Metabolic and Lipid Biomarkers for Pathogenic Algae, Fungi, Cyanobacteria, Mycobacteria, Gram-Positive Bacteria, and Gram-Negative Bacteria

The utilization of metabolomics and lipidomics analytical platforms in the study of pathogenic microbes is slowly expanding. These research approaches will significantly contribute to the establishment of microbial metabolite and lipid databases of significant value to all researchers in microbiology. In this review, we present a high-level overview of some examples of biomarkers that can be used to detect the presence of microbes, monitor the expansion/decline of a microbe population, and add to our understanding of microbe biofilms and pathogenicity. In addition, increased knowledge of the metabolic functions of pathogenic microbes can contribute to our understanding of microbes that are utilized in diverse industrial applications. Our review focuses on lipids, secondary metabolites, and non-ribosomal peptides that can be monitored using electrospray ionization high-resolution mass spectrometry (ESI-HRMS).


Introduction
Metabolomics and the subfield of lipidomics are technologies that provide valuable data regarding microbial strain identification, metabolism, pathogenicity, drug-resistance, structural adaptations, and cell signaling [1,2].Microbial lipids are significantly different from those of mammals, containing very long and very short fatty acyl chains attached to assorted headgroups.These headgroups include glycerol, sugars, fatty alcohols, amino acids, and peptides [3].This is a review of these lipids and secondary metabolites unique to pathogenic microbes.We highlight a number of areas where high-resolution mass spectrometry has been utilized to assess the members of complex lipid or metabolite families.References to more detailed studies of individual families and to the mass spectral and tandem mass spectral data are provided.
It is important to raise a key issue at this point.There is only a limited number of studies in the literature regarding microbial lipidomics, and more research needs to be undertaken to increase our understanding of the biomarkers reviewed here.This is essential to define the mechanisms of microbial and polymicrobial biofilm formation.Biofilms are considered critical pathogenic factors in many acute and chronic infections and encompass a large variety of lipid molecules.The biomarkers in this review have all been verified by MS/MS and NMR analyses.
This review presents established lipid and metabolic biomarkers of pathogenic microbes.The utility of these biomarkers remains to be more fully explored.Our focus was on biomarkers that can be monitored in biofluids to assess microbial infections.Our review also focuses on electrospray high-resolution mass spectrometric (ESI-HRMS) data since this methodology provides both sensitivity and specificity to monitor even trace microbial infections.The need for HRMS is also stressed since lipid isobars can confound mass spectrometric data that are obtained with low resolution [3].

Pathogenic Algae, Cyanobacteria, and Fungi
Prototheca spp.and Chlorella spp.algae are opportunistic eukaryotes that enter damaged tissue surfaces and mucous membranes.Cyanobacteria are prokaryotes universally present in fresh and marine waters.Ingestion of these bacteria results in the absorption of a variety of toxins produced by cyanobacteria.The cell wall of algae is composed of polysaccharides and glycoproteins, while cyanobacteria lack a cell wall.
Pathogenic fungi are eukaryotes encompassing approximately 300 pathogenic species that possess a number of unique metabolic and lipid biomarkers.Fungi do not possess chloroplasts, and their cell wall is unique in that it includes a chitin-glucan polysaccharide complex.
Other possible functional roles include the regulation of DNA synthesis [9] and stimulation of glycosyltransferases involved in monohexosyldiacylglycerol (MHDG) and dihexosyldiacylglycerol (DHDG) synthesis [10].

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Glycerolipids, which includes MHDG and DHDG, are also localized to the thylakoid membrane of chloroplasts [3,4].The hexosyl substituent can be glucose or galactose (Figure 1).However, these glycolipids are also essential lipids in the membranes of Gram-positive bacteria.

