Claims:I/We Claim:

1) A genetically modified cyanobacteria in which a gene encoding a non-cysteine lyase protein has been knocked out.

2) The genetically modified cyanobacteria according to claim 1, wherein the cyanobacteria is marine cyanobacterium.

3) The genetically modified cyanobacteria according to claim 1, wherein the cyanobacteria is selected from a group comprising Cyanobacterium sp., Geminocystis sp., Myxosarcina sp., Pleurocapsa sp., Hydrococcus sp., Arthrospira sp. and combinations thereof.

4) The genetically modified cyanobacteria according to claim 1, wherein the cyanobacteria is selected from a group comprising Cyanobacterium aponinum, Cyanobacteria bacterium J149, Cyanobacterium stanieri PCC 7202, Geminocystis sp. strain NIES-3709, Myxosarcina sp. GI1, Pleurocapsa sp. PCC 7319 and Hydrococcus rivularis.

5) The genetically modified cyanobacteria according to claim 1, wherein the cyanobacterium is Cyanobacterium aponinum PCC10605 (Accession No. CCAP 1455/1).

6) The genetically modified cyanobacteria according to any of the preceding claims, wherein the gene encoding the non-cysteine lyase protein corresponds to a nucleotide sequence selected from a group comprising SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13 and combinations thereof; or wherein the gene encoding the non-cysteine lyase protein corresponds to a nucleotide sequence which is at least 60%, 70%, 80%, 90% or 95% identical to SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11 or SEQ ID No. 13.

7) The genetically modified cyanobacteria according to any of the preceding claims, wherein the non-cysteine lyase protein corresponds to an amino acid sequence selected from a group comprising SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14 and combinations thereof; or wherein the non-cysteine lyase protein corresponds to an amino acid sequence which is at least 60%, 70%, 80%, 90% or 95% identical to SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12 or SEQ ID No. 14.

8) The genetically modified cyanobacteria according to any of the preceding claims, wherein a wild-type Cyanobacterium aponinum PCC10605 comprises nucleotide sequence set forth as SEQ ID No. 1, a wild-type Cyanobacteria bacterium J149 comprises nucleotide sequence set forth as SEQ ID No. 3, a wild-type Cyanobacterium stanieri PCC 7202 comprises nucleotide sequence set forth as SEQ ID No. 5, a wild-type Geminocystis sp. strain NIES-3709 comprises nucleotide sequence set forth as SEQ ID No. 7, a wild-type Myxosarcina sp. GI1 comprises nucleotide sequence set forth as SEQ ID No. 9, a wild-type Pleurocapsa sp. PCC 7319 comprises nucleotide sequence set forth as SEQ ID No. 11, a wild-type Hydrococcus rivularis comprises nucleotide sequence set forth as SEQ ID No. 13;
and wherein said nucleotide sequences set forth as SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, or SEQ ID No. 13 is knocked out to obtain the corresponding genetically modified cyanobacteria.

9) The genetically modified cyanobacteria according to any of the preceding claims, wherein said genetically modified cyanobacteria is the Cyanobacterium aponinum PCC10605 comprising SEQ ID No. 1 knocked out, the Cyanobacteria bacterium J149 comprising SEQ ID No. 3 knocked out, the Cyanobacterium stanieri PCC 7202 comprising SEQ ID No. 5 knocked out, the Geminocystis sp. strain NIES-3709 comprising SEQ ID No. 7 knocked out, the Myxosarcina sp. GI1 comprising SEQ ID No. 9 knocked out, the Pleurocapsa sp. PCC 7319 comprising SEQ ID No. 11 knocked out, or the Hydrococcus rivularis comprising SEQ ID No. 13 knocked out.

10) The genetically modified cyanobacteria according to any of the preceding claims, wherein the genetically modified cyanobacteria comprises a vector set forth as SEQ ID No. 15 for the knockout of the gene encoding the non-cysteine lyase protein.

11) The genetically modified cyanobacteria according to any of the preceding claims, wherein the knockout of the gene encoding the non-cysteine lyase protein prevents phycobilisome (PBS) degradation during stress conditions of growth of the genetically modified cyanobacteria.

12) The genetically modified cyanobacteria according to any of the preceding claims, wherein the genetically modified cyanobacteria has enhanced photosynthetic capacity and achieves enhanced biomass production relative to corresponding wild-type cyanobacteria.

13) A method of obtaining the genetically modified cyanobacteria as defined in any of the preceding claims, comprising knocking out the gene encoding the non-cysteine lyase protein in the corresponding wild-type cyanobacteria.

14) The method of obtaining the genetically modified cyanobacteria according to claim 13, comprising:
constructing a vector for the knock-out of the gene encoding the non-cysteine lyase protein; and
transforming wild-type cyanobacteria with said vector, to obtain the genetically modified cyanobacteria.

15) The method of obtaining the genetically modified cyanobacteria according to claim 13, wherein the vector is set forth as SEQ ID No. 15.

16) A lyase knock out vector set forth as SEQ ID No. 15.

