Exploring the potential of two Pseudomonas species to produce vincristine from vinblastine via biotransformation

Abstract

A biotransformation pair consisting of vinblastine: vincristine present in the Catharanthus roseus plant is of immense pharmacological significance. In this study, we successfully transformed vinblastine into vincristine outside the plant using Pseudomonas aeruginosa 8485 and Pseudomonas fluorescens 2421 and evaluated the antiangiogenic potential of thus produced vincristine through the CAM assay. The toxicity assay showed that both Pseudomonas spp. can tolerate varying concentrations (25–100 µl of 1 mg/ml) of vinblastine. The biotransformation was performed in a liquid nutrient broth medium containing vinblastine (25–100 µl), and Pseudomonas spp. inoculums (50–150 µl) by incubating at 30 °C and 37 °C, respectively for 8 days. The process was optimized for substrate and culture concentrations, pH, temperature, and rotation speed (rpm) for the highest conversion. Analysis using LC–MS/MS confirmed the presence of vincristine as a product of the vinblastine biotransformation by two Pseudomonas spp. P. fluorescens 2421 showed a faster conversion rate with 95% of vinblastine transformed within 24 h than P. aeruginosa 8485, which demonstrated a conversion rate of 92% on the 8th day. From LC–MS/MS analysis, the optimal conditions for the reaction were determined as vinblastine (25 µl), microbial inoculums (150 µl or 200 × 10⁶ and 210 × 10⁶ CFU/ml), pH 7.4, rotation speed of 180 rpm, and temperatures of 30 °C and 37 °C with incubation time of 8 days. The vincristine produced exhibited potent antiangiogenic activity in the CAM assay reducing the thickness and branching of blood vessels in a dose-dependent manner. The study concludes that both Pseudomonas spp. showed promise for vincristine production from vinblastine, without compromising its antiangiogenic properties.

Full text

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Exploring the potential of two
Pseudomonas species to produce
vincristine from vinblastine
via biotransformation
Gauri Srivastava , Ruchika Mittal , Nidhi Srivastava & Deepak Ganjewala
*
A biotransformation pair consisting of vinblastine: vincristine present in the Catharanthus roseus plant
is of immense pharmacological signicance. In this study, we successfully transformed vinblastine
into vincristine outside the plant using Pseudomonas aeruginosa 8485 and Pseudomonas uorescens
2421 and evaluated the antiangiogenic potential of thus produced vincristine through the CAM
assay. The toxicity assay showed that both Pseudomonas spp. can tolerate varying concentrations
(25–100 µl of 1 mg/ml) of vinblastine. The biotransformation was performed in a liquid nutrient
broth medium containing vinblastine (25–100 µl), and Pseudomonas spp. inoculums (50–150 µl)
by incubating at 30 °C and 37 °C, respectively for 8 days. The process was optimized for substrate
and culture concentrations, pH, temperature, and rotation speed (rpm) for the highest conversion.
Analysis using LC–MS/MS conrmed the presence of vincristine as a product of the vinblastine
biotransformation by two Pseudomonas spp. P. uorescens 2421 showed a faster conversion rate with
95% of vinblastine transformed within 24 h than P. aeruginosa 8485, which demonstrated a conversion
rate of 92% on the 8th day. From LC–MS/MS analysis, the optimal conditions for the reaction were
determined as vinblastine (25 µl), microbial inoculums (150 µl or 200 × 106 and 210 × 106 CFU/ml), pH
7.4, rotation speed of 180 rpm, and temperatures of 30 °C and 37 °C with incubation time of 8 days.
The vincristine produced exhibited potent antiangiogenic activity in the CAM assay reducing the
thickness and branching of blood vessels in a dose-dependent manner. The study concludes that both
Pseudomonas spp. showed promise for vincristine production from vinblastine, without compromising
its antiangiogenic properties.
Keywords Antiangiogenic, Biotransformation, CAM assay, Vinblastine, Vincristine, Pseudomonas spp
Two monoterpene indole alkaloids vinblastine (VBL) and vincristine (VCR) produced in the Catharanthus roseus
(Madagascar Periwinkle) plant are of immense pharmacological signicance. Structurally, VBL contains a methyl
group while VCR contains a formyl group on the indole nitrogen of the vindoline skeleton (Fig.1)1. ese com-
pounds known as mitotic poisons, display powerful anticancer properties by disrupting microtubule functions
and arresting the cell cycle in the G2/M phase2–6. VCR, in particular, is more potent than VBL and is gaining
recognition as a wonder drug in cancer treatment, especially for blood cancer and other malignancies3. VBL is
commonly used in the treatment of Hodgkin’s lymphoma, lung cancer, bladder cancer, brain cancer, melanoma,
and testicular cancer. Both compounds are included in the World Health Organization’s essential medicines list
and are among the few monoterpene indole alkaloids on the market, with annual sales exceeding €1 billion7.
Biosynthesis of these compounds involves thirty-one enzymatic steps through the 2-C-methyl-D-erythritol
4-phosphate (MEP) and shikimic acid pathways8. ese pathways provide the terpenoid moiety secologanin and
indole moiety tryptamine, which condensed to form catharanthine and vindoline9–11. e subsequent dimeriza-
tion of these compounds by a peroxidase enzyme leads to the formation of VBL and VCR12. To date, 26 genes
have been identied, which are involved in the biosynthesis of catharanthine and vindoline synthesis. However,
the enzymes responsible for converting an intermediate α-3′,4′-anhydrovinblastine into VBL and VBL into VCR
remain unidentied13. A recent study using RNA-seq data has identied two candidate genes possibly alpha/beta
hydrolases, that play a role in the biosynthesis of VCR and VBL14.