Cyanobacterial and Fungal Non-Ribosomal Peptide Synthesis (NRPS)
Bacteria and fungi possess non-ribosomal peptide synthetases (NRPSs), which are multi-modular enzyme complexes responsible for the biosynthesis of secondary metabolites.These include a number of linear or cyclic end-products [12][13][14].In cyanobacteria, NRPSs generate a number of cyclic 5 amino acid peptide families (Table 1; Figure 2) with an exocyclic amino acid attached via a ureido linkage.The D-lysine in position 2 enables the cyclic structure via type III polyketide synthase.Amino acids can be modified by methylation, acetylation, and/or acylation via fatty acyl AMP ligases (FAALs).These secondary metabolites are potent enzyme inhibitors (proteases, carboxypeptidases, and phosphatases).
Fungal peptides are diverse in nature and unique from bacterial peptides but not as widespread as polyketides, alkaloids, and terpenoids.Examples of fungal antimicrobial cyclic peptides include (Figure 3) the following:

Large Member Families
Small Member Families

Cyanobacterial and Fungal Non-Ribosomal Peptide Synthesis (NRPS)
Bacteria and fungi possess non-ribosomal peptide synthetases (NRPSs), which are multi-modular enzyme complexes responsible for the biosynthesis of secondary metabolites.These include a number of linear or cyclic end-products [12][13][14].In cyanobacteria, NRPSs generate a number of cyclic 5 amino acid peptide families (Table 1; Figure 2) with an exocyclic amino acid attached via a ureido linkage.The D-lysine in position 2 enables the cyclic structure via type III polyketide synthase.Amino acids can be modified by methylation, acetylation, and/or acylation via fatty acyl AMP ligases (FAALs).These secondary metabolites are potent enzyme inhibitors (proteases, carboxypeptidases, and phosphatases).

Large Member Families
Small Member Families Fungal peptides are diverse in nature and unique from bacterial peptides but not as widespread as polyketides, alkaloids, and terpenoids.Examples of fungal antimicrobial cyclic peptides include (Figure 3) the following:

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Fatty acid esters: Chlorosphaerolactylates; Nocuolactylates; The actions of lactylates remain to be elucidated since they only demonstrate weak antimicrobial activity [22].

Cyanobacterial and Fungal Indole Alkaloids
Cyanobacteria produce a variety of complex indole alkaloid families [19][20][21].Several examples are presented in Table 2 along with their biological activities.These alkaloid families are characterized by reactive isonitrile functional groups.Most, but not all, family members are also halogenated with a chlorine substitution (Figure 5).
Fungal indole alkaloids are mycotoxins with complex ring extensions of the core indole nucleus.Examples include the fumitremorgins, fumigaclavines, and fumiquinazolines of Aspergillus sp.(Figure 6).Fungi produce a diverse array of other complex alkaloids (Figure 7).Several examples from Aspergillus sp.include sesquiterpenoid pyripyropenes and the complex isoprenoid fumagillin [25].

Cyanobacterial and Fungal Indole Alkaloids
Cyanobacteria produce a variety of complex indole alkaloid families [19][20][21].Several examples are presented in Table 2 along with their biological activities.These alkaloid families are characterized by reactive isonitrile functional groups.Most, but not all, family members are also halogenated with a chlorine substitution (Figure 5).

Ambiguine B
Hapalinole A Fischerindole L Welwitindolinone A  Fungal indole alkaloids are mycotoxins with complex ring extensions of the core indole nucleus.Examples include the fumitremorgins, fumigaclavines, and fumiquinazolines of Aspergillus sp.(Figure 6).Fungi produce a diverse array of other complex alkaloids (Figure 7).Several examples from Aspergillus sp.include sesquiterpenoid pyripyropenes and the complex isoprenoid fumagillin [25].

Carotenoids
Algae and cyanobacteria contain high levels of several carotenoids not present in bacteria or fungi.The biosynthesis of carotenoids utilizes geranylgeranyl pyrophosphate as a precursor [27], generating halocynthiaxanthin-3-acetate and phoenicoxanthin in algae and echinenone in cyanobacteria (Figure 8).

