Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae

Rodrı́guez, Eduardo; Ward, Shannon L.; Fu, Huimin; Revill, W. Peter; McDaniel, Robert; Katz, Leonard

doi:10.1007/s00253-004-1658-7

Cited by 29 publications

(35 citation statements)

References 22 publications

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“…Researchers are now focusing on PPTs within alternative heterologous hosts, such as Pseudomonas (16) and Streptomyces (20,40) species. The ability to distinguish between the Sfp-like PPT subfamilies and the likelihood of association with either PK or NRPS biosynthetic pathways has important implications in heterologous expression.…”

Section: Discussionmentioning

confidence: 99%

The Phosphopantetheinyl Transferase Superfamily: Phylogenetic Analysis and Functional Implications in Cyanobacteria

Copp

Neilan

2006

Appl Environ Microbiol

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Phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds including fatty acid, polyketide, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. PPTs display low levels of sequence similarity but can be classified into two major families based on several short motifs. The prototype of the first family is the broad-substrate-range PPT Sfp, which is required for biosynthesis of surfactin in Bacillus subtilis. The second family is typified by the Escherichia coli acyl carrier protein synthase (AcpS). Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were utilized in this research to reveal novel subfamily groupings, including two subfamilies within the Sfp-like family. In the present study degenerate oligonucleotide primers were designed for amplification of cyanobacterial PPT gene fragments. Subsequent phylogenetic analyses suggested a unique, function-based PPT type, defined by the PPTs involved in heterocyst differentiation. Evidence supporting this hypothesis was obtained by sequencing the region surrounding the partial Nodularia spumigena PPT gene. The ability to genetically classify PPT function is critical for the engineering of novel compounds utilizing combinatorial biosynthesis techniques. Information regarding cyanobacterial PPTs has important ramifications for the ex situ production of cyanobacterial natural products.

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Section: Discussionmentioning

confidence: 99%

The Phosphopantetheinyl Transferase Superfamily: Phylogenetic Analysis and Functional Implications in Cyanobacteria

Copp

Neilan

2006

Appl Environ Microbiol

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“…and Streptomyces spp. (22,36), are being investigated for heterologous expression and biochemical characterization of secondary metabolite gene clusters from cyanobacteria.…”

Section: Discussionmentioning

confidence: 99%

Characterization of PPT _Ns , a Cyanobacterial Phosphopantetheinyl Transferase from Nodularia spumigena NSOR10

et al. 2007

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The phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds, including fatty acids, polyketides, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by transfer of a phosphopantetheinyl moiety. The diverse PPT superfamily can be divided into two families based on specificity and conserved sequence motifs. The first family is typified by the Escherichia coli acyl carrier protein synthase (AcpS), which is involved in fatty acid synthesis. The prototype of the second family is the broad-substraterange PPT Sfp, which is required for surfactin biosynthesis in Bacillus subtilis. Most cyanobacteria do not encode an AcpS-like PPT, and furthermore, some of their Sfp-like PPTs belong to a unique phylogenetic subgroup defined by the PPTs involved in heterocyst differentiation. Here, we describe the first functional characterization of a cyanobacterial PPT based on a structural analysis and subsequent functional analysis of the Nodularia spumigena NSOR10 PPT. Southern hybridizations suggested that this enzyme may be the only PPT encoded in the N. spumigena NSOR10 genome. Expression and enzyme characterization showed that this PPT was capable of modifying carrier proteins resulting from both heterocyst glycoplipid synthesis and nodularin toxin synthesis. Cyanobacteria are a unique and vast source of bioactive metabolites; therefore, an understanding of cyanobacterial PPTs is important in order to harness the biotechnological potential of cyanobacterial natural products.Phosphopantetheinyl transferases (PPTs) are enzymes that are required for the activation of carrier proteins in the pathways for synthesis of fatty acids and a wide range of diverse metabolites, including nonribosomal peptides and polyketides (24). The PPT superfamily can be separated into two families of enzymes based on sequence and substrate specificity. The first family includes the acyl carrier protein (ACP) synthase (AcpS)-type PPTs (120 amino acids), which are involved in activating carrier proteins involved in primary metabolism, including carrier proteins involved in fatty acid synthesis (FAS). The majority of microorganisms harbor an AcpS-type PPT, which typically has a limited range of specificity for carrier proteins involved in secondary metabolism. The second group of PPTs is the Sfp-like family (230 amino acids), which exhibit similarity to the Bacillus subtilis PPT Sfp, which is responsible for the activation of carrier proteins in the biosynthetic pathway for surfactin (24, 34). Microorganisms which require activation of carrier proteins involved in secondary metabolism pathways, such as carrier proteins involved in nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways, require the activity of an Sfp-like PPT. Some members of the Sfp-like family, which can be further divided into the W/KEA and F/KES subfamilies (11), exhibit a wide range of activity with noncognate carrier proteins. T...

