A MODIFIED MICROORGANISM HAVING ENHANCED BIOMASS
SYNTHESIS CAPACITY AND A METHOD THEREOF
FIELD
The present disclosure relates to a modified microorganism having enhanced biomass
synthesis capacity and a method for manufacturing the modified microorganism.
BACKGROUND
Microorganisms such as algae are constantly exposed to harsh environmental
conditions during their life cycle. The harsh environmental conditions include abiotic
stresses such as ultraviolet radiation (UV), salinity, light, unfavorable temperature,
alkalinity, nutrient limitation, oxidative stress, senescence, sulfur deficiency, carbon
deficiency, nitrogen use inefficiency and the like. Biotic stresses include infection by
virus, bacteria, fungus or other stress causing pathogens. These conditions pose a
constant threat to the DNA integrity of these microorganisms and cause damage to
their DNA, such as modified bases, mispaired bases, intrastrand crossbinding,
interstrand crossbinding, pyrimidine dimers, single stranded breaks and double
stranded breaks (DSBs). This consequently leads to cell death, thereby preventing the
survival of these microorganisms in harsh environments. In order to overcome stress,
these microorganisms are required to physiologically adapt themselves to such harsh
environmental conditions. This may lead to loss of the unique traits of interest in these
organisms. The low survival capacity and loss of unique traits of interest in these
organisms are the major difficulties in exploiting their capabilities for industrial
purposes.
The repair of DNA double strand breaks (DSBs) is essential to maintain the integrity
of the genome. Un-repaired or improperly repaired DNA damage may result in
genomic instability and eventually in cell death.
Many proteins are necessary for such DNA DSB repair. One such important protein is
Rad52 protein, which is necessary for accurate repair of DSBs and is highly conserved
among eukaryotes, including animals, fungi and yeast. It also plays an auxiliary role
with the replication protein A (RPA) in the action of Rad51 protein. Furthermore,
Rad52 protein has an annealing activity and promotes the formation of D-loops in
super helical DNA.
Disruption of RAD52 gene may cause severe recombination phenotype including
extreme X-ray sensitivity, increased chromosome loss and failure to produce viable
spores. Research on Rad52 mutants in Saccharomyces cerevisiae has revealed a
critical role of Rad52 protein in double-strand break repair and meiosis. It has been
revealed that in Saccharomyces cerevisiae, homologous recombination provides a
major mechanism for eliminating DNA double-stranded breaks which may be induced
by ionizing radiations or may be associated with injured DNA replication forks; and
Rad52 protein plays a fundamental role in homologous recombination pathway and
DNA double strand break repair.
WO2003089573 suggests a method of identifying compounds that induce a DNA
repair pathway and/or inhibit retroviral cDNA integration into a host genome.
US200401 11764 suggests an expression cassette comprising a meiotically active
promoter operably linked to a polynucleotide encoding a recombinational DNA repair
polypeptide or its fragment, which is capable of stimulating plant meiotic
recombination when expressed into RNA and/or said polypeptide.
However, these conventional methods are typically incapable of enhancing the
survival capacity of microorganisms along with enhanced biomass synthesis and
simultaneously maintaining their genomic integrity in an effective and efficient
manner, wherein confirmed stable transgene integration is being achieved.
Additionally, some conventional methods employ a large number and/or quantity of
chemicals in order to facilitate enhanced biomass synthesis.
Therefore, there exists a need to develop an efficient and effective method for
enhancing the biomass synthesis capacity of a microorganism.
OBJECTS
Some of the objects of the present disclosure, which at least one embodiment herein
satisfies, are as follows:
It is an object of the present disclosure to provide a modified microorganism having
enhanced biomass synthesis capacity.
It is another object of the present disclosure to provide a method for manufacturing a
modified microorganism having an enhanced biomass synthesis capacity.
It is yet another object of the present disclosure to provide a method for manufacturing
a modified microorganism having an enhanced DNA repair capacity in response to
stress for achieving increased biomass.
