AMINO ACID BIOSYNTHESIS

Amino acids are synthesized from the intermediates in Glycolysis and Krebs cycle.

Aminoacids and their respective Glycolysis/Krebs cycle intermediates are given below:

• Histidine is synthesized through from Glucose-6-posphate through Ribose-5-posphate.
• Serine, Glycine, Cysteine are synthesized from the common precursor 3-Posphoglycerate.
• Posphoenolpyruvate along with erythrose-4-posphate are the precursors for aromatic aminoacids viz., Phenylalanine, tryptophan, tyrosine.
• Alanine, Valine, Leucine, Isoleucine are synthesized from Pyruvate.
• Aspartate, Asparagine, methionine, threonine and Lysine are synthesized from Oxaloacetate.
• Glutamate, Glutamine, Arginine, Proline are aynthesized from alpha-Ketoglutarate

Histidine Synthesis:

PRPP, ATP, Glutamine are the 3 presursors in Histidine synthesis.

PRPP - Posphorybosyl pyroposphate

The addition of ATP to PRPP is catalysed by ATP Posphorybosyl transferase
L-Histidinol is converted to Histidine by Histidinol dehydrogenase

Serine, Glycine and Cysteine Synthesis :

The synthesis of Cysteine varies in bacteria, Plants and mammals.
Plants and animals produce reduced sulfur from sulfates in the environment. This reduced sulfur is used in the synthesis of Cystein, and Methionine ('sulfur conataining amino acids' ).

Mammals synthesize cysteine from two aminoacids, methionine and serine. Methionine provides the sulfur atom and serine provides the carbon skeleton.

I will Place the synthesis of remaining aminoacids in the next posts

METABOLISM, METABOLITE,.................

Metabolism is defined as the entire set of enzyme catalysed transformations of organic molecules in a cell.
Metabolism is the sum of anabolism and catabolism

Anabolism is the Phase of intermediary metabolism concerned with the energy requiring biosynthesis of cell components from smaller precursors

Catabolism is the phase of intermediary metabolism concerned with the energy yielding degradation of nutrient molecules.

Metabolite is a chemical intermediate in the energy catalysed reactions of a cell.

DIFFERENT FORMS OF FEED BACK CONTROL

I had mentioned in my earlier posts the difference between Feedback inhibition and Feedback repression. But both can be considered under a single name i.e, Feedback Control.

Feedback Control in Unbranched Pathways

Unbranched pathways give only one end product.

In unbranched pathways control is achieved in two ways:

• The end product inhibiting the first enzyme of the pathway.
• The end product or its derivatives repressing all the enzymes involved in the pathway

Feedback Control in Branched Pathways

In branched pathways there will be more than one end product. So control cant be as simple as in unbranched pathways. Each of the end product has its own physiological significance and its own levels of optimum concentration. If one of the end product exerts control on an enzyme common to two or more end products then the organism may suffer from the lack of those end products whose synthesis has been blocked. So different control mechanisms have evolved that avoid these sort of problems. They include:

• Concerted/ Multivalent Feedback control
• Co-operative Feedback Control
• Cumulative Feedback Control
• Sequential Feedback Control
• Control by Isoenzymes

Concerted/Multivalent Feedback control:
When all the end products are present in excess, they togather exert control over the first enzyme of the pathway. When either of them is not present in excess control can't be exerted.

Co-operative Feedback control:
This is similar to concerted feed back control. The difference is that in Co-operative feed back control when when of the endproducts is in excess, it can exert a weak control independent of the other endproducts. When both the endproducts occur in excess they exert a synergistic control.

Cumulative Feedback control:
Each of the end products exert a certain proportion of control over the first enzyme of the pathway independently. They donot exert a synergistic control as in case of the co-operative feedback control.

In both Co-operative feedback control and Cumlative feedback control, each of the end products exert control immediately after the branch point so that the common intermediate for the end products is diverted away from the pathway of the product in excess.


Sequential Feedback control:
Each endproduct controls the enzyme immediately after the branchpoint leading to the end product. Due to this the intermediates build up that inturn exerts control on previous enzymes.