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“…Floss et al (7) subsequently showed that during ansamitosin biosynthesis, O-methylation occurs before incorporation by the PKS; therefore, MM-ACP is the in vivo extender unit, not HM-ACP. Whereas genetic experiments support the hypothesis that these enzymes are involved in the incorporation of a MM-ACP extender unit (7,10,11) and a crystal structure of FkbI has been determined (9), biochemical evidence for MM-ACP has yet to be provided.…”

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confidence: 99%

Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units

Chan

Boyne

Podevels³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

126

128

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Combinatorial biosynthesis of type I polyketide synthases is a promising approach for the generation of new structural derivatives of polyketide-containing natural products. A target of this approach has been to change the extender units incorporated into a polyketide backbone to alter the structure and activity of the natural product. One limitation to these efforts is that only four extender units were known: malonyl-CoA, methylmalonyl-CoA, ethylmalonyl-CoA, and methoxymalonyl-acyl carrier protein (ACP). The chemical attributes of these extender units are quite similar, with the exception of the potential hydrogen bonding interactions by the oxygen of the methoxy moiety. Furthermore, the incorporated extender units are not easily modified by using simple chemical approaches when combinatorial biosynthesis is coupled to semisynthetic chemistry. We recently proposed the existence of two additional extender units, hydroxymalonyl-ACP and aminomalonyl-ACP, involved in the biosynthesis of zwittermicin A. These extender units offer unique possibilities for combinatorial biosynthesis and semisynthetic chemistry because of the introduction of free hydroxyl and amino moieties into a polyketide structure. Here, we present the biochemical and mass spectral evidence for the formation of these extender units. This evidence shows the formation of ACP-linked extender units for polyketide synthesis. Interestingly, aminomalonyl-ACP formation involves enzymology typically found in nonribosomal peptide synthesis.antibiotics ͉ combinatorial biosynthesis

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Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae

Cited by 29 publications

References 22 publications

The Phosphopantetheinyl Transferase Superfamily: Phylogenetic Analysis and Functional Implications in Cyanobacteria

The Phosphopantetheinyl Transferase Superfamily: Phylogenetic Analysis and Functional Implications in Cyanobacteria

Characterization of PPT _Ns , a Cyanobacterial Phosphopantetheinyl Transferase from Nodularia spumigena NSOR10

Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units

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Engineered biosynthesis of 16-membered macrolides that require methoxymalonyl-ACP precursors in Streptomyces fradiae

Cited by 29 publications

References 22 publications

The Phosphopantetheinyl Transferase Superfamily: Phylogenetic Analysis and Functional Implications in Cyanobacteria

The Phosphopantetheinyl Transferase Superfamily: Phylogenetic Analysis and Functional Implications in Cyanobacteria

Characterization of PPT Ns , a Cyanobacterial Phosphopantetheinyl Transferase from Nodularia spumigena NSOR10

Hydroxymalonyl-acyl carrier protein (ACP) and aminomalonyl-ACP are two additional type I polyketide synthase extender units

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Characterization of PPT _Ns , a Cyanobacterial Phosphopantetheinyl Transferase from Nodularia spumigena NSOR10