Rose M. Sheridan

Optimizing natural products by biosynthetic engineering: Discovery of nonquinone Hsp90 inhibitors

A biosynthetic medicinal chemistry approach was applied to the optimization of the natural product Hsp90 inhibitor macbecin. By genetic engineering, mutants have been created to produce novel macbecin analogues including a nonquinone compound (5) that has significantly improved binding affinity to Hsp90 (Kd 3 nM vs 240 nM for macbecin) and reduced toxicity (MTD > or = 250 mg/kg). Structural flexibility may contribute to the preorganization of 5 to exist in solution in the Hsp90-bound conformation.

Roles of rapH and rapG in Positive Regulation of Rapamycin Biosynthesis in Streptomyces hygroscopicus

Rapamycin is an important macrocyclic polyketide produced by Streptomyces hygroscopicus and showing immunosuppressive, antifungal, and antitumor activities as well as displaying anti-inflammatory and neuroregenerative properties. The immense pharmacological potential of rapamycin has led to the production of an array of analogues, including through genetic engineering of the rapamycin biosynthetic gene cluster. This cluster contains several putative regulatory genes. Based on DNA sequence analysis, the products of genes rapH and rapG showed high similarities with two different families of transcriptional activators, LAL and AraC, respectively. Overexpression of either gene resulted in a substantial increase in rapamycin biosynthesis, confirming their positive regulatory role, while deletion of both from the chromosome of S. hygroscopicus resulted in a complete loss of antibiotic production. Complementation studies indicated an essential role of the RapG regulator for rapamycin biosynthesis and a supportive role of RapH. A direct effect of rapH and rapG gene products on the promoter of the rapamycin polyketide synthase operon, rapA-rapB, was observed using the chalcone synthase gene rppA as a reporter system.

Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: insights into nitrile formation †

The 18‐membered polyketide macrolide borrelidin exhibits a number of important biological activities, including potent angiogenesis inhibition. This has prompted two recent total syntheses as well as the cloning of the biosynthetic gene cluster from Streptomyces parvulus Tü4055. Borrelidin possesses some unusual structural characteristics, including a cyclopentane carboxylic acid moiety at C17 and a nitrile moiety at C12 of the macrocyclic ring. Nitrile groups are relatively rare in nature, and little is known of their biosynthesis during secondary metabolism. The nitrile group of borrelidin is shown here to arise from the methyl group of a methylmalonyl‐CoA extender unit incorporated during polyketide chain extension. Insertional inactivation of two genes in the borrelidin gene cluster, borI (coding for a cytochrome P450 monooxygenase) and borJ (coding for an aminotransferase), generated borrelidin non‐producing mutants. These mutants accumulated different compounds lacking the C12 nitrile moiety, with the product of the borI‐minus mutant (12‐desnitrile‐12‐methyl‐borrelidin) possessing a methyl group and that of the borJ‐minus mutant (12‐desnitrile‐12‐carboxyl‐borrelidin) a carboxyl group at C12. The former but not the latter was converted into borrelidin when biotransformed by an S. parvulus mutant that is deficient in the biosynthesis of the borrelidin starter unit. This suggests that 12‐desnitrile‐12‐methyl‐borrelidin is a competent biosynthetic intermediate, whereas the carboxylated derivative is a shunt metabolite. Bioconversion of 12‐desnitrile‐12‐methyl‐borrelidin into borrelidin was also achieved in a heterologous system co‐expressing borI and borJ in Streptomyces albus J1074. This bioconversion was more efficient when borK, which is believed to encode a dehydrogenase, was simultaneously expressed with borI and borJ. On the basis of these findings, a pathway is proposed for the formation of the nitrile moiety during borrelidin biosynthesis.

Rapamycin biosynthesis: elucidation of gene product function

The function of gene products involved in the biosynthesis of the clinically important polyketide rapamycin were elucidated by biotransformation and gene complementation.

