Enhanced biosynthesis of coated silver nanoparticles using isolated bacteria from heavy metal soils and their photothermal-based antibacterial activity: integrating Response Surface Methodology (RSM) Hybrid Artificial Neural Network (ANN)-Genetic Algorithm (GA) strategies.

This study explores the biosynthesis of silver nanoparticles (AgNPs) using the Streptomyces tuirus S16 strain, presenting an eco-friendly alternative to mitigate the environmental and health risks of chemical synthesis methods. It focuses on optimizing medium culture conditions, understanding their physicochemical properties, and investigating their potential photothermal-based antibacterial application. The S16 strain was selected from soils contaminated with heavy metals to exploit its ability to produce diverse bioactive compounds. By employing the combination of Response Surface Methodology (RSM) and Artificial Neural Network (ANN)-Genetic Algorithm (GA) strategies, we optimized AgNPs synthesis, achieving an improvement of nearly 2.45 times the initial yield under specific conditions (Bennet's medium supplemented with glycerol [5 g/L] and casamino-acid [3 g/L] at 30 °C for 72 h). A detailed physicochemical characterization was conducted. Notably, the AgNPs were well dispersed, and a carbonaceous coating layer on their surface was confirmed using energy-dispersive X-ray spectroscopy. Furthermore, functional groups were identified using Fourier-transform infrared spectroscopy, which helped enhance the AgNPs' stability and biocompatibility. AgNPs also demonstrated efficient photothermal conversion under light irradiation (0.2 W/cm2), with temperatures increasing to 41.7 °C, after 30 min. In addition, treatment with light irradiation of E. coli K-12 model effectively reduced the concentration of AgNPs from 105 to 52.5 µg/mL, thereby enhancing the efficacy of silver nanoparticles in contact with the E. coli K-12.

Three transcriptional regulators positively regulate the biosynthesis of polycyclic tetramate macrolactams in Streptomyces xiamenensis 318.

Polycyclic tetramate macrolactams (PTMs) are a widely distributed class of structurally complex natural products, and most of them exhibit multiple biological activities. However, the transcriptional regulators (TRs) involved in the regulation of PTM production have seldom been reported. Here, we identified three TRs, i.e., Sxim_22880, CvnABCSx, and WblASx, and revealed their positive roles in the regulation of PTM biosynthesis in mangrove-derived Streptomyces xiamenensis 318. This strain produces a considerable amount of PTMs at 30 °C, but the production of PTMs is mostly blocked at 37 °C. Quantitative real-time PCR analysis confirmed that the transcriptions of PTM biosynthetic genes were downregulated. We determined that the transcriptions of several putative TRs, i.e., WblASx, Sxim_22880, and CvnCSx, were significantly downregulated under such heat-shock conditions. We showed that the transcription of PTM biosynthetic genes and the production of PTMs could be restored at 37 °C if the impaired transcriptions of wblASx, sxim_22880, and cvnABCSx were restored. Electrophoretic mobility shift assays showed that none of these TRs could bind to the promoter region of the PTM gene cluster, suggesting their indirect but positive involvement in the regulation on PTM production. Moreover, concurrent overexpression of the three TRs in S. xiamenensis 318 resulted in a 242.5% increase in PTM production when the strain was cultured at 30 °C. Furthermore, overexpression of these three TRs in Streptomyces sp. FR-008 and S. albus J1074 stimulated the production of new secondary metabolites, indicating that these conserved TRs could be used to activate cryptic secondary metabolite gene clusters in Streptomyces.

Enhanced doxorubicin production by Streptomyces peucetius using a combination of classical strain mutation and medium optimization.

Doxorubicin (DXR), which is produced by Streptomyces peucetius, is an important anthracycline-type antibiotic used for the treatment of various cancers. However, due to the low DXR productivity of wild-type S. peucetius, it is difficult to produce DXR by one-step fermentation. In this study, a DXR-resistance screening method was developed to screen for DXR high-producing mutants. Then, S. peucetius SIPI-11 was treated several times with UV and ARTP (atmospheric and room temperature plasma) to induce mutations. Treated strains were screened by spreading on a DXR-containing plate, isolating a mutant (S. peucetius 33-24) with enhanced DXR yield (570 mg/L vs. 119 mg/L for the original strain). The components of the fermentation medium, including the carbon and nitrogen sources, were optimized to further enhance DXR yield (to 850 mg/L). The pH of the fermentation medium and culture temperature were also optimized for effective DXR production. Finally, DXR production by S. peucetius 33-24 was investigated in flask culture and a fermenter. The yield of DXR was as high as 1100 mg/L in a 5-L fermenter, which is the highest DXR productivity reported thus far, suggesting that S. peucetius 33-24 has the potential to produce DXR by direct fermentation.

