofUS 7,795,207 B2 Sep. 14,2010 Lipopeptide compositionsUS 2010/0184649 A1 Jul. 22, 2010 Novel antibacterial agents for the treatmen

Gram-positive infectionsUS 7,671,011 B2 Mar. 2, 2010 Antimicrobial and anticancer lipopeptidesUS 7,655,623 B2 Feb. 2, 2010 Dab9 derivatives of lipopeptide antibiotics

methods of making and using the sameUS 2009/0233870 A1 Sep. 17, 2009 Antimicrobial peptidesUS 2009/0202519 A1 Aug. 13, 2009 Compositions and methods for treating Gr

positive bacterial infectionin a mammalian subject

US 7,408,025 B2 Aug. 5, 2008 Lipopeptides as antibacterial agentsUS 6,911,525 B2 Jun. 28, 2005 Lipopeptides as antibacterial agentsUS 6,750,199 B2 Jun. 15, 2004 Antimicrobial sulfonamide derivatives of

lipopeptide antibioticsUS 6,696,412 B1 Feb. 24, 2004 High purity lipopeptides, lipopeptides mic

and processes for preparing sameUS 6,624,143 B1 Sep. 23, 2003 Calcium salts of lipopeptide antibiotics,

method for producingsame and their use

US 6,511,962 B1 Jan. 28, 2003 Derivatives of laspartomycin and preparatand use thereof

5. Lipopeptides in the cosmetic industry

In last few decades, lipopeptides have been extensively used inthe cosmetics industry due to their exceptional surface properties,having anti-wrinkle and moisturizing activities on human skin(Kanlayavattanakul and Lourith, 2010). Several lipopeptide applicationshave been well-documented. Among them, surfactin has received mostattention with its multipurpose application in the industry (Nissen et al.,1997;Nitschke andCosta, 2007). Surfactin is themost active biosurfactant(Rosenberg and Ron, 1999), with excellent surface properties whichlower the surface tension of water from 72 to 27.9 mN m−1(Arima etal., 1968) and the interfacial tension of 0.36–34 mN m−1 at the criticalmicelles concentration (CMC) of 1–240 μM(Deleua et al., 1999). Interest-ingly, a low CMC value of a compound is particularly suitable for topicaldermatological application (Kanlayavattanakul and Lourith, 2010).Surfactins are highly biocompatible with very low cytotoxicity to

Source Inventors

Modified friulimicin Boyce et al.Engineered lipopeptide synthetase polypeptide Reznik et al.

vatives Amphomycin or aspartocin derivative Cameron et al.

Cyclodextrin derivatives Labischinski et al.t of Isolated combined compounds MetCalf,III et al.

Conjugate lipopeptide Shai et al.and Synthetic amphomycin-type Fardis et al.

Synthetic Blondelle et al.am Synthetic Beutler et al.

Synthetic Hill et al.Synthetic Hill et al.Lipopeptide derivatives Curran et al.

elles Daptomycin Kelleher et al.

Synthetic Vértesy et al.

ion Laspartomycin Borders et al.

Table 3Lipopeptide based vaccines in clinical trial.

Identificationno.

Lipopeptide Clinical application Status Source Reference

NCT00002353 P3C541b To evaluate an HIV lipopeptide immunotherapeutic, at two doselevels administered subcutaneously in HIV-seropositive patients.

Phase 1completed

Synthetic NIH AIDS Clinical TrialsInformation Service

NCT00121121 LIPO-4 To evaluate the safety and immunogenicity of LIPO-4 vaccine in-tradermally compared to intramuscular administration.

Phase 1completed

HIV lipopeptides including 4peptides from Gag, Pol, RTand Nef HIV-1 proteins.

Odile Launay,

NCT00121758 LIPO-5 To test the safety and immune response to an experimental HIVvaccine, LIPO-5, in healthy volunteers.

Phase 2continue

Five lipopeptides from gag,nef and pol with more than50 epitopes.

