Biosynthesis of rapamycin and its regulation: past achievements and recent progress

The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzymesubstrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs.The assembly line architecture of multimodular polyketide synthases (PKSs) 5 represents a promising catalytic framework for combinatorial biosynthesis (1). A particularly well studied example of this family of multienzyme systems is the 6-deoxyerythronolide B synthase (DEBS), which produces 6-deoxyerythronolide B (Fig. 1), the parent aglycone of the macrolide antibiotic erythromycin (2). DEBS is comprised of three distinct homodimeric proteins: DEBS1, DEBS2, and DEBS3, each containing two PKS modules, with each module being responsible for a distinct round of polyketide elongation and modification. DEBS uses propionyl-CoA to prime the loading didomain (LDD) of module 1 and six methylmalonyl-CoA-derived extender units in catalysis of the six cycles of polyketide chain elongation, followed by terminal release of the mature polyketide chain by thioesterase (TE)-catalyzed macrolactone formation.Since the original genetic characterization of DEBS over two decades ago (3, 4), extensive in vivo and in vitro analysis has revealed that specific protein-protein interactions play a critical role in the proper vectorial channeling of biosynthetic intermediates from one PKS module to the next (5). A particularly effective mini-assembly line for mechanistic analysis has been a simple bimodular derivative of DEBS (Fig. 2) harboring modules 1 and 2 with a fused TE domain. This bimodular PKS has served as a prototype for many analogous constructs.The potential for engineering chimeric PKSs by recombining modules from paralogous polyketide biosynthetic pathways has been explored for nearly two decades. Early studies revealed the importance of ACP-KS interactions (6, 7), as well as the utility of intermodul...

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“…3). The predicted structures of each of the corresponding triketide products (structures 2-8 (17)(18)(19)) are shown in Fig. 3.…”

Section: Engineering Of Chimeric Bimodular and Trimodular Pkss-mentioning

confidence: 99%

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Klaus

Ostrowski

Austerjost

et al. 2016

Journal of Biological Chemistry

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“…Its immunosuppressive activity due to inhibition of T-cell activation and proliferation has led to its potential use in clinical treatment of graft rejection in organ transplant (Weber et al 2005) and autoimmune diseases such as rheumatoid arthritis (Foroncewicz et al 2005). In addition to its therapeutic usefulness, semisynthetic derivatives of rapamycin has also been shown to display activities against cancer (Park et al 2010), Parkinson’s disease (Tain et al 2009), and AIDS (Nicoletti et al 2009). Its anti-aging activity has been published recently when scientists observed its ability to extend the life span of mice (Zou and Li 2013).…”

Section: Introductionmentioning

confidence: 99%

Kinetics of rapamycin production by Streptomyces hygroscopicus MTCC 4003

et al. 2013

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Research work was carried out to describe the kinetics of cell growth, substrate consumption and product formation in batch fermentation of rapamycin using shake flask as well as laboratory-scale fermentor. Fructose was used as the sole carbon source in the fermentation media. Optimization of fermentation parameters and reliable mathematical models were used for the maximum production of rapamycin from Streptomyces hygroscopicus MTCC 4003. The experimental data for microbial production of rapamycin fitted well with the proposed mathematical models. Kinetic parameters were evaluated using best fit unstructured models, viz. Andrew’s model, Monod model, Yano model, Aiba model. Andrew’s model showed a comparatively better R2 value (0.9849) among all tested models. The values of maximum specific growth rate (μmax), saturation constant (KS), inhibition constant (Ki), and growth yield coefficient (YX/S) were found to be 0.008 (h−1), 2.835 (g/L), 0.0738 (g/L), and 0.1708 (g g−1), respectively. The optimum production of rapamycin was obtained at 300 rpm agitation and 1 vvm aeration rate in the fermentor. The final production of rapamycin in shake flask was 539 mg/L. Rapamycin titer found in bioreactor was 1,316 mg/L which is 52 % higher than the latest maximum value reported in the literature.

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“…Rapamycin is not only an immunosuppressant but also has antifungal, antitumor, neuroprotective, autoimmune and anti-aging properties. Its biosynthesis, regulation and mutagenic improvement of production have been reviewed by Park et al [84].…”

Section: Immunosuppressantsmentioning

confidence: 99%

Importance of microbial natural products and the need to revitalize their discovery

Demain

2014

Journal of Industrial Microbiology and Biotechnology

342

216

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Microbes are the leading producers of useful natural products. Natural products from microbes and plants make excellent drugs. Significant portions of the microbial genomes are devoted to production of these useful secondary metabolites. A single microbe can make a number of secondary metabolites, as high as 50 compounds. The most useful products include antibiotics, anticancer agents, immunosuppressants, but products for many other applications, e.g., antivirals, anthelmintics, enzyme inhibitors, nutraceuticals, polymers, surfactants, bioherbicides, and vaccines have been commercialized. Unfortunately, due to the decrease in natural product discovery efforts, drug discovery has decreased in the past 20 years. The reasons include excessive costs for clinical trials, too short a window before the products become generics, difficulty in discovery of antibiotics against resistant organisms, and short treatment times by patients for products such as antibiotics. Despite these difficulties, technology to discover new drugs has advanced, e.g., combinatorial chemistry of natural product scaffolds, discoveries in biodiversity, genome mining, and systems biology. Of great help would be government extension of the time before products become generic.

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Biosynthesis of rapamycin and its regulation: past achievements and recent progress

Cited by 69 publications

References 73 publications

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases

Kinetics of rapamycin production by Streptomyces hygroscopicus MTCC 4003

Importance of microbial natural products and the need to revitalize their discovery

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