OPEN
Amity Institute of Biotechnology, Amity University Uttar Pradesh, Sector-125, Noida, UP 201303, India. *email:
deepakganjawala73@yahoo.com
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In C. roseus, the production rates of VBL and VCR are extremely low with only 1g of each compound
extracted from 500 and 2000kg of dried leaves, respectively15. As a result, researchers have been investigating
alternative approaches such as engineering microbial hosts and microbial biotransformation, to produce these
compounds outside the plants. Hamada and Nakazava rst reported the conversion of VBL into VCR using
cell suspension cultures of C. roseus16. Further, Kumar and Ahmad identied an endophytic fungus Fusarium
oxysporum from C. roseus capable of converting VBL into VCR17. Furthermore, it has been reposted that various
endophytic fungi can mimic the plant in producing these compounds. Most recently, Tonk etal. observed that
Fusarium oxysporum acts as a biotic elicitor signicantly enhancing VBL and VCR yields in C. roseus embryos18.
Recently, Zhang etal. have successfully engineered a complex 31-step biosynthetic pathway of vindoline and
catharanthine into Saccharomyces cerevisiae leading to VBL production19. ese studies underline the potential
of microbes in producing and enhancing the yields of VBL and VCR, hence oering promising avenues for
biotechnological advancements. Despite this advancement, further research in this area remains limited.
e microbial biotransformation process is considered a part of the green chemistry approach for the produc-
tion of enantiomerically pure compounds with regio- and stereo-selectivity, eliminating the need for complex
separation and purication processes20–22. is approach could also be very useful for the production of VBL and
VCR, which contain multiple stereo centers, making their synthesis challenging via chemical synthesis. Currently,
VBL production largely depends on the extraction from C. roseus leading to issues of over-harvesting and the risk
of species extinction23. As a result, there is a growing interest in microbial biotransformation to produce VBL and
VCR selectively aiming to address sustainability concerns. Several previous studies have identied endophytic
fungi as valuable sources of alkaloids and capable of biotransformation of morphine, taxol/cephalomannine, and
caeine24–26. Earlier, Neuss etal. reported VBL transformation by Streptomyces albogriseolus and Streptomyces
hamipalus yielding two derivatives indolenine-indoline and 10-hydroxyl vinblastine27. In this study, we explored
the biotransformation capabilities of the two Pseudomonas species in converting VBL to VCR as well as evaluated
its anticancer eects, particularly in terms of antiangiogenic properties using the chick chorioallantoic membrane
(CAM) assay. To our knowledge, this is the rst study to report such biotransformation of VBL into VCR using
Pseudomonas species (led for patent no. 202411039360).
Materials and methods
Chemicals
Vinblastine and vincristine were purchased from Sigma-Aldrich. Methanol and chloroform used were of HPLC
grade from Qualigens, India. Nutrient Broth (NB) media and other chemicals used were of analytical grade and
obtained from Sisco Research Laboratory (SRL), India.
Procurement of microorganisms
Pseudomonas aeruginosa 8485 and Pseudomonas uorescens 2421were procured from Microbial Type Culture
Collection and Gene Bank (MTCC), CSIR-Institute of Microbial Technology, Chandigarh, India.
Sub-culturing was performed twice on nutrient broth (NB) medium by incubating at 30°C and 37°C tem-
peratures, respectively for 24h. Sub-culturing of pure cultures was done on the nutrient agar (NA) medium.
Finally, the cultures were stored at 4°C for further use.
Stock cultures were maintained on NA slants at 4°C and transferred every month. e media (NA) was
prepared and the pH was adjusted to 7.4 ± 0.2. e media was then sterilized in an Erlenmeyer ask at 121°C at
15 psi for 15min. Aseptically 30ml of sterilized NA was poured into pre-sterilized glass petri plates and allowed
to solidify at room temperature.
Toxicity assay
A toxicity assay was conducted to evaluate the tolerance of P. aeruginosa 8485 and P. uorescens 2421 to various
concentrations of VBL28. e sterilized petri plates were lled with 30ml sterilized NA media. en, freshly
prepared pre-grown cultures were evenly spread on the agar plate separately and wells were made in the media
Fig. 1. Chemical structures of (A) vinblastine and (B) vincristine.
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using a sterilized borer (8mm). e wells in each plate were then lled with dierent concentrations 25, 50, and
100µl of VBL separately. e plates were then incubated at 30°C and 37°C for 24h. Aer 24h, the cell growth
on the plates was visually observed to determine if the majority of the cells were still viable aer exposure to
dierent concentrations of substrate.
Further, to test the inhibitory eects of substrate on cell growth, the biomass pellet obtained from each sample
was collected and re-suspended in distilled water. A small amount was then placed on the NA plate and incubated
at 30°C and 37°C for 24h. e experiment was performed in triplicate.
