Biomass Conversion and Biorefinery

https://doi.org/10.1007/s13399-020-00928-2

REVIEW ARTICLE

A comprehensive review on recent trends in production, purification,

and applications of prodigiosin
Tania Paul 1 & Tarun Kanti Bandyopadhyay 1 & Abhijit Mondal 1 & Onkar Nath Tiwari 2 &
Muthusivaramapandian Muthuraj 3 & Biswanath Bhunia 3

Received: 25 January 2020 / Revised: 21 July 2020 / Accepted: 24 July 2020

# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Increasing utilization of artificial chemical dyes as coloring agent and their associated detrimental effects on human health have
instigated the search for novel natural pigments for applications in the food and pharmaceutical industries. A wide range of
advancements have been recorded for utilization of naturally extracted pigments, owing to their assurance in affirmative health
attentiveness in the past few decades. Therefore, it is essential to investigate various potential natural resources such as plants and
microbes from where food-grade colorants can be obtained. Several drawbacks such as low water solubility, instability to light,
heat, or adverse pH are associated with natural pigments extracted from plants. On the contrary, microbial pigments having
higher stability and productivity are considered a potential alternative to synthetic dyes. Prodigiosin is a linear tripyrrole natural
red pigment obtained from Serratia marcescens which is produced as a secondary metabolite. Prodigiosin and their derivatives
act as proapoptotic agents against diverse cancer cell lines and also play a vital role in numerous cellular targets, including multi-
drug resistance cells with reduced or no reported adverse side effects. Owing to its high potential in various sectors, there is an
urgent need to explore and exploit the biosynthesis of prodigiosin using various biotechnological approaches. Furthermore,
multifarious aspects in commercial-scale production of prodigiosin along with its contemporary utilization have been critically
assessed in this article.

Keywords Pigment . Biosynthesis . Microorganism . Prodigiosin . Extraction . Apoptosis . Application

Abbreviations HBC 4-Hydroxyl-2,2′-

PG Prodigiosin or prodiginines. bipyrrole-5-carbaldehyde
MBC 4-Methoxy-2-2′- HBM 4-Hydroxy-2,2′-bipyrrole-5-methanol
bipyrrole-5-carbaldehyde Pig Pigment
MPP 2-Methyl-3-pentylpyrrole UV Ultraviolet

Highlights
• Biosynthesis of prodigiosin and its source
• Extraction and purification of prodigiosin
• Biochemical characterization of prodigiosin

* Muthusivaramapandian Muthuraj Onkar Nath Tiwari

[email protected] [email protected]
* Biswanath Bhunia
[email protected] 1
Department of Chemical Engineering, National Institute of
Tania Paul Technology Agartala, Agartala 799046, India
[email protected] 2
Centre for Conservation and Utilisation of Blue Green Algae,
Tarun Kanti Bandyopadhyay Division of Microbiology, ICAR-Indian Agricultural Research
[email protected] Institute (ICAR-IARI), New Delhi 110012, India
3
Abhijit Mondal Department of Bio-Engineering, National Institute of Technology
[email protected] Agartala, Agartala 799046, India
 Biomass Conv. Bioref.

HCl Hydrogen chloride IPTG Isopropyl-β-D-thiogalactoside

Pig clusters (pigN, pigA, pigF, pigM, pigE, HEp-2 Laryngeal epidermoid carcinoma
pigJ, pigH, pigD, pigB, pigBC)-two HL-60 Human promyelocytic leukemia
Red H Undecylprodigiosin NCIH-292 Lung mucoepidermoid carcinoma
Hap C Prodigiosin MCF-7 Breast adenocarcinoma cell lines
Rph H Prodigiosin R1 HepG2 The human liver cancer cell
Tam Q Tambjamine YPI A549 Human lung carcinoma
YP 1 Pseudoalteromonas tunicate K562 Human bone marrow chronic
Tam Tambjamine producer B-CLL B cell chronic lymphocytic leukemia
Rph Roseophilin producer U937 Human Caucasian histiocytic
LD50 Lethal dose 50% lymphoma derived from malignant cells
DMSO Dimethyl sulfoxide IMR-32 A human neuroblastoma cell line
QSAR Quantitative structure- MCF-7 MCF-7 is a breast cancer cell line
activity relationship isolated in 1970 from a
TRAIL TNF-related apoptosis inducing ligand 69-year-old Caucasian woman
CP-MLR Combinatorial protocol in multiple DLD-1 One of four colorectal
linear regression adenocarcinoma cell
Caspase Cysteine-aspartate proteases family SW-620 SW-620 cell line
Bcl-2 B cell lymphoma 2 expresses carcinoembryonic antigen
Fas FS-7 associated surface antigen P27 Cyclin-dependent kinase
TLC Thin layer chromatography inhibitor 1B (p27Kip1) is an enzyme
TNF Tumor necrosis factor inhibitor that in humans is encoded
HPLC High-performance liquid chromatography by the CDKN1B gene
NMR Nuclear magnetic resonance P53 p53, also known as TP53 or tumour
GC-MS Gas chromatography protein (EC: 2.7. 1.37) is a gene that
mass spectrophotometer codes for a protein that regulates
MS Mass spectrophotometer the cell cycle and hence functions
FTIR Fourier-transform as a tumour suppression
infrared spectrophotometer P73 p73 (encoded by TP73 gene) is
DCW Dry cell weight a p53-related protein that functions
DPPH 2,2-Diphenyl-1-picrylhydrazyl as a transcriptional factor
ABTS 2,2′-Azino-bis 3- P38 P38 mitogen–activated protein kinases
ethylbenzthiazoline-6-sulfonic acid P38 MAP also called RK or CSBP
YPI Pseudoalteromonas tunicata Kinase (cytokinin-specific binding protein),
REDL RED Q, AFA, etc. (MAPK) is the mammalian orthologue of the
DNA Di-oxy ribonucleic acid yeast Hog1p MAP kinase, which
TAM H Tambjamine H participates in a signaling cascade
TAM Q Tambjamine Q controlling cellular responses to
RNA Ribonucleic acid cytokines and stress
ABTS 2,2-Azino-bis Cdk 2 Cyclin-dependent kinase 2
(3-ethylbenzothiazoline-6-sulfonic PKC Protein kinase C
acid) MAPK Mitogen-activated protein kinase
GvHD Graft versus host disease PTP1B Protein-tyrosine phosphatase 1B
IUPAC International Union of Pure and PP2A Protein phosphatase 2
Applied Chemistry JNK Jun N-terminal kinase
LC-HRMS Liquid chromatography- RAD 51 RAD51 gene provides instructions
high-resolution mass spectrometry for making a protein that is essential
DPPH 2,2-Diphenyl-1-picrylhydrazyl for repairing damaged DNA
MRSA Methicillin-resistant c-Jun It is a protein that in humans is
Staphylococcus aureus encoded by the JUN gene
ATP Adenosine triphosphate ΔNp73 ΔNp73-α synergizes with specificity
CP-MLR Combinatorial protocol in protein (Sp1)
multiple linear regressions mTORC1/2 Mammalian target of rapamycin
Biomass Conv. Bioref.

