De novo biosynthesis of terminal alkyne-labeled natural products

Abstract

Abstract:The terminal alkyne is a functionality widely used in organic synthesis, pharmaceutical science, material science, and bioorthogonal chemistry. This functionality is also found in acetylenic natural products, but the underlying biosynthetic pathways for its formation are not well understood. Here we report the characterization of the first carrier proteindependent terminal alkyne biosynthetic machinery in microbes. We further demonstrate that this enzymatic machinery can be exploited for the in situ generation and incorporation of terminal alkynes into two natural product scaffolds in E. coli. These results highlight the prospect for tagging major classes of natural products, including polyketides and polyketide/non-ribosomal peptide hybrids, using biosynthetic pathway engineering.2 Natural products are important small molecules widely used as drugs, pesticides, herbicides, and biological probes. Tagging natural products with a unique chemical handle enables the visualization, enrichment, quantification, and mode of action study of natural products through bioorthogonal chemistry [1][2][3][4] . One prevalent bioorthogonal reaction is the triazole-forming azide-alkyne [3+2] cycloaddition, often referred to as "click" chemistry 5 . This reaction has enabled selective imaging and study of azide-or alkyne-labeled glycans, proteins, nucleic acids and lipids. Despite the success with macromolecules, the labeling of natural products has not been adequately explored. It is often challenging to obtain tagged natural products through total synthesis because of their structural complexity, or through semi-synthesis because of their chemical lability and the limited supply of most natural products. Alternatively, precursor-directed biosynthesis (PDB) may be employed to produce azide-or alkyne-labeled natural products based on the promiscuity of biosynthetic machinery. Mainly used as a tool to introduce structural diversity, PDB has allowed us and other researchers to generate labeled natural products 3,[6][7][8] . However, the coexistence of diffusible precursors and final products 9 with the same chemical handle introduces significant background in the PDB production system, making it incompatible with in situ bioorthogonal chemical transformations. We report here the de novo biosynthesis of alkyne-labeled natural products without the feeding of alkynoic precursors by characterizing and engineering the novel terminal alkyne synthetic machinery that living systems offer.Many acetylenic natural products contain a terminal alkyne functionality, which seems to be crucial for their bioactivity (Fig. 1) 10,11 . Terminal alkynes can be formed by acetylenases, a special family of desaturases that catalyze O 2 -dependent dehydrogenation of C-C bonds in a diiron dependent mechanism 12 . Several membrane-bound acetylenases have recently been 3 identified from plants, insects, fungi, and bacteria 4,10,[13][14][15][16][17] , but all of these reports have been limited to bioinformatics or in vivo studies through mutagenesis or ...

One-pot access to l-5,6-dihalotryptophans and l-alknyltryptophans using tryptophan synthase

“…With the dihalotryptophans in hand, we decided to explore the possibility of using this reaction to process an alkynyl indole (Scheme 3). Incorporation of an alkyne "tag" into a small molecule, protein or natural product can enable selective diversification and tagging, 12 through an alkyneazide cycloaddition reaction, commonly known as "click chemistry". 13 We wanted to see if alkyne-substituted indoles were amenable to the tryptophan synthase reaction.…”

Metabolic Engineering of Microorganisms for the Production of Natural Compounds

“…Especially, investigation of traditional oriental medicines will propose many more novel natural product targets for microbial production. [290] Moreover, with novel enzymes being uncovered, such as the ones in charge of 4+2 cyclic reaction [291] and alkyne biosynthesis, [292] discovery and production of novel natural compounds by metabolic engineering has been greatly facilitated. Overall, cumulated systems biological techniques with recently being developed innovative engineering tools would greatly accelerate this field of research with the objective of providing invaluable natural compounds efficiently.…”

Recent advances in the biosynthesis of unusual polyketide synthase substrates

“…Following subsequent extension, presumably via primary metabolic fatty acid synthase (FAS), and conversion into the malonate derivative via AntE (see below), 5-hexynoylmalonyl-CoA was incorporated into an antimycin analogue ( 19 ) as an extender unit (Figure 3D(ii)). 24 This result paves the way to utilizing this trio of genes to generate “taggable” polyketides without the need for the feeding of alkyne precursors.…”

“…Following this, JamB desaturates the terminal alkene of 18 to yield the alkyne 16 , which is used to prime the PKS (Figure 3D(i)). 24 In vitro investigations suggested that JamB does not exhibit much substrate tolerance. When challenged with alkenoyl-JamC thioesters of varying length (5 to 8 carbons) conversion to the corresponding alkynoyl-JamC was only observed for 16 .…”

“…Such substrate specificity was also observed with regards to the terminal alkene moiety, as no activity was detected when the position of unsaturation was altered in the starting hexenoic acid. 24 The genes responsible for the provision of 16 are arranged in a single operon, forming a convenient cassette which can be utilized as a biosynthetic “tool” in the engineering of PKS pathways. Indeed, introduction of the jamA,B,C cassette into a heterologous Escherichia coli host in which a portion of the antimycin PKS/non-ribosomal peptide synthetase (NRPS) was coexpressed, resulted in the in situ generation of 16 .…”