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From a Natural Product to Its Biosynthetic Gene Cluster: A Demonstration Using Polyketomycin from Streptomyces diastatochromogenes Tü6028
Published on: January 13, 2017
Analysis of the loading and hydroxylation steps in lankamycin biosynthesis in Streptomyces rochei
Kenji Arakawa1, Kazuya Kodama, Satoshi Tatsuno
1Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, Japan.
Abstract:
The biosynthetic gene cluster of lankamycin (LM), a 14-member macrolide antibiotic, is encoded on the 210-kb linear plasmid pSLA2-L in Streptomyces rochei 7434AN4. LM contains a 3-hydroxy-2-butyl group at the C-13 position, which is different from an ethyl group in erythromycin. The following two possibilities could be considered for the origin of this starter moiety of LM biosynthesis: (i) an extra module exists in the biosynthetic gene cluster and loads an additional acetate molecule, or (ii) 3-hydroxy-2-butyrate or its equivalent is loaded and incorporated as a starter. The former possibility was eliminated by the complete sequencing of pSLA2-L, which showed no extra module. On the other hand, the latter was confirmed by incorporation of deuterium in [3-(2)H]dl-isoleucine into the C-14 position of LM. The timing of hydroxylation reactions at the C-15 and C-8 positions of LM was studied by constructing disruptants of two P450 hydroxylase genes, lkmF (orf26) and lkmK (orf37). The lkmF disruptant produced 8-deoxylankamycin, while the lkmK disruptant produced both 15-deoxylankamycin and 8,15-dideoxylankamycin. These results clearly showed that LkmF is a C-8 hydroxylase and LkmK is a C-15 hydroxylase in LM biosynthesis and in addition suggested the order of hydroxylation steps; namely, hydroxylation may occur at first at C-15 by LkmK and then at C-8 by LkmF.
Insights
The origin of the lankamycin (LM) starter unit was identified as 3-hydroxy-2-butyrate, not an additional acetate molecule. Gene studies revealed LkmF as the C-8 hydroxylase and LkmK as the C-15 hydroxylase in LM biosynthesis.
Area of Science:
- Microbiology
- Biochemistry
- Molecular Biology
Background:
- Lankamycin (LM) is a 14-member macrolide antibiotic produced by Streptomyces rochei.
- LM possesses a unique 3-hydroxy-2-butyl group at the C-13 position, distinguishing it from erythromycin.
- The biosynthetic gene cluster for LM is located on the linear plasmid pSLA2-L.
Purpose of the Study:
- To elucidate the origin of the starter moiety in lankamycin biosynthesis.
- To determine the specific roles of P450 hydroxylase genes (lkmF and lkmK) in LM hydroxylation.
- To establish the order of hydroxylation reactions during LM biosynthesis.
Main Methods:
- Complete sequencing of the pSLA2-L plasmid to analyze the biosynthetic gene cluster.
- Deuterium incorporation experiments using [3-(2)H]dl-isoleucine to trace the starter unit.
- Construction and analysis of gene disruptants for lkmF and lkmK.
Main Results:
- The starter moiety of LM biosynthesis was confirmed to be 3-hydroxy-2-butyrate or its equivalent, not an additional acetate molecule.
- Disruption of lkmF resulted in 8-deoxylankamycin, identifying LkmF as the C-8 hydroxylase.
- Disruption of lkmK yielded 15-deoxylankamycin and 8,15-dideoxylankamycin, identifying LkmK as the C-15 hydroxylase.
Conclusions:
- The starter unit for lankamycin biosynthesis originates from 3-hydroxy-2-butyrate.
- LkmF and LkmK are confirmed as the specific hydroxylases for the C-8 and C-15 positions, respectively.
- Hydroxylation likely proceeds sequentially, with C-15 hydroxylation by LkmK preceding C-8 hydroxylation by LkmF.