PI3K-p85 Phosphoinositide 3-kinases requirements for synthetic colors [6]. Extension of nanotech-
SKP2 S-Phase kinase-associated protein 2 nology may be contemplated as a modernistic advancement in
PKB Protein kinase B the field of synthesizing pigments from microbes which re-
HL60 Human acute promyelocytic leukemia sulted in escalating the shelf-life of materials and enhanced the
Hep G2 Human hepatocellular carcinoma solubility of pigments in different solvents [7]. All these ame-
HCT 116 Human colorectal carcinoma liorations have boosted a vital research interest in this partic-
H23-SK-MEL-5 SK-MEL-5 is one of a series of ular field.
melanoma cell lines established Microorganisms produce an expanded spectrum of pig-
from patient-derived tumor samples ments that can be adopted as natural food colorants [8] are
enlisted in Table 1. By virtue of this, a group of microorgan-
isms including bacteria, fungi, and algae are adopted as the
1 Introduction cellular factory for biosynthesis of natural food colors. These
natural pigments have several beneficial roles such as anti-
The freshness of food and its appealing nature depends on the oxidant, anti-bacterial, anti-fungal, and anti-cancer with addi-
color, concentration, and its intensity [1]. Utilization of food tional heat stability and light stability which make them
colors as a selling point clocks back to 1500 BCE when unique. Accordingly, the limelight of food and beverage in-
Roman and Egyptian scripts depicted the color of wines and dustries has bent towards technologies that microbiological in
drugs as standards to identify their quality and taste [2]. origin, for assorted applications. In food industries, the color is
Therefore, in order to meet the selling standards, industries mainly used for several purposes such as baby foods, pasta,
rely on synthetic compounds to have variety of color shades breakfast cereals, sauces, processed cheese, fruit drinks,
in the food. However, the deleterious effects on the use of vitamin-enriched milk products, and a lot of energy drinks.
synthetic color compounds leading to mutagenesis, cancer, Therefore adoption of natural colors in food industry will play
and other allergic reactions have instigated the search for nat- a conduit beneficial role appropriate to human health [9].
ural color compounds that are not toxic to the consumers [3]. Prodigiosin, a nature-derived pigment bearing the chemical
These natural colors are nothing but the pigments derived formula C20H25N30, is a structurally linear tripyrrole red pig-
from plant, and microbial sources which are non-toxic and ment. It is a well-known secondary metabolite that accumu-
have capability to impart its color to the materials in which it lates within the cell membranes and intracellular granules of
is added. These natural pigments encompass a spectrum of both gram-positive and gram-negative bacteria. Prodigiosin
color with several inherent properties which makes them a isolated from bacteria and other compounds synthetically de-
viable substitute for synthetic colors and also used as food rived from this red pigment are very much effective as
additives, color intensifiers, preservatives, and especially as proapoptotic agents and against the cancer cell lines. They
antioxidants [4]. Thus, scientific community has always urged also play an important role in multiple cellular targets, includ-
to delve into the vast source of natural color bearing food- ing multi-drug resistance cells. It is also interesting to note that
grade properties to replace synthetic colors. this particular pigment prodigiosin depicted selectivity against
Almost all living organisms have the capability to synthe- cancer cell with little or no toxicity towards the normal cell
size certain pigments in their system; however, plants and lines. The researchers are very much hopeful that prodigiosin
microbes are the major cell factories that can accumulate high will play a major role in the upcoming future in the field of
levels of pigments and food colorants for industrial-scale pro- new natural pigment synthesis as depicted from the 229 arti-
duction. Plant-acquired natural pigments flaunt a spectrum of cles published on prodigiosin in the past 5 years from 2016
circumspections like sensitivity towards heat or pH, suscepti- (www.Webofknowledge.com). It is also important to note that
ble towards light sensitivity, less solubility in polar solvents, only eleven review articles surfaced the search in which only
and year round unavailability. On the contrary, pigments syn- three articles were discussing the mechanisms of prodigiosin
thesized from microorganism unveil significant stability under biosynthesis and the rest on applications. However, to the best
extreme pH or temperature, with potential solubility in wide of our knowledge, the production strategies, purification
range of solvents, etc. Apart from that, microorganisms can be techniques, and analytical methodologies related to
conveniently cultured in large-scale reactors and in lab-scale prodigiosin synthesis are missing. In addition to that, 98%
reactors with ease [5] utilizing fermentation strategies, and the purified prodigiosin hydrochloride costs 430 US$ for
subsequent downstream processing are facile and effortless. 100 μg (www.sigmaladrich.com) which is not economically
Microorganisms also play an indispensable role in the forma- feasible. Even though several applications have been
tion of natural products as the sources of biologically active identified for this wonderful antibiotic and anti-cancer drug,
pharmaceutical and nutraceutical compounds. Therefore, the commercial-scale synthesis remains unexplored. Thus, the
pigments derived from microbial sources have been one of the present study targets to review the major strategies involved
potential sources for natural colorants that can replace the in the maximization of prodigiosin production, and
Table 1 Few natural pigments identified with potential applications as food coloring agents synthesized by microbes [1, 2]

Pigment Color Microbial Sources Application Bioactivity Reference

Bacteria Fungi Algae

Riboflavin Yellow to Bacillus subtilis and Ashbya gossypii, Candida Chlorella sp., Spirulina sp., Fortification of foods, and Anti-oxidant, anti-cancer, [2, 110,
yellow-- probiotic bacteria famata Nannochloropsis gaditana as coloring agent combats cardiovascular 111]
orange diseases, and assists in
improving vision
beta-Carotene Orange, red, Agrobacterium Mucor spp., Blakeslea trispora, Dunaliella salina Food coloring agent for Anti-oxidant, anti-cancer, [112]
or yellow tumefaciens, Serratia Fusarium sporotrichioides, non-thermally treated precursor for provitamin
marcescens, Escherichia Neurospora crassa, non-alcoholic bever- A synthesis, cholesterol
coli Rhodosporidium diobovatum ages synthesis inhibitor
Astaxanthin Pink red or Agrobacterium Xanthophyllomyces Heamtococcus pulvialis Preservative agent for Anti-oxidant, anti-cancer, [2, 113]
orange-red aurantiacum, dendrorhous meat, delays oxidation light protectant,
Paracoccus of fats and cholesterols anti-cancer, and
carotinifaciens anti-inflammatory
Canthaxanthin Orange and Halobacterium spp., Monascus spp., Cantharellus Chlorella emersonii, Food colorants, cosmetic Anti-oxidant, anti-cancer, [114,
dark pink Haloferax alexandrines, cinnabarinus Chlorella zofingiensis world, and in light protective, 115]
Bardyrhizobium spp., medications anti-inflammatory
and Lactobacillus
pluvalis
Prodigiosin Red Serratia marcescens, - - Coloring yogurt, milk and Anti-bacterial, anti-fungal, [1, 2]
Pseudoalteromonas soft carbonated drinks anti-cancer, anti-malarial
rubra activity
cyclo-Prodigiosin Red Pseudoalteromonas - - Food coloring dye Anti-cancer, anti-microbial, [116]
denitrificans, Vibrio and anti-malarial activity
gazogenes, Alteromonas
rubra, Zooshikela
rubidus
heptyl-Prodigiosin Red Alpha-proteobacteria - - Food coloring dye Anti-malarial activity [1, 2]
Undecylprodigiosin Red Streptomyces spp., - - Food coloring dye Anti-oxidant, [1, 2]
Saccharopolyspora sp., anti-neoplastic, UV light
Actinomadura madurae protective, and
anti-bacterial
Violacein Violet/purple Chromobacterium - - Medicine industry, Anti-fungal, anti-oxidant, [1, 2]
violaceum, cosmetic, food and also and sweeps singlet
Janthinobacterium textiles oxygen
lividum,
Pseudoalteromonas spp.
Phycocyanin Blue Pseudomonas spp. - Spirulina sp., Galdieria Works for gelly Anti-tumor, anti-oxidant, [117]
sulphuraria, other substances, sweets, ice anti-cancer, anti-fungal
cyanobacterial strains and cream, bakery products,
eukaryotic microalgal liqueurs and
strains condiments
Biomass Conv. Bioref.
Biomass Conv. Bioref.

