DESC:FIELD

The present disclosure relates to prokaryote and/or eukaryote strains and a process for the development of prokaryote and/or eukaryote strains with increased/decreased protein synthesis for production of biomass/lipid.

BACKGROUND

Protein biosynthesis is a necessary process in all kingdoms of life. The genetic code is decoded using complex machinery which involves several protein factors and non-coding RNA like ribosomal RNA (rRNA) and transfer RNA (tRNA). Protein length is increased by the addition of amino acids with the help of individual tRNA molecules in a sequential manner dictated by the nucleotides on the messenger RNA (mRNA).

The process of protein biosynthesis involves three distinct steps: initiation, elongation and termination. In prokaryotes and eukaryotic organelles, an additional step called ribosome recycling is involved.

Initiation of protein biosynthesis is a rate-limiting step. The rates of initiation can be altered by altering the amounts of initiation proteins also called initiation factors and/or initiator tRNA in the cell. Initiator tRNA molecules are responsible for initiation of protein synthesis and their levels in a cell directly correlate to the levels of initiation. Earlier studies have shown that the levels of the initiator tRNA are dynamic in their expression, but the physiological significance remained unknown.

Growth of cells depends on the availability of proteins. Thus, increasing the availability of proteins by increasing the rates of one or more of the processes of initiation, elongation, termination, and ribosome recycling of protein synthesis in prokaryotes or eukaryotes should lead to increased growth of the organism and thereby increased biomass and overall productivity for production of desired products such as biodiesel and high value chemicals. The regulation of the rate of initiation is contemplated to be increased by increasing the levels of the translation factors involved in the processes of initiation, elongation, termination and/or ribosome recycling in protein synthesis. The regulation of the rate of initiation is also contemplated to be increased by increasing the levels of cellular RNA molecules involved in the protein synthesis process including mRNA, tRNA and rRNA. Similarly, decreasing the availability of proteins by decreasing the rates of one or more of the processes of initiation, elongation, termination, and ribosome recycling of protein synthesis in prokaryotes or eukaryotes would have major implications in the production of biofuels from prokaryotes/eukaryotes specifically algae/cyanobacteria which is dependent on biomass levels and lipid content.

Therefore, in accordance with the present disclosure there is envisaged a process for controlling and altering the levels of protein synthesis in prokaryotes and eukaryotes, specifically in microorganisms, and more specifically in algae and cyanobacteria.

This disclosure further envisages modified strains of prokaryotes and/or eukaryotes, specifically modified strains of microorganisms such as algae and/or cyanobacteria with increased/decreased protein synthesis for production of biomass/lipids.

OBJECTS

Some of the objects of the present disclosure which at least one embodiment is adapted to provide, are described herein below:

It is an object of the present disclosure to provide a process for increasing the levels of cellular initiator RNA molecules including mRNA, rRNA and tRNA to increase levels of protein synthesis and thereby biomass of a microorganism.

It is an object of the present disclosure to provide a process for decreasing the rates of one or more of the processes of initiation, elongation, termination, and ribosome recycling involved in protein synthesis in prokaryotes or eukaryotes to decrease levels of protein synthesis and thereby increase the lipid of an organism.

It is another object of the present disclosure to provide a process for increasing levels of protein synthesis in an organism to obtain increased overall productivity of products of interest.

It is yet another object of the present disclosure to provide a process for decreasing the levels of cellular initiator RNA molecules including mRNA, rRNA and tRNA to decrease protein synthesis and thereby increase lipid content in a microorganism.

It is an object of the present disclosure to provide a process for decreasing the rates of one or more of the processes of initiation, elongation, termination, and ribosome recycling involved in protein synthesis in prokaryotes or eukaryotes to decrease levels of protein synthesis and thereby increase the lipid of a microorganism.

It is another object of the present disclosure to provide a process for decreasing levels of protein synthesis in an organism to obtain increased lipid content leading to an increased overall productivity of biofuels.