Immobilization of bacterial whole cells
Immobilization of whole cells was performed using the entrapment method29. Sodium alginate (4%) was dis-
solved in 20ml double distilled water on a hot plate with a magnetic stirrer. 5ml of 5% Tris–HCl buer (pH
7.4) was added to 20ml sodium alginate solution. e cell suspension (20ml) of the bacterial strains was then
mixed with this solution separately. e mixture was added dropwise using a syringe onto the surface of 2%
CaCl2 solution. e resulting beads hardened at room temperature for 30min in a CaCl2 solution, washed, and
stored in Tris–HCl (pH 7.4) at 4°C for further use.
Biotransformation of VBL
For biotransformation, full-grown P. aeruginosa 8485 and P. uorescens 2421 cultures (50, 100, and 150µl) were
separately added to 50ml NB in a 150ml Erlenmeyer ask at pH 7.4 and the reaction was started by adding 25,
50, and 100µl of 1mg/ml of VBL dissolved in methanol under sterile conditions. e mixtures were incubated
on a shaker at 30°C and 37°C (180rpm)for 8 days. e biotransformation was routinely monitored by col-
lecting fractions at 1, 3, 5, and 8days, separating the culture from the aqueous component by centrifugation at
8000rpm. e supernatant collected was lyophilized and extracted three times with HPLC grade methanol to
maximize the recovery of the products.
Parallelly, biotransformation using immobilized P. aeruginosa 8485 and P. uorescens 2421(1 gm) was con-
ducted in a 150ml Erlenmeyer ask lled with 50ml NB, and VBL 25µl of 1mg/ml separately. e reactions
were incubated for 24–48h at 30°C and 37°C at 180rpm, with pH adjusted to 7.4. e culture liquid was
ltered and the immobilized beads were recovered, rinsed twice with sterile deionized water, and transferred
into Tris–HCl buer. Optical density (600nm) and pH were measured for comparison. e resulting samples
were then lyophilized, dissolved in HPLC-grade methanol, and stored at4°C. e residues were ltered and
the ltrates were collected for further use. Parallelly, a control experiment was conducted using the NB, and the
bacterial cultures but without substrate.
Thin layer chromatography (TLC) analysis
e biotransformation of VBL was analyzed by thin-layer chromatography (TLC). e samples were loaded on
the TLC plate with the help of a capillary. Subsequently, the plate was air-dried and placed inside a glass chamber
containing a solvent system consisting of chloroform: methanol (9:1 v/v) for the development. Following the
development, the plate was removed and dried using a dryer. To visualize the spots, the plate was sprayed with
the 1%ceric ammonium sulfateprepared in 70% phosphoric acid. Analysis of the chromatogram was carried
out by comparison with the standards of VBL and VCR30.
Liquid chromatography-mass spectrometry (LC–MS/MS) analysis
e samples were analyzed by LC-MS/MS (UPLC-(ESI)-QToF-MS) to conrm the presence ofVBL and VCR
following the determination of the molecular masses. It was performed on an Acquity Ultra Performance Liquid
Chromatography (UPLC), coupled to a Quadruple-Time of Flight mass spectrometer (QToF-MS, Synapt G2
HDMS, Waters Corporation, Manchester, UK). e QToF-MS was operated with electrospray ionization (ESI) at
a nominal mass resolution of 20,000 and controlled by MassLynx 4.1 soware. e data acquisition was done with
the MSE function in continuum mode in the range of m/z 50–1200. e MSE mode provides the full scan of MS
data (low energy, 4V) and MS/MS data (high energy, 10–60V ramping) simultaneously. e source parameters
were set as follows, capillary 3kV, sampling cone 30V, extraction cone 5V, source temperature 120°C, desolva-
tion temperature 500°C, desolvation gas ow 1000L/h, and cone gas ow 50 L/h. For mass spectrometer calibra-
tion, 0.5mM sodium formate was used. e lock spray, the reference mass leucine enkephalin (m/z 556.2771 in
positive and m/z 554.2670 in negative polarity) was used for mass correction with a ow rate of 10µl/min at the
concentration of 2µg/ml at every 10s interval. e chromatographic separation was performed on an ACQUITY
UPLC BEH C18 column (2.1 × 100mm, 1.8µm, Waters India Pvt. Ltd., Bangalore) at 35°C. e mobile phase
consists of A phase, methanol: water (v/v5:95) and B phase, methanol: water (v/v95:5) with 0.1% formic acid
in both phases. A gradient program was used with 0.3ml/min ow rate, with 0–0.5min/ 90% A, 4.5min/ 50%
A, 4.5–8min/ 50–2%A, 8–11min/ 2% A, 11–11.5min/ 2–90% A, 12–14min/ 90% A. e injection volume was
5µl and the samples were maintained at 15°C throughout the analysis.
Chick chorioallantoic membrane (CAM) assay
It was performed in compliance with the national laws relating to the conduct of animal experimentation. Impor-
tantly, the investigations utilizing CAM models typically do not require permissions or approvals from ethics
committees. In this study, eggs used in the CAM assay to evaluate the antiangiogenic potential were between 9
and 13days of incubation and according to the research, chick embryos do not develop pain perception until the
17th day of incubation. Importantly, the investigations utilizing CAM models typically do not require permissions
or approvals from ethics committees. In this study, eggs used in the CAM assay to evaluate the antiangiogenic
potential of VCR between 9 and 13days of incubation19. e antiangiogenic activity of VCR was evaluated using
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an in-vivo CAM assay31. e freshly fertilized chicken eggs procured from Kegg Farms Private Limited, Gurgaon,
India were used in the study. e eggs were cleaned gently with 70% ethanol and then placed in an incubator at
37°C with 85% humidity for 8days. A hypodermic needle was used to create a tiny hole in the shell to conceal
the air sac and another hole was created on the wide side of the egg directly above the vascular region of the
embryonic membrane using candling. By injecting negative pressure via the rst hole, a fake air sac was formed
underneath the second hole, allowing the CAM to detach from the shell. On day 9, a window approximately 2cm
in size was cut above the detached CAM using a sterile surgical blade and forceps. Samples of 100µl, 200µl, and
300µl (at a concentration of 1mg/ml) were loaded onto sterile Whatman lter paper grade-1 disc of 125mm
diameter, while 25µl of standard VCR (1mg/ml) was loaded onto another sterile Whatman lter paper disc.