purification. The study will also cover in detail the various as prodigiosin and undecylprodigiosin or as cyclic derivatives
applications and properties of prodigiosin pigment while s u c h a s c y c lo p r o d i g i o s i n , s t r e p t o r u b i n B , 2 - ( p -
discussing the various analytical techniques and bioactivities hydroxybenzyl) prodigiosin, and cyclo-nonyl-prodigiosin as
identified so far. enlisted in Table 2. Other than that, there are several analogs
of prodigiosin which are synthesized biologically by the in-
corporation of a different chain length of hydrocarbons de-
2 Prodigiosin: sources, derivatives, rived from fatty acids. For instance, the undecylprodigiosin
and characteristic features (C25H35N3O) differs from prodigiosin with its additional car-
bon atoms attributed to the hydrocarbon side chain length, and
Prodigiosin is a secondary metabolite initially identified and hence, it is termed as prodigiosin 25-C [14]. Similarly,
characterized from Serratia marcescens, a gram-negative bac- prodigiosin with different alkyl chain lengths such as propyl,
terium which belongs to the class γ-proteobacteria [10]. butyl, and heptyl prodigiosin are available in nature [15]. For
Furthermore, the presence of prodigiosin pigment was char- example, the Pseudoalteromonas rubra has been reported to
acterized in other γ-proteobacteria such as accumulate 2-(p-hydroxybenzyl) prodigiosin which forms the
Pseudoalteromonas rubra, Vibrio sp., Janthinobacterium, new class of linear acyclic form (Table 2). On the other hand,
Pseudomonas putida, etc., (Table 2). Other than that, the cycloprodigiosin differs from prodigiosin by the oxidative
Streptomyces spp., categorized under actinomycetes, is also cyclization of alkyl chain with the amylpyrrole of the C ring
known to accumulate high levels of prodigiosin pigment. [16]. Similarly, the streptorubin B produced by Streptomyces
Prodigiosin has also been recovered from other bacteria name- sp. is a cyclic red pigment comprising the meta-butyl-cyclo-
ly Streptomyces lividans, Hahella chejuensi, Pseudovibrio heptyl prodigiosin synthesized from undecyl-prodigiosin via
denitrificans, Vibrio psychroerythreus, Serratia plymuthica, oxidative carbocyclization [17]. The other cyclic analogues
and Zooshikella rubidus. Even though several strains have are the meta-cycloprodigiosin and propyl-meta-cyclo-octyl
been shown to accumulate prodigiosin, the major cellular fac- prodigiosin [18] synthesized from Streptomyces coelicolor
tories that synthesize this pigment at high concentrations are M511. Recent studies on the genome and metabolome of a
Serratia marcescens up to 49.5 g L−1 [11] and Streptomyces marine bacterium Vibrio spartinae 3.6 revealed the presence
sp., (138 mg L −1 ) [12]. The variations in the titer of of branched-chain prodigiosin derivative iso-heptyl-
prodigiosin in these strains are due to the fact that these strains prodigiosin synthesized from the pig gene cluster [19]. In
synthesize different derivatives of prodigiosin depending up- addition to that, the strain also found to accumulate
on the cultivation conditions and on the presence of specific cycloprodigiosin, 4″-hexylprodigiosin, and 4″-prodigiosin
prodigiosin-synthesizing gene clusters. For instance, from the pig gene cluster [19]. All these prodiginines can exist
Streptomyces coelicolor A3(2) was found to produce a as different enantiomers with R and S forms. For example, the
tripyrrole which is structurally similar to prodigiosin, named cycloprodigiosin exists in both R and S forms in Pseudomonas
as undecylprodigiosin, and a cyclic derivative, butyl-meta- rubra at a ratio of 83:17. Similarly, the native prodigiosin
cyclo-heptyl-prodiginine in a 2:1 ratio [13]. Similarly, pigment exists in both cis and trans forms based on the solvent
Pseudoalteromonas spp., are known to accumulate pH with trans form exposing the proton group more promi-
cycloprodigiosin and 2-(p-hydroxybenzyl) prodigiosin as de- nently to the media. This cationic charge in prodigiosin helps
rivatives (Table 2). Interestingly, the actinomycetes identified in the binding of Cl− or HCO3− ions more prevalently to the
with undecylprodigiosin were not reported to accumulate pigments and allows its uptake through symport or antiport
prodigiosin, and similarly, the bacterial strains of γ- mechanisms. In addition to that, these pigments in the physi-
proteobacteria that predominantly accumulate prodigiosin ological pH form ionic bonds with both AT and GC of DNA
are not reported to accumulate undecylprodigiosin [14]. To sequences. The pigment is also known to form metallic com-
decipher the variations underlying in their derivatives, the plexes with copper, and zinc ions with the nitrogen available
molecular structure of prodigiosin must be understood. in A, B, and C rings of prodigiosin which assist in the oxida-
The red pigment is a tripyrrole molecule comprising pyr- tive damage of DNA and in subsequent bioactivity [20].
role (A ring), 3-methoxypyrrole (B ring), and 2-methyl-3-
amylpyrrole (C ring) with the IUPAC name (5[3-methoxy-5- 2.1 Analysis of prodigiosin and derivatives
pyrrol-2-ylidene-pyrrol-2-ylidene (-methyl]-2methyl-3-
pentyl-1H-pyrrole). The rings A and B are linked with a Qualitative analysis of the prodigiosin and their derivatives is
bipyrrole unit, whereas the B and C rings are linked with a carried out using UV-visible spectroscopy, NMR, HPLC, GC-
dipyrrin complex [14]. Thus, presence of pyrrolyl-dipyrro- MS, TLC, and FTIR techniques, whereas the quantitative
methene skeleton with 4-methoxy, 2-2 bipyrrole ring system analysis is performed using UV-visible spectroscopy and
may be considered the signature sequence of these com- chromatographic techniques. The absorption maxima for the
pounds. These pigments can either exist as linear form such prodigiosin solubilized in methanol analyzed in the UV-
 Biomass Conv. Bioref.

Table 2 List of organisms that synthesize different prodigiosin derivatives

Organism Type Prodigiosin type Titer Reference

Serratia nematodiphila YO1 Wild-type Prodigiosin 93.2 mg L−1 [21]

Serratia masrcescens UCP Wild-type Prodiogiosin 49.5 g L−1 [11]
1549
Achromobacter denitrificans Wild-type Prodigiosin with 25 carbon atoms 1.314 mg L−1 [118]
SP1
Serratia marcescens TKU011 Wild-type Prodigiosin 4.62 g L−1 [52]
Pseudoalteromonas rubra Wild-type marine bacterium Cycloprodigiosin, 2-(p-hydroxybenzyl) prodigiosin ND [10]
Serratia marcescens Mutated Prodigiosin ND [119]
Streptomyces griseoviridis Wild-type Prodigiosin R2, prodigiosin R1, ND [120]
2464-S5 1,1-methyldodecylprodiginine
Serratia marcescens FZSF02 Wild-type Prodigiosin 15.43 g L−1 [49]
Streptomyces coelicolor Engineered Prodigiosin 96.8 mg g−1 [42]
DCW
Pseudomonas putida KT2440 Engineered Prodigiosin 94 mg L−1 [36]
Zooshikella sp. Wild-type Prodigiosin ND [24]
Streptomyces sp. Wild-type Prodigiosin ND [24]
Serratia rubidaea RAM_Alex Wild-type Prodigiosin 1.6 g L−1 [121]
Pseudomonas putida KT2440 Engineered with 14 derivatives of prodigiosin; prodiginine 1d 10.5 mg L−1 [39]
mutasynthesis
Beneckea gazogenes Wild-type Cyclic prodigiosin ND [14]
Streptoverticillium sp. Wild-type Undecyl-prodigiosin ND [14]
Actinomadura sp. Wild-type Undecyl-prodigiosin ND [14]
Saccharopolyspora sp. Wild-type Undecyl-prodigiosin ND [14]
Streptomyces longisporus ruber Wild-type Metacycloprodigiosin ND [14]
Actinomadura madurae Wild-type Methyl-cyclo-octyl-prodigiosin ND [14]
Actinomadura madurae Wild-type Ethyl-cyclo-nonyl-prodigiosin ND [14]
Streptomyces coelicolor A3 Wild-type Streptorubin B ND [14]
Serratia marcescens JNB5-1 Wild-type Prodigiosin 6.53 g L−1 [43]
Serratia marcescens SK4-72 Engineered Prodigiosin 15.63 g L−1 [43]
Vibrio spartinae Wild-type Cycloprodigiosin and analogues ND [19]

ND not defined

visible spectrophotometer is found to vary between 460 and contents were quantified by obtaining a correlation equation
540 nm which varies based upon the solvent pH [15, 21]. For for absorbance and concentration of the standard pigment.
instance, the prodigiosin remains red in color at pH 7.0 with The FTIR analysis of prodiginine pigments in the frequen-
absorption maxima at 533 nm, whereas at pH 2.0 (pink color) cy range of 4000–400 cm−1 shows maximum absorption at
and 9.0 (orange color), it shows absorption maxima at 540 nm 3.375 m−1 , 2.951 m−1, 1.646 m−1, and 1.556 m−1 for the
and 468 nm respectively [22]. The shift in the absorption prodigiosin pigment in potassium bromide obtained from
maxima under alkaline pH is attributed to the deprotonation Serratia marcescens (10.1007/s12010-014-0921-3). This cor-
of N atoms in pyrrole rings by the alkaline solution [15]. responds to the specific bonds O–H bond, C–H and C=O
Similarly, the undecylprodigiosin and cycloprodigiosin are bonds, N–H bonds and phenyl rings, aromatic C=C, and
found to have absorption maxima at 530 nm and 533 nm NO2 respectively (10.1007/s12010-014-0921-3). The red pig-
respectively in the methanolic extract [12]. The novel pigment ments exhibit similar vibrational energy levels when used with
iso-heptyl-prodigiosin isolated from Vibrio spartinae 3.6 CHCl3. A detailed spectrum captured through Raman spec-
showed absorption maxima at 537 nm [19]. Thus, the crude troscopy and FTIR has been detailed in Jehlicka et al. (2016)
red pigment comprising cyclic or linear derivatives show max- [23]. Another study exhibited a strong absorption at 3.386
imum absorption at 530–540 nm [15]. Therefore, the presence m−1, 2.975 m−1, 1.647 m−1, and 1.087 m−1 for the prodiginine
of prodiginine pigments are confirmed by obtaining the absor- pigments obtained from Zooshikella sp. S2.1 with significant-
bance spectra in the visible wavelength range, and the ly less absorption in the frequency range 2.5 to 1.7 m–1 [24]. In
Biomass Conv. Bioref.

