Metabolic engineering of Streptomyces coelicolor for enhanced prodigiosins (RED) production

Bacterial prodigiosins are red-colored secondary metabolites with multiple activities,such as anticancer,antimalarial and immunosuppressive,which hold great potential for medical applications.In this study,dramatically enhanced prodigiosins(RED) production in Streptomyces coelicolor was achieved by combinatorial metabolic engineering,including inactivation of the repressor gene ohkA,deletion of the actinorhodin(ACT) and calcium-dependent antibiotic(CDA) biosynthetic gene clusters(BGCs) and multi-copy chromosomal integration of the RED BGC.The results showed that ohkA deletion led to a 1-fold increase of RED production over the wild-type strain M145.Then,the ACT and CDA BGCs were deleted successively based on the AohkA mutant(SBJ101).To achieve multi-copy RED BGC integration,artificial ΦC31 attB site(s) were inserted simultaneously at the position where the ACT and CDA BGCs were deleted.The resulting strains SBJ102(with a single deletion of the ACT BGC and insertion of one artificial attB site) and SBJ103(with the deletion of both BGCs and insertion of two artificial attB sites) produced 1.9-and 6-fold higher RED titers than M145,respectively.Finally,the entire RED BGC was introduced into mutants from SBJ101 to SBJ103,generating three mutants(from SBJ104 to SBJ106) with chromosomal integration of one to three copies of the RED BGC.The highest RED yield was from SBJ106,which produced a maximum level of 96.8 mg g^(-1) cell dry weight,showing a 12-fold increase relative to M145.Collectively,the metabolic engineering strategies employed in this study are very efficient for the construction of high prodigiosin-producing strains.

The Effect of Prodigiosin Extract from Serratia rubidea against Citrobacter freundii Infection in Mice

This study was prepared due to a lack of studies on the immune modulatory effects of prodigiosin in mice against bacterial infection and the increased demand for natural resources. This study aimed to extract prodigiosin from Serratia rubidaea against Citrobacter freundii in mice by evaluating its immunomodulatory activity and histological alterations. A total of twenty-four Swiss mice were divided up into four groups of six mice each. C. freundii (1 × 106 cfu/ml) was administered orally to groups (2, 3 and 4) as an infectious dosage, and one milliliter was administered to the first group as a negative control. Following 24 hours from C. freundii infection, the Group 3 and Group 4 groups were given crude prodigiosin extract in the following dosage amounts: the third and fourth groups received (500 and 1000 µg/ml Intraperitoneal) respectively. ELISA test was performed to assess IgM, IL-6, and IL-10 on days 3, 7 and 14. IgM and IL-6 findings demonstrated a significant increase in Group2 and Group4 with differences (P < 0.05), however, G3 showed a significant decrease when compared to the negative control. The results of IL-10 concentration revealed that Group 2 and Group 4 had significant decreases with differences (P < 0.05), with Group 3 having the highest titer. In conclusion, the study’s findings showed that while prodigiosin’s high concentration can boost the immune system and help laboratory animals resist bacterial infection, its low concentration acts as an immune suppressant.

Isolation of a Prodigiosin-producing Strain and Analysis of It's Pigments

[Objective] The aim was to isolate a prodigiosin producing strain and study its pigment fractions.[Method] Red pigment-producing bacteria was identified by physiological and biochemical characteristics after isolation in plate.By using column chromatography and thin-layer chromatography,pigment fractions were separated and purified from the extractives of the strain after fermentation in flask,and then pigment fractions were analyzed via UV-Vis and LC/MS.[Result] A red pigment-producing Serratia marcescens strain NS-17 sampled from soil of Nanchang was isolated and identified.2 pigment fractions showing similar UV-Vis and LC/MS characters were separated and purified,the characters of fraction 1 were identical to those of prodigiosin,while fraction 2 showed a special UV-Vis absorption spectrum that had not been reported.[Conclusion] A prodigiosin-producing Serratia marcescens strain NS-17 and its 2 pigment fractions were isolated.