It is still another object of the present disclosure to provide modified strains of eukaryotes/prokaryotes with increased/decreased protein synthesis for production of biomass/lipid.

Other objects and advantages of the present disclosure will be more apparent from the following description when read in conjunction with the accompanying drawings, which is not intended to limit the scope of the present disclosure.

SUMMARY

The present disclosure provides a modified strain of Synechococcus elongatus PCC 7942 having enhanced biomass synthesis capacity.

In another aspect of the present disclosure there is provided a process for achieving enhanced biomass synthesis in a microorganism, said process characterized by the following steps: cloning at least one gene selected from the group consisting of initiator tRNA gene, initiation factor-3 gene and peptidyl-tRNA hydrolase gene, in a vector; introducing said vector containing said gene into a microorganism; and growing said microorganism on a medium containing a selective agent to facilitate overexpression of said gene in the microorganism.

In another aspect of the present disclosure, there is provided a process for achieving enhanced lipid expression in a microorganism, said process characterized by the following steps: obtaining a knock-out gene construct for knocking out initiator tRNA gene; cloning said knock-out gene construct in a vector; introducing said vector containing the knock-out gene construct into a microorganism; and growing said microorganism on a medium containing a selective agent to facilitate the knock-out of said gene in the microorganism.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

The process of the present disclosure will now be described with the help of the accompanying drawings, in which:

Figure 1 illustrates a schematic process for controlling and regulating protein synthesis in prokaryotes and eukaryotes;
Figure 2 illustrates the pS1s-Ptrc vector containing initiator tRNA-Met1gene cloned into the vector;
Figure 3 illustrates the PCR amplification of wild type Synechococcus elongatus PCC 7942 and modified Synechococcus elongatus PCC 7942 containing extra genes for encoding initiator tRNA-Met1;
Figure 4A illustrates the spot inoculation of Synechococcus elongatus PCC 7942 on NON-IPTG plate and incubated at 30°C;
Figure 4B illustrates the spot inoculation of Synechococcus elongatus PCC 7942 on IPTG plate and incubated at 30°C;
Figure 5A illustrates the spot inoculation of Synechococcus elongatus PCC 7942 on NON-IPTG plate and incubated at 37°C;
Figure 5B illustrates the spot inoculation of Synechococcus elongatus PCC 7942 on IPTG plate and incubated at 37°C;
Figure 6A illustrates the growth curve of Synechococcus elongatus PCC 7942 transformants and control grown at 30°C;
Figure 6B illustrates the growth curve of Synechococcus elongatus PCC 7942 transformants and control grown at 37°C;
Figure 7 illustrates the PCR amplification of wild type Synechococcus elongatus PCC 7942 and modified Synechococcus elongatus PCC 7942 containing extra genes for encoding initiation factor 3 and peptidyl-tRNA hydrolase genes ;
Figure 8 illustrates the PCR amplification of knock-out construct prepared by 4-way Gibson cloning;
Figure 9 illustrates the knock-out construct prepared by deleting initiator tRNA met1;
Figure 10A illustrates the graphical representation of fluorescence observed for Chlamydomonas cells (Day 4) in Tris Acetate Phosphate medium with and without cycloheximide in Nile Red Assay;
Figure 10B illustrates Nile Red stained Chlamydomonas cells from the control sample; and
Figure 10C illustrates Nile Red stained Chlamydomonas cells from the sample containing cycloheximide.

DETAILED DESCRIPTION

Some prokaryote/eukaryote strains have multiple copies of initiator tRNA in their genomes. Increased global protein synthesis in these organisms will lead to increased biomass, which is a vital parameter for overall productivity in terms of the useful biotechnological products that can be obtained from such strains such as biofuels, high value chemicals, biodiesel and so on.

