Very recently with the increasing price of fuel and due to its limited availability, the scientific community and several industries are now concern about finding a different source of fuel generation. Biofuel due its easy generation, cost effectiveness is now the main attraction of scientists and several industries. Among different types of organism cyanobacteria are the perfect choice for biofuel production of their photosynthetic capacity, easy gene manipulation and lack of dependency on fertile land. There are several ways to generate biofuel from cyanobacteria among them the newly developed CRISPR technology is an efficient way to generate biofuel from cyanobacteria. This review highlights the ways through which biofuel can be generated efficiently using CRISPR technology.
In the 21st century demand of biofuel production is really high because of its huge energy generation capacity and many other industrial purposes. Very recently due to excessive requirement and use of fossil fuels, a huge number of environmental as well as economical concerns are arising. So, there is a need of finding alternative sources of fuel production. One of the best solutions to this problem is to increase the use of biomass generated biofuel. Biomass generated biofuel is eco-friendly and as well as cost effective. The production of biofuel is drawing a lot of attention from various industries. The production of biofuel as an alternative of fossil fuel is growing rapidly over the past few years. To produce butanol and ethanol, in the fermentation process starch or sugar as a feed stock can be used. To produce biodiesel, transesterification of lipids (obtained from soybeans, seeds of canola and others) is used.
There are different sources for biofuel production but photosynthetic organisms are considered one of the best sources for it because atmospheric carbons can be easily obtained from the harnessed light energy can be used to direct the obtained carbon towards the biofuel production. Cyanobacteria are highly favored among them because of their excellent amenability towards genetic manipulation.
Over the past decade there are different approaches has been made for production of biofuel in cyanobacteria. However recently with the advancement in Clustered Regularly Interspaced Short Palindromic Repeats (also known as CRISPR) – dependent techniques brought a new breakthrough in the gene manipulation in cyanobacteria thus increasing the biofuel production.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/ Cas9 system is originally a prokaryotic defense system which is used against phage attack (Javed et al., 2019). It has been employed as an important instrument for site directed mutagenesis. CRISPR consists of small repetitive sequence of DNA flanked by small segments of spacer DNA. Spacer DNA generally found in bacteriophage or plasmid and it is integrated into bacterial genome because of its encounter with a bacterial virus or plasmid. The Cas are CRISPR associated genes and these genes translates into nuclease or helicase protein with the task of cutting or unwinding DNA. The CRISPR system functions by integrating phage or plasmid of DNA sequence into his own genome. In future when it again encounters with that phage again, this system will recognize it using the transcribed RNA sequences and a Cas enzyme is directed to cleave that DNA (deoxy ribonucleic acid) at a particular sequence. The Cas9 which is a cleaving enzyme. It has the ability to produce a cut the two active sites of each single strand of a dsDNA.
The mechanism of CRISPR/Cas9 system was discovered by two scientists Doudna and Charpentier (Doudna and Charpentier 2014). According to their report bacteria utilizes the CRISPR/Cas9 system in order to defend themselves from phage attack. After a bacterium gets attacked by a phage, the bacterial DNA produces a RNA which is almost 20nt long and is complementary to the phage DNA. It is known as crRNA. Along with that a protein is also produced which is called Cas9. Depending on the mechanism sometimes along with CRISPR and Cas9 there is another RNA and it is known as trans-activating crispr RNA (also known as tracrRNA). The tracrRNA and crRNA both are linked with a hairpin loop like structure. The actual role of tracrRNA is still now but it was found that it has some sort of role in stabilizing the crRNA with complementary pairing. The crRNA (also known as crisprRNA) binds to the complementary sequence of the target DNA. This complementary region is known as proto spacer sequence. After the binding of crRNA with the target DNA the link between crRNA and tracrRNA is brokrn by RNase3.Interaction between crRNA and target DNA in turn activates the catalytic activity of Cas9 and Cas9 binds and cleaves the dsDNA at a specific site (known as PAM site).There are two ways of repairing the cleaved DNA- one is Non Homologous End Joining (also known as NHEJ) and the other is Homology Directed Repair or HDR. The crRNA and tracrRNA and Cas9, all of these together makes a powerful tool of genome editing. The hairpin loop structure between crRNA and tracrRNA provides the advantage of genome editing. Till now the CRISPR-Cas9 system has been applied in wide range of scientific researches and because of its high accuracy this tool can be used as a method of enhancing the biofuel production in cyanobacteria.
