Rapamycin is a triene macrolide antibiotic produced by Streptomyces hygroscopicus. Besides its wide application as an effective immunosuppressive agent, other important bioactivities have made rapamycin a potential drug lead for novel pharmaceutical development. However, the low titer of rapamycin in the original producer strain limits further industrialization efforts and restricts its use for other applications. Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have rationally designed approaches to generate a rapamycin high producer strain of S. hygroscopicus HD-04-S. These have included alleviation of glucose repression, improved tolerance towards lysine and shikimic acid, and auxotrophy of tryptophan and phenylalanine through the application of stepwise UV mutagenesis. The resultant strain produced rapamycin at 450 mg/L in the shake flask scale. These fermentations were further scaled up in 120 and 20,000 L fermentors, respectively, at the pilot plant. Selected fermentation factors including agitation speed, pH, and on-line supplementation were systematically evaluated. A fed-batch strategy was established to maximize rapamycin production. With these efforts, an optimized fermentation process in the larger scale fermentor was developed. The final titer of rapamycin was 812 mg/L in the 120 L fermentor and 783 mg/L in the 20,000 L fermentor. This work highlights a high rapamycin producing strain derived by mutagenesis and subsequent screening, fermentation optimization of which has now made it feasible to produce rapamycin on an industrial scale by fermentation. The strategies developed here should also be applicable to titer improvement of other important microbial natural products on an industrial scale.
Erythromycins (Ers) are clinically potent macrolide antibiotics in treating pathogenic bacterial infections. Microorganisms capable of producing Ers, represented by Saccharopolyspora erythraea, are mainly soil-dwelling actinomycetes. So far, Actinopolyspora erythraea YIM90600, a halophilic actinomycete isolated from Baicheng salt field, is the only known Er-producing extremophile. In this study, we have reported the draft genome sequence of Ac. erythraea YIM90600, genome mining of which has revealed a new Er biosynthetic gene cluster encoding several novel Er metabolites. This Er gene cluster shares high identity and similarity with the one of Sa. erythraea NRRL2338, except for two absent genes, eryBI and eryG. By correlating genotype and chemotype, the biosynthetic pathways of 3′-demethyl-erythromycin C, erythronolide H (EH) and erythronolide I have been proposed. The formation of EH is supposed to be sequentially biosynthesized via C-6/C-18 epoxidation and C-14 hydroxylation from 6-deoxyerythronolide B. Although an in vitro enzymatic activity assay has provided limited evidence for the involvement of the cytochrome P450 oxidase EryFAc (derived from Ac. erythraea YIM90600) in the catalysis of a two-step oxidation, resulting in an epoxy moiety, the attempt to construct an EH-producing Sa. erythraea mutant via gene complementation was not successful. Characterization of EryKAc (derived from Ac. erythraea YIM90600) in vitro has confirmed its unique role as a C-12 hydroxylase, rather than a C-14 hydroxylase of the erythronolide. Genomic characterization of the halophile Ac. erythraea YIM90600 will assist us to explore the great potential of extremophiles, and promote the understanding of EH formation, which will shed new insights into the biosynthesis of Er metabolites.
Summary Acyltransferase (AT)‐less type I polyketide synthases (PKSs) produce complex natural products due to the presence of many unique tailoring enzymes. The 3‐hydroxy‐3‐methylglutaryl coenzyme A synthases (HCSs) are responsible for β ‐alkylation of the growing polyketide intermediates in AT‐less type I PKSs. In this study, we discovered a large group of HCSs, closely associated with the characterized and orphan AT‐less type I PKSs through in silico genome mining, sequence and genome neighbourhood network analyses. Using HCS‐based probes, the survey of 1207 in‐house strains and 18 soil samples from different geographic locations revealed the vast diversity of HCS‐containing AT‐less type I PKSs. The presence of HCSs in many AT‐less type I PKSs suggests their co‐evolutionary relationship. This study provides a new probe to study the abundance and diversity of AT‐less type I PKSs in the environment and microbial strain collections. Our study should inspire future efforts to discover new polyketide natural products from AT‐less type I PKSs.