It is observed that the initiator tRNA molecules or initiation factor proteins/ribosomal RNA present in certain prokaryotes/eukaryotes are responsible for initiation of protein synthesis. It is possible to increase or decrease the amounts of cellular initiator tRNA molecules by genetic modification of the prokaryote/eukaryote organism to have either more or less copies of initiator tRNA molecules by altering gene expression (up-regulating/down-regulating gene expression). These modified strains will be very useful for the production of biofuels and high value chemicals.

Thus, increasing the availability of proteins by increasing rate of initiation of protein synthesis in prokaryotes/eukaryotes would lead to increased growth of the organisms and thereby increased biomass and overall productivity for production of products of interest such as biodiesel and high value chemicals.

Alternatively, decreased protein synthesis would lead to utilization of fixed carbon in making lipids in photosynthetic organisms. Therefore, decreasing availability of proteins by decreasing the rate of initiation of protein synthesis in prokaryotes/eukaryotes should lead to increased lipid content. This would have implications in biofuel production where amounts of biomass and lipid are major criteria.

Therefore, in accordance with the present disclosure there is envisaged a process for controlling and altering the levels of protein synthesis in prokaryotes and eukaryotes, specifically in microorganisms, and more specifically in algae and cyanobacteria (as depicted in Figure 1).

In an aspect of the present disclosure there is provided a process for increasing the levels of initiator tRNA to increase protein synthesis and thereby biomass in prokaryotes/eukaryotes.
The present disclosure envisages a process for increasing levels of protein synthesis to obtain increased overall productivity of desired products from prokaryotes/eukaryotes, specifically from algae/cyanobacteria.

In an embodiment of the present disclosure, genes involved in protein synthesis are cloned into the microorganism. The overexpression of these extra genes results in increased protein synthesis and thereby increased biomass production.
In the first step, a gene involved in protein synthesis is obtained. In an embodiment of the present disclosure, the gene cloned includes but is not limited to Initiator tRNA gene, Initiation Factor-3 gene and Peptidyl-tRNA hydrolase gene.

Next, the extra copies of the genes encoding for protein synthesis are cloned into a vector. In an exemplary embodiment of the present disclosure, the vector is pS1s-Ptrc.
The vector (as depicted in Figure 2) also contains a selectable marker such as an antibiotic compound. In an embodiment of the present disclosure, the antibiotic compound includes but is not limited to the group consisting of spectinomycin, streptomycin, ampicillin and carbenicillin.

The vector containing the gene to be overexpressed is introduced into the microorganism by direct DNA uptake method. In an embodiment of the present disclosure, the vector containing the gene to be overexpressed is introduced into the microorganism by transformation. The microorganism in accordance with the present disclosure includes prokaryotes/eukaryotes, specifically from algae/cyanobacteria. In an exemplary embodiment of the present disclosure, the microorganism is Synechococcus elongatus PCC 7942.
The initiator tRNA in an organism controls the rate of initiation of protein synthesis.
In an embodiment of the present disclosure two genes encoding initiator tRNA from Synechococcus elongatus PCC 7942 are cloned separately in a vector. In an exemplary embodiment of the present disclosure the vector used is pS1S-pTrc containing spectinomycin and streptomycin as antibiotics for selection (as depicted in Figure 2). The vector pS1S-pTrc is used for the transformation of Synechococcus elongatus PCC 7942 by direct DNA uptake method. The transformants are then confirmed by PCR (as depicted in Figure 3) and the growth analysis is performed (as depicted in Figures 6A and 6B). The transformants show a higher growth as measured by OD as compared to the control samples.

