Biotechnology has always been the pioneer in enhancing the products and increasing the yield. There has been many ways of increasing the yield and with rise of technology the medium and process have also been systematically improved and changed to make it more effective and efficient. In a paper by Beer et al. ,(2009) they have shown the production of bioenergy carriers for fuel cells with help of eukaryotic alage. The genetic manipulation technique used are made such that it gives maximum output by improving and controlling over metabolic pathways, increasing the availability of phenotypes- hence the diversity. Understanding metabolicpathways is by relative studying the complex mechanism of photosynthesis of protein, nucleic acids, carbohydrates, lipids and hydrogen molecules. A complete in depth understanding of the synthesis, the intermediate and end products of this biological system along with understanding all the pathways that connects them helps us in the production of biofuels in optimized way.
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The most recent development in algal genetics is the development of improved gene silencing in C. Reinhardtii. High specific and stable organisms have been reported for high-through output atrificail mi RNA (amiRNA) technique. The new developed amiRNA are likely to emerge as choice for functional genomics and will be applied to other species which are mostly related to biofuel production. High- through output sequencing has played the central role in leading technology in identifying regulatory genes that shows path for algal metabolism in the production of biofuels. For large scale cultivation, the natural diversity of microorganism along with optimized protein synthesis which can be extensively used through hetrologous expression. (Beer et al. ,2009)
Protein engineering have been used in increasing the efficeiecy of lignocelluloses degrading enzyme and also proteins in biofuel synthesis pathway. Lignocelluose represents a sustainable renewable energy source . The protein engineering might help is to overcome the low enzymatic hydrolysis productivity and yield efficiency which results in more active enzyme for the hydrolysisof substrates to sugars and these sugars being converted to biofuels molecules. Single cell genomics were developed to overcome the hurdle of diversity which resulted in exponential increase of carbohydrate relative enzymes and also related biosynthetic and regulation pathways. Developments of expression system that include tRNA synthetase genes with stress response might help improve the enzymes in which S. Cerevisiae has shown preoperty of simultaneously taking up and assembling DNA fragments in one step. This property greatly increases and breakdowns the process and helps in bioefuel production where in large pathways can be assembled for consolidated bioprocessing. (Wen et al , 2009 )
Recent advances in genome sequencing, gene annotation, and the ability to rapidly and inexpensively synthesize DNA fragments of sufficient length to encode full-length genes, enzymes, metabolic pathways, and even entire genomes provide the foundation from which synthetic biologists can construct genetic elements for the rational manipulation of microbial phenotypes. The genetic elements for metabolic pathway engineering are now routinely designed, synthesized and assembled from overlapping, chemically synthesized single-stranded oligonucleotides into double-stranded DNA fragments encoding enzymes, reporters, repressors, activators, promoters, terminators, ribosome binding sites, signaling devices, and measurement systems, among others. Several groups are now working to develop a synthetic microbe containing a minimal genome, both to understand the fundamentals of cell biology and to begin the process of developing synthetic organisms for practical applications. In addition to facilitating rational design, the new tools of synthetic biology now enable the construction of high-diversity combinatorial synthetic gene libraries to create and introduce enzymatic diversity into a metabolic pathway. As such, this emerging field offers great promise for advancing the development of microbial systems for the conversion of biomass to fuels and chemicals. Hence synthetic biology has increased advanced in molecular, system biology along with protein engineering bioinformatics which helps us to degign and arrange genetic materials for manipulating cell phenotypes. With such developments taking place there is chance for microbial organisms in the production of renewable fuels to be successful. (Picataggio, 2009)
Carbon dioxide, the major by-product of ethanol fermentations, is not only reusable but also poses a heavy burden to the environment unless it is sequestered or recaptured. One approach that would meet this goal and also would greatly boost the efficiency of fermentations involves cultivating photosynthetic microalgae, which also are referred to as diatoms, green algae, blue-green algae, and golden algae. Their biomass contains three main components: proteins, carbohydrates, and natural oils. By completing a closed loop for carbon recycling, microalgae could become another source of biomass and oil as part of an overall biomassto- ethanol production process. Microalgae, among the most photosynthetically efficient plants because of their simple cell structure, accumulate high levels of natural oils, use carbon dioxide as their sole carbon source, and grow in both marine and freshwater environments.