Dominique Salmon,Centre des essaisvaccinaux Cochin Pasteur

NCT00712634 CMVpp65-A*0201 To study the side effects and best dose of cytomegalovirusvaccine in healthy participants

Phase 1completed

CMV-pp65 peptide-specific Tcells

Don Diamond, BeckmanResearch Institute

NCT00003977 hpv16 E7 To study of immunization with alternating human papillomavirus e7lipopeptide epitope vaccine and dendritic cells presenting the e7epitope for the treatment of recurrent or persistent cervical cancer.

Phase 1completed

Human Papillomavirus E7Lipopeptide Epitope

Michael A Steller, MDsteward St. Elizabeth'sMedical Center of Boston.

NCT01144026 TUTI-16 To study of TUTI-16 in HIV-1 infected and uninfected subjects Phase 2completed

Synthetic-Thymon UniversalTat Immunogen

Mardik Donikyan,Clinilabs.

NCT00001790 FK463 To evaluate the safety, tolerance, and pharmacokinetics of FK463,a novel echinocandin (cell wall-active antifungal lipopeptide),as early empirical therapy for prevention of fungal infections inimmunocompromised children.

Phase 1completed

Echinocandin (cellwall-active antifungallipopeptide)

National Institutes ofHealth Clinical Center(CC)

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mammalian cells (Kanlayavattanakul and Lourith, 2010), haveattested safe application, and minor irritation to human skin. Somecompanies have used several surfactin derivatives in dermatologicalproducts and in cleansing cosmetics with a highly washable capability(Kanlayavattanakul and Lourith, 2010). A high-quality cosmetic is mainlycharacterized by properties which stimulate the production of new colla-gen and elastin. With the increasing age of the individual and decreasinglevels of growth factors, the skin loses elastin causing finewrinkles to ap-pear. A few USA-based cosmetic companies have already developed andmarketed their lipopeptide-formulated products that directly help tostimulate collagen and elastin production as anti-aging agents. Antioxi-dant supplementation to cosmetics is themost effective way for delayinglipid peroxidation. Antioxidants protect the skin from free radical damagefor smoother and softer skin.

Indeed, lipopeptides are well-characterized for their antioxidantproperties (Singh and Cameotra, 2004). In addition to use for cleansing,anti-wrinkle, stimulation of collagen and elastin production, and freeradical scavenging and moisturizing agents, they have broad-spectrumantimicrobial activity (Sun et al., 2006). Thus, lipopeptides supporthealthy skin physiology with several types of facial cosmetics, lotions,and beautywashes. To expand biotechnology to a profitable industry, ge-netic manipulation of organisms is essential for high yield and high qual-ity production of lipopeptides in the future.

6. Lipopeptides in the food industry

Lipopeptides are well-characterized in terms of their anti-adhesive,antimicrobial, antiviral and antitumor activities, which ensure theirposition in the pharmaceutical and cosmetic industries. In the foodindustry, lipopeptides are used as emulsifiers in the processing ofraw materials. In the baking industry, surfactins and rhamnolipidsare used to maintain stability, texture, and volume, and also to help inthe emulsification of fat tissue in order to control fat globule agglomer-ation. Recently, some lipopeptides isolated from Enterobactercloaceaehave been introduced into the food industrywith their high emulsifyingproperties, due to ability to enhance viscosity at acidic pH. Every foodmanufacturer uses preservatives during processing to avoid rapid spoil-age. Among bio-preservatives, several antimicrobial compounds (morethan 500) have been accepted. These compounds effectively controlfood microbes (Ricca et al., 2004; Stein, 2005). Antimicrobial peptidesas food preservatives are limited by their sensitivity to proteases. How-ever, this sensitivity can be prevented with the use of ring-structuredpeptides such as lipopeptides. The lipopeptide class of the iturin groupcontains a cyclic heptapeptideacylated with ß-amino fatty acids

