Prodigiosin formation by Serratia marcescens in a chemostat

In a study of the control of metabolite formation, prodigiosin production by Serratia marcescens was used as a model. Specific production rates of prodigiosin formation were determined using batch culture technique. Sucrose as carbon source and NH₄NO₃ as nitrogen source resulted in a specific production rate of 0.476 mg prodigiosin (g cell dry weight)⁻¹ h⁻¹. Prodigiosin formation and productivity was inversely correlated to growth rate when the bacterium was grown under carbon limitation on a defined medium in a chemostat culture. The maximum specific growth rate (μ_max) was 0.54 h⁻¹ and prodigiosin was formed in amounts over 1 mg l⁻¹ up to a growth rate (μ) of 0.3 h⁻¹ at steady state conditions. At a dilution rate of 0.1 h⁻¹ growth at steady state with carbon and phosphate limitation supported prodigiosin formation giving a similar specific yield [1.17 mg prodigiosin (g cell dry weight)⁻¹ and 0.94 mg g⁻¹, respectively], however, cells grown with nitrogen limitation [(NH₄)₂SO₄] did not form prodigiosin. Productivity in batch culture was 1.33 mg l⁻¹ h⁻¹ as compared to 0.57 mg l⁻¹ h⁻¹ in the chemostat.     

Defects in Prodigiosin Formation by L-Forms of Serratia marcescens

An L-form of Serratia marcescens has previously been shown incapable of producing the red pigment, prodigiosin, characteristic of the parent bacteria. Mutants of S. marcesens, unable to form one or the other of the two prodigiosin precursors, 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde or 2-methyl-3-n-amylpyrrole, were used to test the nature of the L-form defect. The L-forms failed to form sufficient amounts of either precursor to be detected by the appropriate mutant, and, when furnished the precursors, failed to couple them to form prodigiosin.     

粘质沙雷氏菌中灵杆菌素合成因素探究进展

灵杆菌素主要由粘质沙雷氏菌产生,其本身是微生物的一种次生代谢产物,属于脂多糖,也是一种典型的生物碱,具有抗癌、抑菌、增强免疫力、抗原虫、抗疟疾等多种生理活性,是化妆品、染料和水体污染治理等领域新兴且不可或缺的成分.随着医药业和工农业等不断发展,灵杆菌素的应用也越来越多,其良好的应用前景使得它的合成和提取工艺成为次生代谢领域的研究热点.但是,目前灵杆菌素的全化学合成和发酵生产受到很多因素的影响,大大阻碍了它的规模化生产和应用,本文重点对影响灵杆菌素生物合成的因素进行了综述,同时对其可能的作用机制进行了总结.本研究表明,微生物发酵生产灵杆菌素时,菌种、营养条件、环境因素(包括培养温度,pH值,溶解氧,光照)、金属离子等因素对灵杆菌素的合成都十分重要,另外再结合基因工程等技术,可大幅度提高灵杆菌素的产量.本综述为灵杆菌素的工业发酵生产提供重要依据.     

Role of Methionine in Biosynthesis of Prodigiosin by Serratia marcescens

Methionine alone did not allow biosynthesis of prodigiosin (2-methyl-3-amyl-6-methoxyprodigiosene) in nonproliferating cells (NPC) of Serratia marcescens strain Nima. However, when methionine was added to NPC synthesizing prodigiosin in the presence of other amino acids, the lag period for synthesis of prodigiosin was shortened, an increased amount of the pigment was formed, and the optimal concentrations of the other amino acids were reduced. Less prodigiosin was synthesized when addition of methionine was delayed beyond 4 h. The specific activity of prodigiosin synthesized by addition of (14)CH(3)-methionine was 40 to 50 times greater than that synthesized from methionine-2-(14)C or (14)COOH-methionine. NPC of mutant OF of S. marcescens synthesized norprodigiosin (2-methyl-3-amyl-6-hydroxyprodigiosene), and the specific activity of this pigment synthesized in the presence of (14)CH(3)-methionine was only 5 to 13 times greater than that synthesized from methionine-2-(14)C or (14)COOH-methionine. A particulate, cell-free extract of mutant WF of S. marcescens methylated norprodigiosin to form prodigiosin. When the extract was added to NPC of mutant OF synthesizing norprodigiosin in the presence of (14)CH(3)-methionine, the prodigiosin formed had 80% greater specific activity than the norprodigiosin synthesized in the absence of the extract. The C6 hydroxyl group of norprodigiosin was methylated in the presence of the extract and methionine. Biosynthesis of prodigiosin by NPC of strain Nima also was augmented by addition of S-adenosylmethionine. Various analogues of methionine such as norleucine, norvaline, ethionine, and alpha-methylmethionine did not affect biosynthesis of prodigiosin by NPC either in the presence or absence of methionine.     