17) A method of enhancing biomass production in cyanobacteria, said method comprising culturing the genetically modified cyanobacteria as defined in any of the preceding claims, to achieve enhanced biomass production.

18) The method according to claim 17, wherein the genetically modified cyanobacteria achieves enhanced biomass production relative to corresponding wild-type cyanobacteria under stress condition(s) of growth.

19) The method according to claim 18, wherein the stress condition is selected from a group comprising nutrient limitation, high light, high salinity, and combinations thereof.

20) The method according to any of the claims 17 to 19, wherein the nutrient limitation comprises deprivation of nutrients achieved by providing culture media only once during the growth, wherein the nutrients deprived is selected from a group comprising nitrogen, phosphorous, micronutrients, and combinations thereof; high light comprises light intensity ranging from about 500 µmol/m2/s to 2100 µmol/m2/s; and high salinity comprises salinity of 4% or more.

21) The method according to any of the claims 17 to 20, wherein the genetically modified cyanobacteria is cultured under a shallow culture depth of about 10 cm or below.

22) The method according to any of the claims 17 to 21, wherein said method is carried out at a temperature ranging from about 25°C to 45 °C and for a time-period ranging from about 6 hours to 300 hours, relative humidity of about 50% to 80%, CO2 air mixture of about 0.05% to 4%, pH of about 7.0 to 8.0, agitation or mixing at about 50-400 rpm, and light intensity of about 20 µmol/m2/s to 2500 µmol/m2/s.
, Description:TECHNICAL FIELD
[001]. The instant disclosure relates to the field of photosynthetic microorganisms and biomass production thereof. Particularly, the present disclosure relates to genetically modified/engineered Cyanobacterium having increased biomass potential for commercial production of algae biofuel and bioproducts.

BACKGROUND
[002]. Photosynthetic microorganisms such as cyanobacteria utilize antenna systems to capture photon to convert sunlight and CO2 into biomass and bioproducts. In particular, large antenna complexes named as phycobilisomes (PBS) harvest light energy in terms of photons and transfer the energy of photon to the reaction centre of photosystem II, where charge separation takes place and reducing equivalents or electrons are generated for further downstream reactions. The electrons further move through a series of protein complexes via carrier molecules in the linear electron transport chain to produce NADPH and ATP molecules to be used in carbon fixation step of Calvin cycle. However, there is a mismatch in the rate of collection of photon, electron transfer through linear electron transport chain and carbon fixation through Calvin cycle. Such mismatch in kinetic rates lead to the bottleneck in photosynthesis process where cyanobacteria fixes carbon using light, water and CO2.

[003]. While the presently known strategies focus on antenna truncation/reduction to improve the photon collection and utilization for efficient photosynthesis in cyanobacteria, such antenna truncation/reduction might create other unwanted cellular effect or reduction of cellular fitness, when grown in outdoor conditions for commercial cultivation.

[004]. Hence, there is a need for an alternate strategy for phycobilisome (PBS) antenna modification in cyanobacteria for efficient use of photons and converting them into biomass. The present disclosure tries to address said need.

SUMMARY
[005]. The present disclosure relates to genetically modified cyanobacteria in which a gene encoding a non-cysteine lyase protein has been knocked out.
[006]. In embodiments of the present disclosure, the genetically modified cyanobacterium is marine cyanobacterium.
[007]. In embodiments of the present disclosure, the genetically modified cyanobacteria is selected from a group comprising Cyanobacterium sp., Geminocystis sp., Myxosarcina sp., Pleurocapsa sp., Hydrococcus sp., Arthrospira sp. and combinations thereof.
[008]. In preferred embodiments of the present disclosure, the genetically modified cyanobacteria is Cyanobacterium aponinum PCC10605 having an Accession No. CCAP 1455/1.
[009]. The present disclosure further relates to a method of obtaining the genetically modified cyanobacteria described above, comprising:
constructing a vector for the knock-out of the gene encoding the non-cysteine lyase protein, and
transforming a wild-type cyanobacteria with said vector, to obtain the genetically modified cyanobacteria.
[0010]. The present disclosure also provides a lyase knock out vector set forth as SEQ ID No. 15, for obtaining the genetically modified cyanobacteria described above.
[0011]. The present disclosure further relates to a method of enhancing biomass production in cyanobacteria, said method comprising culturing the genetically modified cyanobacteria as described above, to achieve enhanced biomass production.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
[0012]. In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where:
[0013]. Figure 1 depicts circular plasmid/vector map for knock-out construction in Cyanobacterium aponinum.
[0014]. Figure 2 depicts biomass measurements in wild type Cyanoabacterium aponinum strain versus lyase knockout Cyanoabacterium aponinum strain. Significant biomass difference (~20%) was observed in lyase deletion strain over wild type strain.
[0015]. Figure 3 depicts ash free biomass analysis between in wild type Cyanoabacterium aponinum strain and lyase knockout Cyanoabacterium aponinum strain. The analysis showed significantly improved biomass in lyase deletion strain in high light conditions of about 1100 µmoles/m2/s.
[0016]. Figure 4 depicts light response curve for wild type and lyase deletion strains of Cyanoabacterium aponinum. The results showed about 10% improvement in maximum photosynthetic rate and improvement in photon quantum efficiency in the lyase deletion strain.
[0017]. Figure 5 depicts growth performance of wild type and lyase deletion strains of Cyanoabacterium aponinum in semi-turbidostat mode of cultivation at OD of 0.4 and at 10 cm shallow depth for 25 days.
[0018]. Figure 6 depicts growth performance of a wild-type Cyanoabacterium aponinum comprising cysteine lyase versus a genetically modified Cyanoabacterium aponinum strain comprising knock-out of cysteine lyase for about 23 days.

