Nature Biotechnology.
Questions
1. Detail the photosynthetic pathway in Synechococcus elongatus and indicate where mutations have been introduced.
Details of the photosynthetic pathway reactions are shown in Figure 1
The mutations introduced are
kivd from Lactobacillus lactis for ketoacid decarboxylase in the equation 1 where kiv decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde (instead of transketolase activity) is introduced
kivd is incorporated into S elongates along with an expression vector containing the Ptrc inducible promoter.
alsS gene for acetolactate synthase, and acetolactate decarboxylase activity from Bacillus subtilis (both involved in acetoin synthesis in Bacillus. Als was able to de carboxylate catalyses kiv like ketoacid decarboxylase (kdc)
ilvC and ilvD genes from Escherichia coli for isoleucine to valine biosynthesis to the above
rbcLS genes from related species strain, S. elongatus strain PCC 6301 catalyzes the enolization into Rubisco complex.
2. Why do Atsumi et, al. choose to isolate isobutyraldehyde
Atsumi et al chose isobutyraldehyde because it is easy and very economic, and energy-efficient to produce isobutanol, in comparison that of hydrogen, ethanol as gasoline substitutes, from many other cellulosic renewable energy sources. Equally important factor is the low boiling point (630 C) and high vapour pressure (66 mm Hg at 40 C) of isobutyraldehyde properties facilitate very easy rapid and cheap separation from microbial cultures.
3. Mathematically show how the productivity (total product divided by volume and time) of isobutyraldehyde compares with biodiesel production
Average Algal Biodiesel production in a well designed production system about 1 x 105 liter per hectare per year.
Assuming that in at 8 hours per day for 292 days 80 of the working days in a year), production would have to be more than 3420 microgram per liter per hectare per year.
In the genetically engineered SA 665 in, an actual production was 1.1 gl the present experiments of isobutyraldehyde over 8 d with a steady state (first 7 d) production rate of 6,230 micrograms per liter per hour. This shows, although at laboratory scale, molecular engineering of Rubisco gene appropriately, would produce the desired results for biodiesel production.
4. (a) Rubisco is believed to be the most abundant protein on earth, where is it found What does it do And how does its structure relate to its function.
Rubisco is an enzyme found abundantly (accounting for more than which is capable of autotrophic nutrition 50 of green leaf proteins in most higher plants, most algae, and many bacteria. This is found in the chloroplasts as a rare unique enzyme and catalyzes carboxylation reaction of 5 carbon sugars at high carbon dioxide concentrations. But when the oxygen product of this reaction increases in concentration, the very same enzyme changes its activity to oxygenation of sugars. Rubisco is usually active in light. Two protein subunits a large (L, about 55 kD) and a small one(S, about 13 kD) together make this unit. Magnesium ions are crucial to the activity of this structure. Magnesium is so positioned in the active part this unit that it facilitates acceptance of carbon dioxide to lysine to form carbamate. Strong binding with carbamate requires an enzyme Rubisco activase to release Rubisco for further carboxylation. Light, pH, temperature, magnesium ion concentration contribute to increased levels of activity of this unit.
Regulation of this unit is in close relationship with all the other enzymes in the Calvins cycle.
5. (a) Several genes are expressed using a Ptrc promoter, discuss how this promoter is activated and what the requirements for expression from it are
Ptrc is synthetic fusion product of two different promoters from E. coli. It is under the control of the isopropyl -d-thiogalactopyranoside (IPTG). It is active even at low concentration of the substrate to activate sufficient levels of expression of the desired gene sequences.