ese discs were then placed aseptically on the growing CAMs, alongside one egg control and one methanol con-
trol. e windows were sealed with sterile paralm and the samples were again placed in the incubator for 3days
at 37°C. e experiment was conducted in triplicate, and the resulting antiangiogenic activity was compared to
the vehicle control (methanol) for each set of samples. e entire process from procurement of microorganisms
to conductingCAM assay is schematically presented in Fig.2.
Results
Screening of microbes for biotransformation of vinblastine
In the initial experiment, various microbes including bacteria and fungi were screened for their ability to convert
VBL into VCR through the toxicity test. e results indicated that P. aeruginosa 8485 and P. uorescens 2421
showed resistance to all concentrations 25–100µl of 1mg/ml VBL (Fig.3). e growth prole analysis indicated
that both strains grew slowly on nutrient agar media with P. aeruginosa 8485 forming translucent shiny white
small colonies and P. uorescens 2421 appeared as white colonies with smooth edges.
e growth proles of these bacteria were further investigated aer immobilizing them with 4% sodium
alginate. Immobilized cells of P. aeruginosa 8485 demonstrated a lower multiplication rate and did not achieve
full growth within 24h as compared to the free cells. eir optical density (0.464) was lower than that of free
cells (1.525) while the pH (7.1) was similar to free cells (7.6). In case of P. uorescens 2421, both immobilized
and free cells exhibited similar multiplication rates and achieved full growth within 24h. e optical density
and pH level of immobilized cells (1.393 and 8.4, respectively) closely matched those of free cells (1.489 and 8.7).
Conversion of VBL to VCR
e progress of VBL biotransformation was assessed by sampling the reaction mixture on days 1, 5, and 8,
followed by analysis using TLC and LC–MS/MS (Figs.4, 5, 6). e thin layer chromatogram from the 1st day
displayed two distinct spots with the Rf values 0.77 and 0.74, resembling standard VBL and VCR, respectively
indicating the initiation of VBL transformation to VCR from day 1 (Fig.4). e chromatogram from the 8th
day exhibited a yellowish-brown spot with the Rf value of 0.74 consistent with VCR standard suggesting the
conversion of VBL to VCR by Pseudomonas species (Fig.4). In contrast, the control ask without VBL showed
no presence of either VBL or VCR.
Analysis of LC–MS/MS data of the reaction fraction of the 1st and 8th day revealed the presence of VBL and
VCR as well as several other unknown compounds. Concurrently, standard VBL and VCR were also analysed
Fig. 2. e workow of methodology from acquiring microorganisms to conductingCAM assay.
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for the identication of anticipatedmetabolites in bacterial reaction fractions (Fig.5). LC–MS/MS spectra and
fragments ions of mass spectroscopy of bacterial metabolite sample of P. aeruginosa 8485 revealed that 79%
of VBL was converted to VCR within 24h, which further increased to 92% by the 8th day (Fig.6A,B). On the
other hand, biotransformation using P. uorescens 2421 resulted in a signicantly higher conversion of 95% of
VBL to VCR compared to 79% of P. aeruginosa 8485 within 24h. However, it decreased to 88% by the 8th day
and anyVBL remained thereaerwas completely degraded (Fig.6C,D). Simultaneously, analysis ofa bacterial
control lacking VBL was performed, which showed absence ofbothVBL and VCR in the bacterial metabolites
and only VBL wasdetectedin the substrate control.
VBL conversion eciency of free and immobilized whole cells
LC–MS/MS analysis of the biotransformation using immobilized bacterial cells showed signicantly lowerVCR
yieldin comparison to free cells on day 1 (Fig.7). Additionally, it was observed that VBL was completely degraded
in the reaction with immbolized cellsas compared to free cells. ese ndings suggested that free cells demon-
strated a higher eciency in transforming VBL into VCR. Comparatively, P. uorescens 2421 more profoundly
converted VBL into VCR than P. aeruginosa 8485 (Table1).
