TY - JOUR
T1 - Construction of a hybrid gene cluster to reveal coupled ring formation-hydroxylation in the biosynthesis of HSAF and analogues from
T2 - Lysobacter enzymogenes
AU - Li, Xue
AU - Wang, Haoxin
AU - Li, Yaoyao
AU - Du, Liangcheng
N1 - Funding Information:
This work was supported in part by the NSFC (81573311 and 81773598) and the Young Scholars Program of Shandong University (2016WLJH31).
Publisher Copyright:
© 2019 The Royal Society of Chemistry.
PY - 2019
Y1 - 2019
N2 - HSAF and analogues are polycyclic tetramate macrolactams (PoTeMs) isolated from Lysobacter enzymogenes. Due to their antifungal activity, distinct chemical structure and new mode of action, PoTeMs have been the subject of several studies for their biosynthetic mechanism. However, polycycle formation is still not well understood. HSAF and several analogues (alteramides) carry a C20-hydroxyl, which is absent in most known PoTeMs such as combamides and pactamides. Previous studies indicated that two genes encoding NAD(P)H-dependent flavin enzymes (OX1/OX2) are responsible for the second five-membered ring formation in HSAF and alteramides. Intriguingly, the products of OX1/OX2 always carry the C20-OH. To test the hypothesis that the formation of the second five-membered ring is coupled with the C20-hydroxylation, we constructed a hybrid PoTeM gene cluster through removing OX1/OX2 in the HSAF cluster and functional complementation by CbmB, which also catalyzes the second five-membered ring formation in combamides but lacking the C20-OH. Two heterologous hosts carrying the hybrid cluster generated the same three PoTeMs, including lysobacterene B (3, the one-ring precursor of HSAF) and combamide D (4, a two-ring product lacking the C20-OH). The third product was not related to either of the clusters and was identified to be pactamide A (5) using mass spectrometry, 1D- and 2D-NMR, and ECD spectroscopy. The results demonstrate the feasibility of producing new PoTeM compounds through combinatorial biosynthesis. More importantly, this study provides the first experimental evidence to support that the second ring formation is coupled with the C20-hydroxylation in the biosynthesis of HSAF and analogues.
AB - HSAF and analogues are polycyclic tetramate macrolactams (PoTeMs) isolated from Lysobacter enzymogenes. Due to their antifungal activity, distinct chemical structure and new mode of action, PoTeMs have been the subject of several studies for their biosynthetic mechanism. However, polycycle formation is still not well understood. HSAF and several analogues (alteramides) carry a C20-hydroxyl, which is absent in most known PoTeMs such as combamides and pactamides. Previous studies indicated that two genes encoding NAD(P)H-dependent flavin enzymes (OX1/OX2) are responsible for the second five-membered ring formation in HSAF and alteramides. Intriguingly, the products of OX1/OX2 always carry the C20-OH. To test the hypothesis that the formation of the second five-membered ring is coupled with the C20-hydroxylation, we constructed a hybrid PoTeM gene cluster through removing OX1/OX2 in the HSAF cluster and functional complementation by CbmB, which also catalyzes the second five-membered ring formation in combamides but lacking the C20-OH. Two heterologous hosts carrying the hybrid cluster generated the same three PoTeMs, including lysobacterene B (3, the one-ring precursor of HSAF) and combamide D (4, a two-ring product lacking the C20-OH). The third product was not related to either of the clusters and was identified to be pactamide A (5) using mass spectrometry, 1D- and 2D-NMR, and ECD spectroscopy. The results demonstrate the feasibility of producing new PoTeM compounds through combinatorial biosynthesis. More importantly, this study provides the first experimental evidence to support that the second ring formation is coupled with the C20-hydroxylation in the biosynthesis of HSAF and analogues.
UR - http://www.scopus.com/inward/record.url?scp=85067612530&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85067612530&partnerID=8YFLogxK
U2 - 10.1039/c9md00154a
DO - 10.1039/c9md00154a
M3 - Article
C2 - 31303988
AN - SCOPUS:85067612530
VL - 10
SP - 907
EP - 912
JO - MedChemComm
JF - MedChemComm
SN - 2040-2503
IS - 6
ER -