The Improvement of Industrial Microorganism · Why the improvement of microorganisms is needed? - the metabolite concentrations produced by wild strains are too low for economical

Strain Improvement

Why the improvement of microorganisms

is needed?

- the metabolite concentrations produced by wild strains are too low for economical processes

- Optimizing the culture medium and growth conditions

could not maximize the organism ability to synthesize products

The major motivation for industrial strain development is economic, since:

Strain Improvement Techniques

Classical techniques

- mutagenesis (UV, mutagen)

Modern techniques

- recombinant DNA technology

Mutagenesis

A process by which the genetic information of an organism is changed in a stable manner, either in nature or experimentally by the use of chemicals or radiation.

base substitutions, insertions, and deletions.

UV light, x-rays, gamma rays, NTG (Nitrosoguanidine) , EMS (Ethyl methanesulfonate )

Ex; Hydrogen production by Bacillus pumilus Lipase production by A. japonicus

Chemical mutagen (base analogs)

Radiation

Chemical mutagens

Radiation

Genetic modification/engineering

by

Recombinant DNA Technology

The manipulation of a trait in

an organism to create a

desired change

What is Genetic Engineering?

Tools

1. DNA (gene target)

2. Restriction endonucleases (molecular scissors)

3. Cloning vector/plasmid (e.g. pGEM, pBR322…)

4. Ligase enzyme (molecular glue)

Cell lysis

Removal of proteins (extraction)

DNA precipitation by ethanol

DNA dilution in water or buffer

Step 1: Isolating the gene

Crude lysate containing

nucleic acids

Mix thoroughly with

an equal volume of

organic solvent

e.g. phenol, chloroform,

or phenol:chloroform

Centrifuge

-The aqueous phase contains water-

soluble molecules, including nucleic acids.

- Proteins and lipids become trapped in

the organic phase, and are thus

separated away.

Perform additional extractions for increased purity

Collect aqueous phase

Extraction/Precipitation Method

Organic extraction

Organic

Aqueous

• Pellet down nucleic acids.

• Wash pellet with 70% ethanol to remove

residual salts and other contaminants.

• Discard ethanol and allow pellet to dry.

After

Add alcohol (100%)

and salt to precipitate

nucleic acids from the

aqueous fraction

Supernatant

Pellet

70% EtOH

Dissolve pellet

(H2O, TE, etc.)

Nucleic Acid Precipitation

Continued

Before After

Centrifuge Wash Centrifuge

DNA Purity and Concentration

Spectrophotometry

Absorbance maximum

for nucleic acids 260 nm

for proteins 280 nm

Concentration of DNA – at 260 nm

Purity of DNA: ratio of 260/280 nm (1.8-2.0)

DNA confirmation by electrophoresis

Step 2: Inserting gene into vector

• Vector – molecule of

DNA which is used to

carry a foreign gene into

a host cell

Promoter gene - A sequence of bases

in a nucleic acid strand, that serves as

a signal to start transcription.

The gene of interest.

Antibiotic resistant gene- Are used as a

marker system for transformed cells.

Cloning vector

Plasmid Vector: pBR322

First modern cloning vector (1976)

pUC Plasmids

Pulser Equipment

Introduction of integration vector

Principle of Electroporation Technique

Recipient cells

Plasmid

DNA

+ -

Current

Pulser

1. Replica plating

Screening the positive clone

2. Blue/White Selection

– Modified E. coli codes for the carboxyl portion of β-galactosidase (β-gal) enzyme. This portion alone cannot cleave X-gal.

– Plasmid codes for amino portion (“LacZ” or α-peptide) of β-gal.

– However, when the two peptides are expressed together in a cell, X-gal is cleaved, and an indigo product stains the bacterial colony blue. This process is called α-complementation.

Blue/White Selection

• Based on the enzymatic reaction of β-galactosidase.

2. Blue/White Selection

kDa

97

66

45

30

14.4

Example: Expression of alanine dehydrogenase

in L. lactis

Strain Specific activity

(mmol min-1 mg protein-1)

Bacillus

sphaericus

IFO3525

0.4

Bacillus subtilis 0.5

L. Lactis

(pNZ8020)

ND

L. Lactis

(pNZ2650,+alaD)

0.39

ND: not detected

Enzyme Gene donor Gene recipient Increase

a-amylase B. amyloliquefaciens B. subtilis 10

a-amylase Bacillus Bacillus 5

stearothermophilus stearothermophilus

a-amylase B. stearothermophilus B. brevis 100

EcoRI E. coli E. coli 50-100

DNA E. coli E. coli 100

Polymerase

Alanine B. sphaericus L. lactis 100

Multiplication of the host cells

Small scale of fermentation (lab scale fermentation) 500ml-shakeflask, 2

L-fermentor

Large scale of fermentation (industrial scale fermentation) 10-L, 100-L

fermentor

Extraction of desired gene product

Purification

Centrifugation

Ammonium sulfate precipitation

Chromatography

- Ionic exchange chromatography

- Affinity chromatography

- Hydrophobic chromatography

- Reverse phase chromatography

SDS- PAGE

M 3 3 2 2 1 1 M

Purification step 3: step 3 2: step 2 1: step 1 M: marker (molecular weight)

Stability of Recombinant Strain

The ability of strain to maintain its high productivity during culture maintenance and fermentation

very important

Yield decay during storage a

proper maintenance techniques

Loss productivity during fermentation difficult to control