Optimization of substrate and culture concentrations
Dierent concentrations of VBL (25, 50, and 100µl) and bacterial cultures (50, 100, and 150µl) were tested to
evaluate their impacts on VBL biotransformation (Table2). e experiment was conducted at temperatures 30°C
for P. aeruginosa 8485 and 37°C for P. uorescens 2421 with constant shaking at 180rpm over 24h.
e maximum conversion (92%) of VBL to VCR was achieved at 25µl of VBL, 150µl of P. aeruginosa contain-
ing 200 × 106CFU/ml and P. uorescens containing 210 × 106CFU/ml), pH 7.4, and rotation speed of 180rpm
during incubation at 37°C for a period of 8days (Table2). Interestingly, VCR formation was started within the
rst 24h of the incubation. e biotransformation also occurred at other concentrations, however, the conver-
sion rates signicantly varied with varying concentrations of VBL and culture volumes. With 50µl VBL, no
conversion was observed on day 1 using P. aeruginosa 8485, which substantially increased to 83% on the 8th day
of incubation. e conversion rate with 100µl of VBL was signicantly higher 42 to 96% between days 1 and 8
of incubation (Table2). In case of P. uorescens 2421, VBL conversion rate at 50µl was 28% on the 1st day, which
signicantlyenhanced to 58% by the 8th day. Similarly, at 100µl of VBL, the conversion rate was 23% on the 1st
day and markedly increased to 71% by the 8th day (Table2).
Similarly, bacterial culture volume also inuenced VBL conversion into VCR. When 50 and 100µl of P. aer-
uginosa 8485 culture were added to the reaction, it resulted in 93% and 88%of VBL conversion, respectively by
the 8th day of the incubation (Table2). However, thebiotransformationwith the similar culture volumes of P.
Fig. 3. Toxicity assay demonstrating tolerance to various concentrations of vinblastine. (A) P. aeruginosa 8485
(B) P. uorescens 2421.
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uorescens 2421 resulted in a comparatively lower conversion of 78% of VBL in 8days. us, it demonstrated a
lower biotransformation eciencyofP. uorescens 2421 compared to P. aeruginosa 8485 under similar conditions.
Antiangiogenic activity of VCR
eVCR producedusing bothPseudomonas species was tested for its potential to inhibit angiogenesis invivo
using the CAM assay and compared to authentic VCR. Treatment ofeggs with 100µl of VCR produced using P.
aeruginosa 8485 did not showanyinhibition ofangiogenesis. However, when VCRconcentrations were increased
to 200 and 300µl, it exhibited a dose-dependent inhibition of chorioallantoic vessel formation. At 300µl, a
prominent inhibition of vessels similar to that of standard VCR was observed. However, the standardVCR at a
concentration of25µl of 1mg/ml demonstratedpotential toinhibit angiogenesis at 25µl of 1mg/ml(Fig.8).
Similarly, VCR synthesized using P. uorescens 2421 showed impressive inhibitions of vessels in a dose-
dependent manner. However, in contrast to VCR produced by P. aeruginosa, this VCR at 300µl demonstrated
highly toxic eects on vessels.
Fig. 4. (A) Bioconversion reaction of vinblastine into vincristine and (B) a thin layer chromatogram depicting
authentic vinblastine and vincristine and (C) a thin layer chromatogram depicting vincristine in reaction
fractions of days 1, 5, and 8.
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Discussion
In this study, VCR was produced from VBL using two Pseudomonas species, and its antiangiogenic eect was
evaluated using the CAM assay. Naturally, this biotransformation occurs in C. roseus, which is a time-consuming
process with a signicantly low yield of VCR. e aim of our study was to enhance the rate of this biotransforma-
tion using Pseudomonas species leading to higher production of VCR outside the plant. Pseudomonas species
are known for their diverse metabolic capabilities and secrete various extracellular enzymes during their growth
phase, which can be used as biocatalysts in producing industrially relevant compounds32. Moreover, their faster
growth rate and ability to start multiplication in 6–7h makes them more ecient in starting the conversion of
VBL within 24h.
We found that P. aeruginosa 8485 and P. uorescens 2421 were found to convert 80–90% of VBL to VCR within
24h presenting a signicantly faster alternative to the natural process. ere were only two reports available on
the biotransformation of VBL leading to VCR by two dierent methods one using the C. roseus suspension cell
culture16 and another using F. oxysporum17. However, our study demonstrated signicantly higher conversion
rates than previous reports16,17. While the previous study was conducted VBL biotransformation using onlyfree
F. oxysporum cells, we used bothimmobilized andfree cells of twoPseudomonas speciesand determined that
free cells were moreeective in converting VBL into VCR. e immobilization of bacterial cells can have adverse
eect on their growth rate and metabolic capabilities, potentially leading to decreased conversion of VBL to
VCR. Further, immobilization also aects the viability of cells, which can inuence the overall biotransforma-
tion process adversely resulting in lower conversion of VBL to VCR. However, the storage medium and nutrient
availability play a crucial role in maintaining cell viability and metabolic activity. e eects of immobilization
Fig. 5. Mass spectra of (A)vinblastine 811.5 (m/z = 406) and(B) vincristine 825.5 (m/z = 413).
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Fig. 6. (A) LC–MS/MS analysis of biotransformation products from reaction fractions of day 1. [A] P.
aeruginosa 8485 and [B] P. uorescens 2421. e biotransformation assay contains vinblastine (25µl) and
bacterial culture (150µl) incubated at 30 and 37°C, respectively. (B) Mass spectra of biotransformation products
from reaction fractions of day 1. [A] P. aeruginosa 8485 and [B] P. uorescens 2421. (C) LC–MS/MS analysis
of biotransformation products from reaction fractions of day 8. [A] P. aeruginosa 8485 and [B] P. uorescens
2421. e biotransformation assay contains vinblastine (25µl) and bacterial culture (150µl) incubated at 30
and 37°C, respectively. (D) Mass spectra of biotransformation products from reaction fractions of day 8. [A] P.
aeruginosa 8485 and [B] P. uorescens 2421.