Biosynthesis of Prodigiosin, a Secondary Metabolite of Serratia marcescens

Prodigiosenes (prodigiosin and prodigiosin-like pigments) are known to be synthesized by only one genus of Eubacteriales and by two genera of Actinomycetales. Biosynthesis by Serratia marcescens occurs over a relatively narrow range of temperatures, although the bacteria grow over a broad range. When cultures of S. marcescens were incubated at 27 C in 1.0% casein hydrolysate, viable count and protein attained maximal values within 24 to 48 h, whereas prodigiosin did not reach a maximum until 96 h. The greatest amount of pigment was synthesized when cultures were in the senescent phase of growth. Suspensions of nonproliferating bacteria incubated at 27 C in only L-alanine also synthesized prodigiosin, although at a slower rate than growing cultures. Kinetics of growth for the wild-type, red S. marcescens and a white mutant were identical when incubated at 27 C, but the wild type produced abundant pigment. These results plus other data obtained from the literature suggest that prodigiosin is a secondary metabolite. The importance of this proposal to understanding the function of prodigiosin in S. marcescens is discussed.     

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。     

Influence of Dissolved Oxygen Levels on Production of l-Asparaginase and Prodigiosin by Serratia marcescens

The effect of dissolved oxygen concentrations on the behavior of Serratia marcescens and on yields of asparaginase and prodigiosin produced in shaken cultures and in a 55-liter stainless-steel fermentor was studied. A range of oxygen transfer rates was obtained in 500-ml Erlenmeyer flasks by using internal, stainless-steel baffles and by varying the volume of medium per flask, and in the fermentor by high speed agitation (375 rev/min) or low rates of aeration (1.5 volumes of air per volume of broth per min), or both. Dissolved oxygen levels in the fermentation medium were measured with a membrane-type electrode. Peak yields of asparaginase were obtained in unbaffled flasks (3.0 to 3.8 IU/ml) and in the fermentor (2.7 IU/ml) when the level of dissolved oxygen in the culture medium reached zero. A low rate of oxygen transfer was accomplished by limited aeration. Production of prodigiosin required a supply of dissolved oxygen that was obtainable in baffled flasks with a high rate of oxygen transfer and in the fermentor with a combination of high-speed agitation and low-rate aeration. The fermentation proceeded at a more rapid rate and changes in pH and cell populations were accelerated by maintaining high levels of dissolved oxygen in the growth medium.     

Cytotoxic Effect of Prodigiosin,Natural Red Pigment,Isolated from Serratia marcescens UFPEDA 398

Prodigiosin is a secondary metabolite, with red pigmentation, produced by Serratia marcescens. Red pigment is a natural alkaloid whose chemical structure has three pyrrole rings. Prodigiosin has been described for several biological activities, including antitumor, inducing apotosis in T and B lymphocytes. This work aimed to evaluate the cytotoxic activity of prodigiosin in NCHI-292, HEp-2, MCF-7 and HL-60 tumor cell lines. The red pigment was isolated from Serratia marcescens UFPEDA 398 biomass whose fractions were previously separated by column chromatography, purified, identified and further characterized by GC–MS and compared with the computerized library of m/z values. The pigment corresponded to prodigiosin with maximum absorption at 534 nm, molecular weight 323 and structural formula C20H25N3O. During the prodigiosin purification process a purple absorbance fraction at 272.65 nm was also observed. Significant cytotoxic effects of prodigiosin were evidenced for NCHI-292, Hep-2, MCF-7 and HL-60 tumor cell lines. The isolated purple fraction had no cytotoxic effect (IC50 11.3 µg/mL) when compared to prodigiosin (IC50 3.4 µg/mL) for the tumor cell lines studied. The MCF-7 strain was slightly more pigment resistant (IC50 5.1 µg/mL). Therefore, further studies will be needed to elucidate the antitumor mechanisms of prodigiosin action against tumor strains from flow cytometry tests. However, although these data are preliminary, it was evidenced that prodigiosin showed cytotoxic activity in tumor cell lines suggesting promising antitumor properties. In this sense, future studies on the cytotoxic and genotoxic effects of prodigiosin produced by S. marcecsens UFPEDA 398 are suggested.     

