Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli (original) (raw)

Using Chemobiosynthesis and Synthetic Mini-Polyketide Synthases To Produce Pharmaceutical Intermediates in Escherichia coli

Applied and Environmental Microbiology, 2010

Recombinant microbial whole-cell biocatalysis is a valuable approach for producing enantiomerically pure intermediates for the synthesis of complex molecules. Here, we describe a method to produce polyketide intermediates possessing multiple stereogenic centers by combining chemobiosynthesis and engineered mini-polyketide synthases (PKSs). Chemobiosynthesis allows the introduction of unnatural moieties, while a library of synthetic bimodular PKSs expressed from codon-optimized genes permits the introduction of a variety of ketide units. To validate the approach, intermediates for the synthesis of trans -9,10-dehydroepothilone D were generated. The designer molecules obtained have the potential to greatly reduce the manufacturing cost of epothilone analogues, thus facilitating their commercial development as therapeutic agents.

Recent advances in the heterologous expression of microbial natural product biosynthetic pathways

Natural Product Reports, 2013

The heterologous expression of microbial natural product biosynthetic pathways coupled with advanced DNA engineering enables optimisation of product yields, functional elucidation of cryptic gene clusters, and generation of novel derivatives. This review summarises the recent advances in cloning and maintenance of natural product biosynthetic gene clusters for heterologous expression and the efforts fundamental for discovering novel natural products in the post-genomics era, with a focus on polyketide synthases (PKSs) and non-ribosomal polypeptide synthetases (NRPS).

Construction of a Part of a 3‑Hydroxypropionate Cycle for Heterologous Polyketide Biosynthesis in Escherichia coli

Polyketides, an important class of natural products with complex chemical structures, are widely used as antibiotics and other pharmaceutical agents. A clear barrier to heterologous polyketide biosynthesis in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a common substrate of multimodular polyketide synthases. Here we report a route for synthesizing (2S)-methylmalonyl-CoA from malonyl-CoA with a 3-hydroxypropionate cycle in thermoacidophilic crenarchaeon. The engineered E. coli strain produced both propionyl-CoA and methylmalonyl-CoA at intracellular levels similar to those of acetyl-CoA and succinyl-CoA, respectively. This approach may open a way to produce a variety of polyketide drugs in E. coli from renewable carbon sources. Figure 1. Part of a 3-hydroxypropionate cycle. Rapid Report pubs.acs.org/biochemistry

Escherichia coli Allows Efficient Modular Incorporation of Newly Isolated Quinomycin Biosynthetic Enzyme into Echinomycin Biosynthetic Pathway for Rational Design and Synthesis of Potent Antibiotic Unnatural Natural Product

Journal of The American Chemical Society, 2009

Natural products display impressive activities against a wide range of targets, including viruses, microbes and tumors. However, their clinical use is hampered frequently by their scarcity and undesirable toxicity. Not only can engineering Escherichia coli for plasmid-based pharmacophore biosynthesis offer alternative means of simple and easily-scalable production of valuable yet hardto-obtain compounds, but also carries a potential for providing a straightforward and efficient means of preparing natural product analogs. The quinomycin family of nonribosomal peptides, including echinomycin, trtiostin A and SW-163s, are important secondary metabolites imparting antibiotic antitumor activity via DNA bisintercalation. Previously we have shown the production of echinomycin and trtiostin A in E. coli using our convenient and modular plasmid system to introduce these heterologous biosynthetic pathways into E. coli. However, we have yet to develop a novel biosynthetic pathway capable of producing bioactive unnatural natural products in E. coli. Here we . hoik@sci.hokudai.ac.jp. Supporting Information Available: Experimental procedures, two tables of DNA sequence information for the oligonucleotides, a figure for mass spectra of 3, 4, 5, 6, 7 isolated from labeled methionine-feeding experiment, and a table and two figures of spectrometric data on the E. coli-produced 14.

Insights into an Unusual Nonribosomal Peptide Synthetase Biosynthesis

Journal of Biological Chemistry, 2010

The GE81112 tetrapeptides (1-3) represent a structurally unique class of antibiotics, acting as specific inhibitors of prokaryotic protein synthesis. Here we report the cloning and sequencing of the GE81112 biosynthetic gene cluster from Streptomyces sp. L-49973 and the development of a genetic manipulation system for Streptomyces sp. L-49973. The biosynthetic gene cluster for the tetrapeptide antibiotic GE81112 (getA-N) was identified within a 61.7-kb region comprising 29 open reading frames (open reading frames), 14 of which were assigned to the biosynthetic gene cluster. Sequence analysis revealed the GE81112 cluster to consist of six nonribosomal peptide synthetase (NRPS) genes encoding incomplete di-domain NRPS modules and a single free standing NRPS domain as well as genes encoding other biosynthetic and modifying proteins. The involvement of the cloned gene cluster in GE81112 biosynthesis was confirmed by inactivating the NRPS gene getE resulting in a GE81112 production abolished mutant. In addition, we characterized the NRPS A-domains from the pathway by expression in Escherichia coli and in vitro enzymatic assays. The previously unknown stereochemistry of most chiral centers in GE81112 was established from a combined chemical and biosynthetic approach. Taken together, these findings have allowed us to propose a rational model for GE81112 biosynthesis. The results further open the door to developing new derivatives of these promising antibiotic compounds by genetic engineering.

Engineered polyketide biosynthesis and biocatalysis in Escherichia coli

Applied Microbiology and Biotechnology, 2010

Polyketides are important bioactive natural products biosynthesized by bacteria, fungi, and plants. The enzymes that synthesize polyketides are collectively referred to as polyketide synthases (PKSs). Because many of the natural hosts that produce polyketides are difficult to culture or manipulate, establishing a universal heterologous host that is genetically tractable has become an important goal toward the engineered biosynthesis of polyketides and analogues. Here, we summarize the recent progresses in engineering Escherichia coli as a heterologous host for reconstituting PKSs of different types. Our increased understanding of PKS enzymology and structural biology, combined with new tools in protein engineering, metabolic engineering, and synthetic biology, has firmly established E. coli as a powerful host for producing polyketides.

Metabolically engineeredEscherichia colifor efficient production of glycosylated natural products

Microbial Biotechnology, 2008

Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP-4-keto-6-deoxyglucose resulting in increased production of the heterologous sugars TDP-L-mycarose and TDP-D-desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6-deoxyerythronolide B (6-dEB) or 3-a-mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60-and 25-fold more erythromycin D (EryD) than the original strain K207-3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.

Discovery Strategies of Bioactive Compounds Synthesized by Nonribosomal Peptide Synthetases and Type-I Polyketide Synthases Derived from Marine Microbiomes

Marine drugs, 2016

Considering that 70% of our planet's surface is covered by oceans, it is likely that undiscovered biodiversity is still enormous. A large portion of marine biodiversity consists of microbiomes. They are very attractive targets of bioprospecting because they are able to produce a vast repertoire of secondary metabolites in order to adapt in diverse environments. In many cases secondary metabolites of pharmaceutical and biotechnological interest such as nonribosomal peptides (NRPs) and polyketides (PKs) are synthesized by multimodular enzymes named nonribosomal peptide synthetases (NRPSes) and type-I polyketide synthases (PKSes-I), respectively. Novel findings regarding the mechanisms underlying NRPS and PKS evolution demonstrate how microorganisms could leverage their metabolic potential. Moreover, these findings could facilitate synthetic biology approaches leading to novel bioactive compounds. Ongoing advances in bioinformatics and next-generation sequencing (NGS) technologies ...