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Biotechnology > Metabolic Engineering > Flashcards

Metabolic Engineering Flashcards

1
Q

Define Metabolic Engineering

A

it is the improvement of cellular activities by manipulation of enzymatic, transport, and regulatory functions of the cell with the use of recombinant DNA technology

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2
Q

Define Industrial microbiology

A

the use of microorganisms, typically grown in large scale, to produce valuable commercial products or to carry out important chemical transformations

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3
Q

Microorganisms are used for : (3)

A

1- Biomass productions
2- Bioconversion or Biotransformation
3- Metabolite production

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4
Q

Example of each
1- Biomass productions
2- Bioconversion or Biotransformation
3- Metabolite production

A

1- Biomass productions:
production of yeast cells (or supplements eg. yeast extract)

2- Bioconversion or Biotransformation
adding substrate to cells to produce steroid (transforming one molecule to another)

3- Metabolite production
enzymes, amino acids, alcohol, antibiotics, and chemicals

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5
Q

To be industrialized, Microorganisms must : (8)

A

-Produce usuable substances or effects
-be available in pure culture
-be genetically stable, but amenable to genetic
manipulations
-Produce spores (increases their survivability during heat treatment which leads to their presence in a final product) or either reproductive structures to allow easy inoculation (the process of introducing microbes into a culture media so that it reproduces there)
-grow rapidly and produce products quickly in large scale
culture
-grow in such a way that the cells are easily separated from
the product
-not be harmful to humans or agricultural plants and
animals, etc
-GRAS

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6
Q

what if we have a pathogenic bacteria like for example salmonella or staphylococcus pseudomonas that produces useful/imp molecule of interests. What should we do to produce that molecule since we cannot work on pathogenic bacteria?

A

Genetically engineering the bacteria, by isolating the gene of interest that produces the molecule from the pathogenic bacteria and insert it in another bacterial plasmid that is not pathogenic creating rDNA and producing it

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7
Q

The breakdown of large molecules to produce energy

A

Catabolism

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8
Q

Synthesis of small molecules to produce larger molecule by using energy

A

Anabolism

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9
Q

Degradative
Oxidative
Energy Liberated
Converging

A

Catabolism

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10
Q

Biosynthetic
Reductive
Energy Required Energy
Diverging

A

Anabolism

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11
Q

——— are produced during
active cell growth during the log phase and are essential for cell growth

A

primary metabolites

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12
Q

——– are produced near
the onset of stationary phase and not essential for growth

A

secondary metabolites

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13
Q

Explain the graph

A

primary metabolite
the black line shows the inc growth of the cell and the production of ethanol from yeast at the beginning from very small no.

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14
Q

Explain the graph

A

primary metabolite
cells are growing and producing ethanol while sugar are decreasing because its being consumed by the cell as it growths

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15
Q

Explain the graph

A

secondary metabolite
cells are growing and the metabolite produced not at the beginning it synthesized at the end of the log phase (trophophase) meaning at the stationary phase (idiophase) meaning its not imp for cell growth

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16
Q

Explain the graph

A

secondary metabolite
cells are growing and consuming sugar and the metabolite produced not at the beginning

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17
Q

Explain the graph

A

inc in cell conc.
dec in lactose and ammonia as they are carbon and nitrogen sources
inc in the production of secondary metabolite

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18
Q

two types of media

A

1- rich media
2- mineral/minimal media

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19
Q

In nature, secondary metabolites are important for the organisms,
functioning as :

A

(i) sex hormones
(ii) ionophores
(iii) competitive weapons against other bacteria, fungi, amoebae, insects, large animals and plants,
(iv) agents of symbiosis
(v) effectors of differentiation

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20
Q

Nutritional Considerations while prepare a growth media

A
  • Energy source
  • Carbon source
  • Nitrogen, sulfur and phosphorus sources
  • Metallic elements
  • Vitamins
  • Water
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21
Q

Types of Media

A

Complex
Synthetic

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22
Q

————- A microorganism that has the ability
to synthesize all of its amino acids, nucleic acids,
vitamins from inorganic nutrients

A

Prototroph

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23
Q

———– An organism, such as a bacterial
strain, that has lost the ability to synthesize
certain substances required for its growth, like aa.

