Novel Strategies for Overproduction of Microbial Products

Novel Strategies for Overproduction of Microbial Products

Sheetal Raina, Tania Murphy, Daniela De Vizio, Patricia Reffatti, Tajalli Keshavarz*
Applied Biotechnology Research Group, Department of Molecular
and Applied
Biosciences, School of Life Sciences
115 New Cavendish Street, London W1W 6UW, UK
Biotechnology and pharmaceutical industries are in continual quest for the discovery of
novel products and the enhancement of productivity of value products to retain their
global competitiveness. Traditional methods employed to achieve these goals include
microbial strain selection, culture improvement, media development, and, bioreactor
and process design. These methods, however, suffer from severe drawbacks such as the
long time required for successful outcomes, high expenses and, in many cases, low
success rate.
Over the last fifteen years we have introduced two novel strategies for overproduction
of industrially desirable microbial products. These strategies are based on microbial
response to the microbes in their vicinity (quorum sensing) and, to their surrounding
environment (elicitation).
In all these cases, enhancement of productivity was established with several fold
increases: in the elicitation work, addition of oligosaccharide (oligomannuronate and
oligoguluronate blocks) resulted in a 50% increase in penicillin G yield for Penicillium
chrysogenum. While Bacillus licheniformis cultures supplemented with oligoguluronate
resulted in over 19% and 11% increase in bacitracin A yield in the shaken flasks and
bioreactors respectively. In quorum sensing studies addition of spent medium from
Penicillium sclerotiorum IMI 104602 (strain M) - a high producer of multicolic acid- to
P. sclerotiorum IMI 040574 strain S) - a low producer of multicolic acid- led to a 6.4-
fold increase in sclerotiorin yield.
We suggest that exploitation of microbes’ communications to other microbes (selfcommunity)
and to the environment may provide new industrial opportunities for
improved productivity of microbial biomolecules.
1. Introduction
Environmental abiotic and biotic stress factors have been proved to effect variety of
responses in microbes. Elicitors, as stress factors, induce or enhance the biosynthesis of
secondary metabolites added to a biological system. They are classified into various
groups based on their nature and origin: physical or chemical, biotic or abiotic
(Radman, et al., 2003). Initial studies on elicitation of secondary metabolites were
carried out on plant cells (e.g. jasmonic acid supplemented Catharanthus roseus
cultures had an increase in the specific yields of serpentine, ajmalicine, tabersonine, and
lochnericine; Shanks and Bhadra, 1998) and extended, over the years, to bacteria,
animal cell cultures and filamentous fungi. Abiotic stress (abiotic elicitors) imposed by
pH improves pigment production by Monascus purpureus (Orozco and Kilikian, 2008)
and antibiotic production by Streptomyces spp. (Kim, et al., 2000). Traditionally
carbohydrates have been used as carbon sources in fermentation processes. They have
also been used widely in small amounts (mg L-1) as elicitor molecules in bacterial and
fungal fermentations for overproduction of commercially important secondary
metabolites.
In one approach to improve production, we investigated the effect of carbohydrate
biotic elicitors (oligosaccharides, oligomannuronate, oligoguluronate and mannan
oligosaccharides) on variety of fungal systems: Penicillium spp.(Ariyo, et al., 1997),
Ganoderma spp. (Ghorashi, 2004), Corylopsis spp. (Vanhulle, et al., 2007) and bacterial
cultures: Streptomyces spp. (Sangworachat, 2006), Bacillus spp. (Murphy, et al., 2007a)
for production of antibiotics, enzymes, pigments and changes in morphology.
In another approach we used quorum sensing phenomenon. Quorum sensing is the intercell
communication between cells through the release of chemical signals when cell
density reaches a threshold concentration (critical mass). Under these conditions, they
sense the presence of other microbes; change their own genetic expression to find
advantage over their competitors for survival. This process, investigated for more than
30 years, was first discovered in Gram-negative bacteria, and then in Gram-positive
bacteria and dimorphic fungi. The quorum sensing signals differ in different microbial
systems; examples are acyl-homoserine lactones, modified or unmodified peptides,
complex γ -butyrolactone molecules and their derivatives. A number of physiological
activities of microbes (e.g. symbiosis, competence, conjugation, sporulation, biofilm
formation, virulence, motility and the production of various secondary metabolites) is
regulated through the quorum-sensing.
While research into the quorum sensing process has been continuing with an impressive
pace, the activity is limited to research at bench scale mainly in biomedical areas.
However, as the range of quorum sensing-affected physiological activities show, there
is great potential for the use of this communication process for industrial exploitation.
