Natural products comprise a variety of bioactive compounds like bacteriocins, terpenoids, antibiotics, and biosurfactants (Raaijmakers et al., 2010; Anuradha, 2010). Among these, biosurfactants are surface metabolites produced by microbes during the stationary phase of growth. The broad spectrum of structurally different biosurfactants, generally glycolipids, phospholipids, lipopeptides, neutral lipids, and fatty acids, consist of hydrophobic moiety (fatty acids) of varying lengths linked to hydrophilic peptide chains of 7–10 amino acids (Geys et al., 2014). Lipopeptides are one of the major classes of lower molecular weight biosurfactants produced by microorganisms. Around 90 compounds from 26 different lipopeptide families have been identified in the last two decades (Liu et al., 2015). Microbial biosurfactants have gained special interest nowadays because of their surface and interfacial properties, eco-friendly characters, and diverse applications in industrial, health, and environmental sectors. For example, microbial glycoconjugates enhance degradation of toxic organic pollutants by increasing their bioavailability by lowering surface tension and producing a solvent interface (Bhatt et al., 2021).

Biosurfactants are encoded by several genes and synthesized by a variety of microorganisms such as Bacillus sp., Pseudomonas sp., Acinetobacter sp., Candida sp., Cryptococcus sp., Penicillium sp., Aspergillus sp., Kurtzmanomyces sp., Rhodococcus sp., Sphingomonas sp., Arthrobacter sp., Lactococcus sp., and Pseudozyma sp. (Borah et al., 2021). Among these, the genus Bacillus is the most prevalent in biosurfactant production. Carolin et al. (2021) reported on the lipopeptide biosurfactant production by Bacillus sp., and its significant role in the degradation of aromatic amine 4-chloroaniline compound. Similarly, biosurfactants also exhibit a promising potential to efficiently eliminate toxic heavy metal pollutants by using multiple biosurfactant-metal binding stratagems, that is, emulsification, mobilization, complexation, and solubilization (Mishra et al., 2021).

Lipopeptides are biosurfactants mainly comprise a hydrophilic peptide ring and hydrophobic fatty acid moieties. Based on their structure, they are further characterized into cyclic lipopeptides (CLPs) and linear lipopeptides. The cyclic lipopeptides, commonly produced by Bacillus subtilis, include fengycin, surfactin, and iturin. They are composed of a peptide ring of 7 or 10 amino acids with a long hydrophobic fatty acid chain. The fatty acid chain lengths vary in each type, that is, surfactin has a chain length of C₁₃–C₁₆, while iturin’s chain is C₁₄–C₁₇, and that of fengycin is C₁₄–C₁₈ (Hu et al., 2019).

Surfactin has many congeners because of the variation in the length of the fatty acid chain and the types of amino acids. The structure of surfactin offers diverse applications in several global issues such as medicine and environmental protection (Gudiña et al., 2013). It was discovered as a potent fibrin clot-inhibitor and as an antibacterial and antiviral agent. It also showed hypocholesterolemic and anti-tumor activities. Owing to its remarkable surface-, interface-, and membrane-active action, it has the ability to cross plasma membrane barriers and viral envelopes (Santos et al., 2018). It can lower the surface tension of water from 72 to 27 mN/m and is highly thermally stable and salt tolerant. Because of these properties, it possesses huge potential in the microbial enhancement of oil recovery (MEOR) and considered a good candidate for the bioremediation of contaminated soils and sub-surface environments (Hu et al., 2019).

Fengycin is another lipopeptide consisting of a decapeptide linked to a saturated or unsaturated fatty acid (C14–C18). The ring structure of its cyclic peptide is formed by linking the residue at position 3 to the C-terminal–COOH group of the amino acid at position 10 (Fanaei et al., 2021). Fengycins are predominantly produced by Bacillus spp. with potential bioactivities. They are active against phytopathogenic fungi and bind with their plasma membrane, thereby causing cell lysis and leakage (Gimenez et al., 2021).

The biosynthesis of these lipopeptides is governed by a complex set of proteins called non-ribosomal peptide synthetases (Wang et al., 2014). Gene clusters for the synthesis of such compounds have been found in the Bacillus genera (Donio et al., 2013). Several structurally and functionally diverse molecules need to be screened and characterized to create a better understanding of such biomolecules (Lu et al., 2014; Aleti et al., 2015; Singh and Tiwary, 2016).

