directed biosynthesis of epothilones in Escherichia coli [Boddy, C
and polyketides [10 x Precursor-directed biosynthesis of epothilone in ..
Precursor-Directed Biosynthesis of Epothilone in Escherichia c oli
To evaluate these options, the expression levels of the biosynthetically relevant proteins were compared between the two hosts. E. coli HYL3/pBP175 showed an approximately 3-fold higher expression of soluble module 2 than BAP1/pBP175 (). Owing to their large size and codon usage differences between the native and heterologous hosts, type I polyketide synthase proteins are generally not expressed well in E. coli. Previous work has shown the loading of diketide thioester substrates onto DEBS module 2 to be slow compared to subsequent steps, suggesting the increased concentration of module 2 likely relieves a kinetic bottleneck in precursor directed biosynthesis of erythromycin antibiotics.
To test the relevance of the above results for precursor directed biosynthesis of unnatural erythromycin analogs, we targeted preparation of an unnatural macrolide bearing an alkynyl functional group. Alkynes can be used as particularly convenient handles for further semi-synthetic modification using cycloaddition chemistry –. An alkynyl diketide substrate (2, ) was prepared and fed to the various cell lines as detailed in the methods section. The presence of macrolide 4 and absence of compound 3 in liquid cultures that were fed diketide 2 was confirmed by high-resolution mass spectrometry. This result suggests that the bioactivity observed in these experiments does arise from the production of a novel alkynyl erythromycin analog (see ). The bioconversion efficiency of diketide 2 was comparable to that of diketide 1, and the relative productivity trends observed between BAP1/pBP175/pBP130/pHL80*/pHL74 and various mutant strains characterized in this study were also similar for the two precursors (). These results reinforce the utility of E. coli HYL3/pBP175#8/pBP130/pHL80*/pHL74 as a superior cellular biocatalyst for precursor directed biosynthesis of erythromycin analogs.
biosynthesis of epothilone in Escherichia coli.
As shown below, the ability of E. coli HYL3 to effect increased erythromycin production by precursor directed biosynthesis is not exclusive to the wild-type system as it also led to increased titers of 4, an unnatural alkynyl derivative. ().
The above result suggested that DEBS module 2 could be an attractive target for evolution aimed at further enhancing precursor directed biosynthesis of macrolides. In particular, due to the high cost of synthetic precursors and the strong dependence of polyketide production on exogenous precursor concentrations we sought to identify mutants capable of more efficient transformation of low concentrations of synthetic precursors into macrolides. We therefore subjected plasmid pBP175 to random mutagenesis by passing it through the mutator strain of E. coli, XL1-Red , for multiple generations. Plasmid DNA was isolated and co-transformed into HYL3 along with unmutated plasmids pBP130, pHL80* and pHL74. Approximately 500–1000 colonies from the resulting library of transformants were screened in a single-colony assay for mutants that showed larger zones of growth inhibition of B. subtilis than HYL3/pBP175/pBP130/pHL80*/pHL74 in the presence of limiting (10µM) concentrations of diketide 1. Two mutants, designated mutant #1 and mutant #8, were isolated and purified by re-streaking and re-testing. As discussed below, their enhanced ability to convert synthetic precursors into corresponding macrolides is not limited to diketide 1 alone (). By isolating and characterizing the host cell and each plasmid from mutant #1 and mutant #8, it was deduced that both mutants contained phenotypically relevant changes in plasmid pBP175, which were designated pBP175 #1 and pBP175 #8, respectively.
Precursor-directed biosynthesis of epothilone in Escherichia coli
Recently, the entire erythromycin biosynthetic pathway has been reconstituted in E. coli,, enabling the production of active antibacterial agents in this heterologous host. A rapid and sensitive screen for detecting erythromycin from precursor directed biosynthesis was also established . Here, we have modified the erythromycin biosynthetic pathway in a directed fashion increasing titers in E. coli.
