PRINSIP

BIOTEKNOLOGY

Strain Development

Materi

�Pengembangan Strain

�Aplikasi bioteknologi pada tumbuhan

�Aplikasi bioteknologi pada hewan

Aplikasi bioteknologi pada manusia�Aplikasi bioteknologi pada manusia

�Aplikasi bioteknologi pada lingkungan

Pengembangan Strain

� What is strain?

Case: Produksi asam sitrat

Jeruk lemon

dipres

Cairan buah

Kalsium sitrat

ekspor

dipres

presipitasi

Currie dkk (1917)

� Memperkenalkan strain Aspergillus niger mampu

menghasilkan asam sitrat cukup tinggi

� Syarat: ditumbuhkan pada media yang sesuai

- pH media sekitar 2- 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%)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 pHMedia 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 lainMenghambat 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 uvdisinari 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 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.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.lignocellulosic substrates.

Objective: Modify Bacillus genome to

efficiently produce ethanol

Pyruvate

Acetyl-phosphate

Engineering C flow in Bacillus – central carbon

metabolismC from sugar

metabolism

Acetate

Lactate

Pyruvate

oxidase

Acetyl kinase

Lactate

dehydrogenasePyruvateLactate

Formate

Acetyl CoA

TCA Cycle

Acetaldehyde Ethanol

Pyruvate

formate

lyase

Acetyldehydrogenase

Alcohol dehydrogenase

Engineering step 1 – convert pyruvate directly

to ethanol

Pyruvate

Acetyl-phosphate Acetate

Lactate

Acetyl kinase

Pyruvate

oxidaseLactate

dehydrogenase

PyruvateLactate

Acetyl Co

TCA Cycle

Formate Acetaldehyde Ethanol

Pyruvate

formate

lyase

Acetyl

dehydrogenase

Alcohol

dehydrogenase

Add pyruvate

decarboxylase gene

Pyruvate

Acetyl-phosphate Acetate

Lactate

Acetyl kinase

Pyruvate

oxidaseLactate

dehydrogenase

Engineering step 2 – increase C flow to EtOH

PyruvateLactate

Acetyl Co

TCA Cycle

Formate Acetaldehyde Ethanol

Pyruvate

formate

lyase

Acetyl

dehydrogenase

Alcohol

dehydrogenase

Up-regulate Alcohol

dehydrogenase gene

Pyruvate

Acetyl-phosphate Acetate

Lactate

Acetyl kinase

Pyruvate

oxidaseLactate

dehydrogenase

Engineering step 3 – block unwanted C flow by

deleting key genes

PyruvateLactate

Acetyl Co

TCA Cycle

Formate Acetaldehyde Ethanol

Pyruvate

formate

lyase

Acetyl

dehydrogenase

Alcohol

dehydrogenase

Kelemahan

� Menghasilkan mutan yang siftatnya tidak

stabil

� Menghasilkan mutan auxotrof

� Kelebihan?� 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 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 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 • 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

High level

expression

Genetic engineering

Fermentation

Downstream

processingProductApplication

e.g., Biocatalysis

Genetic engineering

– modification of gene by site directed or

random mutagenesis

Protein ‘engineering’

– chemical modification or immobilisation

Reaction engineering

– modification of solvent etc

Reasons for Protein Engineering

� Enhance protein thermostability

� Usually by inserting new intramolecular interactions such as covalent disulphide (S-S) bonds or non-covalent salt bridges.

� Reduce oxidation sensitivity

� By deletion/replacement of oxidation sensitive amino � 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)