Iron transport systems of Serratia marcescens.

Serratia marcescens W225 expresses an unconventional iron(III) transport system. Uptake of Fe3+ occurs in the absence of an iron(III)-solubilizing siderophore, of an outer membrane receptor protein, and of the TonB and ExbBD proteins involved in outer membrane transport. The three SfuABC proteins found to catalyze iron(III) transport exhibit the typical features of periplasmic binding-protein-dependent systems for transport across the cytoplasmic membrane. In support of these conclusions, the periplasmic SfuA protein bound iron chloride and iron citrate but not ferrichrome, as shown by protection experiments against degradation by added V8 protease. The cloned sfuABC genes conferred upon an Escherichia coli aroB mutant unable to synthesize its own enterochelin siderophore the ability to grow under iron-limiting conditions (in the presence of 0.2 mM 2.2'-dipyridyl). Under extreme iron deficiency (0.4 mM 2.2'-dipyridyl), however, the entry rate of iron across the outer membrane was no longer sufficient for growth. Citrate had to be added in order for iron(III) to be translocated as an iron citrate complex in a FecA- and TonB-dependent manner through the outer membrane and via SfuABC across the cytoplasmic membrane. FecA- and TonB-dependent iron transport across the outer membrane could be clearly correlated with a very low concentration of iron in the medium. Expression of the sfuABC genes in E. coli was controlled by the Fur iron repressor gene. S. marcescens W225 was able to synthesize enterochelin and take up iron(III) enterochelin. It contained an iron(III) aerobactin transport system but lacked aerobactin synthesis. This strain was able to utilize the hydroxamate siderophores ferrichrome, coprogen, ferrioxamine B, rhodotorulic acid, and schizokinen as sole iron sources and grew on iron citrate as well. In contrast to E. coli K-12, S. marcescens could utilize heme. DNA fragments of the E. coli fhuA, iut, exbB, and fur genes hybridized with chromosomal S. marcescens DNA fragments, whereas no hybridization was obtained between S. marcescens chromosomal DNA and E. coli fecA, fhuE, and tonB gene fragments. The presence of multiple iron transport systems was also indicated by the increased synthesis of at least five outer membrane proteins (in the molecular weight range of 72,000 to 87,000) after growth in low-iron media. Serratia liquefaciens and Serratia ficaria produced aerobactin, showing that this siderophore also occurs in the genus Serratia.     

Induction of Prodigiosin Biosynthesis after Shift-Down in Temperature of Nonproliferating Cells of Serratia marcescens

Nonpigmented bacteria obtained by growth of Serratia marcescens at 38 C synthesized prodigiosin at 25 C if certain individual amino acids were added to cultures of nonproliferating cells. In order of effectiveness, the amino acids were: DL-histidine, L-proline, L-hydroxyproline, DL-alanine, L-alanine, DL-aspartic acid, D-alanine, DL-proline, L-serine, L-ornithine, L-glutamic acid, and D-proline. DL-Histidine at its optimal concentration (20 mg/ml) induced formation of prodigiosin (198 mug of prodigiosin per mg of bacterial protein) after incubation of cultures for 54 hr. Lower concentrations (10 mg/ml) of the other amino acids usually were optimum but less prodigiosin was synthesized, and the maximal amount of pigment occurred between 36 and 48 hr. DL-Methionine was not effective alone but at a low concentration (40 mug/ml) enhanced and accelerated biosynthesis of prodigiosin in the presence of other suitable amino acids. Addition of 2 mg of L-proline per ml at 0 hr induced formation of only 30 mug of prodigiosin after incubation for 42 hr, but addition at 36 hr of 5 mg more of L-proline per ml increased synthesis to 120 mug at 42 hr. Again, DL-methionine markedly augmented prodigiosin biosynthesis in these cultures. Synthesis of prodigiosin ceased if cultures were shifted from 25 to 38 C. Prodigiosin biosynthesis by the nonproliferating cells was maximum when cultures were aerated, the amount of bacterial protein was about 2.0 mg/ml, and amino acids were added at 0 hr. Bacteria synthesized prodigiosin most efficiently when they were harvested from aerated cultures grown at 38 C for 24 hr in a complete medium in a fermentor.     

