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PRINSIP
BIOTEKNOLOGY
Strain Development
Materi
 Pengembangan Strain
 Aplikasi bioteknologi pada tumbuhan
 Aplikasi bioteknologi pada hewan
 Aplikasi bioteknologi pada manusia
 Aplikasi bioteknologi pada lingkungan
Pengembangan Strain
 What is strain?
Case: Produksi asam sitrat
Jeruk lemon
dipres
Cairan buah
presipitasi
Kalsium sitrat
ekspor
Currie dkk (1917)
 Memperkenalkan strain Aspergillus niger mampu
menghasilkan asam sitrat cukup tinggi
 Syarat: ditumbuhkan pada media yang sesuai
- pH media sekitar 2
- mencegah kondisi lingkungan yang memacu
pertumbuhan vegetatif
- how?
 Selanjutnya produksi asam sitrat didominasi oleh
proses fermentasi
 Upaya peningkatan proses fermentasi al:
Metoda Pengembangan strain
1. Isolasi tipe tertentu
 Isolasi Aspergillus dari alam (tanah)
 Dapat ditingkatkan dengan media diperkaya
(merendam sampel tanah dlm lart tanin)
 Ditumbuhkan pada media standar dg
kandungan KH tinggi (sukrosa/molase 15%)
 Kadar asam sitrat ditentukan dg metoda
titrasi
 Biakan dg hasil baik
disubkultur
 Diulangi bbrp kali
biakan murni
Kelemahan: laborious
Processes involved in production of a commodity
in biotechnology
2.Teknik isolasi spora tunggal
 Dapat mendeteksi dg cepat
Spora jamur
Media dg indikator pH
Zona jernih
 Dihitung unit keasaman dg membagi d zona
jernih dg d zona hambat
3. Passage culture method
 Metoda dg prinsip reaksi biokimia (misal:
resistensi thd kons asam sitrat, suhu, dan
kadar gula tinggi)
 Menghambat pertumbuhan jamur lain
 Diulangi beberapa kali sehingga diperoleh
biakan murni dengan kemampuan yang baik
4. Mutagenesis
 Umumnya dilakukan dengan radiasi
Suspensi spora densitas 107 – 108 spora/ml
disinari dg uv
disebar ke media agar 4% + kasein
diinkubasi
dihitung indeks zona jernih
hasil?
Methods for engineering genes
 Random mutagenesis
 Where the protein structure is not known, or
where the desired change is difficult to define
in structural terms, random mutagenesis of the
gene sequence followed by screening for
functionally enhanced mutants is normally
used
 How to do?
Error Prone PCR (EPP) is most commonly
used for random mutagenesis



The rate of misincorporation of nucleotides by
Taq polymerase in PCR can be greatly
increased by adding Mg2+ or Mn2+ to the PCR
reaction. The misincorporation rate can be
adjusted to obtain 3-10 amino changes per
gene.
Amplicons (with mutations resulting from EPP)
are cloned in expression hosts.
The amplicon library can be screened for a
functional change (e.g., different substrate
specificity, higher reaction rate, better stability
etc.)
Example: Pathway engineering in Bacillus for
production of bioethanol.
 Bacillus species produce little ethanol
naturally, but have some advantages of
ethanologens such as yeast due to their
ability to utilise the C5 carbon sources in
lignocellulosic substrates.
Objective: Modify Bacillus genome to
efficiently produce ethanol
Engineering C flow in Bacillus – central carbon
metabolism
C from sugar
metabolism
Acetyl kinase
Acetyl-phosphate
Acetate
Pyruvate
oxidase
Lactate
dehydrogenase
Lactate
Pyruvate
Pyruvate
formate
lyase
Formate
Acetyl CoA
Acetaldehyde
Ethanol
Acetyl
Alcohol
dehydrogenase
dehydrogenase
TCA Cycle
Engineering step 1 – convert pyruvate directly
to ethanol
Acetyl-phosphate
Lactate
dehydrogenase
Lactate
Acetyl kinase
Pyruvate
oxidase
Pyruvate
Pyruvate
formate
lyase
Formate
Acetate
Acetyl Co
Add pyruvate
decarboxylase gene
Acetaldehyde
Acetyl
dehydrogenase
TCA Cycle
Ethanol
Alcohol
dehydrogenase
Engineering step 2 – increase C flow to EtOH
Acetyl-phosphate
Acetate
Acetyl kinase
Pyruvate
oxidase
Lactate
dehydrogenase
Lactate
Pyruvate
Pyruvate
formate
lyase
Formate
Up-regulate Alcohol
dehydrogenase gene
Acetyl Co
Acetaldehyde
Acetyl
dehydrogenase
TCA Cycle
Alcohol
dehydrogenase
Ethanol
Engineering step 3 – block unwanted C flow by
deleting key genes
Acetyl-phosphate
Acetate
Acetyl kinase
Pyruvate
oxidase
Lactate
dehydrogenase
Lactate
Pyruvate
Pyruvate
formate
lyase
Formate
Acetyl Co
Acetaldehyde
Acetyl
dehydrogenase
TCA Cycle
Ethanol
Alcohol
dehydrogenase
Kelemahan
 Menghasilkan mutan yang siftatnya tidak
stabil
 Menghasilkan mutan auxotrof
 Kelebihan?
 Mutagenesis juga dapat dilakukan dengan
bahan kimia seperti 4-dinitroquinolin-N-oxida
 Note: tahap 1 dan 2 dapat dibalik urutannya.
5. Fusi protoplas
 Cara paling efektif untuk menggabungkan
gen antara strain induk dg karakteristik yg
diinginkan
 Dapat dilakukan secara intraspesifik
(percampuran protoplas dari 2 sp yg berbeda
tapi genus sama) maupun secara
intergenerik (percampuran protoplas dari 2 sp
dg genus berbeda)
 Metoda: menggunakan Polyethilen Glikol
(PEG) solution
the fusion process
•
Electrofusion – protoplasts are aligned in a
special chamber, electric current is applied,
opening channels in cell membrane
• PEG fusion – protoplasts are coated with PEG,
then incubated together; where cell membranes
fuse, channels begin to form
the fusion process
• eventually, cell membrane between is dissolved
and nuclei fuse into 1 nucleus
• in this type of fusion, cytoplasm is mixed
selection of heterokaryons
cell sorting (Cell Facility should be able to do this)
• parental protoplasts are differentially labelled
with fluorescent dyes, one green, one red
• heterokaryons are stained yellow and can be
sorted based on that trait
6. Pendekatan molekuler
 Review on cloning technique
 Engineering options in the product pipeline
Engineering options in the product pipeline
Gene
discovery
Recombinant
gene
Genetic engineering
– modification of gene by site directed or
random mutagenesis
High level
expression
Fermentation
Reaction engineering
Protein ‘engineering’
– modification of solvent etc – chemical modification or immobilisation
Application
e.g., Biocatalysis
Product
Downstream
processing
Reasons for Protein Engineering
 Enhance protein thermostability
 Usually by inserting new intramolecular interactions
such as covalent disulphide (S-S) bonds or noncovalent salt bridges.
 Reduce oxidation sensitivity
 By deletion/replacement of oxidation sensitive amino
acid residues (e.g., cysteine)
 Alter enzyme substrate specificity
 By altering the size and shape of the active site (e.g.,
by removing bulky side chains)
 Increase catalytic activity
 By changing the environment of the active site (by
random mutagenesis and selection)