DESCRIPTION
[0019]. To address the limitations/needs as stated in the background, the present disclosure provides a genetically modified/recombinant Cyanobacterium having enhanced photosynthetic capacity.
[0020]. In an embodiment, the present disclosure relates to a genetically modified Cyanobacterium wherein a lyase gene has been knocked out or deleted.
[0021]. In an exemplary embodiment, the present disclosure relates to a genetically modified Cyanobacterium wherein a lyase gene encoding a non-cysteine lyase protein has been knocked out or deleted.
[0022]. As used herein, the terms/phrases such as ‘non-cysteine lyase’ or ‘non-cysteine lyase protein’ refer to lyase enzyme/protein containing no cysteine amino acid (or lacking cysteine amino acid).
[0023]. As used herein, the terms/phrases such as ‘non-cysteine lyase containing cyanobacteria’ refer to cyanobacteria containing lyase enzyme/protein which lacks a cysteine amino acid.
[0024]. As used herein, the terms ‘knockout’ or ‘knocked out’ refer to the genetic technique in which a gene(s) is deleted or made inoperative.
[0025]. The present invention particularly relates to genetically modified or recombinant cyanobacteria wherein a lyase gene which encodes a non-cysteine lyase protein is knocked out.
[0026]. In an embodiment of the present disclosure, the genetically modified Cyanobacterium is a marine cyanobacterium.
[0027]. In another embodiment of the present disclosure, the genetically modified cyanobacteria is selected from a group comprising Cyanobacterium sp., Geminocystis sp., Myxosarcina sp., Pleurocapsa sp., Hydrococcus sp., Arthrospira sp. and combinations thereof.
[0028]. In another embodiment of the present disclosure, the genetically modified cyanobacteria is selected from a group comprising Cyanobacterium aponinum, Cyanobacteria bacterium J149, Cyanobacterium stanieri PCC 7202, Geminocystis sp. strain NIES-3709, Myxosarcina sp. GI1, Pleurocapsa sp. PCC 7319 and Hydrococcus rivularis, and combinations thereof.
[0029]. In a preferred embodiment of the present disclosure, the genetically modified cyanobacteria is Cyanobacterium aponinum.
[0030]. In an exemplary embodiment of the present disclosure, the genetically modified cyanobacteria is Cyanobacterium aponinum PCC10605.
[0031]. In an embodiment of the present disclosure, the genetically modified cyanobacteria is Cyanobacteria bacterium J149.
[0032]. In another embodiment of the present disclosure, the genetically modified cyanobacteria is Cyanobacterium stanieri PCC 7202.
[0033]. In another embodiment of the present disclosure, the gene encoding the non-cysteine lyase protein is selected from a group comprising SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13 and combinations thereof.
[0034]. In another embodiment of the present disclosure, the non-cysteine lyase protein is selected from a group comprising SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, SEQ ID No. 12, SEQ ID No. 14 and combinations thereof.
[0035]. In an exemplary embodiment of the present disclosure, the genetically modified cyanobacteria is a knockout Cyanobacterium aponinum PCC10605 comprising knockout of a lyase gene encoding a non-cysteine lyase protein.
[0036]. In a preferred embodiment of the present disclosure, the genetically modified cyanobacteria is a knockout Cyanobacterium aponinum PCC10605 wherein SEQ ID No. 1 is knocked out.
[0037]. In an embodiment of the present disclosure, the genetically modified cyanobacteria is a knockout Cyanobacteria bacterium J149 comprising SEQ ID No. 3 knocked-out.
[0038]. In another embodiment of the present disclosure, the genetically modified cyanobacteria is a knockout Cyanobacterium stanieri PCC 7202 comprising SEQ ID No. 5 knocked out.
[0039]. In yet another embodiment of the present disclosure, the genetically modified cyanobacteria is a knockout Geminocystis sp. strain NIES-3709 comprising SEQ ID No. 7 knocked out.
[0040]. In still another embodiment of the present disclosure, the genetically modified cyanobacteria is a knockout Myxosarcina sp. GI1 comprising SEQ ID No. 9 knocked out.
[0041]. In still another embodiment of the present disclosure, the genetically modified cyanobacteria is a knockout Pleurocapsa sp. PCC 7319 comprising SEQ ID No. 11 knocked out.
[0042]. In still another embodiment of the present disclosure, the genetically modified cyanobacteria is a knockout Hydrococcus rivularis comprising SEQ ID No. 13 knocked out.
[0043]. In an embodiment of the present disclosure, the genetically modified cyanobacteria described herein comprises a vector for the knockout of the lyase gene encoding the non-cysteine lyase protein.
[0044]. In another embodiment of the present disclosure, the genetically modified cyanobacteria described herein comprises a vector set forth as SEQ ID No. 15 for the knockout of the lyase gene encoding the non-cysteine lyase protein.
[0045]. In a preferred embodiment of the present disclosure, the genetically modified Cyanobacterium aponinum PCC10605 is obtained by employing the vector set forth as SEQ ID No. 15 for the knockout of the lyase gene encoding the non-cysteine lyase protein.
[0046]. In an exemplary embodiment of the present disclosure, the genetically modified Cyanobacterium aponinum PCC10605 is obtained by employing the vector set forth as SEQ ID No. 15 for the knockout of SEQ ID No. 1.
[0047]. In an exemplary embodiment of the present disclosure, the knockout of lyase gene encoding non-cysteine lyase protein prevents phycobilisome (PBS) degradation during stress conditions of growth of the genetically modified cyanobacteria described herein.
[0048]. In another exemplary embodiment of the present disclosure, the genetically modified cyanobacteria described herein has enhanced photosynthetic capacity and achieves enhanced biomass production relative to corresponding wild-type cyanobacteria.
[0049]. The present disclosure further relates to a method of obtaining the genetically modified cyanobacteria described herein.
[0050]. In an embodiment of the present disclosure, the genetically modified cyanobacteria described herein is obtained by knocking out the lyase gene encoding the non-cysteine lyase protein in the corresponding wild-type cyanobacteria.
[0051]. In another embodiment of the present disclosure, the preparation of genetically modified cyanobacteria described herein comprises:
a) constructing a vector for the knock-out of the gene encoding the non-cysteine lyase protein; and
b) transforming a wild-type cyanobacteria with said vector, to obtain the genetically modified cyanobacteria.
[0052]. In an exemplary embodiment of the method described above, the genetically modified cyanobacteria is obtained by employing a vector set forth as SEQ ID No. 15.
[0053]. The present disclosure further relates to a lyase knock out vector. In an exemplary embodiment, the lyase knock out vector is set forth as SEQ ID No. 15.
[0054]. In an embodiment of the present disclosure, the lyase knock out vector comprises elements including but not limiting to origin of replication, promoter, a sequence corresponding to left homology arm of lyase gene, a selection marker, one or more antibiotic resistance marker/sequences, a sequence corresponding to right homology arm of lyase gene and a terminator.
