A novel medium for the enhanced cell growth and production of prodigiosin from <Emphasis Type="Italic">Serratia marcescens</Emphasis> isolated from soil

Background

Prodigiosin produced by Serratia marcescens is a promising drug owing to its reported characteristics of having antifungal, immunosuppressive and antiproliferative activity. From an industrial point of view the necessity to obtain a suitable medium to simultaneously enhance the growth of Serratia marcescens and the pigment production was the aim of this work. The usage of individual fatty acid as substrate in industries would be cost-effective in the long run and this paved the way for us to try the effect of different fatty acid-containing seeds and oils of peanut, sesame and coconut as source of substrate.

Results

The addition of sugars only showed slight enhancement of prodigiosin production in nutrient broth but not in fatty acid containing seed medium. The powdered peanut broth had supported better growth of Serratia marcescens and higher yield of prodigiosin when compared with the existing nutrient broth and peptone glycerol broth. A block in prodigiosin production was seen above 30°C in nutrient broth, but the fatty acid seed medium used by us supported prodigiosin production upto 42°C though the yields were lower than what was obtained at 28°C. From the results, the fatty acid form of carbon source has a role to play in enhanced cell growth and prodigiosin production.

Conclusion

We conclude by reporting that the powdered and sieved peanut seed of different quality grades were consistent in yielding a fourty fold increase in prodigiosin production over the existing media. A literature survey on the composition of the different media components in nutrient broth, peptone glycerol broth and the fatty acid containing seeds and oils enabled us to propose that the saturated form of fatty acid has a role to play in enhanced cell growth and prodigiosin production. This work has also enabled us to report that the temperature related block of prodigiosin biosynthesis varies with different media and the powdered peanut broth supports prodigiosin production at higher temperatures. The medium suggested in this work is best suitable from an industrial point of view in being economically feasible, in terms of the higher prodigiosin yield and the extraction of prodigiosin described in this paper is simple with minimal wastage.

     

Improved production of teicoplanin using adsorbent resin in fermentations

AIMS: To use adsorbent resins in fermentations to eliminate toxic effects on growth, reduce feedback repression of production and assist in recovery of teicoplanin. METHODS AND RESULTS: An adsorbent resin was added to the culture broth for the adsorption of teicoplanin. Amberlite XAD-16, Diaion HP-20, charcoal and silica gel were investigated as adsorbent resins. The adsorbed teicoplanin was extracted from the resin by 80% methanol after fermentation. Antibiotic activity was quantified by the disc-agar diffusion assay against Bacillus substilis, and qualitative evaluations were based on HPLC using YMC-Pack ODS-A column. Diaion HP-20 was the most effective adsorbent resin when added at a concentration of 5% (w/v) in the inoculation stage. CONCLUSIONS: Addition of Diaion HP-20 in fermentations eliminated toxic effects on growth and reduced feedback repression of teicoplanin by adsorption. There was a 4.2-fold increase in the quantities of teicoplanin. Addition of adsorbent assisted in the recovery of teicoplanin by reducing the recovery steps. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study provide useful information for the production of teicoplanin, a glycopeptide antibiotic produced by Actinoplanes teicomyceticus. Addition of adsorbent in fermentation increased productivity of teicoplanin by more than five times.     

Kinetic analysis of growth rate, ATP, and pigmentation suggests an energy-spilling function for the pigment prodigiosin of Serratia marcescens

Serratia marcescens is a gram-negative environmental bacterium and opportunistic pathogen. S. marcescens expresses prodigiosin, a bright red and cell-associated pigment which has no known biological function for producing cells. We present here a kinetic model relating cell, ATP, and prodigiosin concentration changes for S. marcescens during cultivation in batch culture. Cells were grown in a variety of complex broth media at temperatures which either promoted or essentially prevented pigmentation. High growth rates were accompanied by large decreases in cellular prodigiosin concentration; low growth rates were associated with rapid pigmentation. Prodigiosin was induced most strongly during limited growth as the population transitioned to stationary phase, suggesting a negative effect of this pigment on biomass production. Mathematically, the combined rate of formation of biomass and bioenergy (as ATP) was shown to be equivalent to the rate of prodigiosin production. Studies with cyanide inhibition of both oxidative phosphorylation and pigment production indicated that rates of biomass and net ATP synthesis were actually higher in the presence of cyanide, further suggesting a negative regulatory role for prodigiosin in cell and energy production under aerobic growth conditions. Considered in the context of the literature, these results suggest that prodigiosin reduces ATP production by a process termed energy spilling. This process may protect the cell by limiting production of reactive oxygen compounds. Other possible functions for prodigiosin as a mediator of cell death at population stationary phase are discussed.     