Filamentous fungi are a main microbial source for production of pharmaceutical and
biotechnological products. However, until recently, very little was reported in the
literature regarding quorum sensing phenomena in these fungi. We explored, for the
first time, the possibility of overproduction of fungal metabolites (Raina et al., 2010) in
response to the supplementation of liquid cultures by variety of quorum sensing
molecules.
Bacillus licheniformis is widely present in the environment. Its metabolic diversity has
resulted in its use for production of enzymes, antibiotics and fine chemicals. Bacitracin
produced by B. licheniformis is a polypeptide antibiotic active against Gram positive
and some Gram-negative bacteria. Bacitracin is also used as animal feed additive.
Sclerotiorin synthesized by Penicillium sclerotiorum is a phospholipase A2 inhibitor
and has been classified as an octaketide. Sclerotiorin has also been studied for its
cholesterol ester transfer protein (CETP) inhibitory activity and recently extracts from
Penicillium sclerotiorum have been studied for their activity against methicillin resistant
Staphylococcus aureus (MRSA).
In this paper, we report overproduction of antibiotics using the two methods, elicitation
and quorum sensing. We describe enhancement in the concentration of bacitracin in
response to supplementation of Bacillus licheniformis cultures with oligomanuronate
and overproduction of the fungal antibiotic sclerotiorin in cultures of Penicillium
sclerotiorum (strain S) through the addition of spent medium from a culture of
Penicillium sclerotiorum (strain M) (quorum sensing-based procedure).
2. Materials and Methods
2.1 Microbial strains
Bacillus licheniformis NCIMB 8874 was obtained from Natural Collection of Industrial
and Marine Bacteria, USA. This strain was used for bacitracin elicitation studies.
Penicillium sclerotiorum IMI 104602 (Strain M) and IMI 040574 (Strain S) were
obtained from CABI Biosciences UK Centre, Surrey, United Kingdom. These strains
were used for quorum sensing studies.
2.2 Chemicals and reagents
All chemicals used in this study were obtained from Sigma-Aldrich Company Limited,
Dorset, United Kingdom unless stated otherwise. Analytical grade reagents were used
for quantitative and qualitative assays and HPLC grade reagents were used for high
performance liquid chromatography assays.
2.3 Media and growth conditions
B. licheniformis NCIMB 8874 was grown in a defined medium (no carbohydrates as
carbon source). The medium contained (gL-1): 20 glutamic acid; 1 citric acid; 20
NaH2PO4 . 2H2O; 0.5 Na2SO4; 0.02 MgCl2 . 6H2O; 0.5 KCl, 0.01 CaCl2 . 2H2O; 0.01
MnSO4 . H2O, and 0.01 FeSO4 . 7H2O. Sodium hydroxide was used to adjust the
medium pH to 6.0 before sterilisation. The inoculum as spore suspension (1x107 spores
mL-1) was added to 100 mL the sterile defined medium and incubated at 37°C, 200 rpm
for 16 h. Aliquots of the culture at exponential phase were transferred into shaken flasks
(SF) or fermenters (5 L Fermac 360 Stirred tank reactor, STR). Shaken flasks (500 mL)
contained 90 mL of the defined medium. Incubation was carried out at 37°C at 200 rpm
for 96 h. Growth of B. licheniformis cultures was monitored by measuring their optical
density at 650 nm. For STR experiments, the defined medium was inoculated with a
10% vv-1 of inoculum to make 4 L total. The temperature was 37°C and the stirrer speed
was between 300-600 rpm to keep %DOT above 30% air saturation.
For production of sclerotiorin and study of the effect of the spent medium on
overproduction of this compound, a 2.5 L Stirred Tank Reactor (2.5 L STR,
FerMac360, Electrolab Ltd., UK) with 1.5 L of sterile potato dextrose broth was
inoculated with spores (1x107 mL-1) of P. sclerotiorum Strain S. Two bioreactors were
run simultaneously as control and test. The airflow rate and temperature were set at 1.0
vvm and 27°C respectively. The % DOT (air saturation), pH, and temperature were
monitored throughout the fermentation. The stirrer speed was increased gradually from
initial 120 rpm to 250 rpm during the course of fermentation. Samples were assayed
every 24 h for sclerotiorin production, pH and carbohydrate utilization.
2.4 Preparation of supplements and product assay
Oligoguluronate elicitor (OG): OG was prepared by acid hydrolysis from sodium
alginate as described by Ariyo et al. (1997).
Sterile aliquots of OG, were added to the test SFs and STRs at 24 h for a final
concentrations of 100 mg L-1. Control cultures without addition of OG were used for
comparison.