Prodigiosin from Serratia:Synthesis and potential applications

Prodigiosin is a red pigment with a pyrrolylpyrromethane skeleton.It is mainly produced by bacterial strains belonging to the Serratia genus,but also by some other genera,including Streptomyces and Vibrio.Within the genus Serratia,the pigment is generally produced as a virulence factor.However,it also has many important beneficial biological activities such as immunosuppressive and anti-proliferative activities.Moreover,the pigment has many industrial applications in textile and cosmetics.In this mini-review,we discuss the genetic and molecular mechanisms supporting prodigiosin synthesis and production from the Serratia genus,as well as its potential applications.

Application of prodigiosin as a colorant for polyolefines

Serratia marcescens strain 9986 is a producer of prodigiosin used as a dye of polyolefines (polyethylene, ultratene). The biosynthesis of pigment was closely connected with controlled growth parameters. A prodigiosin yield 0.2 - 0.4 mg per l of culture medium in the batch culture under aerobic conditions was obtained. Prodigiosin was extracted from crude biomass treated by 0.1% of sodium dodecyl sulfate (SDS). This novel elaborated stage is necessary for thermostability a dye in polymer. The consumption of prodigiosin preparation is more economical in the technology of the coloration of polyolefines. The elaborated method has been manufactured for dyeing polyethylene by biological pigment-articles of the common use corresponding to the technological standard.

Continuous Fermentation of a Prodigiosin-Producing <i>Serratia marcescens </i>Strain Isolated from Soil

A red-pigmented bacterial strain NS-17 was isolated from soil, and physiological and biochemical characteristics and 16S rDNA analysis established this organism as a strain of Serratia marcescens. The red pigment isolated from cells of NS-17 was identified as prodigiosin. By analyzing factors affecting the production of prodigiosin, modified medium and culture conditions were set up, and a continuous fermentation method was carried out to take advantage of the mass production of foam during fermentation. Continuous fermentation was achieved in the following conditions: 32℃, 1:1 ventilation ratio, and medium with a nutrient concentration 5-fold higher than that of the fermentation medium (with half the inorganic salt concentration) supplied as a feed medium at a flow rate of 8 mL/min. For the first time of continuous fermentation of Serratia marcescens coupling with foam flotation, a high yield of prodigiosin was obtained. After 56 h of culturing, the total harvest of cells was enhanced 2.33-fold compared to that of batch fermentation, the total amount of prodigiosin was enhanced 2.70-fold compared to that of batch fermentation, and cells and prodigiosin were concentrated in the efflux broth automatically.

Egads It’s Enterobacteriaceae: Serratia rubidaea Urinary Tract Infection &Enterobacter aerogenes Bacteremic Urinary Tract Infection

We present the 3rd known case of a Serratia rubidaea urinary tract infection in a 49-year-old male with past medical history of bilateral lower extremity paraplegia who presented to the emergency room with fever, lethargy, and red tinted urine. The Serratia genus, of the family Enterobacteracia, in particular the species marcescens, are important causes of infection in humans, animals, and insects, however, until the mid-1950s, this was not the case, and the organism was considered non-pathogenic and frequently used in medical experiments. Serratia are facultatively anaerobic, gram-negative rods, the majority with peritrichous flagella primarily inhabiting soil, water, and plant surfaces. Serratia marcescens is now a known pathogen, however, less frequently isolated species, including Serratia rubidaea, are worthy of discussion, especially due to its characteristic red color and rarity. We aim to increase the awareness Serratia rubidaea including its presentation, inherent antimicrobial resistance, and treatment options.