柴达木沙漠链霉菌S10T阿糖腺苷的大孔树脂分离工艺优化与结构鉴定

为了获得具有药用价值的活性天然产物,采用4种大孔吸附树脂对柴达木沙漠链霉菌(Streptomyces qaidemensis)S10^T发酵液进行静态吸附和解吸实验,优化分离工艺。结果显示,AB-8型树脂具有良好的吸附和解吸性能,该树脂对柴达木沙漠链霉菌S10^T发酵液中的活性天然产物吸附工艺为发酵液pH值9,吸附时间4 h,洗脱液70%甲醇溶液。经正向硅胶、反相硅胶和葡聚糖凝胶Sephadex LH-20分离得到了一个化合物,¹H-NMR和¹³C-NMR结合高分辨质谱(LC-HR-MS)鉴定该化合物为阿糖腺苷(vidarabine),是一种具有抗病毒活性的核苷类抗生素,并简单探究了其在柴达木沙漠链霉菌中的生物合成过程。     

大孔树脂提取链霉菌4903菌株除草活性物质的研究

化感活性物质的提取方法是研究化感作用的重要步骤,对化感链霉菌4903的前期研究证明其具有抑菌及除草活性。本文选用了4种大孔吸附树脂(X-5, NKA-II, AB-8和HP-20)对化感链霉菌4903发酵液的除草活性物质进行提取。实验结果显示:树脂AB-8对链霉菌4903菌株发酵液除草活性成分具有较好的吸附性能和较易被解吸的特性,树脂AB-8对链霉菌4903菌株发酵液的静态吸附量约可达到树脂体积的7倍。用树脂2-3倍体积的80%乙醇溶液则可以把吸附的除草活性物质全部从树脂中解吸出来。AB-8适合用于链霉菌4903菌株发酵液除草活性物质的提取和纯化。     

Enhanced production of insecticidal prodigiosin from Serratia marcescens TKU011 in media containing squid pen

Using fishery-processing wastes of squid pen powder (SPP) as the sole carbon and nitrogen (C/N) source, Serratia marcescens TKU011 produced prodigiosin. The culture was incubated in 50 mL of medium in an Erlenmeyer flask (250 mL) containing 1.5% SPP at 30 °C for 1 day and then changed to 25 °C for 2 more days. The culture broth had high prodigiosin (0.978 mg/mL). S. marcescens TKU011 grown under illumination conditions in a shaking culture exhibited higher prodigiosin production than when grown under dark conditions contrary to previous reports. The culture supernatant reduced surface tension of water, and the surfactant activity increased when prodigiosin production increased. In this study, the fishery-processing waste, squid pen, was used to produce prodigiosin at greater quantities than reported in other studies, and we found that the prodigiosin had a novel property of insecticidal activity. This method has the potential for developing mass production of prodigiosin.     

Strategy for the Improvement of Prodigiosin Production by a Serratia marcescens Mutant through Fed-Batch Fermentation

A Serratia marcescens mutant for prodigiosin production was obtained by u.v. mutation with rational screening methods and a two-step feeding strategy was used to increase its productivity. In flasks, the mutant strain B6 gave a 2.8-fold higher prodigiosin production than that of the parent strain with glycerol as a carbon source. In a 5-l bioreactor, with a two-step feeding strategy in which glucose was selected as the initial carbon source in the fermentation media and glycerol was fed as a ‘prodigiosin inducer’, it gave a 7.8 times higher prodigiosin production (583 mg/l) than the parent stain with the original cultivation mode.     

Incorporation of methionine into prodigiosin

Addition of proline to suspensions of nonpigmented, nonproliferating cells of Serratia marcescens induced biosynthesis of the pigment, prodigiosin. If methionine was included with proline, 4 times as much prodigiosin was formed, although the amount synthesized in the presence of methionine alone was nil. Uniformly ¹⁴C-labelled proline and methionine were incorporated into prodigiosin to about 30% the extent of their incorporation into cellular protein. Experiments with [carboxy-¹⁴C]-, and [Me-¹⁴C] methionine established that isotope from the methyl group was utilized preferentially for biosynthesis of prodigiosin.     