Next-generation sequencing has revolutionized the discovery of natural products, chemicals, and biosynthetic enzymes greatly used in the fields of biotechnology and biomedicine (Luo et al., 2014). Biosurfactant production in various Bacillus species has been widely reported in many studies. Moreover, many Bacillus species are known for their metabolic capability, environmental versatility, and their ability to remove xenobiotic compounds and heavy metals via biosurfactant production (Arora, 2020). However, only limited literature is available on the genetic basis of biosurfactant production in bacteria. To this end, our research is focused on the whole genome sequencing and analysis of biosurfactant-producing and hydrocarbon-degrading indigenous Bacillus subtilis strains MB415 and MB418 isolated from hydrocarbon-contaminated soil. The genome analysis of both strains also indicated the gene clusters for surfactin and fengycin lipopeptides.

In the present study, we investigated the biosurfactant production from the hydrocarbon degraders of the Missa Kiswal oilfield. Two B. subtilis strains were able to produce lipopeptide biosurfactants. Similarly, a study reported the strain B. amyloliquefaciens An6 as a potent biosurfactant producer with diesel oil degradation efficiency (Ayed et al., 2015). Hydrocarbon-degrading bacteria possess an innate potential to produce biosurfactants which aid them in the bioavailability of hydrocarbon substrates (Antoniou et al., 2015).

The genomes of the strains isolated in the present study also decipher Srf operons responsible for the biosynthesis of surfactin and fengycins. In the case of B. subtilis, mostly fengycin and surfactin have been reported in many studies (Pereira et al., 2013). Jadeja et al. (2019) reported the sfp and NPRS gene in Bacillus sp. AKBS9 and emulsan biosynthetic gene cluster in Acinetobactor sp. AKBS16 for biosurfactant production through the whole genome sequence analysis of the aforementioned strains.

B. subtilis is one of the predominant lipopeptide cell factories (Steller et al., 1999). The genetic makeup of biosurfactant-producing organisms is one of the major factors governing the biosynthesis of biosurfactants. Different molecular studies demonstrated the metabolic pathways, operons, and enzymes for the extracellular production of many biosurfactants. Wang et al. (2022) identified entire core synthetic genes for bacillibactin, fengycin, and surfactin production in a novel facultative-halophilic, long-chain hydrocarbon degrader, that is, Bacillus sp. XT-2. Surfactin is a cyclic lipopeptide produced non-ribosomally by surfactin synthetase, a multi-enzyme peptide synthetase complex. The biosynthesis of other lipopeptides is also mediated by similar enzyme complexes. Non-ribosomal peptide synthetases (NRPSs) involved in lipopeptide biosynthesis exhibit a high degree of similarity in structural conformation even in highly distant microbial species (Dhasayan et al., 2015). Fengycin is another cyclic lipodecapeptide containing four d-amino acids and ornithine in the peptide chain. Fengycin looks like a mixture of isoforms that show differences in length and branching of the β-hydroxy fatty acid, and the amino-acid composition of the peptide ring (Hu et al., 2007).

The genomes presented in our study decrypt the B. subtilis sequence variation and detailed comparisons of this vital model species. Comparisons of whole-genome sequences of different bacteria revealed significant genomic variability among phylogenetically interrelated bacterial species. Even sequences with 100% homology exhibit low conservation in the total gene content. B. subtilis is one of the most extensively studied spore forming, non-pathogenic, gram-positive bacteria ubiquitously found in soil (Earl et al., 2007; Chen et al., 2013). Another study demonstrated the complete genome sequence analysis of polystyrene-degrading deep sea B. paralicheniformis G1 strain comprising 4,281,959 bp with 45.88% GC content and encoded 4,213 protein-coding genes. Numerous genes encoding monooxygenase, dioxygenase, peroxidase, esterase, and hydrolase involved in the degradation of synthetic polymers were identified along with the genes associated with flagellum-dependent motility, chemotaxis, biofilm formation, and siderophores biosynthesis (Kumar et al., 2021).

The genetic regulation of lipopeptides is governed by the four open reading frames (ORFs) in the srfA operon directing the surfactin synthesis including srfAA, srfAB, srfAC, and srfAD (Zhu et al., 2021). Surfactin is synthesized by a multi-enzyme synthetase complex mainly comprising SrfA, SrfB, and SrfD subunits. Srf operon for biosurfactant pathways consists of the gene srfAA, -AB, -AC, and–AD in Bacillus spp. The annotation analysis of the genome B. subtilis MB415 and MB418 revealed that both strains also possess this operon (Figure 5). According to previous studies, in the Bacillus subtilis species, the synthesis of surfactin is based on three amino-acid activating components of surfactin synthetase (SrfA, -B, and -C) that are activated by SrfD. The gene srfAD also possesses thioesterase that is required for the last amino acid in the growing peptide chain that results in the cyclic structure of the biosurfactant and B. subtilis (Vedaraman and Venkatesh, 2011).

