Overview

Our team intends to enhance the yield of the Coffea arabica coffee variety by combating one of its most significant obstacles, the plant disease, "Coffee Leaf Rust," caused by the fungi Hemileia vastatrix (HV), which targets leaves and juvenile branches causing premature defoliation, a reduction in photosynthetic capacity, and overall weakening of the coffee trees Coffee Leaf Rust (CLR).

We have chosen Bacillus subtilis as our chassis of choice for this project due to its natural presence in the coffee plant microbiota and its proven ability to combat fungal pathogens. This natural association makes B. subtilis a more ecologically balanced option for controlling CLR, caused by HV, and reduces the risk of disturbing the natural balance of the ecosystem.

B. subtilis is a Gram-positive, spore-forming bacterium known for its robustness in various environmental conditions, making it an ideal candidate for use in agricultural settings. Furthermore, B. subtilis is well-documented for its ability to produce secondary metabolites, including the antifungal compound Iturin A, which has shown efficacy against H. vastatrix. This, combined with the bacterium's genetic tractability, allows us to genetically modify Iturin A production via promoter engineering. The use of B. subtilis as our chassis aligns with the project's goal of providing an eco-friendly, sustainable alternative to chemical fungicides while utilizing a microorganism that is both effective and naturally integrated into the coffee plant's biology.

There are various other compounds that may help the plant defend itself either by killing the fungus itself or by triggering the plant defenses​ [1].

Alternative lipopeptides

Kurstakins

Kurstakins are non-ribosomal lipo-heptapeptides that display antifungal activities. They perform general antifungal activity synergistically with other antifungals produced by the bacteria and are secreted during the sporulation phase​[2][17].

Histatin 5

The peptide histatin 5 binds to Ssa1p and is necessary for histatin 5 to be incorporated into cells. Histatin 5 is a surface protein on the fungus Candida albicans, which is responsible for dermatitis, oral thrush, and vaginal yeast infections. It leaks ROS and damages the cell membrane when it is endocytosed. Because humans can manufacture it and turn it into fungal cells like baker's yeast, it is safe for humans to consume​[1].

In non-aqueous settings, histatin 5 enables the peptide to adopt an α-helical shape. The peptide must be translocated in order to have an impact on C. albicans cells, and the polyamine transporters Dur3 and Dur31 are required for this process as well. Histatin 5 damages the integrity of the mitochondrial membrane when the cell is undergoing respiratory metabolism. ATP release and propidium iodide (PI) uptake happen even if the peptide doesn't seem to cause cell lysis. Subsequently, signaling cascades that cause cell death are triggered by ATP binding to surface P2X receptors​[1].

Rs-ARF2

Rs-ARF2 features three stranded β-sheets and an α-helix shape supported by four disulfide bridges and is a 50 amino acid residue plant defensin. The fungus-specific membrane glucosylceramide that induces membrane permeability and leads to Ca2+ uptake, K+ efflux, and medium alkalinization is the target of Rs-ARF2. Additionally, this defensin causes intracellular synthesis of harmful reactive oxygen species. A. flavus, Fusarium solani, Candida albicans, and Candida krusei were reported to be inhibited by Rs-AFP2 and its analogs (which substitute neutral amino acids with arginines). This fungus-specific ceramide is absent from C. glabrata, hence it is not inhibited. At dosages that suppress fungal infections, this peptide and its analogs (e.g., NaD1, Rs-ARF1, SPE10) have minimal cytotoxicity against mammalian cells​[3].

B291

On potato dextrose agar (PDA) plates, B291 effectively inhibits pathogens like Fusarium, Pythium, and Rhizoctonia. It also manages vegetable and melon wilt diseases in the field. This was verified by the study by examining B291's inhibitory effects on species belonging to these taxa. It inhibits the growth of mycelium in all examined species and prevents Fusarium oxysporum from germinating. B291's N-terminal aa sequence is different from that of other antifungal peptides that Bacillus synthesizes. Small random aa acid sequences did, however, match certain regions of other antifungals. Small and stable, antifungal proteins are known not to interfere with other pathways​[4].