[0055]. In an exemplary embodiment of the present disclosure, the lyase knock out vector of SEQ ID No. 15 comprises elements including but not limiting to origin of replication, promoter, ampicillin marker gene under a suitable promoter, a sequence corresponding to left homology arm of lyase gene set forth as SEQ ID No. 16, a kanamycin selection marker sequence set forth as SEQ ID No. 18 under rpsB promoter, a putative terminator, and a sequence corresponding to right homology arm of lyase gene set forth as SEQ ID No. 17.
[0056]. The present disclosure also relates to a method of enhancing biomass production in cyanobacteria, said method comprising culturing the genetically modified cyanobacteria described herein, to achieve said enhanced biomass production.
[0057]. In an embodiment of the present disclosure, the genetically modified cyanobacteria achieves enhanced biomass production relative to corresponding wild-type cyanobacteria under stress condition(s) of growth.
[0058]. In another embodiment of the present disclosure, the stress condition is selected from a group comprising nutrient limitation, high light, high salinity, and combinations thereof.
[0059]. In yet another embodiment of the present disclosure, the nutrient limitation comprises deprivation of nutrients achieved by providing culture media only once during the growth, wherein the nutrients deprived is selected from a group comprising nitrogen, phosphorous, micronutrients, and combinations thereof. In an exemplary embodiment, the nutrient limitation is achieved in batch mode of culturing/growth, wherein the nutrients are not limited intentionally and in the batch cultivation process, the media is added only once i.e. at the beginning of the cultivation. Accordingly, as cells grow, the nutrients become limiting thereby leading to nutrient limiting condition.
[0060]. In still another embodiment of the present disclosure, high light comprises light intensity ranging from about 500 µmol/m2/s to 2100 µmol/m2/s.
[0061]. In still another embodiment of the present disclosure, high salinity comprises salinity of 4% or more.
[0062]. In an embodiment of the present disclosure, the genetically modified cyanobacteria is grown/cultured under a shallow culture depth of about 10 cm or below.
[0063]. In another embodiment, the present method of culturing the genetically modified cyanobacteria for biomass production is carried out at a temperature ranging from about 25°C to 45°C.
[0064]. In another embodiment, the present method of culturing the genetically modified cyanobacteria for biomass production is carried out for a time-period ranging from about 6 hours to 300 hours.
[0065]. In yet another embodiment, the present method of culturing the genetically modified cyanobacteria for biomass production is carried out at a relative humidity of about 50% to 80%.
[0066]. In still another embodiment, the present method of culturing the genetically modified cyanobacteria for biomass production is carried out by employing a CO2 air mixture of about 0.05% to 4%.
[0067]. In still another embodiment, the present method of culturing the genetically modified cyanobacteria for biomass production is carried out pH of about 7.0 to 8.0.
[0068]. In still another embodiment, the present method of culturing the genetically modified cyanobacteria for biomass production is carried out under agitation or mixing at about 50-400 rpm.
[0069]. In still another embodiment, the present method of culturing the genetically modified cyanobacteria for biomass production is carried out at a light intensity of about 20 µmol/m2/s to 2500 µmol/m2/s.
[0070]. In preferred embodiments, the present method of culturing the genetically modified cyanobacteria described herein for biomass production is carried out under batch mode, fed-batch mode, semi-turbidostatic mode, or any combinations thereof.
[0071]. In an exemplary embodiment, the genetically modified cyanobacteria described herein is cultured under batch mode of cultivation.
[0072]. In an exemplary embodiment, the genetically modified cyanobacteria described herein is cultured under semi-turbidostat mode of cultivation.
[0073]. In an exemplary embodiment, the genetically modified cyanobacteria described herein is cultured under fed-batch mode of cultivation.
[0074]. In an embodiment, the present method of culturing the genetically modified cyanobacteria for biomass production employs culture medium selected from a group comprising but not limiting to urea phosphoric acid media, nitrate containing F/2 media, BG11 media with nitrate and optionally sodium bicarbonate, ASN III media, Bold Basal media and combinations thereof.
[0075]. In an exemplary embodiment, the present method of culturing the genetically modified cyanobacteria for biomass production is carried out in batch mode, wherein nutrient limitation is achieved after 3 to 4 days of growth. In particular, during batch process of cultivation, nutrients are added only once i.e. only at the beginning of the cultivation, and as the cells start growing, the nutrients are automatically limited after few days.
[0076]. In another exemplary embodiment, the present method of culturing the genetically modified cyanobacteria for biomass production is carried out in semi-turbidostatic mode, wherein a high salinity, high light or a combination thereof is employed. In a preferred embodiment, the high salinity is 4%. In another embodiment, the high light comprises a light intensity ranging from about 500 µmol/m2/s to 2100 µmol/m2/s.
[0077]. In preferred embodiments of the present disclosure, the enhancement in biomass yield by employing the genetically modified cyanobacteria described herein is about 10% to 20% relative to biomass yield achieved by the corresponding wild-type cyanobacteria.
[0078]. In an exemplary embodiment, the genetically modified cyanobacterial strain described herein was cultivated in daily dilution mode and daily harvesting mode of cultivation in high light and in high sea water salinity conditions for more than 25 days duration which resulted in a concomitant 12% enhancement of harvested biomass compared to the corresponding wild-type strain.
[0079]. Commercial cultivation of cyanobacteria and other related photosynthetic microorganisms require cellular fitness to grow in outdoor light conditions. The currently known strategies include engineering strains with antenna truncation. Said microorganisms are demonstrated to grow only in batch mode of cultivation where media is added only in the beginning of cultivation process and cells grow till the nutrient is not limited. However, for commercial cultivation, it is imperative to also grow the cyanobacteria in daily dilution mode or semi-turbidostat mode where cells are harvested daily and new media is added, which makes the process economically feasible. There is also the likelihood of fitness issue and compromise of biomass production in cyanobacteria with truncated antenna. Further, growth of engineered cyanobacteria with antenna truncation is demonstrated only in fresh water media and not in high salinity or sea water conditions with salinity such as 4% and above. However, growing cyanobacteria under high salinity or sea water is a must for commercial cultivation and economic feasibility for producing biofuel and bioproducts. Hence, there is a need to have engineered cyanobacteria strains which can grow and show improved biomass yields with modulation in photon capturing efficiency and photosynthesis capacity.