Initiation Factor-3 in an organism controls the rate of initiation of protein synthesis.
In an embodiment of the present disclosure the gene encoding initiation factor-3 from Synechococcus elongatus PCC 7942 is cloned in the vector pS1S-pTrc containing spectinomycin and streptomycin as antibiotics for selection. The vector pS1S-pTrc is used for transformation of Synechococcus elongatus PCC 7942 by direct DNA uptake method. The transformants are then confirmed by PCR and the growth analysis is performed. The transformants show a higher growth as measured by OD as compared to the control samples

Peptidyl-tRNA hydrolase (PTH) controls the rate of elongation of protein synthesis in an organism. In an embodiment of the present disclosure the gene encoding PTH from Synechococcus elongatus PCC 7942 is cloned in the vector pS1S-pTrc containing spectinomycin and streptomycin as antibiotics for selection. The vector pS1S-pTrc is used for transformation of Synechococcus elongatus PCC 7942 by direct DNA uptake method. The transformants are then confirmed by PCR and growth analysis is performed. The transformants show a higher growth as measured by OD as compared to the control samples.
Similar experiments were also carried out for E. coli transformed with initiator tRNA, initiation factor-3 and peptidyl-tRNA hydrolase genes. Higher growth as compared to the control samples were observed in case of E. coli also.

Growth analysis of the control and the transformed microorganism containing multiple copies of gene involved in protein synthesis show a clear advantage as depicted in Figures 6A and 6B.

It is known that a major amount of fixed carbon from photosynthesis is channeled to the production of proteins. Therefore, decreased protein synthesis would lead to utilization of this fixed carbon in making lipids, which would have implications in the production of biofuels from prokaryotes/eukaryotes, specifically from algae/cyanobacteria. The protein synthesis can be down-regulated and the carbon flux is redirected to the production of total lipid.

Therefore, in another aspect of the present disclosure there is provided a process for decreasing the levels of initiator tRNA to decrease protein synthesis and thereby increase lipid content in prokaryotes/eukaryotes.

The present disclosure envisages a process for decreasing levels of protein synthesis to obtain increased lipid content leading to an increased overall productivity of biofuels in prokaryotes/eukaryotes.

In the experiment carried out by chemically inhibiting protein synthesis using cycloheximide in Chlamydomonas (Chlamydomonas Resource Center, USA), it is found that the chemical inhibition of protein synthesis results in an increase in neutral lipid (as depicted in Figures 10A, 10B and 10C).

Transfer RNA Met1 and tRNA Met2 are two initiator tRNA genes present in cyanobacteria and both are involved in the global regulation of protein synthesis. These two genes are knocked out separately. A knock-out gene construct in accordance with the present disclosure is prepared by taking out 1 kb flanking sequences of the initiator tRNA gene and cloning in a vector containing antibiotic selective marker. In an embodiment of the present disclosure the selective marker is an antibiotic compound selected from the group including but not limited to kanamycin and chloramphenicol (as depicted in Figure 9). The PCR amplification of the of the knock-out gene constructed in accordance with the present disclosure by 4-way Gibson cloning is depicted in Figure 8; the different lanes indicate independent transformants, marker and positive controls at the end.

In 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/decreased protein synthesis for production of biomass/lipid, particularly, a modified strain in accordance with the present invention can be Synechococcus elongatus PCC 7942 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 1479/16.

The present disclosure is further described in light of the following examples which are set forth for illustration purpose only and not to be construed for limiting the scope of the disclosure.