Control by Isoenzymes:
Isoenzymes are enzymes which catalyse the same reaction but different in their control characteristics.
The critical reaction (usually the first reaction) is catalysed by more than one isoenzymes. In this situation different end products inhibit different isoenzymes. So, when one of the end product is in excess it exerts control on one of the isoenzymes leaving the other isoenzyme active so that the production of the other endproduct is not stopped.

METABOLIC ENGINNERING Question Papers (2008,Reg)

SET : 2

1. How can metabolic pathways genetically controlled explain with any two examples?
2. Explain the metabolic pathway manipulations to improve the productionof 1,3 propanediol.
3. What are precursor effects? Briefly explain the regulation of secondary metabolic pathways.
4. Write short notes on the following : a) How specific rates and yields are related? b) Explain the calculation of yields and specific rates.
5. How gene dosage is evaluated and how does gene dosage affect fermentation process?
6. Explain in detail the role of metabolic flux analysis in metabolic engineering.
7. Free energy is the most useful thermodynamic function in metabolic engineering; illustrate your answer with suitable metabolic pathway.
8. What is RNA interface technology? Explain its role in metabolic manipulations.

SET : 3

1. Define Operon and explain trp operon in detail.
2. Explain the metabolic pathway manipulations to improve the production of 1,3 propanediol.
3. Distuinguish between primary and secondary metabolites; explain the phase during which secondary metabolites are synthesised?
4. Write short notes on : a) Origin of capacity to degrade xenobiotics by microorganisms b) Use of mixed microbial popuations.
5. Write about metabolic pathway manipulations for the enhancement of product yield with reference to ethanol production.
6. Explain briefly how radiolabeled materials are utilized in experimental determination of metabolic flux.
7. Discuss the role of molecular biology and recombinant DNA technology for metabolic pathway improvement.
8. Role of metabolic engineering in the production of Polyhydroxyalkanates(PHA)

BIOCONVERSION OF INSOLUBLE SUBSTRATES

Certain Substrates are insoluble in water. Examples are cholesterols, steroids, hemicellulose, cellulose etc.
In order to achieve their bio-conversion we use organic solvents such as benzene, toulene etc. Addition of detergents is also used.

Examples:

• Oxidation of Cholesterol using Pseudomonas species.
• Dehydrogenation of hydro-cortisone by Arthrobacter simplex.
• Reduction of Androstenedione to testosterone by Saccharomyces cerevisiae.

CO-METABOLISM

Cometabolism is the use of two substrates simultaneously. The term secondary substrate metabolism is also used.
One substrate is a growth promoting substrate and the other isa non growth promoting substrate (it means that the non growth promoting substrate is not used by the microbe for C or N or P.)

During the process of cometabolism both the substrates bind to the enzyme and are metabolized.

Mostly in case of degradation of environmental pollutants, oxygenases (mono and di oxygenases) are used.

CATABOLIC REPRESSION

Catabolite repression is a case in which the enzyme is not produced when a compound is still present in the medium and appears only after the compound disappears.

Example is the Catabolite repression in Lac Operon in E.Coli. When Glucose is present in the medium enzymes for lactose metabolism are not synthesized.

Catabolite repression can be overcome by adding cAMP exogenously.

FEED BACK INHIBITION AND FEED BACK REPRESSION

Feed back Inhibition and Feed back Repression are different.

Feed back repression is a situation in which the end product of the biochemical pathway binds to the regulatory site of the enzyme and stops the binding of the substrate to the enzyme. Simply we can say that the producting is inactivating an enzyme thats alreadt there.

Feed back repression is a case in which the end product or its derivatives inhibit the production of the enzyme at the gene level.

So, you might have understood the difference between feed back repression and feec back regulation.