[0080]. The present disclosure thus provides genetically modified Cyanobacteria as described above and methods for enhancing biomass synthesis in said genetically modified Cyanobacteria. More particularly, in order to achieve commercial feasibility and cultivation of cyanobacteria in outdoor conditions, the genetically modified Cyanobacteria described herein has been engineered by modulating the post translational modification process of phycobilisome (PBS) synthesis. The same is achieved by circumventing the expression of lyase protein by deleting lyase gene which is involved in the post-translational modification of phycobiliproteins. In exemplary embodiments, said lyase gene encodes non-cysteine lyase protein. The genetically modified Cyanobacteria thus obtained can grow under stress conditions including nutrient deficiency, high salinity of 4% and above or sea water salinity, high light intensity, or any combination of said stress conditions. The present genetically modified Cyanobacteria is also shown to grow in shallow water depth of about 10 cm or more.
[0081]. In cyanobacteria, major antenna complexes consist of phycobilisome (PBS). PBS antenna synthesis and organization occurs through series of molecular reactions involving class of enzymes called bilin lyases which carries out creation of thio-ether linkages between specific bilins and phycobiliproteins or chromophores such as phycocyanin, allophycocyanin, phycoerythrins. Bilin lyases are involved in chromophorylation (chromophore attaching activity), chromophore detaching activity and chemical modification activity of phycobillisomes (PBS). During absorption of photon at high light or in stress conditions, PBS undergo lyase mediated degradation to supply nutrient in the form of nitrogen and sulphur. Due to the degradation of the antenna and at the same time high photon impinging rate, cellular fitness gets compromised. The presently developed genetically modified Cyanobacterium comprising knockout of lyase gene encoding lyase protein, more particularly a non-cysteine lyase protein, prevents chromophore detaching activity, and hence PBS is not degraded during stress conditions such as nutrient deficiency, high light and/or high salinity. In particular, the present invention targets post translational modification activity and/or preventing chromophore detaching activity in PBS through deletion/knockout of the lyase gene responsible for chromophore detachment. Without wishing to be bound by any theory, the present inventors hypothesize that the knockout of lyase gene to develop the present genetically modified cyanobacterial strains helps in PBS intactness during stress conditions such as nutrient deficiency, high light, high salinity, and improves photon efficiency, thereby enhancing the biomass production.
[0082]. In an exemplary embodiment of the present disclosure, to obtain a lyase knockout Cyanobacteria, a vector construct was developed using Golden Gate® cloning. The lyase gene to be knocked out was identified and a 2000 base pair polynucleotide sequence upstream and downstream of the lyase gene was isolated by PCR for homologous recombination. A kanamycin antibiotic selection marker under rpsB promoter was then introduced between these homology arms and ligated with origin of replication and ampicillin antibiotics marker for cloning in E. coli strain. The construct obtained was then employed to transform the Cyanobacterial strain by direct DNA uptake method and under kanamycin antibiotic selection marker. The resultant engineered strain has the capability of preventing PBS degradation activity under light stress and salinity stress condition and having better photon utilization efficiency compared to the corresponding wild type strain. When cultivated under shallow depth of 10 cm in daily dilution mode, the engineered strain shows enhanced biomass of more than 12% compared to the wild type strain. In addition to that, batch mode cultivation shows >20% improved biomass where nutrient limitation is evident. Hence, even during stress conditions of nutrient limitation, the engineered strain performs better compared to the wild type strain which reflects that antenna is intact and no degradation of PBS occurs and therefore leading to cellular fitness.
[0083]. The present disclosure thus successfully provides engineered or genetically modified Cyanobacteria having increased biomass potential for commercial production of algae biofuel and bioproducts.
[0084]. ADVANTAGES/BENEFITS:
The engineered/genetically modified cyanobacterial strains of the present disclosure has several advantages/benefits, including, but not limiting to the following:
a) The genetically modified cyanobacterial strains have higher photon quantum yield than the corresponding wild-type strain, higher electron turnover rate, higher chlorophyll a and higher maximum photosynthetic rate compared to the wild-type.
b) The genetically modified cyanobacterial strains can successfully grow under stress conditions of nutrient limitation, high salinity (4% or higher sea water salinity), and/or high light (light intensity ranging from about 500 µmol/m2/s to 2100 µmol/m2/s) even after genetic modification.
c) The genetically modified cyanobacterial strains prevent chromophore detaching activity and thereby prevent PBS degradation process because of deletion of lyase gene.
d) The genetically modified cyanobacterial strains have better photosynthetic capacity in commercial cultivation conditions and result in improvement of at least 10% or more in biomass yield relative to the wild-type cyanobacterial strains.
e) The genetically modified cyanobacterial strains possess antenna integrity in stress conditions. Hence, during stress conditions such as nutrient limitation, high light of about 1000-2100 µmoles/m2/s of PPFD, and/or high salt of about 4% sea water salinity or more, the present strains provide fitness and show an enhanced biomass production of at least about 10% to 20% relative to the wild-type cyanobacteria strain in environmental photobioreactor.
f) In batch mode cultivation under high light conditions in a multi-cultivator system, the present genetically modified cyanobacterial strains show almost 20% higher biomass and could reach 5 g/L of high density compared to the corresponding wild-type strain.
g) The genetically modified cyanobacterial strains show about 12% higher harvested biomass in daily dilution mode of cultivation compared to the corresponding wild-type.
h) The genetically modified cyanobacterial strains show biomass enhancement even at a shallow depth of 10 cm which has a huge commercial potential in terms of reduction in water movement cost through mechanical process and the cost of using fresh water. Globally, where fresh water availability is a major bottleneck and adds to huge costs in cultivation process, the present genetically modified cyanobacterial strains which possess salinity tolerance characteristics along with better photon utilization capacity and photosynthetic capacity will be beneficial for outdoor commercial cultivation for green fuel and bioproduct industry.
[0085]. Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above described embodiments, and in order to illustrate the embodiments of the present disclosure certain aspects have been employed. The examples used herein for such illustration are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein.