Strain improvement by combined UV mutagenesis and ribosome engineering and subsequent fermentation optimization for enhanced 6'-deoxy-bleomycin Z production.

The bleomycins (BLMs) are important clinical drugs extensively used in combination chemotherapy for the treatment of various cancers. Dose-dependent lung toxicity and the development of drug resistance have restricted their wide applications. 6'-Deoxy-BLM Z, a recently engineered BLM analogue with improved antitumor activity, has the potential to be developed into the next-generation BLM anticancer drug. However, its low titer in the recombinant strain Streptomyces flavoviridis SB9026 has hampered current efforts, which require sufficient compound, to pursue preclinical studies and subsequent clinical development. Here, we report the strain improvement by combined UV mutagenesis and ribosome engineering, as well as the fermentation optimization, for enhanced 6'-deoxy-BLM production. A high producer, named S. flavoviridis G-4F12, was successfully isolated, producing 6'-deoxy-BLM at above 70 mg/L under the optimized fermentation conditions, representing a sevenfold increase in comparison with that of the original producer. These findings demonstrated the effectiveness of combined empirical breeding methods in strain improvement and set the stage for sustainable production of 6'-deoxy-BLM via pilot-scale microbial fermentation.

Significance of Heavy-Ion Beam Irradiation-Induced Avermectin B1a Production by Engineered Streptomyces avermitilis.

Heavy-ion irradiation technology has advantages over traditional methods of mutagenesis. Heavy-ion irradiation improves the mutation rate, broadens the mutation spectrum, and shortens the breeding cycle. However, few data are currently available regarding its effect on Streptomyces avermitilis morphology and productivity. In this study, the influence of heavy-ion irradiation on S. avermitilis when cultivated in approximately 10 L stirred-tank bioreactors was investigated. The specific productivity of the avermectin (AVM) B1a-producing mutant S. avermitilis 147-G58 increased notably, from 3885 to 5446 µg/mL, approximately 1.6-fold, compared to the original strain. The mycelial morphology of the mutant fermentation processes was microscopically examined. Additionally, protein and metabolite identification was performed by using SDS-PAGE, 2- and 3-dimensional electrophoresis (2DE and 3DE). The results showed that negative regulation gene deletion of mutants led to metabolic process upregulating expression of protein and improving the productivity of an avermectin B1a. The results showed that the heavy-ion beam irradiation dose that corresponded to optimal production was well over the standard dose, at approximately 80 Gy at 220 AMeV (depending on the strain). This study provides reliable data and a feasible method for increasing AVM productivity in industrial processes.

Reduction of FR900525 using an S-(2-aminoethyl) l-cysteine-resistant mutant.

FK506 (tacrolimus), a macrolide compound with immunosuppressant activity, has been proven to have clinical importance and has been manufactured industrially since 1993 by using mutants with high FK506-production ability; these mutants have been developed from the wild strain Streptomyces tsukubaensis No. 9993. FR900525 is one of the by-products of FK506 production. However, there was no effective industrial method to separate FR900525 from FK506 due to the structural similarity between the two compounds. Therefore, reducing the level of FR900525 was a serious problem in the industrial strain A. In this study, we aimed to reduce the FR900525 production. We first determined that pipecolic acid level was a critical parameter for controlling FR900525 production in strain A. S-(2-Aminoethyl) l-cysteine (AEC)-resistant mutants has been reported to increase lysine productivity successfully in a variety of lysine-producing microorganisms. Therefore, next, we applied a selection of AEC-resistant mutants to enhance pipecolic acid biosynthesis. Finally, four AEC-resistant mutants were obtained from strain A using ultraviolet irradiation, and three of them showed less FR900525 productivity compared to the parental strain A. Our findings indicated that AEC resistance was effective phenotype marker for increasing pipecolic acid productivity and for reducing FR900525 production in S. tsukubaensis. Thus, our study provides an efficient method for reducing FR90025 level during FK506 biosynthesis.

Implication of RuvABC and RecG in homologous recombination in Streptomyces ambofaciens.

