Towards a novel tetraether lipid producer Haloferax volcanii

Presence of Tes across Archaea 5 Haloferax volcanii: ▪ same pathway for isoprenoid synthesis as other Archaea. ▪ produces both saturated and unsaturated DELs as a response to its high saline conditions. ▪ Grows faster than model TELs organisms ( Sulfolobus acidocaldarius). ▪ Methanobrevibacter smithii (from human gut) produces TELs grows faster than S. acidocaldarius. Towards a novel tetraether lipid producer Haloferax volcanii University of Vienna, Department of Analytical Chemistry Carvalho, A.C.: [email protected] /[email protected]t Peeters, E.: Eveline.Peeter[email protected] Zanghellini, J.: juergen.zanghelli[email protected]t Quehenberger, J.: [email protected] References: (1) Rastädter, K., Wurm, D. J., Spadiut, O., & Quehenberger, J. (2020). The Cell Membrane of Sulfolobus spp.—Homeoviscous Adaption and Biotechnological Applications. International Journal of Molecular Sciences, 21(11), 3935tps://doi.org/10.3390/ijms21113935 (2) Santhosh, P. B., & Genova, J. (2023). Archaeosomes: New Generation of Liposomes Based on Archaeal Lipids for Drug Delivery and Biomedical Applications. ACS Omega, 8(1), 1–9. https://doi.org/10.1021/acsomega.2c06034 (3) Caforio, A., Siliakus, M. F., Exterkate, M., Jain, S., Jumde, V. R., Andringa, R. L. H., Kengen, S. W. M., Minnaard, A. J., Driessen, A. J. M., & Van Der Oost, J. (2018). Converting Escherichia coli into an archaebacterium with a hybrid heterochiral membrane. Proceedings of the National Academy of Sciences, 115(14), 3704–3709.g/10.1073/pnas.1721604115 (4) Lloyd, C. T., Iwig, D. F., Wang, B., Cossu, M., Metcalf, W. W., Boal, A. K., & Booker, S. J. (2022). Discovery, structure and mechanism of a tetraether lipid synthase. Nature, 609(7925), 197– 203. https://doi.org/10.1038/s41586-022-05120-2 (5) Zeng, Z., Chen, H., Yang, H., Chen, Y., Yang, W., Feng, X., Pei, H., & Welander, P. V. (2022). Identification of a protein responsible for the synthesis of archaeal membrane-spanning GDGT lipids. Nature Communications, 13(1), 1545. https://doi.org/10.1038/s41467-022-29264-x Ana Cristina Carvalhoa b c *, Eveline Peetersd, Jürgen Zanghellinia b, Julian Quehenberger c a Department of Analytical Chemistry, University of Vienna, Vienna, Austria; bDoctoral School in Chemistry, University of Vienna, Vienna, Austria; cNovoArc GmbH, Vienna, Austria; dResearch Group of Microbiology, Department of Bioengineering, Vrije Universiteit Brussel,Belgium Results Background: Archaeal lipids & Applications 1,2,3 ▪ Archaeal vs conventional lipids (Bacteria and Eukarya) ▪ Advantages of TELs (vs conventional lipids) ▪higher stability against oxidative, hydrolytic, thermal and enzymatic stresses ▪ Application of TELs in pharmaceutical industry, ▪liposomes →oral delivery of small molecules, peptides and proteins ▪lipid nanoparticles →nucleic acid delivery. ▪ TELs are a result from selective pressure from extreme environments. Tetraether synthase ( Tes) 4,5 ▪ Tetraether synthase (Tes) is the enzyme responsible to join two saturated DELs and produce 1 TEL. ▪ The enzyme requires 4 [Fe4S4] cluster and a Fe2+/3+ as cofactors and can be able to join lipids with different types of headgroups. Future perspectives ✓ Screen for optimal induction strength to maintain growth and promote TELs in H.volcanii mutants; ✓ Attempt protein purification and analyse resulting lipid profile; ✓ Explore the effects of other promoters for tes expression; ✓ Integrate tes into genomic DNA. Goals ▪ Develop a workflow for H. volcanii mutant generation; ▪ Introduce the tes gene for expression in H.volcanii; ▪ Characterize mutants phenotypically. Example of a TEL Example of a diether lipid (DEL) Materials and Methods Mechanism of action of Tes. From (4). Gibson assembly tes optimized E. coli MG1655 cultivation harboring pTA1392 :: empty Transformation into: 1-E.coli DH5α 2-E.coli JM110 3H. volcanii H26 Validation: Sequencing Cultivation and characterization of H26 and mutant Tes from M. smithii (Msm_0849) H. volcanii Codon usage table Colony PCR The University of Debrecen has received the funding 2020-2.1.1-ED-2023-00269 from the Hungarian National Research, Development and Innovation Fund to cover the participation of DC10 in PROHITS. 1) Presence of oxygen influences growth 0.0 0.5 1.0 1.5 2.0 010 20 30 40 50 OD600 Process time [h] Hv-min 5 mM Hv-min 3+2mM Hv-min SW Hv-min standard Induction Induction 1) Comparison between one and two step induction Tryptophan addition a) b) H. Volcanii H26 a) and T1 b) grown in 2x YPC medium at 110, 130 and 150 rpm Isoprene chainsEther linkage Capacity to form monolayers - Tetraether lipids (TELs) YPC 1x (literature) YPC 2x (Experiments) Basis Salt water 18% (SW) Yeast Extract (g/L) 510 Peptone (g/L) 1 2 Casamino Acids (g/L) 1 2 1 M KOH (mL/L) 1.7 3.5 1 M CaCl (mL/L) 3 0.0 2.0 4.0 6.0 8.0 10.0 12.0 010 20 30 40 50 OD600 Process hours (h) 2x YPC H26 110 rm YPC 2x H26 130 rpm YPC 2x H26 150 rpm 0.0 2.0 4.0 6.0 8.0 10.0 12.0 010 20 30 40 50 OD600 Process hours (h) old spectofotometer 2x YPC T1 110 rpm 2x YPC T1 130 rpm 2x YPC T1 150 rpm Distribution of Tes homologues in Archaea. Highlight: Organisms used in this study. Black circles: present; White circle: Absent; Gray circles: Not available. From (5). 250 180 95 55 72 43 34 H26 SW 5mM 3+5mM T1 0mM 11112 2 2 2333 3 kDa SDS-PAGE Growth curves