EXAMPLES
[0086]. Cyanobacterium aponinum strain was employed in the present examples which was obtained/sourced from Gagwa, Jamnagar, Gujarat, India.
[0087]. The developed genetically modified Cyanobacterium aponinum PCC10605 strain of the present disclosure comprising SEQ ID No. 1 knock-out was deposited with Scottish Association for Marine Science - Culture Collection of Algae and Protozoa (CCAP), United Kingdom under Accession No. CCAP 1455/1.

EXAMPLE 1:
Development of genetically modified Cyanobacteria
[0088]. To obtain a lyase knockout Cyanobacterium aponinum, a vector construct was made by using Golden Gate cloning. The lyase gene (SEQ ID No. 1) to be knocked out was identified from Cyanobacterium aponinum 10605 genome sequence and a 2000 bp sequence upstream and downstream of the lyase gene was isolated by PCR for homologous recombination. A kanamycin selection marker under rpsB promoter was then introduced between these homology arms and ligated with origin of replication and ampicillin marker for cloning in E. coli. Said vector/plasmid constructed for lyase knockout in Cyanobacterium aponinum is depicted in Figure 1 and the vector sequence is provided as SEQ ID No. 15.
[0089]. The construct obtained was then employed to transform wild-type Cyanobacterium aponinum by direct DNA uptake method and kanamycin selection. Transformation was done using following procedure.
1. Pre-culture: 50 ml of S-BG11 media was inoculated with wild-type Cyanobacterium aponinum colony that was grown on a fresh plate. The inoculum was grown in Percival Chamber with 600-1000 µmol/m2/s of sunlight-spectrum light and 2-5% CO2 for about 1 to 2 days or until OD of the culture reached about 0.5-6.
2. Transformation culture: Pre-culture was used to inoculate 50 ml of fresh S-BG11 to obtain a culture with OD 750 nm of 0.05-0.2. This culture was then grown overnight in 600 uE-1000 µmol/m2/s of sunlight-spectrum light and 2-5% CO2 until OD 750 of 1-6 was achieved, and was transferred to incubator for further growth for about 4 hours to 6 hours.
3. 5 ml worth of culture per each planned transformation in a screw cap conical tube was centrifuged for 10 min at about 3000-4000 relative centrifugal force (rcf) and aspirated leaving 200 µl per transformation volume behind to resuspend the DNA in, or more S-BG11 media was added to obtain 200 µl per transformation by resuspending the obtained cell pellet in S-BG11 media.
4. 1 to 5 µg of DNA was added to the prepared transformation culture. It was vortexed and was incubated on the bench for about 10 to 30 minutes.
5. Everything was plated on 1X BG11+1.5-2.0 Streptomycin/Spectinomycin (10mM-40 mM NaHCO3) plates. The plates were incubated in plate Percival in chemical room for about 3 to 10 days until the colonies were observed.