(b) Comment on the use of the rbcLS gene in this work Why do they use it and what does it show them
rbcLS gene was characterized from Synechococcus elongatus strain PCC 6301 from different mutagenic methods. Genomic region of rbcLS contains genes involved in growth of this organism and chlorophyll biosynthesis at various levels of carbondioxide. (Ronen-Tarazi et al., 1995). Carbon dioxide utilization by Rubisco is not very efficient because of poor turn over rate and differential preferences to CO2 and O2. rbcLS gene from the related species is incorporated into the desired strain, PCC 7942 to achieve higher turn over rates in carbon dioxide fixation and has been achieved as demonstrated in this paper, of this organism
6. What are the uses of isobutyraldehyde
There are several uses of isobutyraldehyde (Matlack, 2001 ). It is converted into isobutanol for use as fuels.
Production of Neopentyl glycol used as polyester, isobutanediol used in personal care industry, Isobutylene dimer urea used as cattle feed for monogastric animals (fowl, pig, rabbits, sheep) and also as low release fertilizers for urea.
Various other industrially useful chemicals such as isobutyric acid, methyl isoaminylketone, and various hydrogenated and esterified compounds.
It finds uses in water testing, in food and flavour industry and for fragrances.
Discuss the pros and cons of the direct bioconversion of CO2 using photosynthesis in to industrially relevant compounds.
The most important argument in favour of direct conversion of CO2
mediated by photosynthesis is that such a process can produce an attractive substitute for use as gasoline consequently reduce dependence on increasing costs of natural gas sources and their transportations. Production of gasoline through such methods would also reduce the need for refining the crudes and their expenses. These also indirectly include taxing on the environmental balances because extraction of isobutanol may not entail process complexities and costs normally involved in refineries.
Assuming that algal flora would be used for such redirection of carbon dioxide, the argument gets appropriately supported by the fact that land flora would not be involved and therefore the currently available cellulosic biomass would continue to be available in future.
Arguments against such a process involves diversion of natural eco-friendly photosynthetic methods to non-cellulosic products. In nature photosynthesis is the key component for maintaining the harmony in ecosystem. .
That this process does not reduce the carbon dioxide levels already accumulated in the atmosphere so far. Increasing magnitude of vehicular uses of gas would change the source of gas. The redeeming point is that natural underground resources would get less disturbed.
1. Detail the photosynthetic pathway in Synechococcus elongatus and indicate where mutations have been introduced.
Details of the photosynthetic pathway reactions are shown in Figure 1
The mutations introduced are
kivd from Lactobacillus lactis for ketoacid decarboxylase in the equation 1 where kiv decarboxylates 2-ketoisovalerate (KIV) to isobutyraldehyde (instead of transketolase activity) is introduced
kivd is incorporated into S elongates along with an expression vector containing the Ptrc inducible promoter.
alsS gene for acetolactate synthase, and acetolactate decarboxylase activity from Bacillus subtilis (both involved in acetoin synthesis in Bacillus. Als was able to de carboxylate catalyses kiv like ketoacid decarboxylase (kdc)
ilvC and ilvD genes from Escherichia coli for isoleucine to valine biosynthesis to the above
rbcLS genes from related species strain, S. elongatus strain PCC 6301 catalyzes the enolization into Rubisco complex.
2. Why do Atsumi et, al. choose to isolate isobutyraldehyde
Atsumi et al chose isobutyraldehyde because it is easy and very economic, and energy-efficient to produce isobutanol, in comparison that of hydrogen, ethanol as gasoline substitutes, from many other cellulosic renewable energy sources. Equally important factor is the low boiling point (630 C) and high vapour pressure (66 mm Hg at 40 C) of isobutyraldehyde properties facilitate very easy rapid and cheap separation from microbial cultures.
3. Mathematically show how the productivity (total product divided by volume and time) of isobutyraldehyde compares with biodiesel production
Average Algal Biodiesel production in a well designed production system about 1 x 105 liter per hectare per year.
Assuming that in at 8 hours per day for 292 days 80 of the working days in a year), production would have to be more than 3420 microgram per liter per hectare per year.
In the genetically engineered SA 665 in, an actual production was 1.1 gl the present experiments of isobutyraldehyde over 8 d with a steady state (first 7 d) production rate of 6,230 micrograms per liter per hour. This shows, although at laboratory scale, molecular engineering of Rubisco gene appropriately, would produce the desired results for biodiesel production.
