Dokument

Summary:
Lasso peptides are a class of natural products that belong to the family of ribosomally‑assembled and posttranslationally‑modified peptides. They are defined by an unique structural motif referred to as the so‑called lariat knot, whose name is derived from the fact that this topology is reminiscent of the knot found in the noose of a lasso. This structure is achieved by the presence of an N‑terminal macrolactam ring that is threaded by the C‑terminal tail of the peptide. The fold in these molecules is then conserved by non‑covalent interactions in the form of bulky amino acids located above and below the macrolactam ring, in this way entrapping the tail inside of the ring. What makes these compounds of interest for research is that their structure, even though it is maintained merely by sterical interactions, often exhibits a tremendous stability against thermal, chemical and proteolytic degradation. Still, up to now little is known about the general function of these compounds for their producing organisms, although there are some interesting biological activities attributed to some of the previously reported lasso peptides. To obtain more information about their physico‑chemical properties, their biosynthesis and to get an idea what role they might play in nature, the primary subject of this thesis was the directed genome mining for and the subsequent isolation and characterization of novel lasso peptides. The results of these projects were published in several studies that will be shown and discussed in the course of this thesis. Amongst other findings, these studies not only include the discovery of a multitude of novel lasso peptides, but through the thorough analysis and characterization of these compounds, several former assumptions of this research area could be overhauled and updated. In addition to this, the bioinformatic data gathered during our genome mining studies furthermore uncovered interesting facts about the distribution of lasso peptides amongst bacteria and about the existence of different subgroups of biosynthetic gene cluster arrangements, which could facilitate future research directed towards identifiying the concrete functions of these compounds. Furthermore, it was also investigated if these compounds are suitable scaffolds for drug development via epitope grafting approaches. In this regard, a previously reported bioactive lasso graft was used as the basis to show that such compounds can indeed be further optimized and improved upon by rational approaches that utilize the information obtained from research done with simple linear or cyclic peptides that are, in contrast to lasso peptides, easily accessible by synthetic means.