EXAMPLE 2:
Growth Assessment and Biomass Production
[0090]. I. For growth assessment of the Cyanobacterium aponinum lyase knock-out strain developed according to Example 1 versus the wild type Cyanobacterium aponinum, the following procedure was employed. The strains were inoculated overnight in 50 ml conical flask until the OD reached a mid-log phase. The wild-type and the lyase deletion strains were then adjusted to an OD of 0.4 with a total volume of 300 ml. About 80 ml of each in triplicates was then loaded on to the multicultivator and samples were taken at regular intervals for biomass estimation and OD measurement.
[0091]. Growth assessment conditions: Multicultivator, 12:12 Light:Day (L:D), 2% CO2, 1100 µmoles/m2/s light.
[0092]. The results are depicted in Figures 2 and 3, respectively. As seen from Figure 2, significantly improved biomass (~20% more) was observed in Cyanobacterium aponinum lyase deletion strain (D Lyase) when compared to wild type Cyanobacterium aponinum strain. Similarly, as seen from Figure 3, ash free biomass analysis showed significantly improved biomass in the Cyanobacterium aponinum lyase deletion strain when compared to wild-type strain (WT) in high light condition of 1100 µmoles/m2/s.

[0093]. II. The wild-type and the lyase deletion strains were also grown in environmental photobioreactor (ePBR) under very high light conditions. The results are depicted in Figure 4 and Table 1. The method followed was as follows:
a) Strains were evaluated in environmental photobioreactors (ePBR) under summer simulations (light: 2100 µmol/m2/sec; temperature: 280C-380C)
b) Strains were grown in Urea-Phosphoric acid media (UPA). The mode of growth was semi-turbidostat mode.
c) PI (photosynthesis-irradiance) curve measurements were taken using cells with concentration of 2µg/ml of chlorophyll a.
d) The steady-state photosynthesis was measured at incremental light intensities from 20-2000 µmol/m2/sec at a time interval of 5 minutes.
e) Quadratic equation based mathematical model was used to fit the experimental data.