case of the red pigment 2-methyl-3hexyl-prodiginine obtained position of C2, C12, C11, C18, C21, and C22 respectively at
from Streptomyces sp. BSE6.1, broad absorption spectra was the prodigiosin structure [26].
obtained at 3.413 mv1, 2.976 m−1, 1.646 m−1, and 1.086 m−1
[24]. Thus the signature aromatic carbon bonds and amine 2.2 Synthesis of prodigiosin
bonds of prodiginine are known with the absorption at ~ 2.9
m−1 and ~ 1.64 m−1 when analyzed with potassium bromide The biosynthesis of the secondary metabolite prodigiosin fol-
[24, 25]. lows a tortuous pathway involving a bifurcated pathway
Analyses of the prodiginine pigments are usually carried formed by the association of the enzyme-catalyzed condensa-
out using high-performance liquid chromatography, which tion of 4-methoxy-2-2′-bipyrrole-5-carbaldehyde (MBC) with
can provide both the qualitative and quantitative analysis in a mono pyrrole ring. This MBC further reacts with MPP (2-
the presence of mass and UV-visible spectrophotometer as methyl-3-pentylpyrrole) for the fructification of prodigiosin.
detectors. For instance, characterization of prodiginine obtain- The gene clusters involved in the synthesis of prodigiosin vary
ed from Pseudoalteromonas rubra in HPLC fitted with C8 significantly based on the cellular factory involved. For in-
column as solid phase with different ratios of methanol and stance, there is a pig and red cluster which plays a key role
formic acid (1% v/v in water) as mobile phase at a flow rate of in the biosynthesis of prodiginines in Serratia sp., and in
1 mL min−1 [15]. Elutes were analyzed in a photo-diode array Streptomyces sp. respectively [13, 27]. Other than that,
detector and in a mass spectrophotometer. The study showed a Hahella chejuensi contains a hap cluster [28],
variation in retention time of 1.66 to 3.69 min in which over Pseudoalteromonas tunicate possesses a tambajamine cluster
six different prodiginine pigments (2-methyl-3-propyl- named as a tam cluster [29], and the prodigiosin cluster of the
prodiginine, 2-methyl-3-butyl-prodiginine, cycloprodigiosin, rosephilin producer Streptomyces griseoviridis is termed as
prodigiosin, 2-methyl-3-hexyl-prodiginine, and 2-methyl-3- rph cluster [30]. All the groups of clusters have a specific set
heptyl-prodiginine) are eluted and shown to have different of genes that are homologous to each of the enzymes which
absorption maxima ranging from 531 to 537. The mass spec- are all embroiled in the development of MBC in the Serratia
trophotometer analysis showed a maximum m/z ratio of 352.5 marcescens, because of the common route of the biosynthesis
for 2-methyl-3-heptyl-prodiginine, followed by 338.5 for 2- of MBC. Absence of a pig N/red F homolog in the two clus-
methyl-3-hexyl-prodiginine, 324.4 for prodigiosin, 322.4 for ters rph and tam groups of Pseudoalteromonas tunicate and
cycloprodigiosin, 310.2 for 2-methyl;-3-butyl-prodiginine, Streptomyces griseoviridis is an exception. A mixture of
and 296.1 for 2-methyl-3-propyl-prodiginine [15]. Another prodigiosin and norprodigiosin has been reported to be made
study conducted in HPLC with mass detectors exhibited a by pigN mutants of Serratia sp. ATCC 39006, which estab-
retention time of 8.09 min with an m/z value of 324 for lishes that Pig N is not important, but it may catalyze Pig F and
prodigiosin and 9.72 min retention time with m/z value of the methylation of 4-hydroxyl-2,2′-bipyrrole-5-carbaldehyde
338 for 2-methyl-3-hexyl-prodiginine for pigments isolated (HBC) [31]. P. tunicata and S. griseovirdis both contain pig F
from Zooshikella sp., S2.1 and Streptomyces sp. BSE6.1 re- homologs, tam P and rph I. From there, it was found that these
spectively [24]. The study also showed variations in the reten- enzymes are capable of the methylation of MBC in these two
tion time when the analysis was conducted using a gas chro- pathways.
matography system equipped with a mass spectrophotometer The production of MBC has been characterized in Serratia
and a HP-5MS column [24]. Metabolomics study conducted sp. and Streptomyces sp. through a set of analysis which in-
in the novel marine strain Vibrio spartinae 3.6 used LC- cludes homology detection among enzymes, repressing the
HRMS (Liquid Chromatography-High-Resolution Mass genes to identify its function, analysis of intermediate metab-
Spectrometry)-based analysis for the identification of the olites, and complementation experiments [31–33]. From Fig.
prodigiosin pigment and their derivatives [19]. The chromato- 1, it was found that, for the biosynthesis of MBC, there was a
gram identified major peaks at m/z value of 322.1914 and common route that required proline, acetate/malonate, methi-
324.207 1 w hich corresponds to prod igiosin and onine, and serine [31, 33]. The amino acid proline was added
cycloprodigiosin, whereas other minor peaks at m/z values for the formation of pyrrole which is a very common mecha-
of 296.17, 336.20, 338.22, 350.22, and 352.23 remained un- nism like other pyrrole-containing compounds, for example,
identifiable with LC-HRMS analysis [19]. The structural elu- novobiocin, chlorobiocin, coumermycin A1, and pyoluteorin
cidation and the presence of functional groups in the red pig- [32]. For the synthesis of prodigiosin, proline, an amino acid,
ments are usually performed using NMR analysis based on the could reside inside the pyrrolic ring A in a sequence of reac-
proton 1H and 13C. tions catalyzed by enzymes coded by the genes pig A, pig G,
1
H-NMR spectrum of prodigiosin shows the various peaks and pig I and their homologs [32]. In the upcoming steps, a C2
which corresponds the chemical shifts. The chemical shifts are unit from the compound malonyl CoA and a C2N unit from
found at 7.23 ppm, 6.95 ppm, 4.01 ppm, 2.17 ppm, 1.28 ppm, amino acid serine has been incorporated which was catalyzed
and 0.87 ppm which are assigned for the carbon atoms at the by enzyme coded by pig J and pig H. These will form HBM
 Biomass Conv. Bioref.

(4-hydroxy-2,2′-bipyrrole-5-methanol) [31, 33]. The conclud- and (ii) design a novel cellular factory with heterologous ex-
ing step is the biosynthesis of MBC which could be catalyzed pression of the gene clusters involved in prodiginine biosyn-
by Pig M. It involves the oxidation reaction of an alcohol thesis in a safer organism [36, 37]. Among them, the heterol-
group of HBM which forms HBC (4-hydroxyl-2,2′- ogous expression of genes in a non-opportunistic pathogen
bipyrrole-5 carbaldehyde). This reaction is indicated in Fig. such as Pseudomonas putida remains attractive due to sim-
1. HBC then will convert to MBC by methylation [31]. The plicity, well-established and easily available protocols, and
final methylation step was catalyzed by homologs on Pig F. In safety unlike Serratia marcescens which is an opportunistic
some prodiginines precursors, the Pig F could be progressed pathogen [36]. Heterologous expression of the hap gene clus-
by homologs of Pig N. ter involved in prodigiosin biosynthesis obtained from
Two species namely Streptomyces coelicolor and Serratia Hahella chejuensis in E. coli was demonstrated by Kwon
sp. are capable of synthesizing MBC with a different et al. [38]. The study addressed the complex gene regulations
monopyrrole, which requires different enzymes, substrates, that are involved in the expression of hap gene clusters in
and complicated pathways which is also indicated in Fig. 1 E. coli. For instance, the heterologous expression of the hap
[13, 31, 34]. Pig D, Pig E, and Pig B catalyses the biosynthesis gene cluster did not result in the production of prodigiosin
of MPP which is described in Fig. 1. Yet, the specific sub- until the broth from the culture of Hahella chejuensis is added
strates required for MPP biosynthesis are not established. The to the exogenous media [38]. The study revealed the existence
intermediate formed during biosynthesis is known as H2MPP. of two component regulatory system and non-coding region in
It has been observed to concentrate in a delta pig B mutant. So the gene cluster as responsible regulatory elements for
pig B was found to induce the oxidation of H2MPP to the final prodigiosin biosynthesis in Hahella chejuensis and in the re-
product MPP [31]. combinant E. coli system [38]. Domrose et al. (2015) [36]
Therefore, in the biosynthesis of prodigiosin and their sim- established a protocol for heterologous expression of pig gene
ilar compounds, MBC is produced with a monopyrrole MPP cluster genes from Serratia marcescens in Pseudomonas
[31]. This is an exclusive condensation reaction catalyzed by a putida KT2440, generally recognized as a safe strain. The
new family of pyrrole-condensing enzymes and identified by pig gene clusters were integrated with in the chromosome of
Pig C. All the compounds related to prodiginines contain the Pseudomonas putida strain under the control of strong native
Pig C homolog. Again Pig C homologs contain other com- promoter from the host that works based on T7 RNA
pounds such as Red H (undecylprodigiosin), Hap C polymerase-dependent gene expression system. The study re-
(prodigiosin), Rph H (prodigiosin R1), Tam Q (tambjamine ported a maximum prodigiosin titer of 94 mg L−1 with better
YPI), and also a fusion enzyme Pig BC from stability over generations [36]. Another study demonstrated
Janthinobacterium lividum. But the explicit nature of the com- the mutasynthesis strategy to obtain over 13 various structural
pound produced by J. lividum is unrecognized until date. The derivatives of prodigiosin in a recombinant Pseudomonas
combined enzyme Pig BC has been established only as of the putida KT2440 strain [39]. Mutasynthesis is a strategy which
example of a multifunctional enzyme which catalyzes both the involves the feeding of mutasynthon, a synthetic intermediate
terminal condensation reaction and synthesis of monopyrrole or precursor analog in the exogenous media of the recombi-
[35]. Therefore, if more Pig C–like compounds are discov- nant strain leading towards production of synthetic derivatives
ered, more site-directed mutagenesis, sequence alignments, from a biological producer [39]. The study created a mutant
and last of all more crystallography may be done to know strain of P. putida KT2440 deficit for the first enzyme in-
the critically involved residues which are conserved generally. volved in the monopyrrole synthesis pathway and subsequent
All the cyclic derivatives of prodiginines contain one Red G supplementation of the strain with 2-methyl-3-amylpyrrole,
homolog. Each steps discussed above has been portrayed in and its derivatives in the exogenous media generated
the best possible way in Fig. 1. prodiginine pigments effectively without loss in the bioactiv-
ity [39]. Similarly, cycloprodigiosin and derivatives were syn-
thesized using mutasynthesis strategy from P. putida KT2440
3 Recombinant strains for prodigiosin [40]. Another study evaluated the IPTG (isopropyl-β-D-
production thiogalactoside) inducible expression of Pig C enzyme in a
recombinant E. coli DE3 and optimized the media composi-
Even though several proteobacterium are available with intrin- tion which resulted in maximum enzyme activity of 179.3 U/
sic prodiginine production capability, attention on designing a mL [41]. A recent study reported the metabolic engineering
recombinant cellular factory for efficient synthesis of approach to enhance the production levels of the prodiginine
prodiginine has increased attributed to increased complexity pigment from Streptomyces coelicolor M145 [42]. The study
in purification, low titers, and reduced yields. Therefore, the revealed the involvement of a gene ohkA coding for the en-
current strategies target to (i) improve the signature strain of zyme orphan histidine kinase which repressed the prodiginine
Serratia marcescens for increased productivity and yields, production (undecylprodigiosin and streptorubin B).
Biomass Conv. Bioref.