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C(103)-C(104)-C(105)-OH2 -91(4) C26-C16-N17-C57 176.9(4) O113-C113-C213-N113 -32.3(6) O11-C11-C21-N11 -53.1(5) C67-C57-N17-C16 -109.8(4) N114-C113-C213-N113 151.6(4) N22-C11-C21-N11 126.3(4) C47-C57-N17-C16 133.2(4) O113-C113-C213-C313 89.0(5) C11-C21-N11-C17 88.9(5) O37-C67-N18-C28 -3.1(6) N114-C113-C213-C313 -87.1(5) O11-C11-N22-C22 2.8(7) C57-C67-N18-C28 171.6(4) N113-C213-C313-C413 -73.3(5) C21-C11-N22-C22 -176.5(4) C67-N18-C28-C38 176.5(4) C113-C213-C313-C413 164.9(4) C11-N22-C22-C12 127.6(4) C67-N18-C28-C18 -60.0(5) C213-C313-C413-O313 86.9(6) O12-C12-C22-N22 1.4(6) O18-C18-C28-N18 143.2(4) C213-C313-C413-N213 -91.0(5) N13-C12-C22-N22 -179.8(4) N19-C18-C28-N18 -40.7(5) O113-C113-N114-C214 -2.2(7) O12-C12-N13-C53 -178.8(4) O18-C18-C28-C38 -95.0(5) C213-C113-N114-C214 173.9(4) C22-C12-N13-C53 2.4(6) N19-C18-C28-C38 81.1(5) C113-N114-C214-C114 -69.3(5) O12-C12-N13-C23 10.8(7) N18-C28-C38-C48 -177.8(4) C113-N114-C214-C314 167.8(4) C22-C12-N13-C23 -168.0(4) C18-C28-C38-C48 59.4(6) O114-C114-C214-N114 -33.8(6) C12-N13-C23-C13 -77.8(6) C28-C38-C48-C58 -140.6(5) N115-C114-C214-N114 151.1(4) C53-N13-C23-C13 110.7(5) C38-C48-C58-O28 -17.1(9) O114-C114-C214-C314 89.9(6) C12-N13-C23-C33 160.8(5) C38-C48-C58-O38 162.4(5) N115-C114-C214-C314 -85.2(5) C53-N13-C23-C33 -10.7(5) O18-C18-N19-C29 -6.0(7) N114-C214-C314-C514 60.6(6) O13-C13-C23-N13 175.5(5) C28-C18-N19-C29 178.1(4) C114-C214-C314-C514 -62.5(6) N14-C13-C23-N13 -5.5(7) C18-N19-C29-C19 -118.8(4) N114-C214-C314-C414 -66.4(7) O13-C13-C23-C33 -67.8(7) C18-N19-C29-C39 120.2(5) C114-C214-C314-C414 170.4(6) N14-C13-C23-C33 111.2(6) O19-C19-C29-N19 -70.7(5) O114-C114-N115-C515 170.0(4) N13-C23-C33-C43 29.4(6) N110-C19-C29-N19 108.5(4) C214-C114-N115-C515 -14.8(7) C13-C23-C33-C43 -96.6(6) O19-C19-C29-C39 51.3(5) O114-C114-N115-C215 2.1(7) C23-C33-C43-C53 -37.9(7) N110-C19-C29-C39 -129.5(4) C214-C114-N115-C215 177.2(4) C12-N13-C53-C43 176.9(5) N19-C29-C39-C79 179.2(4) C114-N115-C215-C315 156.5(4) C23-N13-C53-C43 -11.9(6) C19-C29-C39-C79 60.0(5) C515-N115-C215-C315 -13.0(5) C33-C43-C53-N13 30.4(7) N19-C29-C39-C59 -54.1(5) C114-N115-C215-C115 -82.7(5) O13-C13-N14-C24 3.1(8) C19-C29-C39-C59 -173.3(4) C515-N115-C215-C115 107.7(4) C23-C13-N14-C24 -175.9(4) C79-C39-C59-C69 62.1(6) O115-C115-C215-N115 -112.3(5) C13-N14-C24-C34 104.9(5) C29-C39-C59-C69 -63.3(6) N116-C115-C215-N115 68.3(5) C13-N14-C24-C14 -126.4(5) O19-C19-N110-C210 -2.7(6) O115-C115-C215-C315 2.3(7) O14-C14-C24-N14 63.3(5) C29-C19-N110-C210 178.1(4) N116-C115-C215-C315 -177.2(4) N15-C14-C24-N14 -118.6(4) C19-N110-C210-C110 70.7(5) N115-C215-C315-C415 31.7(5) O14-C14-C24-C34 -169.9(4) O110-C110-C210-N110 23.3(6) C115-C215-C315-C415 -87.2(5) N15-C14-C24-C34 8.2(6) N111-C110-C210-N110 -157.8(4) C215-C315-C415-C515 -39.3(5) N14-C24-C34-C44 -65.8(6) O110-C110-N111-C211 -0.2(6) C114-N115-C515-C415 -179.7(4) C14-C24-C34-C44 169.2(4) C210-C110-N111-C211 -179.1(4) C215-N115-C515-C415 -10.7(5) C24-C34-C44-C64 175.7(5) C110-N111-C211-C111 144.2(4) C315-C415-C515-N115 30.4(5) C24-C34-C44-C54 -59.0(7) O111-C111-C211-N111 14.4(6) O115-C115-N116-C216 3.3(8) O15-C15-C25-N15 -7.1(6) N112-C111-C211-N111 -167.8(4) C215-C115-N116-C216 -177.2(4) N16-C15-C25-N15 172.0(4) O111-C111-N112-C212 1.3(6) C115-N116-C216-C116 -100.0(6) O15-C15-C25-C35 113.9(5) C211-C111-N112-C212 -176.5(4) O116-C116-C216-N116 -130.1(5) N16-C15-C25-C35 -67.0(5) C111-N112-C212-C112 -120.5(4) O216-C116-C216-N116 49.9(6) O14-C14-N15-C25 4.6(6) C111-N112-C212-C312 113.2(4) C523-N117-C217-C117 92.9(5) C24-C14-N15-C25 -173.3(4) O112-C112-C212-N112 -165.3(4) O117-C117-C217-N117 -52.8(6) C35-C25-N15-C14 100.9(5) N113-C112-C212-N112 15.7(6) N118-C117-C217-N117 126.6(4) C15-C25-N15-C14 -137.4(4) O112-C112-C212-C312 -39.0(6) O117-C117-N118-C218 6.3(7) O15-C15-N16-C26 1.0(7) N113-C112-C212-C312 141.9(4) C217-C117-N118-C218 -173.0(4) C25-C15-N16-C26 -178.0(4) N112-C212-C312-C412 -77.6(5) C117-N118-C218-C118 126.1(4) C15-N16-C26-C16 129.9(5) C112-C212-C312-C412 155.0(4) O118-C118-C218-N118 -4.6(6) O16-C16-C26-N16 151.4(4) C212-C312-C412-C512 -92.5(6) N119-C118-C218-N118 176.6(4) N17-C16-C26-N16 -28.0(6) C212-C312-C412-C912 85.2(6) O118-C118-N119-C519 180.0(4) C21-N11-C17-O17 9.0(6) C912-C412-C512-C612 0.4(8) C218-C118-N119-C519 -1.3(7) C21-N11-C17-C27 -170.7(4) C312-C412-C512-C612 178.3(5) O118-C118-N119-C219 11.8(6) O17-C17-C27-C47 137.3(4) C412-C512-C612-C712 0.6(9) C218-C118-N119-C219 -169.4(4) N11-C17-C27-C47 -43.1(5) C512-C612-C712-C812 -0.9(9) C118-N119-C219-C119 -74.5(6) C17-C27-C47-C57 170.8(3) C612-C712-C812-C912 0.2(9) C519-N119-C219-C119 115.9(5) C27-C47-C57-N17 -55.6(5) C712-C812-C912-C412 0.8(8) C118-N119-C219-C319 165.5(4) C27-C47-C57-C67 -174.5(4) C512-C412-C912-C812 -1.2(7) C519-N119-C219-C319 -4.0(5) N17-C57-C67-O37 -9.7(6) C312-C412-C912-C812 -178.9(5) O119-C119-C219-N119 164.2(5) S58 Supplementary Table S14b. Torsion angles [°] for the -4 aa truncation of xanthomonin I. N120-C119-C219-N119 -21.4(7) C323-C423-C523-N117 -44.9(5) O128-C128-N129-C429 3.7(7) O119-C119-C219-C319 -79.9(6) O123-C123-N124-C224 1.4(6) C228-C128-N129-C429 -179.9(4) N120-C119-C219-C319 94.5(5) C223-C123-N124-C224 175.4(3) C429-C229-C329-O229 90.4(6) N119-C219-C319-C4A19 24.1(7) C123-N124-C224-C324 172.8(4) C429-C229-C329-N229 -88.2(5) C119-C219-C319-C4A19 -99.8(7) C123-N124-C224-C124 -61.1(5) C128-N129-C429-C229 130.4(4) N119-C219-C319-C4B19 -17.1(9) O124-C124-C224-N124 151.5(4) C128-N129-C429-C129 -108.9(5) C119-C219-C319-C4B19 -141.1(9) N125-C124-C224-N124 -28.9(5) C329-C229-C429-N129 -76.9(5) C118-N119-C519-C4A19 174.7(6) O124-C124-C224-C324 -84.0(5) C329-C229-C429-C129 161.5(4) C219-N119-C519-C4A19 -16.5(7) N125-C124-C224-C324 95.6(4) O129-C129-C429-N129 -34.0(6) C118-N119-C519-C4B19 -146.0(8) N124-C224-C324-C424 -69.0(5) N130-C129-C429-N129 148.3(4) C219-N119-C519-C4B19 22.8(8) C124-C224-C324-C424 165.5(4) O129-C129-C429-C229 87.6(5) C4B19-C319-C4A19-C519 62.8(10) C224-C324-C424-C524 -174.9(4) N130-C129-C429-C229 -90.1(5) C219-C319-C4A19-C519 -36.2(9) C324-C424-C524-O224 -28.5(7) O129-C129-N130-C230 -2.3(7) N119-C519-C4A19-C319 32.5(9) C324-C424-C524-O324 154.9(5) C429-C129-N130-C230 175.4(4) C4B19-C519-C4A19-C319 -63.9(12) O124-C124-N125-C625 -12.8(6) C129-N130-C230-C130 -59.9(5) C4A19-C319-C4B19-C519 -60.8(10) C224-C124-N125-C625 167.6(4) C129-N130-C230-C330 176.9(4) C219-C319-C4B19-C519 31.4(12) C325-C225-C425-C525 53.6(6) O130-C130-C230-N130 -47.7(6) N119-C519-C4B19-C319 -32.4(11) C625-C225-C425-C525 -72.0(5) N131-C130-C230-N130 134.6(4) C4A19-C519-C4B19-C319 58.7(10) C124-N125-C625-C125 -130.6(4) O130-C130-C230-C330 74.5(6) O119-C119-N120-C220 6.8(8) C124-N125-C625-C225 108.6(4) N131-C130-C230-C330 -103.1(5) C219-C119-N120-C220 -167.3(4) O125-C125-C625-N125 -62.0(5) N130-C230-C330-C430 -62.3(6) C119-N120-C220-C320 120.5(5) N126-C125-C625-N125 119.6(4) C130-C230-C330-C430 176.2(5) C119-N120-C220-C120 -111.5(5) O125-C125-C625-C225 60.4(5) N130-C230-C330-C530 173.6(5) O120-C120-C220-N120 59.1(5) N126-C125-C625-C225 -118.0(4) C130-C230-C330-C530 52.1(6) N121-C120-C220-N120 -120.9(4) C425-C225-C625-N125 -60.9(5) O130-C130-N131-C531 175.2(5) O120-C120-C220-C320 -175.6(4) C325-C225-C625-N125 172.6(4) C230-C130-N131-C531 -7.1(7) N121-C120-C220-C320 4.4(6) C425-C225-C625-C125 179.9(4) O130-C130-N131-C231 5.5(7) N120-C220-C320-C420 -59.0(6) C325-C225-C625-C125 53.4(5) C230-C130-N131-C231 -176.8(4) C120-C220-C320-C420 175.8(4) O125-C125-N126-C226 -5.3(6) C130-N131-C231-C131 -64.3(6) C220-C320-C420-C620 -61.7(7) C625-C125-N126-C226 173.0(4) C531-N131-C231-C131 124.6(5) C220-C320-C420-C520 175.2(5) C125-N126-C226-C126 78.6(5) C130-N131-C231-C331 176.2(5) O120-C120-N121-C221 8.4(7) O126-C126-C226-N126 7.7(6) C531-N131-C231-C331 5.1(6) C220-C120-N121-C221 -171.6(4) N127-C126-C226-N126 -170.9(3) O131-C131-C231-N131 -37.6(7) C120-N121-C221-C121 -134.4(4) O126-C126-N127-C227 0.9(7) N132-C131-C231-N131 147.0(4) C120-N121-C221-C321 104.0(5) C226-C126-N127-C227 179.6(4) O131-C131-C231-C331 78.1(7) O121-C121-C221-N121 -17.5(5) C126-N127-C227-C127 144.6(4) N132-C131-C231-C331 -97.2(6) N122-C121-C221-N121 164.4(4) O127-C127-C227-N127 14.4(6) N131-C231-C331-C4A31 24.3(7) O121-C121-C221-C321 104.6(5) N128-C127-C227-N127 -167.1(4) C131-C231-C331-C4A31 -98.1(7) N122-C121-C221-C321 -73.5(5) O127-C127-N128-C228 2.2(6) N131-C231-C331-C631 -21.0(8) O121-C121-N122-C222 0.9(6) C227-C127-N128-C228 -176.2(4) C131-C231-C331-C631 -143.4(7) C221-C121-N122-C222 178.9(4) C127-N128-C228-C128 -121.0(4) C130-N131-C531-C631 -155.6(7) C121-N122-C222-C122 139.7(4) C127-N128-C228-C328 113.4(5) C231-N131-C531-C631 14.7(8) O122-C122-C222-N122 142.5(4) O128-C128-C228-N128 -172.7(4) C130-N131-C531-C4A31 164.5(6) N123-C122-C222-N122 -38.4(5) N129-C128-C228-N128 10.7(6) C231-N131-C531-C4A31 -25.1(6) O122-C122-N123-C223 0.5(6) O128-C128-C228-C328 -47.4(6) N131-C531-C631-C331 -25.5(9) C222-C122-N123-C223 -178.6(4) N129-C128-C228-C328 136.0(4) C4A31-C531-C631-C331 47.3(7) C122-N123-C223-C123 -102.2(4) N128-C228-C328-C428 -71.4(5) C4A31-C331-C631-C531 -65.3(8) C122-N123-C223-C323 140.9(4) C128-C228-C328-C428 161.4(4) C231-C331-C631-C531 28.6(9) O123-C123-C223-N123 -12.9(5) C228-C328-C428-C528 92.6(6) C231-C331-C4A31-C531 Supporting Information References [1] J. D. Hegemann, M. Zimmermann, X. Xie, M. A. Marahiel, J Am Chem Soc 2013, 135, 210-222.
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26. Supplementary Table S14a. Torsion angles [°] for the -4 aa truncation of xanthomonin I.
27. Strasse 4 and LOEWE-Center for Synthetic Microbiology, D-35032, Marburg, Germany *marahiel@staff.uni-marburg.de Supporting Information Supporting figure 1 – heat stability of (a) caulonodin IV and (b) caulonodin VI, heat and carboxypeptidase stability of (c) caulonodin V and (d) caulonodin VII shown as UV traces and mass signals measured on the low-resolution HPLC-MS system References S17 12.0 ± 1.6
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