Feedback Repression uses Isoenzymes

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METABOLIC ENGINEERING Question Papers (Supple, 2007)

SET :1

1. Explain the Jacob Monod model of induction and also regulation of the operon by CAMP. [16]

2. What do you understand by feed back regulation? Explain this with special reference to amino acid biosynthetic pathways. [16]

3. How can you overcome feed back regulation? Explain some strategies by which one can maximize the production of a metabolite of interest. [16]

4. Define primary and secondary metabolites. What is the difference between the two? Give examples for both of them. [16]

5. What are bioconversions? How are these important in synthesis? Explain with specific examples. [16]

6. Explain two of the following:
(a) Cometabolism
(b) Precursor effects in metabolite synthesis [16]

7. Discuss the different strain improvement methods that are currently being used. [16]

8. Write notes on:
(a) Repression
(b) Induction
(c) Regulation of enzyme synthesis
(d) Alteration in permeability. [16]

SET :2

1. Explain the operon concept using Lac and Trp operons as examples. [16]

2. What do you understand by membrane transport? Explain the different transport processes in detail. [16]

3. What is Feed back regulation? How can one overcome feedback regulation? [16]

4. Explain any two of the following:
(a) Catabolite repression
(b) Alteration in cell permeability
(c) Cometabolism. [16]

5. Explain in detail the different strategies that can be adopted for maximizing the yields of secondary metabolite. [16]

6. Distinguish between primary and secondary metabolites. Give at least four examples each for each of them. 1[16]

7. What are the different methods used for strain improvement? [16]

8. Write notes on:
(a) Gene dosage
(b) Mixed or sequential bioconversions
(c) Advantages of bioconversions
(d) Isozymes. [16]

SET :3

1. Explain in detail the regulation of amino acid biosynthetic operons. [16]

2. Explain the following:
(a) Passive diffusion
(b) Active transport
(c) Feed back regulation. [16]

3. What are primary and secondary metabolites. Distinguish between the two and give at least four examples for each type of metabolite. [16]

4. What are Bioconversions? What are the factors that effect bioconversions. Discuss its advantages over chemical synthesis. [16]

5. Write about the following in detail:
(a) Mutations
(b) Gene dosage. [16]

6. Comment on:
(a) Importance of altering membrane permeability.
(b) Isoenzymes and their role in regulation. [16]

7. What are the different strategies used for strain improvement? Discuss in detail [16]

8. Write short notes on:
(a) Mutants resitant to repression
(b) Induction
(c) Catabolite repression.
(d) Conversion of insoluble substances. [16]

SET :4

1. Explain in detail the Jacob Monod model of gene regulation by using the example of Lac and Trp operons. [16]

2. Explain the different modes of membrane transport. [16]

3. What are primary metabolites? How do you formulate different strategies for enhancing the production of primary metabolites.

4. What are secondary metabolites? Explain the phase during which it is synthesized and the methods. [16]

5. Discuss the factors influencing bioconversions. [16]

6. How can one improve the yields of a desired product by both genetic and fermentation methods. [16]

7. Write short notes on:
(a) Bioconversion of insoluble products
(b) Feed back inhibition. [16]

8. Comment on:
(a) Catabolite repression
(b) Gene dosage. [16]

METABOLIC ENGINEERING Question Papers (Regular, 2007)

SET : 1

1. Describe in detail positive and negative control of gene expression by citing the example of lac operon.

2. Write about different enzymes required for the synthesis of cAMP.

3. How do you apply the principle of metabolic engineering to the synthesis of tryptophan. Explain in detail.

4. Discuss the different parameters required for limiting end product accumulation.

5. Write notes on :

a) Secondary metabolites

b) Idiophase.

6. How does carry out the bioconversion reaction for insoluble substrate. Discuss in detail.

7. Discuss different kinds of bioconversions with suitable examples.

8. Explain the phenomena of feed back repression in resistant mutants.

METABOLIC ENGINEERING Question Papers (Regular, 2006)

SET : 1

1. Describe the primary screening involved in strain selection with example.

2. Write notes on :

a) Co-metabolism during bio-conversion

b) Concerted feed back regulation

3. Define bioconversion and list out in detail advantages of bio conversion in pharmaceutical industry with suitable examples

4. What is enzyme inhibition and detail various modes of enzyme inhibition.

5. Compare and contrast direct and indirect fermentations citing amino acid synthesis as example?

6. List out the biotechnological applications of enzymes (e.g. protease, amylase etc) produced by fermentation.

7. Detail on (with example)

a) Active transport

b) Group transport

8. List out organisms involved in production of secondary metabolites with detail on one secondary metabolite as example.