Fig. 1 Biosynthetic pathway of prodigiosin and its derivative

 Biomass Conv. Bioref.

Repression of the ohkA gene resulted in a recombinant strain urea) ceases the production of prodigiosin. The underly-
synthesized two-folds higher prodiginine pigments as com- ing mechanism may be believed as the poor electron-
pared with the wild-type strain (7.6 mg g−1 DCW) with no donating capability of nitrogen which results in the pro-
mutation. Furthermore, knockout of act, cda gene clusters duction of some intermediate substances, which are toxic
involved in the actinorohodin biosynthesis and calcium- to the microorganisms [46, 48]. Therefore researchers are
dependent antibiotic synthesis with simultaneous overexpres- in constant exploration for a new and economical organic
sion of red gene clusters responsible for prodiginine pigment nitrogen source. Few literature reports revealed that pep-
production resulted in over 12-fold increment with 96.8 mg tone could be an excellent alternative in this particular
g −1 DCW as compared with the wild-type strain [42]. regard as they contain a wide spectrum of amino acids.
Similarly, the prodigiosin production in the signature strain For instance, screening of over eight different organic
Serratia marcescens has been reported to be negatively regu- nitrogen sources such as peanut powder, soya peptone,
lated by the D-Ala-D-Ala carboxypeptidase encoded by the yeast extract, beef extract, fish meal, corn steep liquor,
BVG90_02415 gene [43]. Selective repression of the enzyme tryptone, and soya powder at 10 g L −1 concentration
activity through knockout mutation resulted in 1.46 increment showed the highest prodigiosin titer of 3.76 g L−1 in pea-
in prodigiosin titer as compared with that of the wild-type nut powder followed by soya peptone with 1.77 g L−1 and
strain. Detailed analysis of the mutant strain showed a signif- beef extract with 1.69 g L−1 [49]. Furthermore, study on
icant leakage of prodigiosin resulting in augmented expres- the combination of beef extract and peanut powder as dual
sion levels in genes under pig cluster [43]. Thus, development nitrogen sources enhanced the prodigiosin titer up to ~
of a novel producer strain with high titer and prodiginine pig- 35% with 5.06 g L −1 of prodigiosin. The study also
ment yield is still awaited. showed reduced or no synthesis of prodigiosin in the pres-
ence of corn steep liquor, soya powder, and fish meal
while sub-optimal production of prodigiosin was evi-
4 Factors modulate prodigiosin production denced with tryptone and yeast extract used as sole nitro-
gen sources [49]. In many studies, casein has been used as
Prodigiosin is a secondary metabolite that is generally the nitrogen source for the growth of Serratia marcescens
formed as a secondary metabolite in the sub-sequential and associated with prodigiosin production. Screening of
stages of bacterial advancement. The production of tryptone, peptone, ammonium nitrate, ammonium sulfate,
prodigiosin depends upon many parameters, such as tem- and urea on the growth and production of prodigiosin
perature, pH, environmental factors, inorganic phosphate from Serratia marcescens showed peptone as the best
availability, the composition of media, and other factors source in combination with glycerol yielding 610 units
[31]. The literature revealed that prodigiosin could also be per cell of prodigiosin pigment [50]. For the strain
found from economic and environmentally benign ante- Serratia nematodiphila RL2, yeast extract was found to
cedents such as vegetable oils [44–46], ethanol, cassava be the optimal nitrogen source supporting growth and
wastewater [46], corn steep liquor [11], and squid pen prodigiosin production [50]. Thus, a strain-dependent var-
powder [47]. A lot of differentials and selective media iation in the production titer based on nitrogen source is
options have been designed for the production of evidenced. In addition to that, addition of amino acids to
prodigiosin. Few important media components that have the media especially L -proline and glycine has been
been found pragmatic in the production of prodigiosin, known to improve the prodigiosin production in many
among which nutrient broth (0.52 mg mL−1) [44] and strains as enlisted in Table 3. The amino acid proline is
peptone glycerol broth (0.302 mg mL−1), are eminent. a direct precursor molecule that incorporates the backbone
An experimental study portrayed admirable growth of required for MBC involved in prodigiosin biosynthesis.
Serratia marcescens that was carried out by using the Thus, addition of proline amino acid is expected to in-
powdered seed of sesame in water, peptone glycerol crease the prodigiosin titer in these organisms.
broth, and nutrient broth [44]. Compared with other eco- Carbon sources play a key role in determining the
nomic sources, namely sesame oil, peanut oil, and coco- prodigiosin production in these strains. Screening of different
nut oil, and sesame seed portrayed a better yield of carbon sources such as dextrin, sucrose, lactose, maltitol,
prodigiosin in nutrient broth at 28–30 °C. Powdered pea- starch, glucose, citrate, and glycerol for the growth of
nut broth at 37 °C portrayed an excellent outcome in Serratia marcescens showed glucose as the best carbon source
terms of prodigiosin production, which is very much com- with 38% improvement in prodigiosin titer as compared with
mensurable with the pigment that has been produced in that of the control conditions [49]. Another study showed
nutrient broth at 30 °C [44]. Literature reported that the glycerol as the optimal carbon source with maximum
presence of inorganic nitrogen sources (e.g., ammonium prodigiosin titer of 560 units per cell among the various other
salts like (NH 4) 2SO 4, NH 4 Cl, and NH 4NO 3 , and also carbon sources screened such as cotton seed cake, black seed
Biomass Conv. Bioref.

Table 3 Organisms synthesizing Prodigiosin under different growth conditions

Organism Prodigiosin titer (mg Growth conditions Reference

L−1)

Serratia nematodiphila YO1 7.80 Luria Bertani (LB) broth [21]

Hahella chuejuensis KCTC2396 28.10 Marine broth 2216 [116]
Zooshikella rubidus S1-1 47.80 Marine broth 2216 [116]
Serratia nematodiphila YO1 89.10 Peanut oil 3% + LB broth [21]
Serratia nematodiphila YO1 89.50 Olive oil 3% + LB broth [21]
Serratia nematodiphila YO1 93 Palm oil 3% + LB broth [21]
Serratia marcescens BMJ 816 and AMJ 103 Starch 1.60%, 1% trace compounds, 1.60% peptone [37]
817
Serratia marcescens MO-1 278 Yeast extract 0.40%, D-mannitol 1.00%, 0.40% ram horn peptone [122]
Serratia marcescens FZSF02 353 N2 Source: tryptone 10 g L−1 [49]
Serratia marcescens FZSF02 380 N2 Source: yeast extract 10 g L−1 [49]
Serratia nematodiphila RL 2 760 Nutrient broth medium supplemented with 1.00% lactose and 1% yeast [123]
extract
Serratia marcescens 870 Kitchen wastewater supplemented with peptone and proline [124]
Hahella chejuensisKCTC2396 1500 Sucrose 10 g L−1, peptone 4 g L−1, yeast extract 1 g L−1 [116]
Serratia marcescens FZSF02 1508 N2 source: beef extract 10 g L−1 [49]
Serratia marcescens FZSF02 1589 N2 source: soya peptone and peanut powder 10 g L−1 [49]
Serratia marcescens FZSF02 1601 Carbon source: lactose 5 g L−1 [49]
Serratia marcescens FZSF02 1675 Carbon source: glycerol 5 g L−1 [49]
Serratia marcescens FZSF02 1744 Carbon source: sucrose 5 g L−1 [49]
Serratia marcescens FZSF02 1774 N2 source: 10 g L−1 soya peptone [49]
Serratia marcescens FZSF02 1939 Carbon source: starch 5 g L−1 [49]
Serratia marcescens FZSF02 2040 Carbon source: dextrin 5 g L−1 [49]
Serratia marcescens FZSF02 2117 Carbon source: maltitol 5 g L−1 [49]
Serratia marcescens FZSF02 2397 Carbon source: glucose 5 g L−1 [49]
Serratia marcescens TKU011 2480 N2 source: squid pen powder 1.50% [125]
Serratia marcescens SS-1 2500 N2 source: yeast extract 5 g L−1, proline 10 g L−1 [126]
Serratia marcescens FZSF02 3762 N2 source: peanut powder 10 g L−1 [49]
Serratia marcescens TKU011 4620 pH 5.65–6.15; 1% alpha chitin, 0.6% casein [52]
Serratia marcescens FZSF02 5062 N2 source: 10 g L−1 beef extract and peanut powder [49]
Serratia marcescens JNB5-1 6350 Sucrose 2%, beef extract 1.50%, L-proline 0.75% [43]
Serratia marcescens JX 1 6500 Peptone 16 g L−1, glycerol 20 g L−1, glycine 2 g L−1 [127]
Serratia marcescens SK4-72 15630 Sucrose 2%, beef extract 1.50%, L-proline 0.75% [43]
Serratia marcescens 38750 Powdered peanut broth [44]
Streptomyces sp. NRCF69 47000 20% dairy processing water and mannitol with salts [128]
Serratia marcescens UCP 1549 49500 6.00% cassava wastewater and 2% mannitol [11]
Serratia marcescens FZSF02 0 Carbon source: citrate 5 g L−1 [49]
Serratia marcescens SMΔR Yeast extract 5 g L-1, proline 10.10 g L−1, 6% sunflower oil [129]