It is still another object of the present disclosure to provide a method for overexpressing
yeast RAD52 gene in a microorganism which is effective in inducing DNA
repair in the microorganism.
It is yet another object of the present disclosure to provide an expression vector
comprising a yeast RAD52 gene and a promoter capable of promoting the expression
of said gene in DNA damaging conditions.
Other objects and advantages of the present disclosure will be more apparent from the
following description, which is not intended to limit the scope of the present
disclosure.
SUMMARY
In an aspect of the present disclosure there is provided a modified strain of
Chlamydomonas reinhardtii CC125-45-03 having enhanced biomass synthesis
capacity.
In another aspect of the present disclosure there is provided a method for enhancing
biomass synthesis capacity in a microorganism, the method comprises the following
steps: synthesizing a gene encoding a protein capable of inducing DNA repair;
cloning said obtained gene along with a promoter capable of regulating the expression
of said gene in DNA damaging conditions in an expression vector; introducing said
expression vector comprising said gene and said promoter into a microorganism;
growing said microorganism on a medium containing a selective agent; and exposing
said microorganism to stress to facilitate overexpression of said gene and obtaining a
microorganism with enhanced biomass synthesis capacity.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
The method of the present disclosure will now be described with the help of the
accompanying drawing, in which:
Figure 1 illustrates a flow chart depicting the function of RAD52 in the method of the
present disclosure;
Figure 2 illustrates the codon optimized sequence of RAD52;
Figure 3 illustrates the pChlamy_l vector containing the RAD52 gene cloned in the
Notl site, and also containing the Hsp70A-RbcS2 Promoter; and
Figure 4 illustrates the pChlamy_l vector containing the RAD52 gene cloned in the
Notl site and also containing the High Light Inducible (Hli) promoter;
Figure 5 illustrates the growth of wild type and transformants without UV treatment;
and
Figure 6 illustrates the growth of wild type and transformants after UV treatment.
DETAILED DESCRIPTION
The conventional methods for inducing DNA repair in microorganisms are not
effective in enhancing the biomass synthesis along with the DNA repair capacity in
these organisms. Specifically, the conventional methods fail to address prolonged
enhancement of the survival capacity of organisms in harsh environmental conditions
including abiotic and biotic stresses, which pose a major threat to their DNA integrity
and cause DNA damage leading to cell death. In order to overcome stress caused by
such conditions, cells of the organisms are required to physiologically adapt
themselves to the harsh environmental conditions, which may lead to a loss of the
unique traits of interest in these organisms. Alternately, the cells may not be able to
tolerate the stress at all.
The present disclosure therefore, provides a method for manufacturing a modified
microorganism having an enhanced biomass synthesis capacity. The enhanced
biomass synthesis capacity is a result of the overexpression of a gene capable of
inducing DNA repair.
In the modified microorganism of the present disclosure, the DNA repair process
initiates with the nucleolytic processing of the ends of DNA breaks to yield 3'ssDNA
tails, which are bound by recombination factors to form nucleoprotein complex. This
nucleoprotein complex then conducts a search to locate an undamaged DNA
homologue and further catalyzes the formation of a DNA joint called the D-loop, with
the homologue. The proteins encoded by evolutionarily conserved genes of the
RAD52 group catalyze the homologous recombination reaction.
Accordingly, in the present disclosure, a gene encoding a protein capable of inducing
DNA repair is obtained. The function of RAD52 of the present method is illustrated as
a flow chart in Figure- 1. In an embodiment of the present disclosure, the gene is yeast
RAD52 gene encoding Rad52 protein. An example of the yeast in accordance with the
present disclosure is Saccharomyces cerevisiae.
In accordance with one embodiment of the present disclosure, the gene is native yeast
RAD52 gene. The expression of native yeast RAD52 gene is regulated by a suitable
promoter. In accordance with one embodiment of the present disclosure, the promoter
is a constitutive promoter. Alternatively, an inducible promoter is used in the present
disclosure. The inducible promoter is capable of promoting the expression of the
RAD52 gene at DNA damaging conditions.