Fungal Ergosterol
Bacteria and fungi synthesize a wide variety of sterols.Fungi are unique in that the major membrane steroid is ergosterol (Figure 9), not cholesterol, representing a lipid biomarker for fungi [28].The biosynthesis of alkaloids in cyanobacteria and fungi utilizes alkaloid synthesis gene clusters to provide the sequential enzyme activities.An example is the synthesis of fumigaclavine C (Figure 6), which requires a reductase followed by a P450 monooxygenase, followed by an acetyl transferase, and ultimately a prenyltransferase to generate the endproduct, which is a virulence factor in Aspergillus fumigatus [26].

Carotenoids
Algae and cyanobacteria contain high levels of several carotenoids not present in bacteria or fungi.The biosynthesis of carotenoids utilizes geranylgeranyl pyrophosphate as a precursor [27], generating halocynthiaxanthin-3-acetate and phoenicoxanthin in algae and echinenone in cyanobacteria (Figure 8).

Carotenoids
Algae and cyanobacteria contain high levels of several carotenoids not present in bacteria or fungi.The biosynthesis of carotenoids utilizes geranylgeranyl pyrophosphate as a precursor [27], generating halocynthiaxanthin-3-acetate and phoenicoxanthin in algae and echinenone in cyanobacteria (Figure 8).

Fungal Ergosterol
Bacteria and fungi synthesize a wide variety of sterols.Fungi are unique in that the major membrane steroid is ergosterol (Figure 9), not cholesterol, representing a lipid biomarker for fungi [28].

Fungal Ergosterol
Bacteria and fungi synthesize a wide variety of sterols.Fungi are unique in that the major membrane steroid is ergosterol (Figure 9), not cholesterol, representing a lipid biomarker for fungi [28].

Fungal Ergosterol
Bacteria and fungi synthesize a wide variety of sterols.Fungi are unique in that the major membrane steroid is ergosterol (Figure 9), not cholesterol, representing a lipid biomarker for fungi [28].

Fungal Glycosylinositol-Phosphorylceramides (GIPCs)
GIPCs are membrane lipids with a glucuronic acid-inositol-phosphate substituent on a ceramide (Figure 10).These unique lipids are critical in the infection of a host.In filamentous fungi (e.g., Aspergillus fumigatus), these lipids are involved in adhesion, signal transduction, and modulation of the host immune response [30].

Fungal Glycosylinositol-Phosphorylceramides (GIPCs)
GIPCs are membrane lipids with a glucuronic acid-inositol-phosphate subs on a ceramide (Figure 10).These unique lipids are critical in the infection of a h filamentous fungi (e.g., Aspergillus fumigatus), these lipids are involved in adhesion, transduction, and modulation of the host immune response [30].

Mycolic Acids
The cell wall of mycobacteria is extremely complex, with the cytoplasm con within a plasma membrane (lipid bilayer).The subsequent layers include the peri the peptidoglycan layer, the arabinogalactan layer, and the outermost mycomem which is unique to Gram-positive mycobacteria (e.g., Mycobacteria spp.and Salm spp.).Lipids of the mycomembrane that can be monitored in free forms include m acids, trehalose/glucose monomycolates (Figure 11), and dimycocerosates.The inne let of the outer mycomembrane of mycobacteria contains high levels of mycolic acid 33].These fatty acids are composed of a very long chain with a β-hydroxy substitue an α-alkyl side-chain.The long-chain region can also contain cyclopropane rings, a as methoxy or keto substituents.Another lipid family unique to mycobacteria is glyc tidolipids (GPLs; Section 3.3), which are inserted at the cytoplasmic surface of the plasma membrane and the outer mycomembrane.