Spent medium (containing multicolic acid): For preparation of the spent medium for
use as a supplement for production of sclerotiorin by P. sclerotiorum Strain S, P.
sclerotiorum Strain M was grown for 8 days in a medium containing (gL-1): 30 sucrose;
2.6 ammonium tartrate; 0.4 NH4H2PO4; 0.16 (NH4)2SO4; 2.6 tartaric acid; 0.28 MgCO3;
0.4 K2CO3; 0.07 ZnSO4.7H2O; 0.005 CuSO4.5H2O and 0.06 FeSO4.7H2O. The medium
pH was adjusted to 3.5 before sterilisation. The sterile medium (100 mL) was inoculated
with 1 mL of spore suspension (1x107 spores per mL). The cultures were incubated at
26ºC and 150 rpm (2 cm throw) for 9 days. The culture broth was then filtered through a
Whatman filter paper (No.1). The cell free broth was subsequently filter sterilised using
a 0.2 μm cellulose acetate membrane filter and added as the spent medium at 48 h to the
test cultures of P. sclerotiorum Strain S (1.0 % v/v).
Bacitracin A assay: Bacitracin was quantified by gradient HPLC.
Sclerotiorin assay: Quantification of sclerotiorin was carried out using an HPLC
method (Weng et al. 2004).
Total carbohydrate assay: Carbohydrate content of the culture broth was quantified
using the phenol-sulphuric acid method (Chaplin 1994).
3. Results and Discussion
Bacitracin A overproduction: Based on our earlier optimization studies at SF scale
(Murphy et al, 2007b), addition of OG at concentration of 100 mgL-1 to the
B. licheniformis cultures was carried out in the bioreactors after 24 h of culture growth.
Control bioreactors runs were carried out under the same conditions as the test runs but
without OG supplementation. The results of the SF and STR fermentations were
compared. In all cases, the concentration (Figure 1) and the yield of bacitracin A was
higher in the elicited cultures compared to the test. Moreover, the yield increase in the
bioreactor runs was higher than the SF runs and the time to reach the maximum
production was decreased (Table 1). This suggests that scale-up runs under controlled
bioreactor conditions could further increase the productivity.
Table 1: Bacitracin A yield in the controlled and test conditions. Fermentations in
shaken flasks (SF); Fermentations in bioreactors (STR).
SF STR
Control Elicited Control Elicited
Time of max
production (h) 48 42
pH at max 8.01 8.13 8.30 8.30
Bacitracin A
Yield (mg g-1) 168.9 201.5 287.9 321.4
Yield %
increase 19.3 11.6
Figure 1: Bacitracin A concentration in the control and test cultures. Fermentations in
shaken flasks (SF); Fermentations in bioreactors (STR).
Sclerotiorin overproduction Addition of spent medium containing -butyrolactone
molecules from strain M to P. sclerotiorum IMI 040574 (Strain S) resulted in 6.4-fold
increase in the yield of sclerotiorin at 168 h post-inoculation (Table 2). The difference
between the biomass concentration of the test and control cultures was not significant (p
> 0.05) (data not shown). The difference in carbohydrate consumption rate was also
insignificant (p > 0.05). The results suggest that the spent medium from P. sclerotiorum
(strain M) containing multicolic acid (and related derivatives) may be involved in
quorum sensing process in the filamentous fungus P. sclerotiorum. The effects are
similar to those of biotic elicitors as the concentration of multicolic acid and its
derivatives in the spent medium used were small (data not shown) and the concentration
and rate of total carbohydrate consumption between the control and test cultures were
similar.
These results carry potential promise for use in pharmaceutical and biotechnology
industry where microbial communication may be used for the overproduction of
commercially desirable bioproducts.
Table 2: Sclerotiorin maximum production rate (120 – 168 h), maximum carbohydrate
consumptoion rate and yield fold increase in the test and control cultures of
P. sclerotiorum Strain S. The test cultures were supplemented with spent media from
P. sclerotiorum Strain M in volumes of 1.0 %v/v at 48 h post inoculation.
Sclerotiorin production rate
(μg g-1h-1) (120 – 168 h)
Yield
(fold increase)
Sclerotiorin consumption rate
(μg g-1h-1)(0 – 72 h)
Control 38.8 -- 0.140
Test 252.4 6.4 0.151
While the results of the two systems for elicitation and quorum sensing show notable
increases in the productivity of the target products, our studies on other systems (data
not shown) suggest that there are no generic biotic elicitors or quorum sensing
molecules to fit all microbial strains. The time of supplementation, the concentration
and the chemical structure of the molecules are essential in reaching satisfactory results.
Our investigation into the mechanism of these enhancers of productivity has revealed
activity at molecular level covering gene expression and, recently, changes in
intracellular calcium levels and protein phosphorylation.
The minimal requirements for the cultures when biotic elicitors and quorum sensing
molecules are used and the high increases in the productivity of the desired products
make these molecules suitable potential sources to be exploited as alternative methods
for industrial-scale overproduction.
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