In accordance with an embodiment of the present disclosure, the promoter is a nonyeast
light inducible promoter (LIP). The promoter is at least one light inducible
promoter selected from the group comprising Dunaliella, Synechococcus elongatus
PCC 7942 and rbcS promoter.
In accordance with another embodiment of the present disclosure, the gene is
recombinant yeast RAD52 gene.
The DNA sequence of the synthesized RAD52 (recombinant) gene comprises codons
which are optimized for over-expression of said gene in a non-homologous host. The
sequence of the codon optimized RAD52 gene with Sequence ID No. 1 is depicted in
Figure-2. The synthesized gene is then cloned in an expression vector. The expression
vector includes but is not limited to a circular plasmid. Rad52 gene from
Saccharomyces cerevisiae is cloned in the Notl site of pChlamy_l. The pChlamy_l
vector containing the Rad52 gene from Saccharomyces cerevisiae is illustrated in
Figure 3 and Figure 4.
In accordance with one embodiment of the present disclosure, there is provided an
expression vector comprising yeast RAD52 gene driven by either an Hsp70A-RbcS2
promoter (as illustrated in Figure 3) or a high light inducible (Hli) promoter (as
illustrated in Figure 4).
The RAD52 gene-containing vector also contains a selectable marker. The selectable
marker comprises a resistance agent possessing resistance to at least one compound
selected from the group which includes but is not limited to an antibiotic compound,
an antifungal compound and a toxic compound.
The cells of the untransformed microorganism are then transformed with the
expression vector comprising the RAD52 gene to obtain modified microorganism.
The cells of the untransformed microorganism are transformed with an expression
vector by at least one method selected from the group which includes but is not
limited to biolistics, agrobacterium mediated genetic transformation, and
electroporation, preferably electroporation.
In accordance with the present disclosure, the microorganism may be a prokaryotic
microorganism which includes but is not limited to bacteria. The organism may be a
eukaryotic organism which includes but is not limited to plants. The organism may be
a photosynthetic organism which includes but is not limited to plants and algae.
In a preferred embodiment of the present disclosure, the microorganism is an alga.
Alga when used according to the present disclosure is selected from the group
including but not limited to Dunaliella, Chlorella, Nannochloropsis and
Chlamydomonas.
The modified microorganism is cultured on a medium containing at least one selective
agent. The selective agent is at least one compound selected from the group
comprising an antibiotic compound, an antifungal compound and a toxic compound.
The selection agent is at least one antibiotic compound selected from the group which
includes but is not limited to zeocin, kanamycin, chloramphenicol, and hygromycin,
preferably hygromycin. In a preferred embodiment of the present disclosure, the
amount of hygromycin in the medium is 60mg/liter.
Preferably, the culture of the modified microorganism is incubated after
transformation for a time period of 10 to 48 hours before being plated on the medium
containing a selective marker.
The modified microorganisms are selected and isolated based upon the expression of
the selectable marker. The modified microorganisms are screened by molecular
analysis and those resistant to the selective agent are isolated.
In accordance with another embodiment of the present disclosure, the method for
manufacturing a modified microorganism having an enhanced biomass synthesis
capacity in response to stress, further comprises the steps which are described herein
below:
The isolated modified microorganisms are cultured in a selection medium comprising
hygromycin after 12 to 24 hours of isolation, for a time period of 15 to 20 days.
The progeny of the modified microorganism is then isolated to obtain a modified
microorganism having a gene to induce DNA repair in the microorganism in DNA
damaging conditions. The progeny of modified microorganism is analyzed for stable
integration of yeast RAD52 gene by at least one method which includes but is not
limited to Polymerase Chain Reaction, Southern Blot and Northern Blot. This is
followed by isolation of the modified microorganism containing RAD52 gene.