A comparative genome analysis showed gene arrangements of surfactin-producing operon in the draft genome of B. subtilis MB418 shared its neighborhood region showing the same top COG hits with Bacillus subtilis inaquosorum BGSC 3A28 and B. subtilis B4143. With the difference in the SrfAA protein, in Bacillus subtilis MB418 there are 3,586 amino-acid sequences, while it is 3,587 amino acids in the rest of the two closely related genomes. However, in the case of MB415, the operon is entirely different with genes encoding for more proteins and a different arrangement as shown in Figure 6.

The gene srfAB encodes for surfactin family lipopeptide synthetase AB and consists of 3,329 amino acids. A comparison of the gene neighborhood showed maximum similarity with B. subtilis B4071 (Berendsen et al., 2016) having the gene for the protein SrfAB (2695aa) (Figure 6). With the exception, B. subtilis MB415 had an additional gene for non-ribosomal peptide synthase not present in the genome of B4071. The gene operon in the genome of MB415 ends at gene (ycxA) for sugar-phosphate permease, while in the B. subtilis MB4068 (Berendsen et al., 2016) operon, this gene is replaced with another gene for the major facilitator superfamily (Figure 6).

Furthermore, genomes which carry genes for the same proteins vary in sequence length than the ones present in the draft genome of MB415. For example, the srfAA gene of B. subtilis MB415 is of 10,764 bp codes for 3,587 amino acids. However, this gene sequence is relatively small in the previously reported surfactant-producing B. safensis CCMA-560 and B. amyloliqueficiens comprising 10, 713bps (3570aa) and 10, 701bps (3566aa), respectively. The gene srfAC (3828bp) sequence is the same in the genomes of B. subtilis MB418 and MB415 and in the rest of the Bacillus species. The gene encoding thioesterase in the genomes of both B. subtilis MB415 and MB418 comprises 3,834 bps while in B. pumilus, it is encoded by the srfAD gene (732bp) (Jiang et al., 2016). Similarly, surfactin produced by B. subtilis strains isolated from soil consists of seven amino acids and 13–15 lipid chains forming a cyclic lipopeptide as described previously by researchers (Sekhon et al., 2011; Sachdev and Cameotra, 2013).

The genome analysis of both isolates unveiled potential gene operons for the biosynthesis of a secondary metabolite, fengycin. Fengycin, also called plipastatin, is an anti-fungal antibiotic that inhibits filamentous fungi but is ineffective against yeast and bacteria (Volpon et al., 2000). It also has the potential to inhibit phospholipase and biofilm formation in many bacteria (Thaniyavarn et al., 2003). Fengycin is a type of lipodecapeptide with moderate surfactant activity normally produced by several strains of Bacillus spp. Fen operon comprises an N-acyl domain at the N-terminus of FenC and five non-ribosomal peptide synthetase subunits assembled in a co-linear chain in the following order: FenC, FenD, FenE, FenA, and FenB (Guo and Yu, 2014).

The fengycin synthetase gene operon in the draft genome of B. subtilis MB418 exhibited maximum similarity with B. subtilis B4071. However, in the genome of B. subtilis MB415, an esterase-producing gene is identified instead of fenB for fengycin lipopeptide synthetase E as indicated in other closely related Bacillus species (Figure 7). A recent study also reported three genes related to the gene cluster responsible for fengycin biosynthesis (fenBCD) in B. velezensis and B. siamensis (Zeng et al., 2021). Similarly, in another study, five ORFs, namely, FenA, FenB, FenC, FenD, and FenE, were identified for fengycin production in the Bacillus velezensis strain P45 (da Rosa et al., 2022).

Indigenous bacterial strains inhabiting a polluted environment acquire tremendous potential to utilize petroleum hydrocarbons than non-native microbes. The present study demonstrated the production of lipopeptide biosurfactants from B. subtilis MB415 and MB418. The genome analysis of both B. subtilis isolates MB415 and MB418 contain a gene locus that enhanced their proficiency in degrading oil compounds. The presence of genes for flagella and secondary metabolite biosynthesis supported the experimental outcomes of emulsification and oil utilization capacities of both strains.