Iturin

Iturin inhibits the growth of numerous phytopathogens and demonstrates potent antifungal action. It is a lipoheptapeptide cycle. The heptapeptide's seventh position is a Ser connected to a fatty acid chain with 14–17 C atoms by a β-amino acid residue. The heptapeptide has the amino acid sequence (L-)Asn-(D-)Tyr-(D-)Asn-(L-)Gln-(L-)Pro-(D)Asn-(L-)Ser. The fatty acid chains of the homologs A, C, D, and E differ from one another. Mycosubtilin is the name given to this iturin homolog if Asn7 and Ser6 are altered. Bacillomycin and bacillopeptin, which have distinct amino acids at the third, fourth, and fifth positions, are also members of the iturin family​[4].

Fengycin

Fengycin is recognized for its potent antifungal properties, particularly against filamentous fungi, making it a prime candidate for combating CLR due to its membrane-disrupting capabilities​[5][6].

It targets sterols in fungal cell membranes, altering membrane permeability and leading to cell lysis, effectively inhibiting pathogens like HV, the causative agent of CLR​[5][6].

The specificity of Fengycin did not align well with CLR's broader pathogenic mechanisms, and the complexity of its biosynthesis posed challenges for production optimization​[6][7].

Surfactin

Surfactin is a powerful biosurfactant known for its antimicrobial properties across various pathogens, including fungi. Its potential synergistic effects with other antifungal agents made it a candidate for enhanced efficacy against CLR​[6].

It disrupts cell membranes through surfactant properties, causing leakage of cellular contents and inhibiting biofilm formation, which can provide systemic protection in plants​[8][9].

Surfactin's antifungal activity is primarily effective against bacterial infections rather than fungal diseases. Its mechanism as a biosurfactant was not directly applicable to targeting CLR specifically, and the high production costs further limited its viability​[6].

Bacillomycin D & F

Bacillomycin lipopeptides are noted for their antifungal and antiviral properties, showing promise in controlling fungal diseases in plants, including CLR​[5][7].

These compounds disrupt fungal cell membranes by interacting with sterols, leading to cell leakage and eventual death of the fungal cells​[5][7].

Although Bacillomycin D & F exhibited good antifungal activity, they lacked sufficient specificity for CLR. Their complex regulation and biosynthesis would complicate engineering high-producing strains, and their overall efficacy against rust fungi was less impressive compared to alternatives like Iturin A, despite being part of the Iturin family​[6][7].

Mycosubtilin

Mycosubtilin is a potent antifungal lipopeptide with activity against various pathogenic fungi affecting plants. Its strong membrane-disrupting properties were initially attractive for controlling CLR​[8][9].

Similar to other lipopeptides, Mycosubtilin interacts with membrane sterols, causing disruption that leads to cell death across multiple fungal pathogens​[5][6].

While Mycosubtilin has a broad antifungal spectrum, it presented challenges related to stability and toxicity. Higher toxicity levels to plants during initial tests rendered it unsuitable for agricultural applications, alongside difficulties in regulating its production genetically​[6].

Iturin production

Understanding the Function of Iturin: Its Production and Mechanism of Action

The iturin A operon is roughly 38kb long and is composed of four genes: ItuD, ItuA, ItuB, and ItuC, in the respective order that code for their respective enzymes​[8]. ituA is a hybrid of polyketide synthetase (PKS) and NRPS with an additional PCP and C-domain for NRPS​[4], while the other synthetases are non-ribosomal peptide synthetases (NRPS). Each NRPS is made up of modules, each of which functions as a catalytic unit that adds one amino acid to the elongating peptide chain.

ituD: Malonyl coenzyme A transacylase.

ituA: 449kDa PKS-NRPS enzyme.

ituB: A 609kDa NRPS.

ituC: A 297kDa NRPS.

These synthetases possess modules—units that catalyze the incorporation of a specific amino acid into the growing peptide chain. The amino acid sequence of the peptide is usually colinear with the arrangement of the modules of NRPSs​[9]. These modules can be further subdivided into different domains characterized by sets of short, conserved sequence motifs.

  • Adenylation domain (A): This domain recognizes the amino acid, selects it, and adenylates it at the expense of ATP to form an acyl-adenylate intermediate (aminoacyl adenylate), attaching said amino acid to the activated T domain.
  • Condensation domain (C): Peptide bond formation of two consecutively bound amino acids is catalyzed by the condensation domain when two neighboring monomers are activated.
  • Peptidyl carrier domain (PCP) / Thiolation domain: The adenylated amino acid covalently binds to a phosphopantetheine carrier of the adjacent PCP domain. This domain is a transport unit activated by phosphopantetheinyl transferases, which modifies the amino acid adenylate to carry the sulfhydryl group responsible for tethering the amino acid residue via a thioester bond​[10]​ and binding it to conserved serine residues in the carrier domain.
  • Epimerization domain (E): Catalyzes the conversion of l-amino acids to d-isomers and is typically associated with the module that incorporates d-amino acids.
  • Thioesterase domain (T): It attaches to the peptide’s conserved serine residue and then severs the connection using a nucleophile, thus initiating cyclization and the release of the product peptide.