The modified microorganism obtained by the method of the present disclosure
contains RAD52 gene, which is over-expressed to induce DNA repair in the
microorganism in DNA damaging conditions to provide a modified microorganism
having an enhanced biomass synthesis capacity as a result of overexpression of the
gene capable of inducing DNA repair mechanism. The RAD52 gene is expressed
when DNA damage is most and the DNA repair machinery is required.
In accordance with another aspect of the present disclosure, there are provided
modified strains of prokaryotes and/or eukaryotes, specifically modified strains of
algae and/or cyanobacteria with increased biomass synthesis capacity, particularly, a
modified strain in accordance with the present invention can be Chlamydomonas
reinhardtii CC125-45-03 deposited in the Culture Collection of Algae and Protozoa
(CCAP), SAMS Limited, Scottish Marine Institute, Dunbeg, Oban, Argyll, PA37
1QA, UK having CCAP Accession Number 11/172.
The modified microorganism manufactured in accordance with the present disclosure
has a specific promoter which regulates the expression of RAD52; whereby RAD52 is
overexpressed resulting in increased biomass synthesis as a result of the enhanced
DNA repair in the modified microorganism. A 5-fold increase in viability can be
achieved in the modified microorganism. The transformants (RAD52 gene is overexpressed
to induce DNA repair) of the present disclosure and wild type are treated
with UV light and then allowed to grow. An initial decrease in the growth is observed
due to the damage to DNA, but the transformants recover much faster as compared to
the wild type as illustrated by the OD measured at 750nm. The enhanced DNA repair
capacity is due to the over-expression of the RAD52 gene.
The present disclosure will now be described in the light of the following non-limiting
examples:
Example 1: Transformation of Chlamydomonas using electroporation method
Chlamydomonas cells (Invitrogen) were grown in Tris Acetate Phosphate (TAP)
media (Invitrogen) at a temperature of 20°C for 4 to 6 days. After the completion of
growth, 10% Tween 20 was added to 4 x 108 cells of Chlamydomonas. The cells were
centrifuged at 200 rpm at a temperature of 4°C for 5 minutes. The cells were then resuspended
in ice-cold 4ml TAP medium containing 40 mM sucrose solution. 2.5
micrograms of pChlamy_l plasmid (Invitrogen) containing the Rad52 gene along with
High Light Inducible (Hli) promoter was linearised by restriction digestion and the
plasmid DNA was dissolved in 301 TAP medium containing 40 mM sucrose
solution. 50 g of salmon sperm DNA dissolved in 51of water (10 g/ l) was used
as a carrier and denatured by heating at a temperature of 95°C. The salmon sperm
DNA solution (in water) was then added to the cell suspension followed by the
addition of the plasmid DNA prepared as described above to the cell suspension. A
final volume of 250 microliters of the above mixture comprising cell suspension,
plasmid DNA & carrier salmon sperm DNA was taken in a 4 mm cuvette and
electroporation was performed using 720-920 volts, 10 microfarads capacitance using
Biorad electroporator. After electroporation, cells were kept in a water bath at 25°C
for 12 hours. The cells were then plated on TAP media containing 40 mM sucrose
solution. 0.5% agarose, 1ml 20% corn starch, 0.4% polyethylene glycol and 10 g/ml
hygromycin was added to the TAP medium containing 40 mM sucrose solution.
Transformants were obtained and re-streaked on hygromycin containing medium for
further confirmation.
Example 2 : Comparison of transformed Chlamydomonas and control response to
stress
107 cells of transformed Chlamydomonas obtained by the method of Example 1 and
control without Hli-Rad52 were grown in 3ml of Tris Acetate Phosphate (TAP) media
(Invitrogen) for 3 days. After the completion of the growth, both the transformed
Chlamydomonas and the control were exposed to 100,000 lux for 3 hours. After 3
hours, the cells were allowed to grow at normal conditions having a light intensity of
10,000 lux.
After 3 to 4 days, the control culture showed a color change from green to white due
to bleaching/cell death. The control culture could not adapt to the stress and could not
survive after 3 to 4 days. In contrast, the culture of the transformed Chlamydomonas
retained the green color indicating that it was able to adapt to the stress and survive.