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on microbial growth rates can vary among dierent strains and may lead to conicting results in biotransforma-
tion studies33.
e VCR formation varied signicantly with varying concentrations of VBL and bacterial cultures. e
highest conversion rate was attained with a combination of VBL (25µl), bacterial cultures (150µl), pH 7.4, tem-
peratures of 30°C (P. aeruginosa 8485) and 37°C (P. uorescens 2421) with an incubation period of 8days. For P.
aeruginosa 8485, the overall conversion rate over 1–8days of incubation was 92%. In contrast, P. uorescens strain
2421 demonstrated a conversion eciency of 95% within the rst 24h of the incubation, which subsequently
decreased to 89%. Several factors such as fermentation mode, culture media, temperature, humidity, incubation
time, pH, rotation speed, substrate solubility, and co-factors aect microbial transformation processes34,35. Here,
Fig. 6. (continued)
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the optimal substrate and culture concentrations to achieve maximum conversion eciency were determined to
be 25µl and 150µl, respectively. ere are no previous studies to compare these specic ndings. e literature
suggests that alkaloid production can be enhancedbyemploying strategies such as media optimization, the use
of plant growth regulators, and culture practices, culture of high-yielding cell lines, the use of precursors, the
incorporation of elicitors, and the enhancing expression of metabolic pathway enzymes36–38.
e VCR produced in this studyexhibited the potential antiangiogenic eects in vivo using the CAM model.
e microbial biotransformation of VBL to VCR is of great interest due toResearch on VCR is of interest for its
ability to inhibit cell proliferation and angiogenesis39–41. Angiogenesis is a hallmark of cancer and CAM isan
excellent systemfor studying angiogenesis, tumor cell invasion, and metastasis due to its vascularized nature and
Fig. 6. (continued)
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cost-eectiveness42–44. is study’s ndings support the documented anticancer activities of VCR, particularly
its signicant impact onreduction of angiogenesis.
Conclusion
A novel microbial biotransformation system using P. aeruginosa 8485 and P. uorescens 2421 was established
to convert VBL into VCR. is biotransformation system demonstrated promise for ecient production of
VCRoutside the plant, presenting a valuable contribution to the biological production of thistherapeutic com-
pound. erefore, alongside the already known endophytic fungi, Pseudomonas species could be invaluable
Fig. 6. (continued)
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Fig. 7. LC–MS/MS analysis of vinblastinebiotransformation products from reaction fractions of day 1 using
immobilized (A) P. aeruginosa 8485 and (B) P. uorescens 2421 whole cells.
Table 1. Characterization of free and immobilized whole cells of twoPseudomonas species andtheir VBL
conversion eciencyon day 1 of the reaction. VBL vinblastine, VCR vincristine.
Characteristics
Free cells Immobilized cells
P. aeruginosa 8485 P. uorescens 2421 P. aeruginosa 8485 P. uorescens 2421
OD (600nm) 1.525 1.489 0.464 1.393
pH 7.6 8.7 7.1 8.4
Conversion of VBL to VCR(%) 79 95 0.56 0.23
Table 2. Optimization of substrate and bacterial culture concentrations for vinblastineVBL
biotransformation. BS biotransformation system.
Biotransformation assay compositions Incubation time (days)
% VBL conversion to VCR
P. aeruginosa 8485 P. uorescens 2421
Control: Media + Bacterial inoculum (150µl) (No vinblastine) 1-8 No conversion No conversion
BS-1: Media + Bacteria l inoculum (150µl) + Vinblastine (25µl) 1 79 95
8 92 88.74
BS-2: Media + Bacteria l inoculum (150µl) + Vinblastine (50µl) 1 No conversion 27.7
8 83 58.3
BS-3: Media + Bacteria l inoculum (150µl) + Vinblastine (100µl) 1 42 23
8 95.8 71.2
BS-4: Media + Bacteria l inoculum (50µl) + Vinblastine (25µl) 1 No conversion No conversion
8 93.4 78
BS-5: Media + Bacteria l inoculum (100µl) + Vinblastine (25µl) 1 No conversion No conversion
8 88 77.7
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resources for the biotechnological production of VCR. Most importantly, the VCR produced through the
greenprocess showed comparable antiangiogenic ecacy to the standard VCR. In the present study, sophisticated
techniques such aslyophilization and LC–MS/MS were used inextraction and analysis of the biotransformation
products, respectively which may have implications for the operational cost of the system. Additionally, mainte-
nance of these techniques is highly expensive due to the necessity of employingdierent columns for separation
of the metabolites. erefore,further studies in this direction arerequired to enhance the eciency of separation
and purication processesin order to reduce thetotal production costs of VCR. Despite these limitations, this
eco-friendly process presents an alternative strategy for VCR production from VBL.
Data availability
e datasets used and/or analysed during the current study available from the corresponding author on reason-
able request.
Received: 26 March 2024; Accepted: 19 August 2024
References
1. Keglevich, P., Hazai, L., Kalaus, G. & Szantay, C. Modications on the basic skeletons of vinblastine and vincristine. Molecules 17,
5893–5914 (2012).
2. Field, J. J., Waight, A. B. & Senter, P. D. A previously undescribed tubulin binder. Proc. Natl. Acad. Sci. USA 111, 13684–13685
(2014).