Example 1: Transformation of Synechococcus elongatus PCC 7942
The media used was BG11 (Invitrogen) containing 1% agar (HiMedia).
10ml of inoculum containing Synechococcus elongatus PCC 7942 (Institut Pasteur, France) from the culture having an O.D750 of 2 was added in 40ml of fresh BG11 (Invitrogen) media in a 100ml Erlenmeyer flask. 10mM sodium bicarbonate (Sigma) was added to freshly inoculated culture (1ml was added from 0.5 M stock). The culture was incubated overnight at 30°C on a Kuhner Shaker at 100rpm in the presence of 2% CO2.
The culture was streaked on LB (Fluka) agar plate and incubated at 37°C for 24 hours to check for bacterial contamination. The plates were then incubated at room temperature for next 24 hours to check for fungal contamination. No contamination was observed on the plates.
The cells were then harvested in 50ml falcon tube. Culture was centrifuged at 4000rpm for 10 minutes at room temperature. The supernatant obtained was discarded. The pellet was re-suspended in 10ml BG11 media (Invitrogen). 10mM sodium bicarbonate (Sigma) was added (0.2ml was added from 0.5 M stock). The culture was centrifuged at 4000rpm for 10 minutes at room temperature. The supernatant was discarded. Pellet was re-suspended in 2.4ml fresh BG11 media (Invitrogen). 10mM sodium bicarbonate was added (0.048 ml was added from 0.5 M stock).
300µl of culture were distributed in sterile 1.5 ml Eppendorf tubes. 1000ng of DNA to be transformed was added to the tubes. In one of the tubes DNA was not added, which served as control for the experiment. Eppendorf tubes were placed in 50 ml falcon tubes and wrapped with aluminum foil. The tubes were then incubated overnight 30°C on a Kuhner shaker at 100rpm.
BG11 (Invitrogen) media plates containing appropriate antibiotics were prepared and the plates were dried in a biosafety cabinet for 30 minutes. The working stock of the antibiotics prepared is summarized in Table-1.
Table-1: Working stock of the antibiotics used
S. No. Antibiotic Working Stock
1. Carbenicillin (Sigma) 50 mg/ml in sterile distilled water
2. Kanamycin (Sigma) 50 mg/ml in sterile distilled water
3. Streptomycin (Sigma) 50 mg/ml in sterile distilled water
4. Spectinomycin (Sigma) 50 mg/ml in sterile distilled water
5. Chloramphenicol (Sigma) 35 mg/ml in ethanol

150µl from each tube was plated on the selection media containing the antibiotic using glass beads. Plates were incubated at room temperature, having a low light at 12h:12h light: dark cycle. After incubation, tiny colonies were observed on selection plate. 130 colonies were patched on fresh selection plates. 4 single colonies were used for colony PCR with NS primer to check for the integration.
Single colony was suspended in 50µl of sterile distilled water and incubated at 100°C for 15 minutes. This was used as the template for PCR. The concentration of the reagents used for PCR is summarized in Table-2.
Table-2
Working concentration Volume (µl)
Master Mix 2X – Sigma taq pol. 1X 5
Primer 10mM stock – DS5, MP83, MP84, MP86 0.5 mM 0.5 each
Template >250 ng 2
DNAse free water (Sigma) - 1
Reaction volume - 10

PCR was carried out as per the temperatures given below:
1 – 94°C for 5 minutes,
35 – 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 2 minutes,
1 – 72°C for 5 minutes.
The PCR amplification obtained is depicted in Figure 3.
After three generations of the 3 positive clones, single colony was inoculated from both pS1S-pTrc and pS1S-Met1 into 500µl of BG11 (Invitrogen) + Spectinomycin/Streptomycin (Sigma) 2µg/ml and incubated at 30°C with shaking and continuous light (intensity = 125µE/m2/s) for 5 days. The culture was first scaled up to 5ml, then to 50ml with the selective media.
Example 1A: Spot Assay
Neat (undiluted), 10-1, 10-2, and 10-3 dilutions were used for the spot assay,
10µl from each dilution was spot inoculated on 100mM IPTG and NON-IPTG plates in duplicates and were incubated at two different temperatures of 30°C and 37°C for 48 hours.
Growth of the transformants was observed in neat and 10-1 dilutions of the spot assay plates incubated at 30°C as depicted Figures 4A and 4B; and growth of the transformants was observed in neat and 10-1 dilutions of the spot assay plates incubated at 37°C as depicted Figures 5A and 5B.