The transformed Chlamydomonas cells were able to survive for 15 to 20 days,
indicating approximately a 5-fold increase in viability in the transformed
Chlamydomonas.
Example 3 : Comparison of wild type (WT) and transformed Chlamydomonas to
UV light treatment
Five Chlamydomonas transformants obtained by the method of Example 1 and WT
cultures without Rad52 were taken for this study. The five transformants and WT
culture were inoculated in 20ml TAP media (Invitrogen) and incubated under
continuous light till a cell density of 106 cell/ml was obtained (approximately 0.1 OD
at 750nm). After the completion of growth the transformants and the WT culture were
transferred to multi-well plates having 6 wells. The transformants and the WT culture
were illuminated by 2500 of UV. After the cultures were exposed to
UV light, both the transformants and the WT culture were incubated in the dark for 24
hours. Next, the transformants and the WT culture were incubated at 25°C, a light
intensity of 100 2 1 and a light: dark cycle of 12: 12 hours.
Similar plate of transformants and WT culture was incubated without the UV
treatment. The experiment was conducted in duplicate and the average was taken for
plotting graph. Figure 5 illustrates the growth of wild type and transformants without
UV treatment incubated at 25°C, a light intensity of 100 2 1 and a light: dark
cycle of 12: 12 hours. As depicted in Figure 5, both the WT and the transformants
show similar growth rates over a period of 8 days.
Figure 6 illustrates the growth of wild type and transformants given UV treatment
(2500
and a light: dark cycle of 12: 12 hours. As depicted in Figure 6, initially (Day 4) a
decrease in the OD of both WT and transformants was observed. After 4 days the
cultures start recovering and growing resulting in an increase in the OD. The
numerical values of the OD values of the WT and three of the transformants is given
in Table 1 below. It is clearly seen from Figure 6 and Table 1, that three of the
transformants RAD_02, RAD_03 and RAD_05 show an increase in the OD as
compared to the wild type, thereby indicating increased biomass of the transformants
as compared to the wild type. The recovery is faster in transformants as compared to
the wild type as depicted in Figure 6.
Table 1: Numerical OD values as seen in Figure 6 of the drawings
It would be appreciated by a person of ordinary skill in the art that suitable
modifications may be carried out in the method of the present disclosure and still
achieve the results as those achieved by the method of the present disclosure. Such
modifications by virtue of their nature are a part of the present disclosure. The method
provided herein is illustrative and does not rule out such modifications.
ECONOMIC SIGNIFICANCE AND TECHNICAL ADVANCEMENT
The technical advancements offered by the present disclosure are as follows:
• The present disclosure provides a modified microorganism having enhanced
biomass synthesis capacity.
• The present disclosure provides a method for manufacturing a modified
microorganism having enhanced biomass synthesis capacity.
• The method of the present disclosure is simple, efficient and cost-effective due
to use of lesser number of chemicals (as opposed to some conventional
methodologies) and use of efficient strain(s) constructed having enhanced
biomass synthesis capacity.
While considerable emphasis has been placed herein on the preferred embodiments, it
will be appreciated that many embodiments can be made and that many changes can
be made in the preferred embodiments without departing from the principles of the
disclosure. These and other changes in the preferred embodiments as well as other
embodiments of the disclosure will be apparent to those skilled in the art from the
disclosure herein, whereby it is to be distinctly understood that the forgoing
descriptive matter is to be implemented merely as illustrative of the disclosure and not
as limitation.
Throughout this specification the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a stated element, integer
or step, or group of elements, integers or steps, but not the exclusion of any other
element, integer or step, or group of elements, integers or steps.
The use of the expression "at least" or "at least one" suggests the use of one or more
elements or ingredients or quantities, as the use may be in the embodiment of the
invention to achieve one or more of the desired objects or results.
Any discussion of documents, acts, materials or the like that has been included in this
specification is solely for the purpose of providing a context for the invention. It is not
to be taken as an admission that any or all of these matters form part of the prior art
base or were common general knowledge in the field relevant to the invention as it
existed anywhere before the priority date of this application.