Mycobacteria 3.1. Mycolic Acids
The cell wall of mycobacteria is extremely complex, with the cytoplasm contained within a plasma membrane (lipid bilayer).The subsequent layers include the periplasm, the peptidoglycan layer, the arabinogalactan layer, and the outermost mycomembrane which is unique to Gram-positive mycobacteria (e.g., Mycobacteria spp.and Salmonella spp.).Lipids of the mycomembrane that can be monitored in free forms include mycolic acids, trehalose/glucose monomycolates (Figure 11), and dimycocerosates.The inner leaflet of the outer mycomembrane of mycobacteria contains high levels of mycolic acids [31][32][33].These fatty acids are composed of a very long chain with a β-hydroxy substituent and an α-alkyl side-chain.The long-chain region can also contain cyclopropane rings, as well as methoxy or keto substituents.Another lipid family unique to mycobacteria is glycopeptidolipids (GPLs; Section 3.3), which are inserted at the cytoplasmic surface of the inner plasma membrane and the outer mycomembrane.

Glycopeptidolipids (GPLs)
GPLs are complex hybrid molecules [33,34].The peptidolipid scaffold consists of hydroxy or 3-methoxy fatty acid of 28 to 38 carbons linked to a tripeptide-aminoalco (alaninol) core (Figure 12).The tripeptide core consists of D-Phe-allo-Thr-D-Ala. Glyco ation involves a 6-deoxy-α-L-talose linked to D-allo-Thr and α-L-rhamnose linked to ninol.Talose also can be mono-or di-acetylated, while rhamnose can be mono-, di-, or methylated, adding to the complexity and diversity of the GPL family.GPLs at the ex nal surface of the mycomembrane are thought to be involved in both bacterial sliding havior and in the formation of invasive biofilms.

Lipoteichoic Acids (LTAs)
Gram-positive bacteria possess a cytoplasmic membrane and a multilaminar cell w [1].Between the cell membrane and cell wall is a heteropolysaccharide meshwork of p tidoglycans and arabinogalactans.Teichoic and lipoteichoic acids anchor to peptidog cans in the cell wall and, as such, are lipids unique to Gram-positive bacteria, providin strong negative charge to the cell wall [2].However, teichoic and lipoteichoic acids large molecular weight polymers not amenable to simple extraction.Therefore, precurs to LTAs are monitored as biomarkers in Gram-positive bacteria.The specific lipids inclu dihexosyl diacylglycerols (DHDGs; Figure 13) and DHDG-glycerol phosphate (DHD GroP), with phosphoglycerol attached at the 6-hydroxy group of the terminal hexose, a termed an LTA primer (LTAP).LTAP can also be modified by mono-and di-addition alanine at sn-2 and sn-3 of the glycerol [35,36].

Glycopeptidolipids (GPLs)
GPLs are complex hybrid molecules [33,34].The peptidolipid scaffold consists of a 3-hydroxy or 3-methoxy fatty acid of 28 to 38 carbons linked to a tripeptide-aminoalcohol (alaninol) core (Figure 12).The tripeptide core consists of D-Phe-allo-Thr-D-Ala. Glycosylation involves a 6-deoxy-α-L-talose linked to D-allo-Thr and α-L-rhamnose linked to alaninol.Talose also can be mono-or di-acetylated, while rhamnose can be mono-, di-, or tri-methylated, adding to the complexity and diversity of the GPL family.GPLs at the external surface of the mycomembrane are thought to be involved in both bacterial sliding behavior and in the formation of invasive biofilms.

Glycopeptidolipids (GPLs)
GPLs are complex hybrid molecules [33,34].The peptidolipid scaffold consists of a 3hydroxy or 3-methoxy fatty acid of 28 to 38 carbons linked to a tripeptide-aminoalcohol (alaninol) core (Figure 12).The tripeptide core consists of D-Phe-allo-Thr-D-Ala. Glycosylation involves a 6-deoxy-α-L-talose linked to D-allo-Thr and α-L-rhamnose linked to alaninol.Talose also can be mono-or di-acetylated, while rhamnose can be mono-, di-, or trimethylated, adding to the complexity and diversity of the GPL family.GPLs at the external surface of the mycomembrane are thought to be involved in both bacterial sliding behavior and in the formation of invasive biofilms.