Abstract Tiancimycins (TNMs) are a group of 10‐membered anthraquinone‐fused enediynes, newly discovered from Streptomyces sp. CB03234. Among them, TNM‐A and TNM‐D have exhibited excellent antitumor performances and could be exploited as very promising warheads for the development of anticancer antibody‐drug conjugates (ADCs). However, their low titers, especially TNM‐D, have severely limited following progress. Therefore, the streptomycin‐induced ribosome engineering was adopted in this work for strain improvement of CB03234, and a TNMs high producer S . sp. CB03234‐S with the K43N mutation at 30S ribosomal protein S12 was successfully screened out. Subsequent media optimization revealed the essential effects of iodide and copper ion on the production of TNMs, while the substitution of nitrogen source could evidently promote the accumulation of TNM‐D, and the ratio of produced TNM‐A and TNM‐D was responsive to the change of carbon and nitrogen ratio in the medium. Further amelioration of the pH control in scaled up 25 L fermentation increased the average titers of TNM‐A and TNM‐D up to 13.7 ± 0.3 and 19.2 ± 0.4 mg/L, respectively. The achieved over 45‐fold titer improvement of TNM‐A, and 109‐fold total titer improvement of TNM‐A and TNM‐D enabled the efficient purification of over 200 mg of each target molecule from 25 L fermentation. Our efforts have demonstrated a practical strategy for titer improvement of anthraquinone‐fused enediynes and set up a solid base for the pilot scale production and preclinical studies of TNMs to expedite the future development of anticancer ADC drugs.
Significance Leinamycin (LNM) is a promising anticancer drug lead, yet no analog has been isolated since its discovery nearly 30 y ago. By mining bacterial genomes, we discovered 49 potential producers of LNM-type natural products, the structural diversity of which was predicted based on bioinformatics and confirmed by in vitro characterization of selected enzymes and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity. New members of the LNM family of natural products should greatly facilitate drug discovery and development. The LNM-type biosynthetic machineries provide outstanding opportunities to dissect and mimic Nature’s strategies for combinatorial biosynthesis and natural product structural diversity.
Abstract β‐rubromycin (β‐RUB) ( 1 ) is an efficient inhibitor of human telomerase possessing a unique spiroketal moiety as a potential pharmacophore and regarded as a promising anticancer drug lead. But the development of (β‐RUB) ( 1 ) has long been hampered by its low titer and very poor water solubility. By adopting a genome mining strategy, an FAD‐dependent monooxygenase RubN involving with the formation of the spiro system was applied as the probe and Streptomyces sp. CB00271 was screened out from our strain collection as an alternative natural high producer of β‐RUB ( 1 ). After a series of fermentation optimizations, CB00271 could produce 124.8 ± 3.4 mg/L β‐RUB ( 1 ), which was the highest titer up to now. Moreover, the enhanced production of β‐RUB ( 1 ) in fermentation broth also led to the discovery of a new congener β‐RUB acid ( 7 ), which was structurally elucidated as the acid form of β‐RUB ( 1 ). Comparing to β‐RUB ( 1 ), the substituted carboxyl group endowed β‐RUB acid ( 7 ) much better solubility in serum and resulted in its higher activity towards tumor cells. Our work set up a solid base for the pilot‐scale production of β‐RUB ( 1 ) and its congeners to facilitate their future development as promising anticancer drug leads, and also provide an alternative and practical strategy for the exploitation of other important microbial natural products.
'/%3e%3cuse xlink:href='%23a' transform='rotate(23.036 .093 25.536)'/%3e%3cuse xlink:href='%23a' transform='rotate(45.87 1.273 16.18)'/%3e%3cuse xlink:href='%23a' transform='rotate(69.945 .996 12.078)'/%3e%3cuse xlink:href='%23a' transform='rotate(20.66 -19.689 31.932)'/%3e%3c/svg%3e)