Most bacterial organisms rely on homologous recombination to repair DNA double-strand breaks and for the post-replicative repair of DNA single-strand gaps. Homologous recombination can be divided into three steps: (i) a pre-synaptic step in which the DNA 3'-OH ends are processed, (ii) a recA-dependent synaptic step allowing the invasion of an intact copy and the formation of Holliday junctions, and (iii) a post-synaptic step consisting of migration and resolution of these junctions. Currently, little is known about factors involved in homologous recombination, especially for the post-synaptic step. In Escherichia coli, branch migration and resolution are performed by the RuvABC complex, but could also rely on the RecG helicase in a redundant manner. In this study, we show that recG and ruvABC are well-conserved among Streptomyces. ΔruvABC, ΔrecG and ΔruvABC ΔrecG mutant strains were constructed. ΔruvABC ΔrecG is only slightly affected by exposure to DNA damage (UV). We also show that conjugational recombination decreases in the absence of RuvABC and RecG, but that intra-chromosomal recombination is not affected. These data suggest that RuvABC and RecG are indeed involved in homologous recombination in Streptomyces ambofaciens and that alternative factors are able to take over Holliday junction in Streptomyces.

Production of rapamycin in Streptomyces hygroscopicus from glycerol-based media optimized by systemic methodology.

Rapamycin, produced by the soil bacterium Streptomyces hygroscopicus, has the ability to suppress the immune system and is used as an antifungal, anti-inflammatory, antitumor, and immunosuppressive agent. In an attempt to increase the productivity of rapamycin, mutagenesis of wild-type Streptomyces hygroscopicus was performed using ultraviolet radiation, and the medium composition was optimized using glycerol (which is one of the cheapest starting substrates) by applying Plackett-Burman design and response surface methodology. Plackett-Burman design was used to analyze 14 medium constituents: M100 (maltodextrin), glycerol, soybean meal, soytone, yeast extract, (NH4)2SO4, L-lysine, KH2PO4, K2HPO4, NaCl, FeSO4·7H2O, CaCO3, 2-(N-morpholino) ethanesulfonic acid, and the initial pH level. Glycerol, soytone, yeast extract, and CaCO3 were analyzed to evaluate their effect on rapamycin production. The individual and interaction effects of the four selected variables were determined by Box-Behnken design, suggesting CaCO3, soytone, and yeast extract have negative effects, but glycerol was a positive factor to determine rapamycin productivity. Medium optimization using statistical design resulted in a 45% (220.7 ± 5.7 mg/l) increase in rapamycin production for the Streptomyces hygroscopicus mutant, compared with the unoptimized production medium (151.9 ± 22.6 mg/l), and nearly 588% compared with wildtype Streptomyces hygroscopicus (37.5 ± 2.8 mg/l). The change in pH showed that CaCO3 is a critical and negative factor for rapamycin production.

Genome shuffling of Streptomyces roseosporus for improving daptomycin production.

In this work, enhancement of daptomycin production by genome shuffling in Streptomyces roseosporus was conducted. Ultraviolet and NTG were used as mutagenizing agents to improve the volumetric productivity of the wild-type strain. Eight strains with enhanced daptomycin production were screened out as the starting population for genome shuffling. Daptomycin's production increased steadily with each round of genome shuffling. After the fourth round of fusion, a high-production strain (582 mg/L), named F4, was selected as a potential industrial production strain and its heredity stability was stable. Moreover, comparative analysis of the non-ribosomal peptide synthetase (NRPS) genes at the transcript level between the wild and the mutant was studied by RT-PCR in order to explore mechanism of genome shuffling. The transcript levels of NRPS genes dptA, dptBC, and dptD in the mutant were approximately 6.5 to 7 times higher than those in the wild. In summary, it is suggested that this strategy for increasing the daptomycin production in S. roseosporus by genome shuffling may provide an alternative approach to enhance the metabolite production in other Streptomyces.

Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064

Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

[Rational screen of high kelimycin-producing strain by combined conventional mutagenesis and high-throughput screen method].

UNLABELLED: Kelimycin, is a new macrolide antibiotic drug obtained through genetic engineering approaches. With 4"-O-isovalerylspiramycins as the major components, was produced by genetically engineered Streptomyces spiramyceticus transformed with 4"-O-acyltransferase gene from S. mycarofaciens. OBJECTIVE: Improve the efficiency of strain fermentation, to meet the needs of industrial production. METHODS: The enhanced kelimycin-producing strain was obtained by applying various conventional mutagenesis approaches, and high-throughput screen methods, including protoplast mutagenesis by ultraviolet, mutagenesis by diethyl sulfate and UV-reactivation, valine content resistance screen and enrichment of improved strains. A strategy for positive mutant enrichment was developed after mutagenesis and before high-throughput screen. RESULTS: Finally, the high-producing strain WSJ-1-7-49-133-82-18-43 was obtained and its potency in shake flask increased by 56% compared to the original strain. The potency in 500 L pilot fermenter increased by 61%. CONCLUSION: This study shows that the screening industrial production strains can be enhanced effectively by combining multiple conventional mutagenesis and high-throughput screen methods.