cake, and soyabean cake seed [50]. Similarly, for the strain nitrogen, salts, and other process conditions improved the titer
Serratia marcescens B6, glycerol was found as an optimum to a maximum of 4.62 g L−1 [52]. The optimal pH for the
carbon source for maximum prodigiosin production [51]. maximum growth and prodigiosin production was found to
Another study used alpha chitin obtained from shrimp cells be in the range 5.5 to 7 for Serratia sp. as evidenced from
as carbon source along with casein as nitrogen source for the various studies [52]. Temperature is also an important param-
prodigiosin production from Serratia marcescens which eter that influences the production of prodigiosin. For in-
showed maximum 3.35 g L−1 of prodigiosin [52]. Further stance, when the temperature was increased from 30 to 37
optimization of media compositions including the carbon, °C, a reduction in prodigiosin operon expression levels was
 Biomass Conv. Bioref.

evidenced in Serratia marcescens [53]. This confirms the ex-

istence of temperature-controlled operon system for
prodigiosin biosynthesis in this species showing involvement
of HexS transcription factor and catabolite repressor protein
[53].
Being a secondary metabolite, the prodigiosin pigment can
be induced at high levels by exposing high cell density culture
to certain stimulators or modulators. For instance, addition of
fatty acids or lipids to the exogenous media has shown to
increase the prodigiosin biosynthesis in these strains. Several
types of oil were used for the production of prodigiosin, such
as soybean oil, canola oil, olive oil, maize oil, peanut oil, and
tea oil. Among them, olive oil has gained significant attention
attributed to its high prodigiosin production levels. Screening
of different oil types on the growth and prodigiosin levels
showed that the initial addition of palm oil, olive oil, and
peanut oil resulted in a lengthier lag phase as compared with
the control condition without any oil in Serratia
nematodiphila. However, this resulted in over ~ 11-fold incre-
ment in prodigiosin concentration in the cells [21]. This study
also confirmed that optimal production levels and yield were
induced by palm oil [21]. Another study showed that the ini-
tial addition of olive oil to exogenous media resulted in
growth suppression however resulting in ~ 2.24-fold incre-
ment in prodigiosin levels (11.36 g L−1). The study claimed
that the time of oil addition may influence the prodigiosin
production levels [49]. Therefore, the olive oil was added into
exogenous media after allowing the organism to grow in the
media for 24 h which resulted in the highest production of
prodigiosin (15.42 g L−1), thus confirming the influence of
oil addition time on prodigiosin production levels [49]. On
the other hand, a strong association between the growth,
ATP levels, and prodigiosin pigment was reported in
Serratia spp. [54]. Activation of prodigiosin operon transcrip-
tion with reduced cellular ATP levels in high cell density
cultivation confirms the claim [54]. Cultivation at a constant
growth rate in a chemostat revealed the negative influence of
cellular ATP levels with increased prodigiosin production
[55]. The Prodigiosin pigment production is also generally
regulated by the quorum sensing mechanisms. Further studies
on the regulatory mechanisms involved in the production of Fig. 2 Steps involved in downstream processing of prodigiosin
prodigiosin from these organisms are still awaited.

5 Downstream of prodigiosin biomass. During extraction, prodigiosin transfers to organic

phase. At 45 to 50 °C the extract of ethanol is evaporated for
After fermentation, the cells are separated using centrifuga- dryness, and then the residue so obtained is considered
tion. Since, prodigiosin is water insoluble, therefore, organic prodigiosin. The pigment thus obtained was dried in a vacuum
solvent-mediated extraction procedure is usually used for sep- dryer and was resuspended in ethanol. Liquid chromatogra-
aration of prodiogiosin. Figure 2 illustrates the protocol used phy (using glass column and silica gel) is an exquisite choice
for the separation of prodigiosin from cell surface. Here, acid- which can be taken to quantify the amount of obtained
ified ethanol (95%) having pH of 3 serves as the solvent, prodigiosin. Ethanol-suspended pigment obtained from the
which may cause active discoloration of the microbial terminating step of extraction course was poured onto the
Biomass Conv. Bioref.

sorbent of the column chromatography. Hexane-acetone mix- the oxidative stress [63]. Similarly, the anti-oxidant ca-
ture in the ratio of 1:3 followed by acetone can be used to elute pacity up to 62.51% was reported against the mice liver
out the desired pigment from the column. The adjoining step lipid peroxidation and bovine serum albumin protein
of purification associates adoption of thin-layer chromatogra- oxidation by prodigiosin isolated from Streptomyces sp.
phy (TLC) on the glass silica gel 60 F254 plates using hexane- WMA-LM31 [64]. Addition of undecylprodigiosin is
ethyl ether-acetic acid (70:30:1). The corresponding bands of known to inhibit the formation of reactive oxygen spe-
the desired pigment were detected and isolated. The identified cies and simultaneously augment the activity of anti-
pigment was then disengaged from the silica gel using the oxidant enzymes when exposed to UV/gamma irradia-
following solvents on a successive array: 96% ethanol, ace- tions [65]. The studies also show that the anti-oxidant
tone, and chloroform [56]. activity of prodigiosin is very similar to that of ascorbic
For most of the extraction process, polar or non-polar or- acid [62, 63].
ganic solvents are used for extraction of prodigiosin [11, 47]. Studies on the toxicity effects of prodigiosin had varied
Organic solvent–mediated extraction is disadvantageous as results based on the organic solvent medium in which the
most of organic solvents are toxicorganic, and therefore, the pigment is solubilized and on the concentrations. For instance,
process of extraction is not environment friendly [57]. Since evaluation of the prodigiosin solubilized in different solvents
organic solvents are usually expensive and require higher such as methanol, ethanol, acetone, chloroform, and petro-
amount of energy for extraction of prodigiosin, therefore, re- leum ether on chick embryogenesis showed prodigiosin dis-
searchers are looking for environment-friendly low-cost ex- solved in chloroform as the most toxigenic [66]. Toxicological
traction process. Several experiments were carried out by var- assessment of prodigiosin obtained from S. marcescens in a
ious researchers using certain polymers such as chitin and eukaryotic system Caenorhabditis elegans showed no signif-
resin materials for the separation of prodiosin from fermenta- icant toxic activity [67] which exhibited the safe use of
tion broth [58, 59]. prodigiosin and their derivatives as therapeutic agents in eu-
karyotic system.