[0094]. Equation used for plotting light response curve:
Ph. Rate (Pn) = (fI + Pgmax-v{(fI + Pgmax)2- 4? fI. Pgmax})/2?-RD

(Reference: Fitting net photosynthetic light-response curves with Microsoft Excel - a critical look at the models, Lobo et al 2013)

Table 1: Photosynthetic performance
Strain Pmax ? O Ic Ek
Cyanobacterium aponinum Wild type strain 60 0.2 0.7 30 350
Cyanobacterium aponinum Lyase deletion strain 65 0.25 0.7 30 300

[0095]. The results of Figure 4 and Table 1 show that the Cyanobacterium aponinum lyase deletion strain has the capability of preventing PBS degradation activity under light stress and salinity stress condition and has better photon utilization efficiency as depicted by the photosynthesis light response curve having higher photon quantum yield and higher Pmax compared to the wild type strain. In particular, as shown by the photosynthesis light response curve (Figure 4), the Cyanobacterium aponinum lyase deletion strain has larger slope i.e. higher photon quantum yield than the wild-type strain and higher maximum photosynthesis rate than the wild-type. These results demonstrate that the lyase deletion strain has better photosynthetic capacity in commercial cultivation conditions and resulted in more than 10% biomass improvement.

[0096]. III. Growth assessment in semi-turbidostat mode in shallow depth of 10 cm:
Commercial media having urea and phosphoric acid was used for initial inoculum. 20 ml of culture was grown in kuhner shaker for about 2 days (12 hours light, 27°C, 200 RPM, Humidity 70%, 2% CO2) and then sub-cultured according to experimental need. Experiment was started with fresh inoculum in ePBR and acclimatized for 3 days. The same culture was then used for experiment. ePBR conditions were 2100 µmol/m2/sec (about 14 hours), temperature 33±5 ?, 400 rpm, 2% CO2 purging).
[0097]. Experiments were done in duplicates for 25 days in semi turbidostat mode at 10 cm culture depth. Optical density was set and kept at 0.4 (at 750 nm) and harvesting Optimal Density was around 1.4 OD jump. Nutrient was maintained for enough Optimal Density jump.
[0098]. Cyanobacterium aponinum lyase deletion strain showed at least 12% higher biomass productivity (calculated based on average of 25 days) as compared to the corresponding wild-type strain in semi-turbidostat mode of cultivation.
[0099]. The above results thus successfully show enhanced biomass production by the lyase deletion/knock-out cyanobacteria of the present disclosure.
[00100]. Similar to the above examples/results under Examples 1 and 2, non-cysteine lyase knock-out strains of cyanobacterial species such as Cyanobacterium sp., Geminocystis sp., Myxosarcina sp., Pleurocapsa sp., Hydrococcus sp. and Arthrospira sp. which contain genes encoding non-cysteine lyase protein can be developed for achieving enhanced biomass production. For instance, non-cysteine lyase knock-out strains of cyanobacterial species such as Cyanobacterium aponinum, Cyanobacteria bacterium J149, Cyanobacterium stanieri PCC 7202, Geminocystis sp. strain NIES-3709, Myxosarcina sp. GI1, Pleurocapsa sp. PCC 7319 and Hydrococcus rivularisi can be developed based on the gene sequences encoding non-cysteine lyase and the corresponding non-cysteine lyase protein sequences as described under SEQ ID NOs. 3 to 14, respectively. Further, vectors similar to SEQ ID NO. 15/Figure 1 can be employed with minor modifications in right and left arm sequences for developing non-cysteine lyase knock-out strains of cyanobacterial species such as Cyanobacterium aponinum, Cyanobacteria bacterium J149, Cyanobacterium stanieri PCC 7202, Geminocystis sp. strain NIES-3709, Myxosarcina sp. GI1, Pleurocapsa sp. PCC 7319 and Hydrococcus rivularisi. Developing such non-cysteine lyase knock-out strains based on the reading of present disclosure is therefore well within the purview of a person skilled in the art.

EXAMPLE 3:
Growth Assessment of Cysteine containing Cyanobacteria
[00101]. A knock-out strain of cysteine containing NblA lyase in Cyanobacterium aponinum was developed. The growth of said knock-out strain was compared with the growth of wild-type strain for about 23 days. The results are provided under Figure 6. It was seen that no advantage in growth in the cysteine containing NblA lyase knock-out strain was found when compared to the wild-type strain containing cysteine containing NblA lyase. These results further infer that there was no enhancement in the biomass production in said cysteine containing NblA lyase knock-out when compared to the corresponding wild-type strain.
[00102]. The above observations are in sharp contrast to the experimental results obtained in Example 2 which show enhanced biomass production in cyanobacterial strain comprising knock-out of non-cysteine containing NblA lyase vis-à-vis the wild-type strain. Said results further establish the superiority of the presently developed genetically modified cyanobacteria in which the gene encoding a non-cysteine lyase protein was knocked out versus a genetically modified cyanobacteria comprising knock-out of gene encoding cysteine lyase.

[00103]. Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.
[00104]. The foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
[00105]. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” wherever used, 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.
[00106]. With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.
[00107]. Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.
[00108]. While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications 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 modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
[00109]. All references, articles, publications, general disclosures etc. cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication etc. cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.