Improvement of microbial strain and fermentation process of rapamycin biosynthesis.

The purpose of this investigation is to enhance the production of the immunosuppressant drug rapamycin by subjecting the strain CBS 773.23 to ultraviolet (UV) and N-methyl-N'-nitro-N-nitroso guanidine (NTG) mutations. Among all the mutants tested, MTCC 5681 (NRC-CM03/SH) obtained by NTG mutagenesis of strain CBS 773.72 showed the highest activity, 210 mg/L. The effect of different factors including medium composition, pH, temperature, and intensity of mixing on rapamycin production was studied. Based on the study, the optimal concentrations of soluble starch and dry yeast granules were found to be 50 g/L and 1.5 g/L, respectively. Furthermore, optimal values for pH, temperature, and shaking speed were found to be 6.0, 28°C, and 220 rpm, respectively. The production of rapamycin increased 1.6-fold, to 360 mg/L, in shake-flask culture using the optimal combination of factors observed compared with basal cultivation medium using MTCC 5681 mutant strain.

Microwave irradiation is a useful tool for improving isolation of actinomycetes from soil.

Actinomycetes are an important source of novel, biologically active compounds. New methods need to be developed for isolating previously unknown actinomycetes from soil. The objective of this experiment was to study microwave irradiation of soil as a means for isolating previously unknown actinomycetes. Soil samples were collected at ten elevations between 800 and 3670 m on Taibai Mountain, Shaanxi Province, China. Moistened soil samples were irradiated at 120 W heating power (2450 MHz) for 3 min using a household microwave oven. Irradiation increased total actinomycete, streptomycete, and antagonistic actinomycete counts on three types of culture media. Irradiation also increased the number of culturable actinomycete isolates. Some actinomycete isolates were culturable only after the soil was irradiated, whereas other isolates could not be cultured after irradiation. Irradiation of soil from elevations > 3000 m increased actinomycete counts significantly but had little effect on the number of culturable actinomycete isolates. In contrast, irradiation of samples from elevations < 3000 m had relatively little effect on actinomycete counts, but significantly increased the number of culturable actinomycete isolates. We used 16S rDNA sequence analysis to identify 14 actinomycete isolates that were only culturable after irradiation. Microwave irradiation of soil was helpful for isolating Streptomyces spp., Nocardia spp., Streptosporangium spp., and Lentzea spp. Slightly more than 90% of the identified actinomycete species were biologically active. In conclusion, microwave irradiation is a useful tool for isolating biologically active actinomycetes from soil.

Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011.

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24 mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1 U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5 mg/g) with the supplementation of 5 mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5 mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61 mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5 mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44 mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9 mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5 mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.

[Genetic variability of synthesis feature of carotenoids in Streptomyces globisporus 1912].

Seventeen spontaneous and induced mutants, that acquired a new characteristic--the synthesis of beta-carotene and lycopene, were obtained from strain Streptomyces globisporus 1912. It was found that spontaneous mutants inherited more stably the acquired carotenogenesis as compared to induced ones. Synthesis of carotenoids by all isolated Crt+ Lcp+ cultures is a constitutive feature. It was shown that Crt(+)-mutants (4Crt, 6Crt, 7Crt, RVCrt and R3Crt) synthesized beta-carotene and lycopene, while Lcp(+)-mutants (TpS16-1, TpS16-2, 4Lcp and R3Lcp)--only lycopene. The obtained mutants and transformants of S. globisporus 1912, synthesizing carotene were characterized by a simultaneous change of two or three phenotypic characteristics: synthesis of the antibiotic landomycin E. sporullation and carotenogenesis. It can be assumed that the high instability of this characteristic (carotenogenesis) in strain S. globisporus 1912 was caused by localization of the crt-genes cluster close to a TIR-element in a chromosome terminal region, frequent structural reorganization of DNA here were reported in the literature.

Clavulanic acid production by the MMS 150 mutant obtained from wild type Streptomyces clavuligerus ATCC 27064.

