Single cell mutant selection for metabolic engineering of actinomycetes

Akhgari, Amir; Baral, Bikash; Koroleva, Arina; Siitonen, Vilja; Fewer, David P.; Melançon, Charles E.; Rahkila, Jani; Metsä‐Ketelä, Mikko

doi:10.1016/j.ymben.2022.07.002

“…To verify whether LugOI has a similar function as PgaE 34 in angucycline biosynthesis, a construct for constitutive expression of pgaE was generated. To do so, pgaE was cloned under the control of the SP44 promoter in the pS-GK plasmid, lacking the kanamycin reporter gene 63 . This plasmid comprises the T7 terminator, the SP44 promoter, the ribosomal binding site (RBS-SR41) and the reporter gene, sf gfp (super folder green fluorescent protein), cloned into pSET152.…”

Section: Methodsmentioning

confidence: 99%

Unravelling key enzymatic steps in C-ring cleavage during angucycline biosynthesis

Elsayed,

van der Heul,

Xiao

et al. 2023

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Angucyclines are type II polyketide natural products, often characterized by unusual structural rearrangements through B- or C-ring cleavage of their tetracyclic backbone. While the enzymes involved in B-ring cleavage have been extensively studied, little is known of the enzymes leading to C-ring cleavage. Here, we unravel the function of the oxygenases involved in the biosynthesis of lugdunomycin, a highly rearranged C-ring cleaved angucycline derivative. Targeted deletion of the oxygenase genes, in combination with molecular networking and structural elucidation, showed that LugOI is essential for C12 oxidation and maintaining a keto group at C6 that is reduced by LugOII, resulting in a key intermediate towards C-ring cleavage. An epoxide group is then inserted by LugOIII, and stabilized by the novel enzyme LugOV for the subsequent cleavage. Thus, for the first time we describe the oxidative enzymatic steps that form the basis for a wide range of rearranged angucycline natural products.

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“…Structures of 1 a and 1 b , which we name herein mutaxanthene F and G, respectively, are structurally similar to mutaxanthenes A–C, which were previously isolated from a soil‐derived Nocardiosis sp., [20] as well as Amycolatopsis orientalis NRRL F3213 [21] . Although the gene clusters ( mux cluster in Nocardiosis sp.…”

Section: Resultsmentioning

confidence: 99%

“…[21] Although the gene clusters (mux cluster in Nocardiosis sp. and mut cluster in A. orientalis NRRL F3213) for mutaxanthene biosynthesis have been reported, [20,21] the mtx cluster in S. setonesis NAK80 only showed moderate sequence identity (30-60 %) to its counterparts in the mux and mut clusters (Table S4). Notably, the common 12,12a-dihydro-1H-benzo[b]xanthene scaffold in mutaxanthenes is very rare among aromatic polyketides.…”

Section: In Vivo Characterization Of the Biosynthetic Pathway For Mut...mentioning

confidence: 99%

“…Structures of 1 a and 1 b, which we name herein mutaxanthene F and G, respectively, are structurally similar to mutaxanthenes A-C, which were previously isolated from a soil-derived Nocardiosis sp., [20] as well as Amycolatopsis orientalis NRRL F3213. [21] Although the gene clusters (mux cluster in Nocardiosis sp. and mut cluster in A. orientalis NRRL F3213) for mutaxanthene biosynthesis have been reported, [20,21] the mtx cluster in S. setonesis NAK80 only showed moderate sequence identity (30-60 %) to its counterparts in the mux and mut clusters (Table S4).…”

Section: In Vivo Characterization Of the Biosynthetic Pathway For Mut...mentioning

confidence: 99%

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Total Biosynthesis of Mutaxanthene Unveils a Flavoprotein Monooxygenase Catalyzing Xanthene Ring Formation

Xiang

¹

,

Shi

²

,

Zhu

³

et al. 2023

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Flavoprotein monooxygenases (FPMOs) play important roles in generating structural complexity and diversity in natural products biosynthesized by type II polyketide synthases (PKSs). In this study, we used genome mining to discover novel mutaxanthene analogues and investigated the biosynthesis of these aromatic polyketides and their unusual xanthene framework. We determined the complete biosynthetic pathway of mutaxathene through in vivo gene deletion and in vitro biochemical experiments. We show that a multifunctional FPMO, MtxO4, catalyzes ring rearrangement and generates the required xanthene ring through a multistep transformation. In addition, we successfully obtained all necessary enzymes for in vitro reconstitution and completed the total biosynthesis of mutaxanthene in a stepwise manner. Our results revealed the formation of a rare xanthene ring in type II polyketide biosynthesis, and demonstrate the potential of using total biosynthesis for the discovery of natural products synthesized by type II PKSs.

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“…To do so, pgaE was cloned under the control of the SP44 promoter in the pS-GK plasmid, lacking the kanamycin reporter gene. 58 This plasmid comprises the T7 terminator, the SP44 promoter, the ribosomal binding site (RBS-SR41) and the reporter gene, sfgfp (super folder green fluorescent protein), cloned into pSET152. The pgaE gene was amplified from the genome of Streptomyces sp.…”

Section: Generation Of Constructs For the Complementation Of Lugoi Lu...mentioning

confidence: 99%

Unravelling key enzymatic steps in C-ring cleavage during angucyclines biosynthesis

Elsayed,

Heul,

Xiao

et al. 2023

Preprint

0

View full text Add to dashboard Cite

Angucyclines are type II polyketide natural products, often characterized by unusual structural rearrangements through B- or C-ring cleavage of their tetracyclic backbone. While the enzymes involved in B-ring cleavage have been extensively studied, little is known of the enzymes leading to C-ring cleavage. Here, we unravel the function of the oxygenases involved in the biosynthesis of lugdunomycin, a highly rearranged C-ring cleaved angucycline derivative. Targeted deletion of the oxygenase genes, in combination with molecular networking and structural elucidation, showed that LugOI is essential for C12 oxidation and maintaining a keto group at C6 that is reduced by LugOII, resulting in a key intermediate towards C-ring cleavage. An epoxide group is then inserted by LugOIII, and stabilized by the novel enzyme LugOV for the subsequent cleavage. Thus, for the first time we describe the oxidative enzymatic steps that form the basis for a wide range of rearranged angucycline natural products.

show abstract

Single cell mutant selection for metabolic engineering of actinomycetes

Cited by 8 publications

References 58 publications

Unravelling key enzymatic steps in C-ring cleavage during angucycline biosynthesis

Unravelling key enzymatic steps in C-ring cleavage during angucycline biosynthesis

Total Biosynthesis of Mutaxanthene Unveils a Flavoprotein Monooxygenase Catalyzing Xanthene Ring Formation

Unravelling key enzymatic steps in C-ring cleavage during angucyclines biosynthesis

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