The assembly of the lipopeptide begins at ItuA. The acyl-CoA-ligase domain couples coenzyme A to a long-chain fatty acid through an ATP-dependent reaction. This activated fatty acid gets transferred to the 4-phosphopantetheine cofactor of the initial PCP domain​[11]. The reaction of a malonyl-CoA to the second PCP domain is catalyzed by malonyl coenzyme A transacylase, encoded by ItuD​[4]. In the next step, the β-ketoacyl synthetase domain catalyzes the condensation of malonyl and acyl thioester to give a β-ketoacyl thioester, which is subsequently converted into a β-amino fatty acid. It is then transferred through a PCP domain to be coupled with asparagine, both steps of which are catalyzed by condensation domains. The nascent lipopeptide passes into ituB and ituC wherein more amino acids are incorporated into the precursor’s structure till it reaches the thioesterase domain at the end of ituC, which releases the lipopeptide via cleavage and initiates cyclization ​[12]​[13].

Fig. Overview of the operons encoding for the nonribosomal peptide synthetases for the lipopeptide Iturin​[4].

Conclusion

Iturin is a lipopeptide produced from the iturin operon that makes it too big to insert in a practical vector (38kb).

The mode of action of Iturin A

Iturin A interacts electrostatically with the charged head groups ​[14]​ of sterol components in fungal membranes through, directing the readjustment of the membrane’s organization by causing changes in the micro-domain membrane fluidity, thus altering its permeability and disrupting it ​[15][16]​. This occurs due to the insertion of the hydrophobic tail into the lipid bilayer, causing the asymmetrical lateral expansion of the bilayer’s upper leaflet and subsequently destabilizing the membrane by perturbing the hydration degree ​[17][18]​. These activities are directly dependent on the intrinsic curvature of the target membrane and the concentration of Iturin with respect to its CMC ​[17]​. Very low concentrations of Iturin A induce the formation of short-lived voltage-independent anion selective pores that grow larger over time, supporting the hypothesis that Iturin A molecules penetrate the lipid layer and self-associate to form conducting structures through lateral diffusion ​[19]​. These pores formed due to the damaging of the cell membrane ​[20]​ cause the loss of essential macromolecular compounds from the cell and the uncontrolled ion efflux causes the cell membrane to lose its integrity . Iturin A’s destabilizing properties were correlated to its cone-shape which introduces a positive curvature strain in the membrane ​[19]​. Recently, this lipopeptide was shown to specifically interact with the fungal voltage gated potassium ion channel protein kvac exothermally while not directly interacting with ergosterol ​[21]​, explaining the elevated potassium ion levels so commonly observed in tandem. It also downregulates the expression of genes coding for various efflux and transport molecules ​[22]​, with its inhibiting effects lasting for several weeks ​[23]​.
Fig. Cell membrane degradation by the action of LPs.

Iturin Natural Promoter

Promoter Analysis of the ituD Gene

The operon is controlled by the promoter Pitu, which is fairly conserved in different strains of Bacillus subtilis. The transcription start site of ituD was found to be an A residue 56 bp upstream of the first residue of the ituD initiation codon. Upstream of this start site is a TATACACA-16 bp-TAGGAT sequence that exhibits low levels of homology to the consensus −10 and −35 (TTGACA-17 bp-TATAAT) sequences of ςA​[8].

Fig. Promoter region of ATCC 13592 annotated on Benchling..

Pitu is an inducible promoter triggered by stress from the entry into sporulation phases either due to Quorum sensing (QS), natural growth phase, or external stimulus which includes unknown stress caused by HV​[24].

Stress triggers the ComXQPA QS system causing a cascade of events that end with Iturin production. The ComX pheromone is produced in an immature form and is activated by ComQ. ComP and ComA form a combined ComP/ComA that’s then split, and ComP is phosphorylated when ComX exits the cell, activating ComA, which in turn activates DegQ and DegU, regulatory factors that then trigger the Iturin operon. DegU, a response regulator in a two-component system (DegU-DegQ), when phosphorylated, triggers the transcription of genes responsible for Iturin production, thereby boosting their expression​[4].