A

Auxotroph

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24
Q

Types of Reproduction

A
  • Binary fission
  • asexual reproduction by a separation of the body into two new bodies.
  • Budding
  • asexual reproduction in which a new organism develops from an outgrowth or bud due to cell division at one particular site.
  • Fragmentation
  • asexual reproduction or cloning, where an organism is split into fragments. Each of these fragments develops into mature, fully grown individuals that are clones of the original organism.
    – More common in fungi
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25
Q

two ways to measure the growth of the cell

A
  • Turbidity
    . Spectrophotometer
    . scale (%T/ OD or Abs)
  • Filteration
26
Q

Why are amino acids
important in biotechnology?

A

Amino acids are used by the body to produce
peptides, Proteins, hormones and neurotransmitters
that are essential in regulating physiological
functions.

27
Q

—— regulates
metabolic rate

A

Tyrosine
Tyrosine»Thyroxin

28
Q

——– regulates blood
sugar levels

A

amino acids
Amino acids»>insulin

29
Q

Role of enzymes in metabolic pathways

A

Enzymes facilitate each step of metabolic pathway
They are proteins acting as chemical catalysts
Accelerate conversion of substrate to product

30
Q

————– Compound that participates in the chemical reaction that produces another compound

A

Precursor or intermediate

31
Q

The yield of penicillin can be increased for
example by:

A
  • Improvement in composition of the medium
  • Isolation of better penicillin producing mold sp.
    Penicillium chrysogenum which grows better in
    huge deep fermentation tank
  • Development of submerged culture technique
    for cultivation of mold in large volume of liquid
    medium through which sterile air is forced.
32
Q

—————— cross-feeding is the phenomenon of one species feeding on the metabolic products of another species to cope up with the energy limitations by electron transfer

A

syntrophy

33
Q

Complex media

A

– Most commonly used
– Exact composition is unknown
* Beef extract, yeast extract, tryptone, peptone, salt

34
Q

Synthetic media

A
  • Exact composition is known
    – To determine growth requirements
  • Glucose, salt, magnesium, potassium, etc.
    -Minimal medium: contains: Glucose + Mineral source of
    Nitrogen + salt, magnesium, potassium, etc
35
Q

————- are defined as mutants that cannot make
certain nutrients.

A

Auxotrophic mutants cannot make a specific nutrient, so they require the relevant nutrient in their growth medium

36
Q

process of Isolation of auxotrophic mutants tyrosine-

A
  • Plating of a big number of bacterial cells on:
    Minimal medium + glucose, containing the 19 aa but lacking Tyr,+ penicillin»»ON incubation

-Since penicillin stops the wall synthesis only once it has begun, all protrophic cells will die, but not auxotrophic mutants that are Tyr-, because they cannot grow and synthesize the cell wall without Tyrosin.

-transfer the mutants to a rich medium»»>growth

-Use these auxotrophic mutants to identify the tyrosin pathway intermediates or precursors of synthesis

-After having isolated a big number of mutants tyr-, use the latter for the identification of the precursors of the metabolic pathway: Culture of each mutant in MM+glucose+ 19 aa + a small qty of Tyr // accumulation of precursors

37
Q

why do we need to add penicillin in the media with both the 19aa and Tyr-?

A

penicillin stops the wall synthesis only once it has begun, all protrophic cells will die, but not auxotrophic mutants that are Tyr-, because they cannot grow and synthesize the cell wall without Tyrosin.

38
Q

Identification of precursors using biochemical techniques :

A

Chromatography HPLC High Performance Liquid Chromatography, Mass spectrometry LCMS (liquid), GCMS (Gas),….