SET : 2

1. How can metabolic pathway be genetically controlled? Explain with two examples.

2. Detail

a) Passive diffusion

b) Isozymes

3. Explain gene regulation by Jacob and Monad model citing lac operon as example.

4. Detail how gene dosage is evaluated and how does gene dosage effect fermentation process.

5. Describe in detail the various modes of diffusion?

6. Explain fermentation parameter involved in production of wine from yeast.

7. How does mutation effect the enzyme production? List out the factors involved in the optimization of mutants for high yield production?

8. Distinguish and differentiate concerted feed back regulation and cumulative feed back regulation with examples.

SET : 3

1. List out and describe the parameters involved in scale up of fermentation (large scale) from pilot scale.

2. Describe

a) Gene dosage

b) Aminoacid regulation of RNA synthesis

3. Explain mixed OG sequential bioconversion with suitable example.

4. Describe catabolic repression with tryptophan operon as example.

5. Define mutation and various modes of generating mutations in improving industrial biotechnology of an organism.

6. Describe induction and repression phenomena with E.coli as an example.

7. How can permeability of an organism be altered in focus with primary metabolism.

8. Detail on

a) Glucose effect

b) Active transport

SET : 4

1. Describe in detail the secondary screening involved in strain selection with example.

2. Detail

a) CAMP

b) Feed back repression

3. Define bioconversion and describe factors involved in bioconversion with example(s).

4. What is antibiotic fermentation and explain its methodology in penicillin production.

5. List out and detail factors contributing to catalytic efficiency of enzymes.

6. How do mutations effect the enzyme production. List out aspects involved in optimization of mutants for high yield.

7. Describe in detail conversion of insoluble substance by mixed sequential bioconversion.

8. Detail

a) Fed batch fermentation

Continuous fermentation.

METABOLIC ENGINEERING Question Papers (Supple, 2006)

SET : 1

1. What is bio-conversion? Discuss the advantages of bioconversion.

2. What is permeability? Discuss the alteration of permeability.

3. Discuss the biosynthesis of secondary metabolites.

4. What is growth cycle? How do you recognize growth cycle peaks?

5. Explain bio-conversion of insoluble substances.

Write about cAMP deficiency

6. Write short notes on the following:

a) Jacob Monod model

b) Passive diffusion

c) Resistant mutants

d) Catbolic repression

7. Describe the amino acid regulation of RNA synthesis.

8. Write short notes on :

a) Mutation

b) Cometabolism

c) Gene dosage

d) Feed back regulation

SET : 2

1. Define bio-conversion and what are the factors important to bio-conversions?

2. What is catabolic repression? Discuss mutants resistant to repression.

3. Describe the catabolite regulation in secondary metabolism?

4. Discuss the synthesis of primary metabolites.

5. Wtite about :

a) Differential regulation by isozymes.

b) Explain feed back regulation.

6. Write short notes on the following :

a) Precursor effect

b) Gene dosage

c) Co-metabolism

d) Permeability

7. Describe the metabolic regulation in branched pathways.

8. Write short notes on the following :

a) Resistant mutants

b) Diffusion

c) Mutation

d) Enzyme induction

SET : 3

1. Discuss the regulation of enzyme synthesis.

2. What is fermentation? What steps do you suggest for improving fermentation.

3. Discuss the biosynthesis of secondary metabolites.

4. What is permeability? Discuss the alteration of permeability.

5. Write about the following :

a) Feed back repression

b) Bioconversion of insoluble substances

6. Write short notes on the following :

a) Jacob Monod model

b) Bio-conversion

c) Gene dosage

d) Precursor effect

7. Describe the amino acid regulation of RNA synthesis.

8. Write short notes on the following :

a) Co-metabolism

b) Mutation

c) Diffusion

d) Strain selection.

SET : 4

1. Discuss the avoidance of product inhibition.

2. Discuss the role of strain in improving fermentation with some examples.

3. Explain the procedures of secondary metabolites.

4. What is permeability? Discuss the alteration of permeability.

5. Write about differential regulation by isozymes.

Define diffusion? Explain passive diffusion and facilitated diffusion.

6. Write short notes on the :

a) Jacob Monod model

b) Precursor effect

c) Catabolic repression

d) Gene dosage

7. Describe amino acid regulation of RNA synthesis.

8. Write short notes on the following :

a) Feed back regulation

b) Mutation

c) Bio-conversions

d) cAMP deficiency