(chain length C14-C16) and the Fengycin class consists of a ß-hydroxyfatty acid with unusual amino acids such as ornithine andallo-threonine. The surfactin family also contains a cyclic heptapeptidethat forms a lactone bridgewith ß-hydroxy fatty acids. They are proteaseresistant, exhibiting strong growth inhibition of a wide range of plantand fungal pathogens (Fusarium graminearum, Rhizoctonia solani andAspergillus flavus) or post-harvest pathogens such as Botritis cinereaand Penicillium expansum (Asaka and Shoda, 1996; Toure et al., 2004).Gandhi and Skebba (Skebba, 2007) recommended 0.10% rhamnolipidsurfactant in formulations ofmuffins and croissants in order to improvethe moisture content, texture, and freshness for longer periods.Lipopeptides also prevent corrosion agents on stainless steel surfaces.Considering this, Dagbert et al. (2006) provided evidence that AISI304 stainless steel corrosion is delayed in the presence of thebiosurfactant produced by a P. fluorescens strain. Thus, lipopeptidessatisfy several characteristics of a beneficial additives for emulsifying,antiadhesive, and antimicrobial activities which suggest their applica-tion as multipurpose ingredients or additives. As food additives in theglobal market, their sales values are continually growing at a rate ofabout 2%–3% per year. In terms of market increase, the most significantgrowth rates in food additives were observed in emulsifiers (up 10.5%)and hydrocolloids (up 6.0%) (Freire et al., 2009). Hence, lipopeptides inthe near future will represent a significant percentage of themarket forfood additives.

7. Lipopeptides in the biotech industry

The multipurpose applications of lipopeptides are compelling in amarket where production is highly expensive. Attempts were madewith great emphasis on the utilization of various cheap agro-industrial substrates and large-scale production. A review article bySaharan et al. (2011) eloquently described the utilization and evalua-tion of raw materials with different approaches for their commercialproduction and current developments. They also provided insightsinto the role of various developed processes such as fermentation,optimization, product recovery, and substrate utilization for theirmanagement of cost-effective production and limitations. Biosurfactantsare microbial surface-active agents, a product of the biotechnology usedin various industrial, environmental and medical applications (Koglin etal., 2010; Mulligan, 2005). Increasingly, the demand for biosurfactantproducts is due to their biodegradability, low toxicity, biocompatibility,digestibility and excellent surface activity, and effectiveness under ex-treme temperature and pH conditions (Mulligan, 2005). Solubilization,emulsification, dispersion, wetting, foaming and detergent capacity, and

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antimicrobial activity are the major functions of biosurfactants (Banat etal., 2010). Biosurfactants are produced from cheap raw materials whichare very easily available in large quantities, typically from hydrocarbons,carbohydrates and lipids. Biotechnology markets accept the challengesfor lowering the product cost by the utilization of the cheapest rawmaterials (Winterburn andMartin, 2012). They are trying to geneticallymanipulate or alter the lipopeptide structure to selectively enhanceantimicrobial and other activities (Koglin et al., 2010). Recently,lipopeptide AB has been marketed by a company based in Solna,Sweden. The company has developed a pharmaceutical product inwhich LL-37 is integrated into a thin film and lipopeptide AB operatesas a subsidiary of pergamumAB. Several lipopeptides have beenmanip-ulated for alteration of physicochemical extremities,which is also usefulduring formulation. Now, several small biotech industries are growingwith the ability to focus on the development of engineered strains forcost-effective production. Furthermore, novel structurally alteredlipopeptides produced by site-directedmutations are also in the industrypipeline (Mukherjee and Das, 2010).

8. Conclusions and future perspectives

Due to their huge number of applications, lipopeptides are extremelyuseful molecules. Nowadays lipopeptides have been applied in severalindustries, such as pharmaceutical, cosmetic, food and biotechnology,where antimicrobial, surfactant, and emulsifying chemical propertiesare used. The production and application of thesemolecules are very im-portant in world commerce, mainly in the pharmaceutical industry.However, some lipopeptides, due to the size of the molecule, have ahigh production cost,which does not allow large-scale synthesis. In addi-tion, there are limitations on the bioavailability of lipopeptides since theycan be degraded by the body's peptidases. Thus, investments in researchand development of new lipopeptides, whether natural or synthetic, areextremely important, and the knowledge of lipopeptide chemical varia-tions can be very useful to achieve this end.

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