Metabolic engineering applications to renewable resource utilization

Lignocellulosic materials containing cellulose, hemicellulose, and lignin are the most abundant renewable organic resource on earth. The utilization of renewable resources for energy and chemicals is expected to increase in the near future. The conversion of both cellulose (glucose) and hemicellulose (hexose and pentose) for the production of fuel ethanol is being studied intensively, with a view to developing a technically and economically viable bioprocess. Whereas the fermentation of glucose can be carried out efficiently, the bioconversion of the pentose fraction (xylose and arabinose, the main pentose sugars obtained on hydrolysis of hemicellulose), presents a challenge. A lot of attention has therefore been focused on genetically engineering strains that can efficiently utilize both glucose and pentoses, and convert them to useful compounds, such as ethanol. Metabolic strategies seek to generate efficient biocatalysts (bacteria and yeast) for the bioconversion of most hemicellulosic sugars to products that can be derived from the primary metabolism, such as ethanol. The metabolic engineering objectives so far have focused on higher yields, productivities and expanding the substrate and product spectra.

Application of metabolic engineering

A fermentation process was developed for production of indigo from glucose using recombinant Escherichia coli. This was achieved by modifying the tryptophan pathway to cause high-level indole production and adding the Pseudomonas putida genes encoding naphthalene dioxygenase (NDO). In comparison to a tryptophan-over-producing strain, the first indigo-producing strain made less than half of the expected amount of indigo. Severe inactivation of the first enzyme of aromatic biosynthesis, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase (the aroGfbr gene product), was observed in cells collected from indigo fermentations. Subsequent in vitro experiments revealed that DAHP synthase was inactivated by exposure to the spontaneous chemical conversion of indoxyl to indigo. Indigo production was thereafter improved by increasing the gene dosage of aroGfbr or by increasing substrate availability to DAHP synthase in vivo by either amplifying the tktA (transketolase) gene or inactivating both isozymes of pyruvate kinase. By combining all three strategies for enhancing DAHP formation in the cell, a 60% increase in indigo production was achieved. Metabolic engineering was then further applied to eliminate a byproduct of the spontaneous conversion of indoxyl to indigo, thereby solving a serious problem with the use of bio-indigo in the final denim dyeing application.

CO METABOLISM

Cometabolism is an approach to Biological degradation of hazardous solvents. Using methane as the primary energy source, some microbes release enzymes that degrade the chlorinated solvents.
Cometabolism is defined as the simultaneous metabolism of two compounds, in which the degradation of the second compound (the secondary substrate) depends on the presence of the first compound (the primary substrate). For example, in the process of degrading methane, some bacteria can degrade hazardous chlorinated solvents that they would otherwise be unable to attack.




Co-metabolism can be defined as:
The transformation of an organic compound by a microorganism incapable of using the substrate as a source of energy or of one of its constituent elements
(Alexander, 1967. Agriculture and the Quality of Our Environment (N.C. Brady, Ed.) pp 331-342. Am. Soc. Adv. Sci. Washington D.C.)
What this means is that the organism in question derives no benefit from the co-metabolism of the compound. No energy is available, nor are any of the (C, N, P, S, etc) elements in the compound used as a significant source of material for biosynthetic activity.
The term "secondary substrate metabolism" is also in the literature - this means that the organism is actually growing on a second substrate and is transforming another substrate at the same without gaining benefit. This undoubtedly occurs, but is very difficult to detect and prove in natural environments, even though it can be demonstrated in pure cultures.
For this reason, co-metabolism is taken to be either case:
1. The organism in question is NOT growing on another substrate - it may not even be proliferating at all.
2. The organism IS growing on another substrate (known or unknown) but is also co-metabolizing the compound in question.
Mechanisms of co-metabolism
1. Initial enzyme or enzymes change the substrate to a product that is not further transformed by the other enzymes in the organism to anything that can be further metabolized. Do enzymes have many different substrates ?
2. The original substrate is transformed to a product that inhibits later enzymes in a metabolic sequence or .inhibits the growth of the organism.
3. The organism needs a second substrate to achieve some later reaction - that substrate is missing.
The first explanation is the most likely in most cases.
It implies that co-metabolism is "accidental" or "fortuitous". An enzyme can react with a compound, produce no energy for the cell, nor incorporate any of the elements of the compound into biosynthetic processes, but still achieve partial transformation of the compound. It may be that many of the compounds co-metabolized are similar to normal substrates of the cells, differing sufficiently so that their immediate products cannot be further metabolized.
For example:
Enzyme A ----------> Enzyme B -------------> Enzyme C
Substrate A ----------> Product B ------------> Product C
Substrate Ax-----------> Product Bx [not metabolized by enzyme C]
Substrate Ax is "sufficiently similar" to Substrate A that Enzyme A can transform it to Bx, but Bx is "sufficiently different" to B so as to prevent further metabolism by Enzyme C.