39
Q

Non-Essential Amino Acids
in Humans can be formed from……

A

a-keto acids by transamination and subsequent reactions

40
Q

Metabolic regulation:
Genetic level

A

control transcription of genes (repression, induction and catabolic repression (glucose effect)) * Catabolic repression: In the presence of Glucose , there is repression of certain sugar-metabolizing operons (eg lac) in favour of glucose utilization when glucose is the predominant carbon source in the environment of the cell

41
Q

Metabolic regulation:

A

Genetic level
Cellular level:

42
Q

Metabolic regulation:
Cellular level

A

Enzyme activity: feedback inhibition

43
Q

——- The end product or all end products repress the enzymes of the pathway

A

Feedback inhibition

44
Q

Fermentation….

A

is a series of biological Ox/Red reactions yielding energy, in which organic chemicals serve as both the electron donor and the final electron acceptor

Anaerobic process that does not use the E.T.S. Usually involves the incomplete oxidation of a carbohydrate which then becomes the final electron acceptor. But it can occur in presence of oxygen

45
Q

Fermentation pathways:

A

a. Homolactic acid F.
b. Alcoholic F.

46
Q

FERMENTATION CAN YIELD:

A

SINGLE PRODUCTS (HOMOFERMENTATIONS)
Pyruvate»»lactate
OR
MULTIPLE PRODUCTS (HETEROFERMENTATIONS)
Pyruvate»»lactate + ethanol, CO2

47
Q

DEFINITION of Industrial fermentations

A
  • The industrial fermentation is the production of
    microbial products (biomass or metabolites)

-process for the production of useful products
through mass culture of micro-organisms. The end products or the various intermediate products (primary and secondary metabolites) are siphoned off & purified for commercial use

48
Q

Types of fermenters

A

a batch (or closed )process
a continuous (or open) process

49
Q

Batch process

A

Here the bioreactor is filled with sterile nutrient medium, the microbe is mixed in & no further additions or removals are made during the period of incubation (ie a closed system).

50
Q
A

antibiotics such as Penicillin

51
Q

Disadvantages and Advantages of Batch fermenter

A

Disadvantages of the process are obviously the time spent in cleaning, sterilizing, filling ..etc with each run, as well as the fact that there is no continuous supply of the product.

Advantages are that the process involves a relatively short fermentation time, is relatively easy to keep under control and allows all stages of growth to occur (some products only form at particular stages eg penicillin at end of exponential phase).

52
Q

Continuous process

A

Here nutrients are added & cells are harvested at a given steady rate.
This is done by running in fresh sterile nutrient medium (substrate) & withdrawing an equivalent amount of culture medium together with the fermentation products

53
Q

Continuous process,,, This technique is used in the production of —————

A

mycoproteins and also of metabolites such as lactic acid and also vitamin C

54
Q

Disadvantages and Advantages of Batch fermenter continuous process

A

Advantage is that :
* the process can run day & night so there is increased
productivity , no time wasted cleaning out & sterilizing the fermenter.
* The technique naturally gives higher product volume relative to batch process of equivalent size.
*there is a continuous supply of the product to meet demand , you don’t need to wait ‘till the next batch is ready.

Disadvantage is that the process is more helpless to contamination by unwanted micro-organisms

55
Q

Cooling of the fermenter is achieved by

A

water jacket or cooling coils inside the fermenter.

56
Q

Types of Products Produced in Microbes

A

*Amino Acids
*Vitamins
*Food Additives
*Enzymes
*Recombinant Protein Drugs
*Antibiotics
*Fuels
*Plastics

57
Q

Enzymes Uses

A

Detergents
Baking
Starch Derived Syrups
Protein Processing

58
Q

————- converts starch to oligosaccharides

A

b-amylase

59
Q

——- converts oligosaccharides to glucose monomers

A

Glucoamylase

60
Q

———– converts glucose to fructose (Sweetener)

A

Glucose isomerase

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