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Some experts consider algal biodiesel fuel the most energy-positive and environmentally beneficial end-product possible from highvolume carbon dioxide reuse schemes. Microalgae systems also use far less water than do traditional oilseed crops. Some species of algae appear to be particularly well suited for producing biodiesel due to their high oil content, with some varieties containing more than 50% oil, exceeding terrestrial plants. Moreover, some algal varieties grow extremely fast. Typically classified according to pigmentation, life cycle, and cellular structure, the four most abundant groups of algae are Bacillariophyceae, Chlorophyceae, Cyanophyceae, and Chrysophyceae. Establishing indoor and outdoor large-scale growth facilities for evaluating various means of harvesting the algae via low-energy-requiring processes and with minimal loss of water. As part of this program, we characterized five species of microalgae from tropical habitats that produce oils with chain lengths that meet the requirements for making jet fuel. We are also evaluating algae for their capacity to produce hydrogen. (Govind and Sen, 2009)
Amino acid biosynthetic pathways are complex and tightly regulated. Thus, microorganisms normally do not produce the desired levels of amino acids. A traditionally used method of random mutation and selection for the development of amino acid producers is now complemented by rational metabolic engineering, which became more powerful as omics tools have become available. Furthermore, systems metabolic engineering based on ntegrated analysis of omics data and genomescale metabolic model allows development of superior strains having well-defined genotypes. This approach will prevail in the future as our understanding of the cell as a system continuously advances. Multiple high-throughput omics data are being integrated to provide additional insights into the global metabolic and regulatory networks. Recent multi-level analyses of E. coli indicate how flexible the regulatory network is in response to the environmental and genetic perturbations, so as to maintain its metabolic integrity, and might provide possible implications of systems metabolic engineering. Even though significant advances have been made in bioinformatics and systems biology to decipher the complex omics data, new methods need to be developed for their integrated analysis. Also, algorithms for the more accurate and realistic simulation of in silico genome-scale metabolic and regulatory networks need to be developed to suggest optimal strain design strategies. In addition, methods to incorporate metabolic engineering strategies considering the fermentation and recovery processes at the early stage of strain development would be beneficial. When these are accomplished, systems metabolic engineering will become a more powerful strategy for strain development.(Park and Lee, 2008)
Increased knowledge of the precursor and riboflavin synthesis pathways allows the development of rational approaches by the overexpression or disruption of genes encoding enzymes which either limit important steps in the flux or catalyze undesirable reactions.
The development of molecular tools, the second requirement for that approach, has reached a sophisticated level for B. subtilis, and has been started by Peter Philippsen's group for A. gossypii but has not been published for C. famata. While the impact of overexpressing all six rib genes is still under investigation in A. gossypii, three gene manipulations (two in earlier steps of the pathway and one concerning the transport of riboflavin led to the desired changes in metabolic flux. A second copy of AgICL1 can improve riboflavin overproduction when soybean oil is the carbon source . This enzyme, catalyzing the key step of the glyoxylate cycle, was found to be the second regulated enzyme after the extracellular lipase. Although this lipase, which is strongly repressed by its fatty acid and glycerol reaction products, was found to become rapidly inactivated in a fermentor culture, the capacity for enzyme formation by the fungus is far beyond that needed in the process. A further example of increased riboflavin formation was shown by overexpression of AgGLY1, encoding threonine aldolase . Control of the gene by the AgTEF promoter and terminator resulted in a tenfold increase of threonine aldolase specific activity. A greater increase in riboflavin production was obtained by feeding threonine than by feeding glycine, which was caused by a better uptake of threonine. The third example of metabolic design deals with the very last step of the pathway. In A. gossypii the riboflavin formed is transported via specific carriers either into the cellular vacuole or the extracellular medium. A knock-out of the vacuolar ATPase subunit AgVMA1 resulted in the complete excretion of the product into the medium. This was expected because detailed cytological and biochemical studies revealed a vacuolar accumulation caused by an active transport mechanism In B. subtilis overexpression of the rib genes, which are organized in a cluster, is achieved by replacement of the two regulated promoters by constitutive ones derived from a phage . Despite multiple copies of this four-gene construction having been inserted at two different sites in the genome, a separate overexpression of ribA was necessary to reach maximal productivity . The ribA gene encodes a bifunctional protein with dihydroxybutanone phosphate synthetase activity at the N-terminal and GTP cyclohydrolase II activity at the C-terminal. This showed that the initial steps of ribo¯avin synthesis limited productivity in the last published stage of strain improvement. Next the oxidative branch of the pentose phosphate pathway, which was found to be increased in the production strain can become rate-limiting.The beneficial effect of an adequate vitamin supply in food or feed is undisputed. The short-term perspective is the transformation of plant products, e.g. oils or sugars, into vitamins by the use of microorganisms. This enables cheap production, which means an increasing it with economic and ecological demands, and a product in pure form. At the moment it is impossible to predict whether the use of one specific organism will be more advantageous and displace the others.While B. subtilis is certainly the fastest-growing organism, its growth-linked riboflavin production bears the risk of selecting non-producing mutants. In A. gossypii this risk is absent because riboflavin production is not growth-linked; however, the growth time is lost for production. An advantage is a good production on plant oil as substrate. Triglycerides, which can be used as oil or solid fat, makepossible a high substrate charge in the fermentor, because of their osmotic neutrality. C. famata, as a yeast, is easier to distribute in a 100 m3 fermentor than a filamentous fungus growing in mycelial pellets. But keeping the iron concentration in the substrate below 15 lM to prevent its negative effect requires extra effort. A long-term scientific prospect might be the metabolic design of the food and feed plants themselves to increase their nutritional value. This approach is already in progress with provitamin A in rice . For pure form applications, a next step could be the redirection of the plant's metabolic flux to produce and store the vitamin instead of sugar or oil. It is unlikely that such approaches will be taken by a commercial company because the riboflavin market is much too small.
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