Lipoteichoic Acids (LTAs)
Gram-positive bacteria possess a cytoplasmic membrane and a multilaminar cell wall [1].Between the cell membrane and cell wall is a heteropolysaccharide meshwork of peptidoglycans and arabinogalactans.Teichoic and lipoteichoic acids anchor to peptidoglycans in the cell wall and, as such, are lipids unique to Gram-positive bacteria, providing a strong negative charge to the cell wall [2].However, teichoic and lipoteichoic acids are large molecular weight polymers not amenable to simple extraction.Therefore, precursors to LTAs are monitored as biomarkers in Gram-positive bacteria.The specific lipids include dihexosyl diacylglycerols (DHDGs; Figure 13) and DHDG-glycerol phosphate (DHDG-GroP), with phosphoglycerol attached at the 6-hydroxy group of the terminal hexose, also termed an LTA primer (LTAP).LTAP can also be modified by mono-and di-additions of alanine at sn-2 and sn-3 of the glycerol [35,36].

Gram-Positive Bacteria 4.1. Lipoteichoic Acids (LTAs)
Gram-positive bacteria possess a cytoplasmic membrane and a multilaminar cell wall [1].Between the cell membrane and cell wall is a heteropolysaccharide meshwork of peptidoglycans and arabinogalactans.Teichoic and lipoteichoic acids anchor to peptidoglycans in the cell wall and, as such, are lipids unique to Gram-positive bacteria, providing a strong negative charge to the cell wall [2].However, teichoic and lipoteichoic acids are large molecular weight polymers not amenable to simple extraction.Therefore, precursors to LTAs are monitored as biomarkers in Gram-positive bacteria.The specific lipids include dihexosyl diacylglycerols (DHDGs; Figure 13) and DHDG-glycerol phosphate (DHDG-GroP), with phosphoglycerol attached at the 6-hydroxy group of the terminal hexose, also termed an LTA primer (LTAP).LTAP can also be modified by mono-and di-additions of alanine at sn-2 and sn-3 of the glycerol [35,36].large molecular weight polymers not amenable to simple extraction.Therefore, precursors to LTAs are monitored as biomarkers in Gram-positive bacteria.The specific lipids include dihexosyl diacylglycerols (DHDGs; Figure 13) and DHDG-glycerol phosphate (DHDG-GroP), with phosphoglycerol attached at the 6-hydroxy group of the terminal hexose, also termed an LTA primer (LTAP).LTAP can also be modified by mono-and di-additions of alanine at sn-2 and sn-3 of the glycerol [35,36].

Modified Diacylglycerols
Trihexosyl diacylglyceriols (triHDGs) and acyl-triHDGs are lipids unique to some Clostridium spp.and Romboutia spp.and are useful biomarkers to identify bacterial subspecies [40,41].Another unique modified DG family in these bacterial species is Nacetylglucosaminyl-DGs [40,42].These lipids are also further modified by the addition of a phosphoethanolamine group to the carbohydrate moiety [42].

Modified Diacylglycerols
Trihexosyl diacylglyceriols (triHDGs) and acyl-triHDGs are lipids unique to some Clostridium spp.and Romboutia spp.and are useful biomarkers to identify bacterial subspecies [40,41].Another unique modified DG family in these bacterial species is N-acetylglucosaminyl-DGs [40,42].These lipids are also further modified by the addition of a phosphoethanolamine group to the carbohydrate moiety [42].