The numerical values mentioned for the various physical parameters, dimensions or
quantities are only approximations and it is envisaged that the values higher/lower
than the numerical values assigned to the parameters, dimensions or quantities fall
within the scope of the disclosure, unless there is a statement in the specification
specific to the contrary.
CLAIMS
1. A method for enhancing biomass synthesis capacity in a microorganism,
said method characterized by the following steps:
a. synthesizing a gene encoding a protein capable of inducing DNA repair;
b. cloning said synthesized gene along with a promoter capable of
regulating the expression of said gene in DNA damaging conditions in
an expression vector;
c. introducing said expression vector comprising said gene and said
promoter into the microorganism;
d. growing said microorganism in a medium containing a selective agent;
and
e. exposing said microorganism to stress to facilitate overexpression of
said gene and obtaining the microorganism with enhanced biomass
synthesis capacity.
2. The method of claim 1, wherein said gene is a yeast RAD52 gene encoding
Rad52 protein, selected from a group comprising native yeast RAD52 gene
and recombinant yeast RAD52 gene.
3. The method of claim 2, wherein said yeast is Saccharomyces cerevisiae and
wherein yeast RAD52 gene is a codon optimized gene with Sequence ID
No. 1.
4. The method of claim 1, wherein said microorganism in step (c) is selected
from the group comprising prokaryotes and eukaryotes, preferably a
photo synthetic microorganism.
5. The method of claim 1, wherein said microorganism is an alga, selected
from the group comprising Dunaliella, Chlorella, Nannochloropsis and
Chlamydomonas.
6. The method of claim 1, wherein said promoter is at least one light inducible
promoter selected from the group comprising Dunaliella, Synechococcus
elongatus PCC 7942 and rbcS promoter.
7. The method of claim 1, wherein said expression vector is pChlamy_l.
8. The method of claim 1, wherein said selective agent in the medium is at
least one compound selected from the group comprising antibiotic
compound, antifungal compound and toxic compound.
9. The method of claim 1, wherein said antibiotic compound is at least one
selected from the group comprising zeocin, kanamycin, chloramphenicol
and hygromycin, preferably hygromycin.
10. The method of claim 1, wherein in step (e), said microorganism is exposed
to at least one stress selected from the group comprising ultraviolet
radiation (UV), salinity, light, unfavorable temperature, alkalinity, nutrient
limitation, oxidative stress, senescence, sulfur deficiency, carbon
deficiency, nitrogen use inefficiency, virus, bacteria and fungus.
11. A method for manufacturing a modified microorganism having enhanced
biomass synthesis capacity, said method characterized by the following
steps:
a. synthesizing a gene encoding a protein capable of inducing DNA repair;
b. cloning said synthesized gene along with a promoter capable of
regulating the expression of said gene in DNA damaging conditions in
an expression vector;
c. introducing said expression vector comprising said gene and said
promoter into the microorganism;
d. growing said microorganism on a medium containing a selective agent;
and
e. exposing said microorganism to stress to facilitate overexpression of
said gene to obtain the microorganism having enhanced biomass
synthesis capacity,
wherein, the biomass synthesized by the modified microorganism is
increased relative to the unmodified microorganism.
12. A method for increasing algal biomass; said method characterized by the
following steps:
a. synthesizing a gene encoding a protein capable of inducing DNA repair;
b. cloning said synthesized gene along with a promoter capable of
regulating the expression of said gene in DNA damaging conditions in
an expression vector;
c. introducing said expression vector comprising said gene and said
promoter into an alga
d. growing said alga in a medium containing a selective agent; and
e. exposing said alga to stress to facilitate overexpression of said gene to
obtain enhanced algal biomass.
13. A modified microorganism manufactured by the process claimed in claim
11.
14. A modified strain of Chlamydomonas reinhardtii CC125-45-03 having
CCAP Accession Number 11/172.