Clavulanic acid (CA) is a powerful inhibitor of the beta-lactamases, enzymes produced by bacteria resistants to penicillin and cefalosporin. This molecule is produced industrially by strains of Streptomyces clavuligerus in complex media which carbon and nitrogen resources are supplied by inexpensive compounds still providing high productivity. The genetic production improvement using physical and chemical mutagenic agents is an important strategy in programs of industrial production development of bioactive metabolites. However, parental strains are susceptible to loss of their original productivity due genetic instability phenomenona. In this work, some S. clavuligerus mutant strains obtained by treatment with UV light and with MMS are compared with the wild type (Streptomyces clavuligerus ATCC 27064). The results indicated that the random mutations originated some strains with different phenotypes, most divergent demonstrated by the mutants strains named AC116, MMS 150 and MMS 54, that exhibited lack of pigmentation in their mature spores. Also, the strain MMS 150 presented a larger production of CA when cultivated in semi-synthetics media. Using other media, the wild type strain obtained a larger CA production. Besides, using the modifed complex media the MMS 150 strain showed changes in its lipolitic activity and a larger production of CA. The studies also allowed finding the best conditions for a lipase activity exhibited by wild type S. clavuligerus and the MMS150 mutant.

A combined approach of classical mutagenesis and rational metabolic engineering improves rapamycin biosynthesis and provides insights into methylmalonyl-CoA precursor supply pathway in Streptomyces hygroscopicus ATCC 29253.

Rapamycin is a macrocyclic polyketide with immunosuppressive, antifungal, and anticancer activity produced by Streptomyces hygroscopicus ATCC 29253. Rapamycin production by a mutant strain (UV2-2) induced by ultraviolet mutagenesis was improved by approximately 3.2-fold (23.6 mg/l) compared to that of the wild-type strain. The comparative analyses of gene expression and intracellular acyl-CoA pools between wild-type and the UV2-2 strains revealed that the increased production of rapamycin in UV2-2 was due to the prolonged expression of rapamycin biosynthetic genes, but a depletion of intracellular methylmalonyl-CoA limited the rapamycin biosynthesis of the UV2-2 strain. Therefore, three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA were evaluated to identify the effective precursor supply pathway that can support the high production of rapamycin: propionyl-CoA carboxylase (PCC), methylmalonyl-CoA mutase, and methylmalonyl-CoA ligase. Among them, only the PCC pathway along with supplementation of propionate was found to be effective for an increase in intracellular pool of methylmalonyl-CoA and rapamycin titers in UV2-2 strain (42.8 mg/l), indicating that the PCC pathway is a major methylmalonyl-CoA supply pathway in the rapamycin producer. These results demonstrated that the combined approach involving traditional mutagenesis and metabolic engineering could be successfully applied to the diagnosis of yield-limiting factors and the enhanced production of industrially and clinically important polyketide compounds.

Kinetic analysis and modeling of daptomycin batch fermentation by Streptomyces roseosporus.

In this study, Streptomyces roseosporus was subjected to helium-neon (He-Ne) laser (632.8 nm) irradiation to improve the production ability of extracellular antibiotic daptomycin. Under the optimum irradiation dosage of 18 mW for 22 min, a stable positive mutant strain S. roseosporus LC-54 was obtained. The maximum A21978C (daptomycin is a semisynthetic antimicrobial substance derived from the A21978C complex) yield of this mutant strain was 296 mg/l, which was 146% higher than that of the wild strain. The mutant strain grew more quickly and utilized carbohydrate sources more efficiently than the wild strain. The batch culture kinetics was investigated in a 7 l bioreactor. The logistic equation for growth, the Luedeking-Piret equation for daptomycin production, and Luedeking-Piret-like equations for carbon substrate consumption were established. This model appeared to provide a reasonable description for each parameter during the growth phase and fitted fairly well with the experiment data.

[Obtaining and characteristic of Streptomyces globisporus 1912 mutants with high biosynthesis of landomycin e].

Eight highly productive UV-induced mutants of Streptomyces globisporus 1912 with high biosynthesis of landomycin E were isolated, their isolation frequency being 2 x 10(-3). Mutants 1-1, 1-4-1, 1-1y, 1-2y, 4-1, 1-2 produced 175-187.5 mg of landomycine E and 1-1b, 4-1-1 and 1-3--80-100 mg per 1 liter of soybean agar after 48 hours of cultivation The mutant 4-1-1 had the highest antibiotic activity and middle level of landomycin E production.

Generation of high rapamycin producing strain via rational metabolic pathway-based mutagenesis and further titer improvement with fed-batch bioprocess optimization.

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.