Aminoacyl Fatty Acyls of Hydroxy Fatty Acids (FAHFAs)
Gram-negative bacteria lack the cell wall characteristic of Gram pid A is a major membrane lipid in the cell envelope, composed membrane with an intermediate peptidoglycan layer (e.g., Bacteroid romonas gingivalis).Intact lipid A molecules are large and tethered quiring acid hydrolysis prior to mass spectrometric analyses [49].
By contrast, a number of lipid A precursors are readily anal lipid extraction procedures.Modified fatty acyls of hydroxy fatty a example of these lipid A constituents that are absent from Gram-po Examples of this are the family of lipodipeptides, Gly-Ser-FAHFA ( Gly-Ser-FAHFA precursor/metabolite Gly-Ser-HFAs [3,52,53], and ily, Gly-Ser-Orn-FAHFA, all of which are 3-HFA in the FAHFAs [5 all generate the MS 2 products Gly, Ser, and Gly-Ser [51].The 3-H Ser-3-HFAs is validated with MS 2 loss of the 3-HFA as an aldehyde, from 2-HFAs.

Aminoacyl Fatty Acyls of Hydroxy Fatty Acids (FAHFAs)
Gram-negative bacteria lack the cell wall characteristic of Gram-positive bacteria.Lipid A is a major membrane lipid in the cell envelope, composed of an inner and outer membrane with an intermediate peptidoglycan layer (e.g., Bacteroiddetes spp.and Porphyromonas gingivalis).Intact lipid A molecules are large and tethered to the membrane, requiring acid hydrolysis prior to mass spectrometric analyses [49].

Glucosaminylphosphatidylglycerol (GlcN-PG)
Phosphatidylgycerols (PGs) are dominant glycerophospholipids in the membranes of Gram-negative bacteria and are involved in protein translocation across membranes.Pseudomonas aeruginosa modifies these GPLs by glycosylation to generate glucosaminyl-PGs ( [58]; Figure 17).These modified PGs may be involved in biofilm formation, which is a major characteristic of P. aeruginosa infections.This suggestion is bolstered by recent observations that poly (acetyl,arginyl)glucosamine disrupts biofilm formation by P. aeruginosa and resulted in microbial clearance in a rat model of lung infection [59].A family of Gly-Ser-FAHFA-phosphatidic acids (Gly-Ser-FAHFA-PAs) has also been isolated from some Gram-negative bacteria (e.g., P. gingivalis) [51,55].These lipopeptides all reside in the outer bacterial membrane [54].

Glucosaminylphosphatidylglycerol (GlcN-PG)
Phosphatidylgycerols (PGs) are dominant glycerophospholipids in the membranes of Gram-negative bacteria and are involved in protein translocation across membranes.Pseudomonas aeruginosa modifies these GPLs by glycosylation to generate glucosaminyl-PGs ( [58]; Figure 17).These modified PGs may be involved in biofilm formation, which is a major characteristic of P. aeruginosa infections.This suggestion is bolstered by recent observations that poly (acetyl,arginyl)glucosamine disrupts biofilm formation by P. aeruginosa and resulted in microbial clearance in a rat model of lung infection [59].
Since P. aeruginosa infections are associated with high morbidity and mortality; increased understanding of the dynamics and function of GlcN-PGs may be a new approach for the development of antimicrobial therapeutic strategies.
GlcN-PGs appear to be unique to P. aeruginosa, in that we have not monitored these lipids in a number of other microbes, presented as follows: Bacilli

Conclusions
This paper presents a high-level review of unique microbial biomarkers that are useful in microbiological research.Increasing our knowledge base in this area will lead to the improved identification of microbial infections, increased understanding of the complex roles of microbial lipids and secondary metabolites in cellular function and pathogenicity, and the development of new antimicrobial therapies.The importance of HRMS relates to the significant number of lipid and metabolite isobars.Utilizing ESI-HRMS, investigators will be able to evaluate all of the described lipids with a single organic extract [76].The teria to regulate their population density and associated biofilm formation (e.g., Pseudomonas aeruginosa).Interestingly, Escherichia spp.and Salmonella spp.can sense AHLs produced by other bacterial species but do not synthesize them [74].These bacteria produce alternate QS molecules like autoinducer-2 ([75]; Figure 23).