3. Skubník, J., Pavlíčková, V. S., Ruml, T. & Rimpelová, S. Vincristine in combination therapy of cancer: Emerging trends in clinics.
Biology 10, 849. https:// doi. org/ 10. 3390/ biolo gy100 90849 (2021).
4. Almagro, L., Fernández-Perez, F. & Pedreno, M. A. Indole alkaloids from Catharanthus roseus: Bioproduction and their eect on
human health. Molecules 20, 2973–3000 (2015).
5. Gajalakshmi, S., Vijayalakshmi, S. & Devi, R. V. Pharmacological activities of Catharanthus roseus: A perspective review. Int. J.
Pharm. Biol. Sci. 4, 431–439 (2013).
6. Dhyani, P. et al. Anticancer potential of alkaloids: A key emphasis to colchicine, vinblastine, vincristine, vindesine, vinorelbine
and vincamine. Cancer Cell Int. 22, 1–20 (2022).
Fig. 8. e CAM assay illustrating antiangiogenic eects. (A) vincristine control (B) egg control (C,D) 100
and (E,F) 300µl of vincristine produced from vinblastine using P. aeruginosa 8485 and P. uorescens 2421,
respectively.
Content courtesy of Springer Nature, terms of use apply. Rights reserved

14
Vol:.(1234567890)
Scientic Reports | (2024) 14:19652 | https://doi.org/10.1038/s41598-024-70571-8
www.nature.com/scientificreports/
7. Prasad, V. & Mailankody, S. Research and development spending to bring a single cancer drug to market and revenues aer
approvall. JAMA Intern. Med. 177, 1569–1575 (2017).
8. Qu, Y., Safonova, O. & De Luca, V. Completion of the canonical pathway for assembly of anticancer drugs vincristine/vinblastine
in Catharanthus roseus. Plant J. 97, 257–266 (2019).
9. Zhu, X., Zeng, X., Sun, C. & Chen, S. Biosynthetic pathway of terpenoid indole alkaloids in Catharanthus roseus. Front. Med. 8,
285–293 (2014).
10. Sharma, A., Verma, P., Mathur, A. & Mathur, A. K. Genetic engineering approach using early vinca alkaloid biosynthesis genes led
to increased tryptamine and terpenoid indole alkaloids biosynthesis in dierentiating cultures of Catharanthus roseus. Protoplasma
255, 425–435 (2018).
11. Mistry, V., Darji, S., Tiwari, P. & Sharma, A. Engineering Catharanthus roseus monoterpenoid indole alkaloid pathway in yeast.
Appl. Microbiol. Biotechnol. 106, 2337–2347 (2022).
12. Sottomayor, M. & Ros Barceló, A. Peroxidase from Catharanthus ros eus (L.) G. Don and the biosynthesis of alpha-3’,4’-anhydrovin-
blastine: A specic role for a multifunctional enzyme. Protoplasma 222, 97–105. htt ps:// do i . o r g/ 10. 1007/ s00709- 003- 0003-9 (2003).
13. Zhu, J., Wang, M., Wen, W. & Yu, R. Biosynthesis and regulation of terpenoid indole alkaloids in Catharanthus ros eus. Pharmacogn.
Rev. 9, 24–28. https:// doi. org/ 10. 4103/ 0973- 7847 (2015).
14. Caputi, L. et al. Missing enzymes in the biosynthesis of the anticancer drug vinblastine in Madagascar periwinkle. Science 360,
1235–1239. https:// doi. org/ 10. 1126/ scien ce. aat41 00 (2018).
15. Ferreres, F. et al. Simple and reproducible HPLC-DAD-ESI-MS/MS analysis of alkaloids in Catharanthus roseus roots. J. Pharm.
Biomed. Anal. 51, 65–69. https:// doi. org/ 10. 1016/j. jpba. 2009. 08. 005 (2010).
16. Hamada, H. & Nakazawa, K. Biotransformation of vinblastine to vincristine by cell suspension cultures of Catharanthus roseus.
Biotechnol. Lett. 13, 805–806 (1991).
17. Kumar, A. & Ahmad, A. Biotransformation of vinblastine to vincristine by the endophytic fungus Fusarium oxysporum isolated
from Catharanthus roseus. Biocatal. Biotransform. 31, 89–93 (2013).
18. Tonk, D. et al. Fungal elicitation enhances vincristine and vinblastine yield in the embryogenic tissues of Catharanthus roseus.
Plants 12, 3373 (2023).
19. Zhang, J. et al. A microbial supply chain for production of the anti-cancer drug vinblastine. Nature 609, 341–347. https:// doi. org/
10. 1038/ s41586- 022- 05157-3 (2022).
20. Borges, K. B. et al. Stereoselective biotransformations using fungi as biocatalysts. Tetrahedron 20, 385–397 (2009).
21. Hegazy, M. E. F. et al. Microbial biotransformation as a tool for drug development based on natural products from mevalonic acid
pathway: A review. J. Adv. Res. 6, 17–33 (2015).
22. Naik, S. B. Potential roles for endophytic fungi in biotechnological processes: A review. In Plant and Human Health Volume 2:
Phytochemistry and Molecular Aspect 327–344 (Springer, 2019).
23. Mishra, S., Sahu, P. K., Agarwal, V. & Singh, N. Exploiting endophytic microbes as micro-factories for plant secondary metabolite
production. Appl. Microbiol. Biotechnol. 105, 6579–6596 (2021).