6 Bioactivity of prodigiosin 6.1 Anti-protozoan and anti-larval activity

Increased attention on the red pigments and their derivatives is The anti-malarial equity of both natural as well as synthetic
due to their inherent bioactivity against several strains of bac- prodiginines has been quantified with the help of chemometric
teria, algae, larva, and parasites [24, 60]. They are also well- descriptors. To spell out further the anti-malarial activity
known for their higher immunomodulating capability and cy- against Plasmodium falciparum D6 strain (involved in cere-
totoxicity against cancerous cells [61]. Being a pigment, the bral malaria and resistant to chloroquinone), researchers can
prime activity expected from these molecules is their anti- use the statistically corroborated quantitative structure-activity
oxidant capability. For instance, prodigiosin obtained from relationship models. A wide array of chemometric 2-D de-
Serratia marcescens exhibited anti-oxidant activity of ~ 25– scriptors has been contrived via combinatorial protocol with
30% even at very low concentrations of 2 ppm in both DPPH- the help of multiple linear regression (CP-MLR) analysis
and ABTS-based assay [62]. The maximum oxygen- methodology. Literature reports that there are 53 fabricated
scavenging capacity up to 99% was shown with 10 ppm and prodiginines commercially accessible for in vitro anti-
8 ppm of prodigiosin against ABTS and DPPH respectively malarial activity against Plasmodium falciparum pan-
[62]. The anti-oxidant capability of prodigiosin against the sensitive D6 which can be compared with that of chloroquine
microcystin-LR toxicity was evaluated in a hepatic carcinoma [1]. For instance, the metacycloprodigiosin showed relatively
cell line termed as HepG2 [63]. In general, addition of less IC50 values of 1.7 nM against the malarial parasite and
microcystin-LR to hepatic cells results in the generation of found to be the best among the various derivatives of
reactive oxygen species. The study showed significant reduc- prodiginine pigments [68]. These pigments have also shown
tion in the levels of reactive oxygen species in HepG2 cells better activity in the cases with chloroquinone-resistant strains
reated with microcystin-LR in the presence of of Plasmodium falciparum. Similarly, the prodigiosin derived
prodigiosin as compared with that of the control with from Serratia marcescens has shown activity against
no prodigiosin thereby proving the anti-oxidant capabil- P. falciparum with IC50 value of 1.1 μg mL−1 which was ~
ity of prodigiosin. Furthermore, an IC 50 value of 3-fold lesser in the presence of nanoparticles as compared to
1.124 μM was proposed by the study conducted on the control with no additional nanoparticle intervention [69].
the determination of anti-oxidant activity [63]. The The undecylprodigiosin and streptorubin B derived from
study also depicted the activation of signaling pathway Streptomyces coelicolor have shown IC50 values of 7.7 nM
related to nuclear factor erythroid-2-related factor 2 and 7.8 nM respectively when tested against P. falciparum
(keap1/Nrf2) which in turn activates the defense against [68]. The recent studies target to design novel synthetic
 Biomass Conv. Bioref.

analogues of prodigiosin with enhanced activity reduced IC50 inhibitory concentration of 125 mg mL−1 [78]. In certain
values and increased stability with less cytotoxicity. In line cases, these pigments are also known to have algicidal activ-
with this, three prodigiosin analogs designed with modifica- ity. For example, the growth of a harmful alga Phaeocystis
tions in the C ring showed varied IC50 values of 4.6 nM, 1.7 globola was inhibited by the prodigiosin pigment obtained
nM, and 0.9 nM. The lowest IC50 value was obtained for the from Hahella sp. KA22 by increasing the reactive oxygen
prodigiosin analog with a six-carbon alkyl replaced at α- species concentration within the cells [79]. The red pigment
position and aryl incorporation at β-position of the C ring has also been reported to show sluggish activity against cer-
[70]. Similarly [71], Marchal et al. identified that the presence tain bacterial pathogens (constitutes both gram-positive as
of nitrogen in the A ring is necessary for maximal activity of well as gram-negative) and fungal pathogens namely
prodigiosin against the protozoa. The study also showed a Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa,
prodigiosin analog with modification in A ring and C ring Candida albicans, Trichoderma koningi, and Penicillium
which resulted in the IC50 value of 5.6 μM [71]. These pig- notatum [80]. The intracellular anti-bacterial red pigment ob-
ments have also found to be active against the different pro- tained from Serratia marcescens IBRL USM 84 showed in-
tozoans such as Entamoeba histolytica, Schitosoma, creased anti-bacterial activity against Gram-positive bacteria
Trypanosoma cruzi, and Trypanosoma brucei [70]. The (Staphylococcus aureus, Staphylococcus saprophyticus,
mechanism involved in the inhibition of Trypanosoma strains Bacillus subtilus, Enterococcus avium, and Streptococcus
was found to be the inactivation of oxidative phosphorylation pyogenes) as compared with gram-negative (Escherichia coli,
mechanism that occurs in the mitochondria thereby resulting Pseudomonas aeruginosa, Aeromonas hydrophila, Proteus
in ~ 80% reduction in respiration and associated energy bio- mirabilis, and Klebsiella pneumonia) bacterial pathogens
synthesis (10.1186/1756-3305-4-66). In addition, the study [81]. Another study evaluated the potentials of prodigiosin
also proposed prodigiosin as one of the broad-spectrum anti- pigment as an anti-microbial agent against a set of bacteria
parasitic drug such as anisomycin, obatolclax, and nithiamide and fungi which includes Bacillus sp., Salmonella sp.,
[72]. Pseudomonas sp., E. coli, Staphylococcus aureus, Candida
The prodigiosin pigment has shown activity against larva sp., Mucor sp., Aspergillus sp., and Penicillium sp. [60].
in different growth stages and to nematodes [73, 74]. Anti- This study also reported maximum activity of prodigiosin
larval activity against Aedes and Anopheles mosquito by against gram-positive bacteria as compared with that of
prodigiosin obtained from Serratia marcescens NMCC46 gram-negative bacteria with only a marginal effect against
was evidenced with over 50% mortality of Aedes and the fungal pathogens [60]. Activated carbon functionalized
Anopheles was observed at 41.65 mg L−1 and 51.12 mg L−1 with iron-oxide and conjugated with prodigiosin showed
for the 2nd instars respectively [73]. The anti-larval activity of highest anti-bacterial activity against the antibiotic-resistant
prodigiosin (100 mg L−1) was evaluated against Aedes aegypti bacterial strains with minimum inhibition concentration of 4
mosquito which showed mortality of about 76% after 48 h of and 5 mg mL−1 for E. coli and B. cereus respectively [82]. The
incubation [60]. Similarly, the LC50 value of 14 to 27 μg mL−1 red pigment prodigiosin was also established to be a right
was obtained for prodigiosin against pupal stage and the 2nd, curing agent on plasmids of E. coli HB 101 and S. aureus.
3rd, and 4th instars of Aedes, whereas it was 19.7 to 32.2 μg But it also failed in some cases such as to cure the plasmid of
mL−1 for Anopheles stephensi [75]. some bacteria such as Proteus mirabilis and Enterococcus
avium [83]. The ability of prodigiosin to go through the
6.2 Anti-microbial activity outer membrane and inhibiting target enzymes such as
DNA gyrase and topoisomerase IV, which inhibited cell
Prodigiosin and associated derivatives are renowned for its growth, was the proof of anti-bacterial activity of
anti-bacterial and anti-fungal activity [10]. For instance, 2-p- prodigiosin [84]. Assessment of the anti-bacterial activity
hydroxybenzyl-prodigiosin derived from Pseudoalteromonas of prodigiosin on different food pathogens such as Bacillus
rubra has shown anti-bacterial activity against MRSA (meth- cereus, Staphylococcus aureus, Clostridium botulinum,
icillin-resistant Staphylococcus aureus), wild-type Vibrio vulnificus, Escherichia coli, and Salmonella enterica
Staphylococcus aureus, and E. coli and exhibited anti-fungal showed maximum activity against all the strains when com-
activity against Candida albicans [76]. Similarly, the pared with the results in a positive control with sodium
streptorubin B, derived from Streptomyces sp. MC11024, is nitrite as preservative [62]. The prodigiosin pigment
also known to have anti-bacterial activity against MRSA showed antifungal activity against Fusarium oxysporium,
strain with minimum inhibitory concentration of 32 μg Aspergillus flavus, and Penicillium notatum with minimum
mL −1 [77]. The prodigiosin pigment obtained from a inhibitory concentration of 8, 10, and 21 μg mL−1 respec-
Serratia marcescens strain isolated from soil showed in- tively [25]. Recent studies on the synthetic prodigiosin
creased anti-bacterial activity against the oxacillin-resistant against fungal strains of Batrachochytrium dendrobatidis,
strains of Staphylococcus aureus at a slightly higher minimum and B. salamandrivorans showed minimum inhibitory
Biomass Conv. Bioref.

