Epothilone biosynthesis

Scheme 10.8 Biosynthesis of epothilone. Individual PKS domains are represented as circles and individual NRPS domains as hexagons. Acyl carrier proteins (ACPs) and thiola-tion domains (T) are posttranslationally modified by a phos-phopantetheinyl group to which the biosynthetic intermediates are covalently bound throughout the chain assembly. The thioesterase domain (TE) cyclizes the fully assembled carbon chain to give the 16-membered lactone. Following dehydration of Cl 2—Cl 3 to give epothilones C and D, the final step in epothilone biosynthesis is the epoxidation of the C12=C13 double bond by the cytochrome P450 enzyme P450epol<. KS ketosyn-thase KS(Y) active-site tyrosine mutant of KS AT acyltransfer-ase C condensation domain A adenylation domain ...

Using precursor-directed biosynthesis, Boddy et a/. were able to effectively reconstitute epothilone biosynthesis in E. coli. A pentaketide NAC thioester intermediate... 

L. Elucidating the mechanism of cis double bond formation in 47. epothilone biosynthesis. J. Am. Chem. Soc. 2004 126 46-47. 

To see whether the unusual lack of substrate selectivity of the AT domain of module 4 could be mimicked in other modules of the epo PKS, AT4 was used to replace the native AT domain in module 3 in an M xanthus host that contained the epothilone biosynthesis gene cluster. The exchange of the DNA encoding AT3 for the DNA segment encoding AT4 was accomplished employing a two-step recombination process (Fig. 3). The resulting strain was fermented and the epothilone products were isolated and analyzed. As found in the parent strain... 

Figure 19 Epothilone biosynthesis and novel derivatives generated by genetic engineering. Domains with an asterisk are assumed to be inactive. The hydroxyl group formed by the KR domain in module 4 is dehydrated by the DH domain found in module 5. Module 4 incorporates either malonyl-CoA or methylmalonyl-CoA extender units (the figure only shows the incorporation of malonyl-CoA). Epothilone derivatives generated by genetic engineering are shown in boxes (see text) and the sites of mutagenesis are indicated.

Figure 11 Chemoenzymatic synthesis of epothilone C with pathway-specific TE domain. This TE was excised from the last PKS module involved in epothilone biosynthesis. Its function in production of epothilone C (28) further suggests the versatility of TE domains from PKS, NRPS, PKS/NRPS hybrid, and NRPS/PKS hybrid systems.

C. Walsh, N. Kelleher, Mass spectrometric interrogation of thioester-bound intermediates in the initial stages of epothilone biosynthesis, Chem. Biol. 2004, 11, 327-335. 

Figure 3.114 Proposal for the formation of the 2-methyl-4-carboxythiazole starter unit during epothilone biosynthesis.

H.W. Chen, S. O Connor, D.E. Cane, C.T. Walsh, Epothilone biosynthesis assembly of the methylthiazolylcarboxy starter unit on the EpoB subunit. Chem. Biol. 8, 899-912 (2001)... 

In NRPS, the cyclization domain catalyzes cyclization of the side-chain nucleophile from a dipeptide moiety such as AA-Ser or AA-Cys (AA = amino acids) to form a tetrahedral intermediate, followed by dehydration to form oxazolines and thiazolines (Scheme 7.1) [20]. The synthesis of a 2-methyl oxazoline from threonine follows a similar mechanism. Once a heterocycle is formed, it can be further modified by reductase to form tetrahydro thiazolidine in the case of pyochelin biosynthesis. Conversely, oxidation of the dehydroheterocycles lead to heteroaro-mahc thiazoles or oxazoles as in the case of epothilone D (Figure 7.2) [21]. 

While there are many natural products that are either NRPS or PKS derived, there are multidomain megasynthases that contain both NRPS and PKS biosynthetic features. Examples of such biosynthetic motifs are epothilone, jamaicamide, and the endiyne C-1027.33-35 At NRPS-PKS interfaces in these systems, the condensation of malonyl takes place with an amino acid that was activated by an A domain. Alternatively, an amino acid loaded on a T domain condenses with an elongating polyketide. Since the substrates and intermediates in NRPS, PKS, or hybrid NRPS/PKS biosynthesis introduce mass changes on T domains, they are ideal candidates for investigation by MS. 

Figure 5. Proposed scheme for the biosynthesis of epothilone intermediates from modules 3-5 in E. coli. The segment containing modules 3. 4, 5 and the TE domain were comtructed in an expression vector and expressed as described in the text.

These findings provide evidence that the DH domain of module 5 is responsible for the introduction of two successive double bonds in epothilones C and D the c/s-I2,13 and the trans- Q,, the latter of which, in the normal course of biosynthesis, is reduced by the action of ER5. 10,11-... 

Escherichia coli has also been engineered for epothilone production. The genes encoding the entire gene cluster were redesigned in silica and then synthesized to enable optimal tuning of the codon usage for expression in E. coli Expression of the cluster in a strain of E. coli modified to enable polyketide biosynthesis resulted in the production of epothilones C and D. 

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