Exploring the bioactive potential of Serriatia marcescens VITAPI （Acc： 1933637） isolated from soil

BACKGROUND： Serratia is one of the most important groups of bacteria which produces proteolytic enzymes effectively and known to possess anti-inflammatory properties. The main focus of the current study was to extract the enzyme serratiopeptidase and pigment prodigiosin from Serratia mascescens. Prodigiosin is a red colored pigment produced by the bacterium Serratia marcescens. It is emerging as a valuable molecule because of its large applications. It has already been proved that pigmented strain of Serratia marcescens is less virulent than non-pigmented strains. Moreover the strain we have obtained is from farm soil which indicates that prodigiosin production can be carded safely using this strain. METHODS： In the present study, the isolate VITASP strain was confirmed by morphological, biochemical and molecular studies. The enzyme and pigment were analyzed for anti-oxidant, anti-inflammatory and cytotoxic properties. RESULTS： The isolate was further confirmed and identified as Serratia marcescens with 99% similarity. The extracted pigment showed potent radical scavenging effect with 86% and the enzyme was found to inhibit 83%, which was significant in comparison to ascorbic acid standard. The in vitro anti-inflammatory effect of pigment in controlled experimental conditions revealed its protection at 88% and the enzyme with 90%. Aspirin was used as the reference drug. The present findings exhibited a concentration dependent inhibition. The cytotoxic bioassay of pigment showed the IC5o value as （50） ~tg/mL with 63% cytotoxicity which was statistically significant compared to positive control. CONCLUSIONS： Therefore, it appears to be an essential remedial and application research. It may turn out to be highly beneficial to mankind in solving many problems associated with human health.

Comparative transcriptome analysis reveals metabolic regulation of prodigiosin in Serratia marcescens

Prodigiosin is a secondary metabolite mainly produced at 30°C in Serratia marcescens,but it can hardly be synthetized at 37°C or higher.In this study,we provide insight into the metabolic regulation of prodigiosin synthesis in response to temperature through transcriptome sequencing.The analysis of the function of differentially expressed genes suggested that temperature resulted in significant alteration of the metabolic pathways between 30 and 37°C.Specifically,30°C favored transcriptional expression of the pig gene cluster.At the same time,the carbon flux was redistributed to pathways of pyruvate,proline,serine,especially homoserine,cystathionine,homocysteine,methionine,and s-adenosylmethionine metabolism,all involved in prodigiosin biosynthesis,and was finally increased towards the prodigiosin synthesis pathway in S.marcescens at 30°C.Interestingly,results further confirmed increased transcriptional level of five regulators(LuxS,RpoS,Hfq,EepR,CRP),and decreased content of hexS through qPCR.Finally,successful co-overexpression of mmuM and metK,related to homocysteine,methionine,and s-adenosylmethionine metabolism,in the chromosome of JNB5-1(JNB5-1/MK)resulted in increased prodigiosin titer up to 7.57 g/L in JNB5-1/MK at 30°C,which was 41.2%higher than that in JNB5-1.Our transcriptome analysis provides further insight into the strain’s response to temperature changes at the transcription level,which is of great significance for improving the production of prodigiosin.

Studies on culture condition of new marine bacterium Zooshikella sp. SY01

New marine bacterium Zooshikella sp.SY01,producer of prodigiosin,was isolated from the seawaters of Sanya Bay.The culture conditions of this bacterium were investigated.Zooshikella sp.SY01 was cultured in 2216E media which contained tryptophan,histidine,lac-tonic acid,camphor,limonene,casein,diphenyl guani-dine,coumarin and 1,3-dinitrobenzene,respectively.After 5 days cultivation,the extracts of different culture broths were detected by direct infusion mass spectroscopy using positive ESI mode.As the results,tryptophan,his-tidine and casein didn't show any observable influences on the biosynthesis of prodigiosin.Lactonic acid,camphor,limonene,diphenyl guanidine,coumarin could inhibit the bacterium growth and prodigiosin biosynthesis to a cer-tain extent,slower the culture broth to turn red.However,1,3-dinitrobenzene inhibited the bacteria to produce pro-digiosin completely.MS data suggested that various metabolites with chemodiversity were produced in differ-ent culture media.In particular,a series of high-molecu-lar-weight compounds with high relative abundances were observed in the medium containing limonene.To further optimize the culture condition,more new prodigiosin ana-logues and lead compounds can be obtained and the goal of"one strain-many compounds"can be achieved.