MOLECULAR BIOLOGY OF CANCER Question Papers (Supple, 2006)

SET : 1

1. Describe the phase of cell cycle and the events that 0ccur in each. Name the important phases that check cell cycle progression.

2. Write short notes on any two of the following viruses as causative agents for human cancer :

a) Hepatitis B virus

b) SV 40

c) Adenovirus

d) Papilloma virus.

3. P53 is designated as the guardian of the genome. Support the concept.

4. Discuss the role of different environmental substances as causative agents of cancers providing some specific examples.

5. What are ionizing radiations and what type of damages could they produce in the DNA molecule? Discuss in detail.Correlate DNA damage with carcinogenesis.

6. The international comparison of clinical staging is standardized in 1959 under “TNM” system. Explain in detail the method followed in staging the cancer by the TNM method.

7. What is the main difference between normal Radiographic Imaging and Magnetic Resonance Imaging? Which is superior in diagnosis and why?

8. What are alkylating drugs? What is their basic mechanism of action to suppress or kill cancer cells? Name some of the most commonly used drugs of this category mentioning their specificity against selected cancers.

SET : 2

1. Describe the phases of cell cycle and the events that occur in each. Name the important phases that check cell cycle progression.

2. Mutation in proto oncogenes that change them to oncogenes are dominant. Provide a correct explanation with suitable examples.

3. Describe the function of the normal Rb gene in controlling cell cycle progression.

4. Describe the tests commonly used in screening chemicals for their carcinogenic potentials.

5. Briefly discuss the different agents that could be possible carcinogens.

Mention the source of these agents that enter human environment.

In general, what biological effects can these agents cause?

6. The American Cancer Society has listed some very common warning signals as cautions. List these signals. Why is it important to check for these signals? Discuss.

7. Enumerate the different biopsy techniques with reference to their specific usefulness.

8. What is the basis of gene therapy in cancer treatment? It is expected to be more potent and specific against specific cancers – discuss this in the light of available information and trail phases of the treatment.

SET : 3

1. Describe the phases of cell cycle and the events that occur in each. Name the important phases that check cell cycle progression.

2. Name any three different types of proteins that control growth in mammalian cells.

Describe oncogenes resulting from mutations in these protein coding genes. Name the tumors they induce.

3. Which gene involvement is indicated in patient with breast and ovarian carcinomas?

Discuss in detail the mechanism controlling the induction of these cancers.

4. Name a few base analogues and the mechanism of action of mutagenesis.

5. Name the different units that measure radiation energy.

Describe the rate of release of energy and its biological effects.

6. The American Cancer Society has listed some very common warning signals as cautions. List these signals. Why is it important to check for these signals? Discuss.

7. Name the tests useful in early detection & staging of cancer. Briefly describe them.

8. Describe the different types of radiations and their sources that are used in radiotherapy for the treatment of cancers. Also describe the radiation units that measure the dose.

SET : 4

1. Enumerate in detail the pathway by which Ras protein checks cell cycle progression in response to environmental factors.

2. Write short notes on any 2 of the following oncogenes :

a) erb A

b) erb B

c) H – ras

d) Src

3. Discuss the relationship between BCL – 2 & P53 genes and how does mutation in either of them effect the normal cellular process.

4. Discuss the role of different environmental substances as causative agents of cancers providing some specific examples.

5. Briefly discuss the different agents that could be possible carcinogens.

Mention the source of these agents that enter human environment.

In general, what biological effects can these agents cause?

6. The American Cancer Society has listed some very common warning signals as cautions. List these signals. Why is it important to check for these signals? Discuss.

7. What is aspiration biopsy? Describe the techniques, its usefulness and the risk involved.

8. Cyclophosphamide (Endoxan) is the most commonly used drug, the drug of choice.

Support your answer by giving its special activity and its broad spectrum usage.