Conclusions
This paper presents a high-level review of unique microbial biomarkers that are useful in microbiological research.Increasing our knowledge base in this area will lead to the improved identification of microbial infections, increased understanding of the complex roles of microbial lipids and secondary metabolites in cellular function and pathogenicity, and the development of new antimicrobial therapies.The importance of HRMS relates to the significant number of lipid and metabolite isobars.Utilizing ESI-HRMS, investigators will be able to evaluate all of the described lipids with a single organic extract [76].The

Conclusions
This paper presents a high-level review of unique microbial biomarkers that are useful in microbiological research.Increasing our knowledge base in this area will lead to the improved identification of microbial infections, increased understanding of the complex roles of microbial lipids and secondary metabolites in cellular function and pathogenicity, and the development of new antimicrobial therapies.The importance of HRMS relates to the significant number of lipid and metabolite isobars.Utilizing ESI-HRMS, investigators will be able to evaluate all of the described lipids with a single organic extract [76].The more polar metabolites can be analyzed utilizing extraction with acetonitrile/methanol/formic acid [77].These analytical approaches enable the acquisition of a broad range of lipid and metabolite information with two analytical platforms.

Figure 5 .Figure 6 .
Figure 5. Structures of representative cyanobacterial indole alkaloids.These alkaloids are characterized by the reactive isonitrile functional group in all family members.

Figure 5 .
Figure 5. Structures of representative cyanobacterial indole alkaloids.These alkaloids are characterized by the reactive isonitrile functional group in all family members.

Figure 5 .Figure 6 .
Figure 5. Structures of representative cyanobacterial indole alkaloids.These alkaloids are characterized by the reactive isonitrile functional group in all family members.

Figure 7 .
Figure 7. Structures of fungal sesquiterpenoid pyripyropene A and the complex isoprenoid fumagillin.

Figure 7 .
Figure 7. Structures of fungal sesquiterpenoid pyripyropene A and the complex isoprenoid fumagillin.

Figure 7 .
Figure 7. Structures of fungal sesquiterpenoid pyripyropene A and the complex isoprenoid fumagillin.

Figure 9 .
Figure 9. Structures of ergosterol and a representative sophorolipid.Figure 9. Structures of ergosterol and a representative sophorolipid.

Figure 9 .
Figure 9. Structures of ergosterol and a representative sophorolipid.Figure 9. Structures of ergosterol and a representative sophorolipid.

Figure 12 .
Figure 12.General outline of the structure of GPLs.Variants include replacement of the Phe by ine, valine, or leucine.VLCFA: very-long-chain fatty acids, which are 3-hydroxy or 3-metho Amino acids are D isomers.

Figure 12 .
Figure 12.General outline of the structure of GPLs.Variants include replacement of the Phe by serine, valine, or leucine.VLCFA: very-long-chain fatty acids, which are 3-hydroxy or 3-methoxy.Amino acids are D isomers.

Figure 12 .
Figure 12. outline of the structure of GPLs.Variants include replacement of the Phe by serine, valine, or leucine.VLCFA: very-long-chain fatty acids, which are 3-hydroxy or 3-methoxy.Amino acids are D isomers.

Figure 20 .
Figure 20.Structure of sulfobacin A. Flavocristamide A, with 2 methy substituents on the fatty acid chains, is an isobar.Chromatography is needed to distinguish these two isobars.

Figure 20 .
Figure 20.Structure of sulfobacin A. Flavocristamide A, with 2 methy substituents on the fatty acid chains, is an isobar.Chromatography is needed to distinguish these two isobars.

Figure 20 .
Figure 20.Structure of sulfobacin A. Flavocristamide A, with 2 methy substituents on the fatty acid chains, is an isobar.Chromatography is needed to distinguish these two isobars.

Figure 20 .
Figure 20.Structure of sulfobacin A. Flavocristamide A, with 2 methy substituents on the fatty acid chains, is an isobar.Chromatography is needed to distinguish these two isobars.

Figure 20 .
Figure 20.Structure of sulfobacin A. Flavocristamide A, with 2 methy substituents on the fatty acid chains, is an isobar.Chromatography is needed to distinguish these two isobars.