Spo0A is activated by one of five histidine kinases that are autophosphorylated upon perceived stress, then transfer their phosphorus to Spo0A, converting it to Spo0A~P, which regulates about 121 genes in Bacillus, including various genes responsible for sporulation and biofilm formation​[8].

The transcriptional regulator CodY senses nutrient availability. In nutrient-rich conditions, CodY binds to the promoters of genes related to secondary metabolism, including Iturin biosynthetic genes, repressing their expression​[25].

Fig. Overview of molecular interactions in regulation of genes related to lipopeptide biosynthesis.

The sequence for ATCC 13592 was then annotated for convenience, and further research for improving production was done. We were hoping to replace the natural promoter with a stronger inducible promoter, such as PbacA, to modulate the time of production and amount needed enough to kill HV, provided appropriate nutrient.

References

  1. M. Fernández de Ullivarri, S. Arbulu, E. Garcia-Gutierrez, and P. D. Cotter, “Antifungal Peptides as Therapeutic Agents,” Front Cell Infect Microbiol, vol. 10, Mar. 2020, doi: 10.3389/fcimb.2020.00105.
  2. M. Béchet et al., “Structure, biosynthesis, and properties of kurstakins, nonribosomal lipopeptides from Bacillus spp.,” Appl Microbiol Biotechnol, vol. 95, no. 3, pp. 593–600, Aug. 2012, doi: 10.1007/s00253-012-4181-2.
  3. M. Strieker, A. Tanović, and M. A. Marahiel, “Nonribosomal peptide synthetases: structures and dynamics,” Curr Opin Struct Biol, vol. 20, no. 2, pp. 234–240, Apr. 2010, doi: 10.1016/j.sbi.2010.01.009.
  4. M. Geissler, K. M. Heravi, M. Henkel, and R. Hausmann, “Lipopeptide Biosurfactants From Bacillus Species,” in Biobased Surfactants: Synthesis, Properties, and Applications, Elsevier, 2019, pp. 205–240. doi: 10.1016/B978-0-12-812705-6.00006-X.
  5. A. Hanif et al., “Fengycin Produced by Bacillus amyloliquefaciens FZB42 Inhibits Fusarium graminearum Growth and Mycotoxins Biosynthesis,” Toxins (Basel), vol. 11, no. 5, p. 295, May 2019, doi: 10.3390/toxins11050295.
  6. P. Zihalirwa Kulimushi, A. Argüelles Arias, L. Franzil, S. Steels, and M. Ongena, “Stimulation of Fengycin-Type Antifungal Lipopeptides in Bacillus amyloliquefaciens in the Presence of the Maize Fungal Pathogen Rhizomucor variabilis,” Front Microbiol, vol. 8, May 2017, doi: 10.3389/fmicb.2017.00850.
  7. J. Xiao, X. Guo, X. Qiao, X. Zhang, X. Chen, and D. Zhang, “Activity of Fengycin and Iturin A Isolated From Bacillus subtilis Z-14 on Gaeumannomyces graminis Var. tritici and Soil Microbial Diversity,” Front Microbiol, vol. 12, Jun. 2021, doi: 10.3389/fmicb.2021.682437.
  8. K. Tsuge, T. Akiyama, and M. Shoda, “Cloning, sequencing, and characterization of the iturin A operon,” J Bacteriol, vol. 183, no. 21, pp. 6265–6273, 2001, doi: 10.1128/JB.183.21.6265-6273.2001.
  9. E. H. Duitman et al., “The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase,” Proc Natl Acad Sci U S A, vol. 96, no. 23, pp. 13294–9, Nov. 1999, doi: 10.1073/pnas.96.23.13294.
  10. C. R. Harwood, J.-M. Mouillon, S. Pohl, and J. Arnau, “Secondary metabolite production and the safety of industrially important members of the Bacillus subtilis group,” FEMS Microbiol Rev, vol. 42, no. 6, pp. 721–738, Nov. 2018, doi: 10.1093/femsre/fuy028.
  11. L. M. P. Heinilä, “Cyanobacterial specialized metabolites: biosynthesis, bioactivity and structure,” Dec. 2021, Accessed: Oct. 02, 2024. [Online]. Available: http://hdl.handle.net/10138/336461.