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.     

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.     

Prodigiosin synthesis in mutants of Serratia marcesens

Morrison, D. A. (Harvard College, Cambridge, Mass.). Prodigiosin synthesis in mutants of Serratia marcescens. J. Bacteriol. 91:1509-1604. 1966.-Exchange of biosynthetic intermediates through the culture medium was used to characterize several hundred new color mutants of Serratia marcescens. The general scheme of prodigiosin synthesis as a bifurcated pathway, in which monopyrrole and bipyrrole precursors are synthesized separately and then coupled to form pigment, was confirmed and extended. Mutants of one new class excreted a product likely to be a new intermediate in monopyrrole synthesis, those of a second excreted a new product in the bipyrrole pathway, and those of a third were blocked at early steps in both pathways. Two novel classes of mutants were isolated, in each of which a lack of some product present in Serratia and Escherichia cultures resulted in loss of all steps in prodigiosin biosynthesis.     

Cation transport in Serratia marcescens and Serratia marinorubra

The sodium, potassium, and magnesium ion contents of Serratia marcescens and those of its salt-tolerant relative, S. marinoruba, were determined by atomic-absorption spectrometry. The intracellular K(+) and Mg(2+) contents of both microorganisms were found to be dependent on the ionic strength of the growth or suspending medium. The Mg(2+) content of S. marinoruba was generally greater than that of S. marcescens. The Na(+) content of the cells was normally low and did not increase as the cells aged or when the cells were grown in media of high ionic strength. The transport of K(+) by resting cells suspended in hypertonic solution was studied by chemical and light-scattering techniques and was found to be more rapid in S. marcescens than in S. marinorubra. The slower rate of K(+) transport in S. marinorubra is probably related to the lower glycogen reserves found in resting cells of this microorganism. K(+) transport was found to have a pH optimum of 5.5 to 6.1 for S. marcescens, and the K(m) for K(+) was approximately 1.6 mm. Na(+) and Mg(2+) were not taken up by the cells, although the presence of Mg(2+) tended to decrease rates of K(+) uptake. Tris-(hydroxymethyl)aminomethane, routinely used for resuspending the cells, was apparently taken up by the cells at pH >7.     

Concomitant Synthesis of Bacteriocin and Bacteriocin Inactivator from Serratia marcescens

We have found that Serratia marcescens strain P & S is bacteriocinogenic. However, the phenotypic expression of bacteriocin activity depends upon the temperature at which the cells are grown. When the organism is grown at 30 to 37 C, no bacteriocin activity can be demonstrated, whereas when it is grown at 39 C bacteriocin activity is readily observed. It appears that the P & S strain concomitantly synthesizes a bacteriocin and a substance which not only can inactivate the bacteriocin but also has a high activation energy for inactivation. This inactivator readily loses its activity when heated at 39 C for 1 hr. Two mutants were isolated from the P & S strain which can produce active bacteriocin when grown at temperatures from 30 to 39 C. It is significant that these mutants have considerably less bacteriocin inactivator. The data suggest that the inactivator is an extracellular protease. The ability of one of these mutants, JF58-12, to produce active bacteriocin at temperatures between 30 and 39 C is a stable property, whereas in the other mutant, JF48W, this property is unstable. JF48W was selected from the P & S strain in two steps: first a streptomycin-resistant variant (strain A-10) was isolated and from this mutant a strain (JF48W) was isolated which not only synthesized little of the inactivator but also did not synthesize the red pigmnet prodigiosin. This latter pleiotropic mutant appears to revert in one step to a phenotype similar to the P & S strain, since it is streptomycin-sensitive and produces prodigiosin and normal amounts of inactivator and the demonstration of bacteriocin activity is temperature-dependent.