The feasibility of producing inactive macrolides aglycones in E. coli by precursor directed biosynthesis was originally demonstrated using an engineered host BAP1 containing plasmids pBP175, which encodes for DEBS module 2 and propionyl-CoA carboxylase, and pBP130, which encodes for DEBS2 and DEBS3 (; ) . Subsequently, we engineered two additional compatible plasmids pHL80, which encodes mycarose biosynthetic and glycosyl transfer functions, and pHL74, which encodes desosamine biosynthetic and glycosyl transfer functions () to produce bioactive glycosylated macrolides . E. coli BAP1/pBP175/pBP130/pHL80/pHL74 produces the antibiotic 6-deoxyerythromycin D (6d-EryD) when fed with compound 1 (), a synthetic cell permeable mimic of the natural diketide intermediate in erythromycin biosynthesis . Through directed evolution, we also isolated a mutant plasmid pHL80*, which substantially improves mycarosylation of the aglycone compared to wild-type pHL80, facilitating improved precursor directed biosynthesis . A simple, visual single colony screening assay was used to isolate a mutant of E. coli BAP1/pBP175/pBP130/pHL80*/pHL74 (designated Mutant D) which showed a >3-fold improvement in the amount of 6-deoxyerythromycin D production with high (>0.3 mM) concentrations of diketide 1. Here we have analyzed Mutant D with the goal of understanding the molecular basis of this phenotype, and have extended this finding through additional rounds of directed evolution toward improved mutants. We identified and analyzed several new mutants to explain the improved erythromycin biosynthesis from simple precursors, and demonstrated their utilities for the engineered biosynthesis of a novel derivative of erythromycin with promising antibacterial properties.
coli by precursor directed biosynthesis was ..
of epothilone in Escherichia coli.
21/12/2015 · The feasibility of producing inactive macrolides aglycones in E
agent epothilone C in Escherichia coli.
08/01/2018 · Total Synthesis and Precursor Directed Biosynthesis of ..
01/10/2017 · Figure 3
using recombination in Escherichia coli
Notwithstanding its promise, there are significant limitations to precursor directed biosynthesis. The feeding of labile precursors to Streptomyces or Saccharopolyspora bacteria that naturally produce bioactive polyketides is fraught with challenges ,–. By contrast, incorporation of precursors into heterologous polyketide biosynthetic pathways in E. coli is relatively efficient , but such systems typically yield inactive polyketide intermediates which have not undergone post-PKS modifications. Furthermore, due to the low affinity of the acceptor PKS module for unnatural thioester substrates , precursors are often required to be fed in high concentration to the engineered strains to effectively transform precursors into polyketide products. The substrate specificity of one or more downstream PKS modules may also cause kinetic bottlenecks in the production of polyketides by precursor directed biosynthesis ,.
“Improved precursor-directed biosynthesis in E
Erythromycin and related macrolide antibiotics are widely used polyketide natural products. We have evolved an engineered biosynthetic pathway in Escherichia coli that yields erythromycin analogs from simple synthetic precursors. Multiple rounds of mutagenesis and screening led to the identification of new mutant strains with improved efficiency for precursor directed biosynthesis. Genetic and biochemical analysis suggested that the phenotypically relevant alterations in these mutant strains were localized exclusively to the host-vector system, and not to the polyketide synthase. We also demonstrate the utility of this improved system through engineered biosynthesis of a novel alkynyl erythromycin derivative with comparable antibacterial activity to its natural counterpart. In addition to reinforcing the power of directed evolution for engineering macrolide biosynthesis, our studies have identified a new lead substance for investigating structure-function relationships in the bacterial ribosome.
Engineered polyketide biosynthesis and ..
In summary, we have evolved a system capable of improved production of erythromycin-type macrolide antibiotics by precursor directed biosynthesis in E. coli. The parent producer of 6-deoxyerythromycin D was subjected to two rounds of random mutagenesis and with the improved mutants identified via activity-based single colony screening procedures. Phenotypic characterization revealed no changes of the biosynthetic proteins themselves, but rather a genetically more stable system in which altered protein expression relieved a kinetic bottleneck present in the wild-type system. The best of these mutants is not only able to produce four-fold more antibiotic, but also does so at a relatively low precursor concentration. Finally, we have demonstrated the utility of this system by, for the first time, reporting the production of a novel erythromycin analog derived from a simple synthetic precursor in a single E. coli cell line. This new antibiotic not only displays promising antibacterial activity, but also possesses an attractive handle for semi-synthetic modification.
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