  12. Md. A. Shishir et al., “Non-ribosomally synthesized lipopeptides: Promising novel therapeutics for cancer treatment,” Cancer Plus, vol. 5, no. 2, p. 2569, Jun. 2023, doi: 10.36922/cp.2569.
  13. N. Roongsawang, K. Washio, and M. Morikawa, “Diversity of nonribosomal peptide synthetases involved in the biosynthesis of lipopeptide biosurfactants,” Int J Mol Sci, vol. 12, no. 1, pp. 141–72, Dec. 2010, doi: 10.3390/ijms12010141.
  14. Md. A. Shishir et al., “Non-ribosomally synthesized lipopeptides: Promising novel therapeutics for cancer treatment,” Cancer Plus, vol. 5, no. 2, p. 2569, Jun. 2023, doi: 10.36922/cp.2569.
  15. H. Yue et al., “Optimization of iturin A production from Bacillus subtilis ZK-H2 in submerge fermentation by response surface methodology,” 3 Biotech, vol. 11, no. 2, Feb. 2021, doi: 10.1007/s13205-020-02540-7.
  16. S. M. Sreedharan, N. Rishi, and R. Singh, “Microbial lipopeptides: Properties, mechanics and engineering for novel lipopeptides,” Microbiol Res, vol. 271, p. 127363, 2023, doi: 10.1016/j.micres.2023.127363.
  17. D. Balleza, A. Alessandrini, and M. J. Beltrán García, “Role of Lipid Composition, Physicochemical Interactions, and Membrane Mechanics in the Molecular Actions of Microbial Cyclic Lipopeptides,” J Membr Biol, vol. 252, no. 2–3, pp. 131–157, Jun. 2019, doi: 10.1007/s00232-019-00067-4.
  18. F. Besson, F. Peypoux, G. Michel, and L. Delcambe, “Mode of action of iturin A, an antibiotic isolated from Bacillus subtilis, on Micrococcus luteus,” Biochem Biophys Res Commun, vol. 81, no. 2, pp. 297–304, Mar. 1978, doi: 10.1016/0006-291X(78)91532-2.
  19. J.-M. Bonmatin, O. Laprevote, and F. Peypoux, “Diversity Among Microbial Cyclic Lipopeptides: Iturins and Surfactins. Activity-Structure Relationships to Design New Bioactive Agents,” Comb Chem High Throughput Screen, vol. 6, no. 6, pp. 541–556, Nov. 2012, doi: 10.2174/138620703106298716.
  20. J. Wang et al., “Identification of Lipopeptide Iturin A Produced by Bacillus amyloliquefaciens NCPSJ7 and Its Antifungal Activities against Fusarium oxysporum f. sp. niveum,” Foods, vol. 11, no. 19, Sep. 2022, doi: 10.3390/foods11192996.
  21. S. Yang et al., “Insights into the antifungal mechanism of Bacillus subtilis cyclic lipopeptide iturin A mediated by potassium ion channel,” Int J Biol Macromol, vol. 277, p. 134306, Oct. 2024, doi: 10.1016/j.ijbiomac.2024.134306.
  22. M. Hua et al., “Iturin A Strongly Inhibits the Growth and T-2 Toxin Synthesis of Fusarium oxysporum: A Morphological, Cellular, and Transcriptomics Study,” Foods, vol. 12, no. 6, p. 1278, Mar. 2023, doi: 10.3390/foods12061278.
  23. M. A. Klich, A. R. Lax, and J. M. Bland, “Inhibition of some mycotoxigenic fungi by iturin A, a peptidolipid produced by Bacillus subtilis,” Mycopathologia, vol. 116, no. 2, pp. 77–80, Nov. 1991, doi: 10.1007/BF00436368.
  24. D. Cai et al., “A novel strategy to improve protein secretion via overexpression of the SppA signal peptide peptidase in Bacillus licheniformis,” Microb Cell Fact, vol. 16, no. 1, p. 70, Dec. 2017, doi: 10.1186/s12934-017-0688-7.
  25. J. Li, Q. Yang, L. Zhao, S. Zhang, Y. Wang, and X. Zhao, “Purification and characterization of a novel antifungal protein from Bacillus subtilis strain B29,” J Zhejiang Univ Sci B, vol. 10, no. 4, pp. 264–272, Apr. 2009, doi: 10.1631/jzus.B0820341.