Example 2B: Growth Curve Assay
Fresh inoculum having an initial OD of 0.2 - 0.3 was inoculated into 50ml BG11+ SP/SM 2µg/ml. The culture was incubated at two different temperatures of 30°C and 37°C with shaking at 100rpm shaking and in the presence of light for 72 hours. pS1s-pTRC was used as the control strain and the experimental strain was pS1S-pTrc-Met1. The experiment was carried out in triplicates with induction and without induction for both test and control.
The OD was monitored for 72 hours. Average of the triplicates was used for plotting the graphs.
Figure-6A illustrates the graphical representation of the OD at 30°C wherein Line-4 depicts pS1S-pTrc uninduced, Line-3 is pS1S-pTrc induced, Line-2 is pS1S-pTrc-Met1 uninduced and Line-1is pS1S-pTrc-Met1 Induced.
Figure 6B illustrates the graphical representation of the ODs at 37°C wherein Line-3 depicts pS1S-pTrc induced, Line-4 is pS1S-pTrc uninduced, Line-2: pS1S-pTrc-Met1 uninduced and Line-1 is pS1S-pTrc-Met1 Induced.
As illustrated in the Figures 6A and 6B comparatively higher OD was observed for the transformants as compared to the control samples, when incubated at 30°C and 37°C.

TECHNICAL ADVANCEMENT
The technical advancements offered by the present disclosure are as follows:

-The process of the present disclosure provides a process for increasing the levels of cellular initiator tRNA molecules to increase levels of protein synthesis and hence, the biomass of a microorganism.

-The process of the present disclosure provides a process for decreasing the levels of cellular initiator molecules to decrease protein synthesis and hence, increase lipid content of the microorganism.

-The process of the present disclosure provides modified microorganisms with increased/decreased protein synthesis for production of biomass/lipid.

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.
While certain embodiments of the inventions have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Variations or modifications in the process or compound or formulation or combination of this invention, within the scope of the invention, may occur to those skilled in the art upon reviewing the disclosure herein. Such variations or modifications are well within the spirit of this invention. ,CLAIMS:1. A modified strain of Synechococcus elongatus PCC 7942 having enhanced biomass synthesis capacity having CCAP Accession Number 1479/16.
2. A process for achieving enhanced biomass synthesis in a microorganism, said process characterized by the following steps:
a. cloning at least one gene selected from the group consisting of initiator tRNA gene, initiation factor-3 gene and peptidyl-tRNA hydrolase gene, in a vector;
b. introducing said vector containing said gene into a microorganism; and
c. growing said microorganism on a medium containing a selective agent to facilitate the overexpression of said gene and obtain enhanced biomass in the microorganism.
3. The process as claimed in claim 2, wherein said selective agent is an antibiotic compound.
4. The process as claimed in claim 3, wherein said antibiotic compound is at least one selected from the group consisting of spectinomycin, ampicillin, carbenicillin and streptomycin.
5. The process as claimed in claim 2, wherein said microorganism is selected from the group comprising prokaryotes and eukaryotes, preferably a photosynthetic microorganism.
6. The process as claimed in claim 5, wherein said photosynthetic microorganism is Synechococcus elongatus PCC 7942.
7. The process as claimed in claim 2, wherein said vector is pS1s-Ptrc.
8. A modified microorganism having enhanced lipid synthesis capacity.
9. A process for achieving enhanced lipid expression in a microorganism, said process characterized by the following steps:
a. obtaining a knock-out gene construct for knocking out initiator tRNA gene;
b. cloning said knock-out gene construct in a vector;
c. introducing said vector containing the knock-out gene construct into a microorganism; and
d. growing said microorganism on a medium containing a selective agent to facilitate the knock-out of said gene in the microorganism.
10. The process as claimed in claim 9, wherein said selective agent is an antibiotic compound.
11. The process as claimed in claim 10, wherein said antibiotic compound is at least one selected from the group consisting of kanamycin and chloramphenicol.
12. The process as claimed in claim 9, wherein said microorganism is selected from the group comprising prokaryotes and eukaryotes, preferably a photosynthetic microorganism.