24. Rathbone, D. A. & Bruce, N. C. Microbial transformation of alkaloids. Curr. Opin. Microbiol. 5, 274–281 (2002).
25. Chen, T. S., Li, X., Bollag, D., Liu, Y. & Chang, C. Biotransformation of taxol. Tetrahedron Lett. 42, 3787–3789 (2001).
26. Asano, Y. Development of new microbial enzymes and their application to organic synthesis. J. Synth. Organ. Chem. 57, 1064–1072
(1999).
27. Neuss, N. et al. Vinca alkaloids XXXIII [1]. Microbiological conversions of vinca leukoblastine (VLB, vinblastine), an antitumor
alkaloid from Vinca rosea. Linn. Helv. Chim. Acta 57, 1886–1890 (1974).
28. Bouaziz, A. et al. Antibacterial and antioxidant activities of Hammada scoparia extracts and its major puried alkaloids. South
Afr. J. Bot. 105, 89–96 (2016).
29. Zheng, Y. G., Zhang, X. F. & Shen, Y. C. Microbial transformation of validamycin A to valienamine by immobilized cells. Biocatal.
Biotransform. 23, 71–77 (2005).
30. Volkov, S. K. & Grodnitskaya, E. I. Methods for raising the eciency of chromatographic purication of vinblastine preparations.
Pharm. Chem. J. 29, 801–803 (1995).
31. Tayal, N., Srivastava, P. & Srivastava, N. Anti angiogenic activity of Carica papaya leaf extract. J. Pure Appl. Microbiol. 13, 567–571
(2019).
32. Molina, G., Pimentel, M. R. & Pastore, G. M. Pseudomonas: A promising biocatalyst for the bioconversion of terpenes. Appl.
Microbiol. Biotechnol. 97, 1851–1864. https:// doi. org/ 10. 1007/ s00253- 013- 4701-8 (2013).
33. Żur, J., Wojcieszyńska, D. & Guzik, U. Metabolic responses of bacterial cells to immobilization. Molecules 21, 958 (2016).
34. Cardillo, A. B. et al. Production of tropane alkaloids by biotransformation using recombinant Escherichia coli whole cells. Biochem.
Eng. J. 125, 180–189 (2017).
35. Dhiman, S., Mukherjee, G. & Singh, A. K. Recent trends and advancements in microbial tannase-catalyzed biotransformation of
tannins: A review. Int. Microbiol. 21, 175–195 (2018).
36. Singh, N. R., Rath, S. K., Behera, S. & Naik, S. K. Invitro secondary metabolite production through fungal elicitation: An approach
for sustainability. In Fungal Nanobionics: Principles and Applications 215–242 (Springer Singapore, 2018).
37. Satheesan, J. & Sabu, K. K. Endophytic fungi for a sustainable production of major plant bioactive compounds. In Plant Derived
Bioactives: Production, Properties and erapeutic Applications 195–207 (Springer Singapore, 2020).
38. Mujib, A., Tonk, D., Gulzar, B., Maqsood, M. & Ali, M. Quantication of taxol by high performance thin layer chromatography
(HPTLC) in Taxus wallichiana callus cultivated invitro. BioTechnologia 101, 337–347 (2020).
39. Fischer, I., Gagner, J. P., Law, M., Newcomb, E. W. & Zagzag, D. Angiogenesis in gliomas: Biology and molecular pathophysiology.
Brain Pathol. 15, 297–310 (2005).
40. Irie, N., Matsuo, T. & Nagata, I. Protocol of radiotherapy for glioblastoma according to the expression of HIF-1. Brain Tumor
Pathol. 21, 1–6 (2004).
41. Jung, Y. J., Isaacs, J. S., Lee, S., Trepel, J. & Neckers, L. Microtubule disruption utilizes an NFkappa B-dependent pathway to stabilize
HIF-1alpha protein. J. Biol. Chem. 278, 7445–7452 (2003).
42. Nowak-Sliwinska, P., Segura, T. & Iruela-Arispe, M. L. e chicken chorioallantoic membrane model in biology, medicine and
bioengineering. Angiogenesis 17, 779–804 (2014).
43. Tayal, N. et al. Anti-angiogenic activity of columbin: A diterpenoid from Tinospora cordifolia. Int. J. Biol. Pharm. Allied Sci. 9,
540–552 (2020).
44. Kaur, A. et al. Novel biogenic silver nanoconjugates of Abrus precatorius seed extracts and their antiproliferative and antiangiogenic
ecacies. Sci. Rep. 13, 13514 (2023).
Acknowledgements
e authors express their gratitude to founder presidentDr. Ashok K Chauhan and Chancellor Mr. Atul Chauhan
Amity University Uttar Pradesh, Noida, India for providing the necessary support and facilities. e authors
also would like to acknowledge the Amity Institute of Microbial Technology, Noida for granting permission to
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utilize the lyophilizer. Special thanks are extended to the Council of Scientic and Industrial Research (CSIR),
New Delhi for their invaluable technical support.
Author contributions
GS: Experimental work, literature collection, analysis of results, and preparation of the manuscript’s main dra.
RM: Literature collection, support in manuscript preparation. NS: CAM assay and its analysis. DG: Conceptu-
alization, supervision,evaluation, editing, reviweingand approvalof the nal manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 024- 70571-8.
Correspondence and requests for materials should be addressed to D.G.
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