concentrations of 1.2, and 8.8 μg mL−1 respectively [85]. several multi-drug resistance efflux pumps which can give
Another study showed the anti-fungal activity of cyclic resistance to other anti-cancer agents [92, 93]. Prodiginines
prodigiosin derived from Pseudomonas putida through have been found as proapoptotic anti-cancer agents. It also
mutasynthesis against Saccharomyces cerevisiae and has multiple functions; it can induce cellular stress and
Pichia pastoris with minimum inhibitory concentration of DNA damage, and change intracellular pH; all of these
0.4 and 1.7 μM respectively [40]. It is also interesting to can induce apoptosis. The presence of prodigiosin inside
note that the red pigment has also shown activity against the the cell controls its function, and it was found that it was
replication of herpes simplex virus type 1 and type 2 in present in the nucleus [93], also mainly situated in the cy-
ex vivo conditions [86]. toplasm of the cells [94]. It was also found inside the gran-
Various mechanisms can explain the way through which ules near to the nucleus [95], and it was also present inside
the prodigiosin pigment affects the growth of bacterial and the mitochondrial membrane [96]. This proapoptotic activ-
fungal cells. For instance, during the logarithmic growth phase ity of prodigiosin fully supported the hypothesis of DNA
of Bacillus subtilis, Bacillus mycoides, and Bacillus cleavage as proapoptotic activity. Obatoclax is a compound
licheniformis, the prodigiosin pigment obtained from Vibrio found in the mitochondrial membrane, which plays a major
ruber DSM 14379 caused the leakage of plasma membrane role with the anti-apoptotic compound Bcl-2 family mem-
and activation of autolysins resulting in cell lysis [87]. The bers staying in the mitochondrial membrane [97]. The two
induction of cell lysis was found to be dependent on the main apoptotic pathways assemble as effector caspases. The
growth phase which showed the highest lytic activity in the intrinsic pathways also can be described as the cell death
mid-logarithmic growth phase, whereas in the stationary pathway of mitochondria. It was established that it was
phase, it resulted in a bacteriostatic activity [87]. Detailed found to be activated by some factors such as cellular stress,
molecular dynamics and localization studies showed that the radiation, and withdrawal of growth factor or cytotoxic
prodigiosin crosses the plasma membrane and interacts with drugs. Another pathway named an extrinsic cell death path-
the membrane proteins and the hydrophillic phospholipid bi- way also has been established. Its activity depends upon the
layer thereby resulting membrane disruption [88]. activation of the initiator caspase, then CASPASE-8, next
Furthermore, these pigments are known to cause increased Bid cleavage and activation of C ASPASE-3 and
reactive oxygen species within the cells, leading to fragmen- CASPASE-7 independently of mitochondria. Another
tation of DNA [22]. The study also showed an involvement of two-cell death receptors are Fas and apoptosis-related tu-
caspase-like enzyme in the autolysis of bacterial cells in the mour necrosis factor which induces the ligand receptor
presence of high concentrations of prodigiosin, whereas at low known as TRAIL. These two receptors were directly related
concentrations of prodigiosin, the motility of bacteria is re- to the induction process of the extrinsic cell death pathway.
stricted [22]. Thus, the bactericidal activity is due to mem- Prodigiosin is the major prodiginine which is famous as an
brane leakage and further induction of cell lysis. anti-cancer agent. It intercalates into the double-stranded
DNA, and in the presence of copper, it catalyzes the cleav-
6.3 Anti-cancer activity age of the double-stranded DNA [98, 99]. So these connec-
tions between cytotoxicity and cleavage of double-stranded
Prodigiosin and its related compound were tested in an ex- DNA established a strong hypothesis. It tells that cleavage
periment against more than 60 cancer cell lines. Table 4 of both strand of a double-stranded DNA is crucial for
details the list of various cell lines tested with their probable proapoptotic anti-cancer activity of the prodiginines. Also,
minimum inhibitory concentration. The average inhibitory the prodigiosin has been observed to bind by intercalation,
concentration was determined to be 2.1 μM [89, 90]. mostly at the AT sequences from the minor groove [98].
Evaluation of the cytotoxicity on laryngeal epidermoid car- Malignant cells have 3.5-fold higher concentration of Cu
cinoma (HEp-2), human promyelocytic leukemia (HL-60), (II) than non-malignant cells. So these copper-mediated
lung mucoepidermoid carcinoma (NCIH-292), and breast DNA cleavages of prodigiosin have a major role in the pro-
adenocarcinoma (MCF-7) cell lines was carried out with gression of apoptosis of cancer cell lines. But in non-
prodigiosin obtained from Serratia marcescens UFPEDA malignant cells, this has no activity. The drug prodigiosin
398 strain [91]. The study showed varied results with IC50 and its similar compound create major compounds for the
value of 3.4 μg mL−1 for prodigiosin in NCHI-292, HL-60, rescue deficiencies in the P53 pathway inside the cancer
and Hep-2 cell lines with reduced effects on MCF-7 cell line cells. It is done by upregulation of the P73 pathway and
evidenced from an increased IC50 value of 5.1 μg mL−1 interaction of the mutant P 53 /P 73 inside the cell [100].
[91]. The cytotoxic effects and genotoxicity mechanisms Molecular docking studies were conducted to determine
of prodigiosin is not well-understood attributed to its mul- the anti-cancer activity of the prodigiosin pigment against
tiple applications in the literature. Prodiginines are also the RAF-1 protein (oncogene). The study revealed the interaction
found as a better option because they are not affected by of prodigiosin with the glutamate residue at the 348th position of
 Biomass Conv. Bioref.

Table 4 Natural prodigiosin and its similar compound which has several anti-cancer activities

SL. no. Name of the organ Test cell line Mechanism behind IC50 References

1. Blood B-CLL Apoptosis 0.116 μM

Jurkat TopoI inhibition/p27/p21/cdk2/ 4.48 μM [130]
cyclin-E/apoptosis PKC/p38/MAPK
K562 PKC/PTP1B/PP2A 2.5 μM [130, 131]
U937 PKC/PTP1B/PP2A 0.7 μM
2. Brain IMR-32 ATP production 0.7 ± 0.1 μM [131]
3. Breast MCF-7 JNK/MAPK/RAD51 4.0 μM [132]
4. Colorectal DLD1 c-Jun/ΔNp73/p73/apoptosis; Less than 1.6 μM [133, 134]
lysosomal acidification
SW-620 Apoptosis 0.273 μM [134]
5. Lung A549 PI3K-p85/Akt/mTOR; PKB/SKP2/p27 0.42 μM [135, 136]
H23 PKB/SKP2/p27 0.42 μM [136]
6. Skin SK-MEL-5 mTORC1/2 inhibition 1.02 μM [137]
7. Lung Human lung carcinoma A549 ND 3740 nm [49]
8. Bone Marrow Human bone marrow chronic K562 ND 283 nm [49]
9. Blood Human acute promyelocytic leukemia HL60 ND 79.6 nm [49]
10. Human hepatocellular carcinoma Hep G2 ND 6160 nm [49]
11. Human colorectal carcinoma HCT 116 ND 1399 nm [49]

RAF-1 protein and yielded binding energy of − 5.0 [60]. In and collagen-induced arthritis (which were very common)
another study, the prodigiosin pigment has been found to inhibit was successfully carried out in mouse model [104].
the autophagy of colorectal cancer thereby enabling effective Undecylprodigiosin, m etacyclo prodigiosin, and
caspase-dependent apoptosis under in vivo conditions when cycloprodigiosin all are different forms of prodigiosin-
administered in combination with 5-fluorouracil [101]. The study inhibited proliferation of T cells. Yet, various levels of
showed that the prodigiosin pigment repressed the in vivo toxicity were reported [105].
autophagosome-lysosome fusion and cathepsin-dependent
maturation of lysosomes in the colorectal cancer cells [101]. In
another study, a conjugate of prodigiosin and silver nanoparticles 6.5 Role on cell cycle
was constructed for evaluating their potential in anti-cancer
activity against the human liver cancer cell (HepG2). The study Prodigiosin plays an active role in different inhibition of the
showed increased anti-cancer activity for the prodigiosin-silver cell cycle at different stages (Fig. 3). Prodiginines have
nanoparticle conjugate under in vitro conditions as compared been found to inhibit cell cycle at multiple steps. In
with that of unconjugated prodigiosin molecule [102]. The in different types of structures and within some experimental
silico analysis of the molecular docking between the prodigiosin system, this effect was found [106]. Also, the prodiginines
conjugate and cancer cell line proteins identified the inhibitory have been found to stop the phosphorylation of retinoblas-
activity against the mitogen-activated protein kinase in cancer toma RB protein [107]. It is a vital factor for the progression
cells [102]. of the G1 phase to the S phase inside the cell. Prodigiosin
was found to inhibit P21, cyclin E, CDK 2, P27, and RB
phosphorylation in the leukemic Jurkat cells. These all lead
6.4 Immunosuppressive activity to apoptosis, which means programmed cell death [106].
Prodigiosin also has been found to affect the accumulation
The inhibition of the cell cycle is a unique property of of P53 and induction of NAG-1, specifically in the human
prodigiosin, which is found and established at non-apoptotic breast cancer cell line [108]. But, prodiginines and its
doses, like an immunosuppressant. Prodigiosin has been related compounds have not been found to affect the
found to reduce graft versus host disease (GvHD). For this, expression of another component IL-2, which makes a
no remarkable sign of toxicity was found in mouse models complex with its receptor IL-2R. It is also a very much
[103]. In addition to that, prodigiosin also has another impor- important checkpoint for the proliferation of T cells. Also,
tant effect, which is the delay in the progression of autoim- a big range of effects of these prodiginines has been
mune diabetes. Literature reported that prevention of GvHD reported in different stages of the cell cycle. In the picture
Biomass Conv. Bioref.

Fig. 3 Role of prodigiosin in cell cycle

below, we can find a very good description of the effects of description and the below picture can co-relate the effects
prodiginines inside the cell cycle. The picture is very much of inhibition of the cell cycle, which is a major function of
clear and understandable to all. The connection between the the compound prodiginines.
 Biomass Conv. Bioref.

7 Conclusion Funding information The authors received financial assistantship in the

form of fellowship to conduct doctoral research from the National
Institute of Technology and Ministry of Human Resource
So prodigiosin was found as a significant pigment, which Development, Government of India.
belongs to a family of natural red dye. It was found in various
types of bacteria, but first, it was isolated from Serratia References
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