4. (a) Rubisco is believed to be the most abundant protein on earth, where is it found What does it do And how does its structure relate to its function.
Rubisco is an enzyme found abundantly (accounting for more than which is capable of autotrophic nutrition 50 of green leaf proteins in most higher plants, most algae, and many bacteria. This is found in the chloroplasts as a rare unique enzyme and catalyzes carboxylation reaction of 5 carbon sugars at high carbon dioxide concentrations. But when the oxygen product of this reaction increases in concentration, the very same enzyme changes its activity to oxygenation of sugars. Rubisco is usually active in light. Two protein subunits a large (L, about 55 kD) and a small one(S, about 13 kD) together make this unit. Magnesium ions are crucial to the activity of this structure. Magnesium is so positioned in the active part this unit that it facilitates acceptance of carbon dioxide to lysine to form carbamate. Strong binding with carbamate requires an enzyme Rubisco activase to release Rubisco for further carboxylation. Light, pH, temperature, magnesium ion concentration contribute to increased levels of activity of this unit.
Regulation of this unit is in close relationship with all the other enzymes in the Calvins cycle.
5. (a) Several genes are expressed using a Ptrc promoter, discuss how this promoter is activated and what the requirements for expression from it are
Ptrc is synthetic fusion product of two different promoters from E. coli. It is under the control of the isopropyl -d-thiogalactopyranoside (IPTG). It is active even at low concentration of the substrate to activate sufficient levels of expression of the desired gene sequences.
(b) Comment on the use of the rbcLS gene in this work Why do they use it and what does it show them
rbcLS gene was characterized from Synechococcus elongatus strain PCC 6301 from different mutagenic methods. Genomic region of rbcLS contains genes involved in growth of this organism and chlorophyll biosynthesis at various levels of carbondioxide. (Ronen-Tarazi et al., 1995). Carbon dioxide utilization by Rubisco is not very efficient because of poor turn over rate and differential preferences to CO2 and O2. rbcLS gene from the related species is incorporated into the desired strain, PCC 7942 to achieve higher turn over rates in carbon dioxide fixation and has been achieved as demonstrated in this paper, of this organism
6. What are the uses of isobutyraldehyde
There are several uses of isobutyraldehyde (Matlack, 2001 ). It is converted into isobutanol for use as fuels.
Production of Neopentyl glycol used as polyester, isobutanediol used in personal care industry, Isobutylene dimer urea used as cattle feed for monogastric animals (fowl, pig, rabbits, sheep) and also as low release fertilizers for urea.
Various other industrially useful chemicals such as isobutyric acid, methyl isoaminylketone, and various hydrogenated and esterified compounds.
It finds uses in water testing, in food and flavour industry and for fragrances.
Discuss the pros and cons of the direct bioconversion of CO2 using photosynthesis in to industrially relevant compounds.
The most important argument in favour of direct conversion of CO2
mediated by photosynthesis is that such a process can produce an attractive substitute for use as gasoline consequently reduce dependence on increasing costs of natural gas sources and their transportations. Production of gasoline through such methods would also reduce the need for refining the crudes and their expenses. These also indirectly include taxing on the environmental balances because extraction of isobutanol may not entail process complexities and costs normally involved in refineries.
Assuming that algal flora would be used for such redirection of carbon dioxide, the argument gets appropriately supported by the fact that land flora would not be involved and therefore the currently available cellulosic biomass would continue to be available in future.
Arguments against such a process involves diversion of natural eco-friendly photosynthetic methods to non-cellulosic products. In nature photosynthesis is the key component for maintaining the harmony in ecosystem. .
That this process does not reduce the carbon dioxide levels already accumulated in the atmosphere so far. Increasing magnitude of vehicular uses of gas would change the source of gas. The redeeming point is that natural underground resources would get less disturbed.
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