杀线虫芽孢杆菌发酵条件优化及大孔树脂筛选

芽孢杆菌 SMrs28分离自草原毒草瑞香狼毒根际土壤,其代谢产物有明显的线虫触杀活性。为了确定该菌株的最优发酵条件及初步纯化活性成分的树脂类型,采用单因素试验对发酵条件进行优化,通过静态吸附试验对大孔树脂类型进行筛选。结果表明: SMrs28菌株的最优发酵条件为:以葡萄糖和酵母粉作为最佳碳源和氮源,发酵时间48 h,接种量10%,温度28 ℃,转速180 r·min^-1,150 mL的三角瓶装液量30 mL,初始pH 7.2。静态吸附试验表明,大孔吸附树脂D101对发酵液中活性成分的吸附和解吸效果显著优于XAD-4、HP20和AB-8,其解吸液的杀线虫活性明显提高。发酵条件的优化和大孔吸附树脂的筛选,明显提高了发酵液的杀线虫活性,为进一步分离纯化该菌株的活性成分奠定了基础,为微生物杀线虫剂的开发和应用提供了理论依据。     

Enhancement by Sodium Dodecyl Sulfate of Pigment Formation in Serratia marcescens O8

Three methods were used to determine the enhancement by sodium dodecyl sulfate (SDS) of prodigiosin formation in Serratia marcescens O8. The results of the agar disk diffusion method indicated that pigment formation was dependent upon the concentration of SDS. Diameters of the pigment zones were proportional to the logarithm of SDS concentrations of 300 to 1,500 μg/ml. When bacteria were grown in broth containing SDS from 0 to 800 μg/ml and the pigment extracts were analyzed spectrophotometrically, a similar enhancement of pigment formation was observed. Finally, these results were confirmed by high-performance liquid chromatographic analysis of the extracts. Prodigiosin appeared to be the sole component with increased synthesis. The possible mechanism of the SDS enhancement effect could be explained by an increase in negative binding sites by the association of SDS with a cell envelope component(s). These binding sites may be required for prodigiosin synthesis.     

粘质沙雷氏菌产灵菌红素培养基的筛选

目的：确定菌株S418产生灵菌红素的最优培养基配方及其的分类地位。方法：以花生粉为基础培养基,通过单因素试验和四因素三水平正交试验筛选出了菌株S418产灵菌红素的最佳培养基配方;根据该菌株的16S rRNA基因序列系统发育树分析初步确定了菌株S418的分类地位。结果：培养基最优配方为：花生粉2%,花生油0.5%,L-脯氨酸1%,硫酸镁0.025%。在28℃、pH7.5、250r/min振荡培养24h,灵菌红素产量达67.92mg/L。菌株S418初步鉴定为粘质沙雷氏菌（Serratia marcescensS418）。结论：花生粉培养基是一种适合粘质沙雷氏菌产灵菌红素的优良培养基。     

灵杆菌合成灵菌红素的转录调控

灵菌红素是一种具有多种生物活性的红色素,具有巨大的经济价值和广阔的应用前景。灵杆菌是灵菌红素的生产菌株,同时也是研究灵菌红素合成的模式菌株。本文综述了转录水平上调控灵杆菌合成灵菌红素的研究进展,总结了双(多)组分调控系统、群体感应系统、σ因子和转录因子在调控灵杆菌合成灵菌红素过程中发挥的作用,并对未来的研究方向进行了展望。     

Strain improvement of Serratia marcescens ECU1010 and medium cost reduction for economic production of lipase

Industrial strain improvement plays a central role in the commercial development of microbial fermentation processes. The strain of Serratia marcescens ECU1010, a wild-type lipase-producer capable of stereospecific synthesis of a Diltiazem precursor, was subjected to physical mutation involving treatment by UV-irradiation for 30 s. A mutant strain, no. UV-01, showed enhanced lipase production, but lost the capability of producing red pigment (prodigiosin). The variant strain UV-01 had a 2.3-fold higher activity than the wild type and was stable in its enzyme production for ten serial transfers. For reduction of the fermentation medium cost, dried powder of corn steep liquor was used as an inexpensive substitute for beef extract in the medium. Dextrin as an organic carbon source and Tween-80 as an important element were further optimized, respectively. The high primary biodegradation of the Tween-80 by S. marcescens ECU1010 and its variant demonstrated their potential ability of degrading alkyl polyethoxylates to remove harmful nonionic surfactants from polluted effluents and streams. The optimal cultivation time for lipase biosynthesis was 24 h. These optimized compositions resulted in an economic production of lipase by S. marcescens ECU1010 var. UV-01, with a dramatically reduced cost (1/8–1/7 of the initial one) which is more suitable for industrial application.     

Prodigiosin is not a determinant factor in lysis of Leishmania (Viannia) braziliensis after interaction with Serratia marcescensd-mannose sensitive fimbriae

In this paper, the lytic activity of two variants of Serratia marcescens against promastigotes of Leishmania braziliensis was studied. In vitro assays showed that S. marcescens variant SM365 lyses L. braziliensis promastigotes, while the variant DB11 did not. Scanning electron microscopy (SEM) revealed that S. marcescens SM365 adheres to all cellular body and flagellum of the parasite. Several filamentous structures were formed and identified as biofilms. After 120 min incubation, they connect the protozoan to the developing bacterial clusters. SEM also demonstrated that bacteria, adhered onto L. braziliensis promastigote surface, formed small filamentous structures which apparently penetrates into the parasite membrane. d-mannose protects L. braziliensis against the S. marcescens SM365 lytic effect in a dose dependent manner. SM365 variant pre cultivated at 37 °C did not synthesize prodigiosin although the adherence and lysis of L. braziliensis were similar to the effect observed with bacteria cultivated at 28 °C, which produce high concentrations of prodigiosin. Thus, we suggest that prodigiosin is not involved in the lysis of promastigotes and that adherence promoted by bacterial mannose-sensitive (MS) fimbriae is a determinant factor in the lysis of L. braziliensis by S. marcescens SM365.     