Results & Discussion
Recent advancement in synthetic-biology allows us to modify, edit heterologous host and by doing so increase the productivity of biofuels, increase their yield at industrial scale with a very low cost. The yield of biofuel production can be increase with the help of metabolic engineering, a high yield can be achieved by optimizing the metabolic flux. Very recently to enhance the biofuel production, synthetic biology and metabolic engineering have been implemented in cyanobacteria.
Few years back a synthetic pathway of isobutanol was genetically engineered by Astumi et al in synechococcus7942 (Astumi et al., 2009) to produce isobutanol and isobutaldehyde directly from Carbon dioxide (CO2) and the productivity was increased due to overexpression of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) enzyme. Similarly, during ethanol production pyruvate decarboxylase (also known as pdc) and alcohol dehydrogenase (adh) are the two most important genes. In 2009, pdc and adh genes set were integrated at the psbA2 locus of synechococcus 6803 and it was exposed under the control PpsbA2, a light inducible promoter (Singh et al., 2009). These genetically engineered cyanobacteria produced 550mg/L ethanol under high intensity of light (~1000 μE/m2 /s). Ethanol now a days is the most common biofuel globally.
Recently biodiesel has drawn a lot of attention. Biodiesel which contains long chain of alkyl esters and are refers to as animal fat-based diesel fuel. Biodiesel production can be achieved by reaction of lipids (animal fat and vegetable oil) with esters of alcohol producing fatty acids. Cyanobacteria are a good source of diacylglycerol (DAG) and triacylglycerol (TAG). After the extraction of DAG and TAG they can be used as biodiesel (Radakovits et al., 2010; Sheng et al., 2011). In spite of all these advantages in genetically engineered cyanobacteria there is a major disadvantage of it is that while studying the growth curve of genetically engineered cyanobacteria, it has been observed that these genetically engineered strains are very weak during lag phase and that could reduce the yield of fatty acids in an industrial bioreactor.
In a recent study, it has been observed that photosynthetic Synechococcus elongatus PCC 7942 and Synechocystis sp. PCC 6803 are the two strains which could convert inorganic carbon to free fatty acids (Roessler et al., 2009). Another approach towards it was the insertion of an acyl-acyl carrier protein (ACP) thioesterase gene into Synechosystis which produce a high yield of free fatty acids (183-211 mg/L) in addition to constrain the metabolic flux for production of free fatty acids (FFA) the acetyl-CoA carboxylase (ACC) was over expressed. Also, fatty acid-activating genes were knocked out to prevent the degradation of free fatty acids (Liu et al., 2011). Recently Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 these two strains were identified that can utilize the exogenous fatty acids and secrete endogenous fatty acids (FA) into the culture medium (Kaczmarzyk et al., 2010). In future these kinds of approach can be further expanded and more modified and optimized strains of cyanobacteria can be produced which will increase the production of free fatty acids.
Cyanobacteria is the one of the oldest photosynthetic microorganisms that are found in nature. When it comes to biofuel production this microorganism has received a bit of special attention than other microorganisms. Short generation, easy to maintain, easy genetic manipulation are the few qualities that makes cyanobacteria special from any other microorganism and an obvious choice for biofuel production.
Biofuel research in cyanobacteria is still very new. Many challenges and opportunities are there while working on issues related to gene manipulation. Light harvesting and CO2 fixation efficiency. To improve efficiency of cyanobacteria towards biofuel production CRISPR (Clustered regularly interspaced short palindromic repeats) – Cas is a very new and promising cutting-edge tool. CRISPR is an effective tool in genome editing in cyanobacteria and have been investigated in Synechocystis sp. PCC6803 (Singh et al., 2016). Synthetic biology and metabolic engineering can be the key of producing eco-friendly biofuels to meet the global energy requirement. Based on recent advanced in CRISPR-Cas technology it is hoped that cleaner and eco-friendly energies will be produced in much amount which will be able to meet the market requirement. Thus, this genome editing technology may pave the way toward fundamental discoveries in biology, with applications in all branches of biotechnology.
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