Biosynthesis of the angiogenesis inhibitor borrelidin: directed biosynthesis of novel analogues

We report the directed biosynthesis of borrelidin analogues and their selective anti-proliferative activity against human cancer cell lines.

Direct production of ivermectin-like drugs after domain exchange in the avermectin polyketide synthase of Streptomyces avermitilis ATCC31272

Ivermectin, a mixture of 22,23-dihydroavermectin B1a9 with minor amounts of 22,23-dihydroavermectin B1b 10, is one of the most successful veterinary antiparasitic drugs ever produced. In humans, ivermectin has been used for the treatment of African river blindness (onchocerciasis) resulting in an encouraging decrease in the prevalence of skin and eye diseases linked to this infection. The components of ivermectin are currently synthesized by chemical hydrogenation of a specific double bond at C22-C23 in the polyketide macrolides avermectins B1a 5 and B1b 6, broad-spectrum antiparasitic agents isolated from the soil bacterium Streptomyces avermitilis. We describe here the production of such compounds (22,23-dihydroavermectins B1a 9 and A1a 11) by direct fermentation of a recombinant strain of S. avermitilis containing an appropriately-engineered polyketide synthase (PKS). This suggests the feasibility of a direct biological route to this valuable drug.

Heterologous expression in Saccharopolyspora erythraea of a pentaketide synthase derived from the spinosyn polyketide synthase

A truncated version of the spinosyn polyketide synthase comprising the loading module and the first four extension modules fused to the erythromycin thioesterase domain was expressed in Saccharopolyspora erythraea. A novel pentaketide lactone product was isolated, identifying cryptic steps of spinosyn biosynthesis and indicating the potential of this approach for the biosynthetic engineering of spinosyn analogues. A pathway for the formation of the tetracyclic spinosyn aglycone is proposed.

Engineered biosynthesis of novel spinosyns bearing altered deoxyhexose substituents

Novel spinosyns have been prepared by biotransformation, using a genetically engineered strain of Saccharopolyspora erythraea, in which the beta-D-forosamine moiety in glycosidic linkage to the hydroxy group at C17 is replaced by alpha-L-mycarose.

Structure guided design of improved anti-proliferative rapalogs through biosynthetic medicinal chemistry

A combination of molecular modelling and rational biosynthetic engineering of the rapamycin polyketide synthase was used to generate rapalogs lacking O- and C-linked methyl groups at positions 16 and 17 respectively. These rapalogs displayed enhanced inhibition of cancer cell lines and were produced at titres close to those of the parent strain. By recapitulating these experiments in higher-producing rapamycin strains, combined with the ectopic expression of gene products acting late in the biosynthetic pathway in order to minimise the accumulation of intermediates, gram-quantities of novel rapalogs bearing multiple structural changes were produced.

Recombinant strains for the enhanced production of bioengineered rapalogs

The rapK gene required for biosynthesis of the DHCHC starter acid that initiates rapamycin biosynthesis was deleted from strain BIOT-3410, a derivative of Streptomyces rapamycinicus which had been subjected to classical strain and process development and capable of robust rapamycin production at titres up to 250mg/L. The resulting strain BIOT-4010 could no longer produce rapamycin, but when supplied exogenously with DHCHC produced rapamycin at titres equivalent to its parent strain. This strain enabled mutasynthetic access to new rapalogs that could not readily be isolated from lower titre strains when fed DHCHC analogs. Mutasynthesis of some rapalogs resulted predominantly in compounds lacking late post polyketide synthase biosynthetic modifications. To enhance the relative production of fully elaborated rapalogs, genes encoding late-acting biosynthetic pathway enzymes which failed to act efficiently on the novel compounds were expressed ectopically to give strain BIOT-4110. Strains BIOT-4010 and BIOT-4110 represent valuable tools for natural product lead optimization using biosynthetic medicinal chemistry and for the production of rapalogs for pre-clinical and early stage clinical trials.