Separation of nattokinase fromBacillus subtilis fermentation broth by expanded bed adsorption with mixed-mode adsorbent

Mixed-mode hydrophobic/ionic matrices exhibit a salt-tolerant property for adsorbing target protein from high-ionic strength feedstock, which allows the application of undiluted feedstockvia an expanded bed process. In the present work, a new type of mixed-mode adsorbent designed for expanded bed adsorption, Fastline PRO^®, was challenged for the capture of nattokinase from the high ionic fermentation broth ofBacillus subtilis. Two important factors, pH and ion concentration, were investigated with regard to the performance of nattokinase adsorption. Under initial fermentation broth conditions (pH 6.6 and conductivity of 10 mS/cm) the adsorption capacity of nattokinase with Fastline PRO was high, with a maximum capacity of 5,350 U/mL adsorbent. The elution behaviors were investigated using packed bed adsorption experiments, which demonstrated that the effective desorption of nattokinase could be achieved by effecting a pH of 9.5. The biomass pulse response experiments were carried out in order to evaluate the biomass/adsorbent interactions betweenBacillus subtilis cells and Fastline PRO, and to demonstrate a stable expanded bed in the feedstock containingBacillus subtilis cells. Finally, an EBA process, utilizing mixed-mode Fastline PRO adsorbent, was optimized to capture nattokinase directly from the fermentation broth. The purification factor reached 12.3, thereby demonstrating the advantages of the mixed-mode EBA in enzyme separation.     

Inhibition of Quorum Sensing in Serratia marcescens AS-1 by Synthetic Analogs of N-Acylhomoserine Lactone

Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C₆-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C₉-CPA), had a strong inhibitory effect on prodigiosin production. C₉-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C₉-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C₆-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C₉-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.     

Separation of Prodigiosenes and Identification as Prodigiosin Syntrophic Pigment from Mutant Pairs of Serratia marcescens

Countercurrent distribution is capable of resolving mixtures of closely related prodigiosene pigments. Syntrophic pigment produced by several pairs of Serratia marcescens color mutants was identified as prodigiosin (2-methyl-3-amyl-6-methoxyprodigiosene) by countercurrent distribution, soda lime pyrolysis, and other techniques. The metabolic block of mutant strain H-462, derived from parent strain HY, was located between the blocks of mutant strains OF and WF, both derived from parent strain Nima.     

Bacterial pigment prodigiosin and its genotoxic effect

A pigment complex has been isolated from the biomass of bacteria Serratia marcescens ATCC 9986 and separated into single fractions by chromatography. An analysis of the fractions by spectrophotometry, thin layer chromatography, NMR spectroscopy and mass spectrometry enabled us to isolate the so-called red fraction, to confirm the identity of the product of the red fraction to the pigment prodigiosin, and to prove its purity. Some features of the toxic action of prodigiosin have been revealed. It has been found for the first time that the pigment is capable of inducing mutations in Salmonella typhimurium TA 100 cells (Ames test) and chromosomal damage in mammalian erythroblasts.     

Pigmentation and Acriflavine Resistance in Serratia marcescens

Stable, orange, acriflavine-resistant variants were selected by treatment of a wild-type, red, acriflavine-sensitive strain of Serratia marcescens with acriflavine. Visible, ultraviolet, infrared, and nuclear magnetic resonance spectra of purified pigment from the red strain were identical to those of the pigment from the orange strain, and the orange mutant was not due to a mutation affecting the structure of the pigment, prodigiosin. The color of the red strain was not affected by variations in pH between 5.0 and 8.0, whereas the color of the orange mutant changed from pink to orange over the same pH range. This variation was mimicked by the pH-induced variation in color of prodigiosin purified from either the red, wild-type or the orange, mutant strains. Density-gradient centrifugation of cell fragments after ultrasonic disintegration resulted in characteristic pigmented bands. Biochemical characterization of these pigmented bands showed that they contained pigment and a protein component, but no lipids, polysaccharides, sugars, glucosamine, or phosphates were detected. Further fractionation of these pigmented bands by zone electrophoresis on a sucrose density gradient indicated that some pigment in S. marcescens was specifically attached to protein components.