PRINSIPBIOTEKNOLOGY

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

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 murniKelemahan: 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 metabolism

C from sugar metabolism

Acetate

Lactate

Pyruvate oxidase

Acetyl kinase

Lactate dehydrogenase

PyruvateLactate

FormateAcetyl 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 kinasePyruvate oxidaseLactate

dehydrogenase

PyruvateLactate

Acetyl Co

TCA Cycle

Formate Acetaldehyde Ethanol

Pyruvate formate lyase

Acetyldehydrogenase

Alcohol dehydrogenase

Add pyruvate decarboxylase gene

Pyruvate

Acetyl-phosphate Acetate

Lactate

Acetyl kinasePyruvate oxidaseLactate

dehydrogenase

Engineering step 2 – increase C flow to EtOH

PyruvateLactate

Acetyl Co

TCA Cycle

Formate Acetaldehyde Ethanol

Pyruvate formate lyase

Acetyldehydrogenase

Alcohol dehydrogenase

Up-regulate Alcohol dehydrogenase gene

Pyruvate

Acetyl-phosphate Acetate

Lactate

Acetyl kinasePyruvate oxidaseLactate

dehydrogenase

Engineering step 3 – block unwanted C flow by deleting key genes

PyruvateLactate

Acetyl Co

TCA Cycle

Formate Acetaldehyde Ethanol

Pyruvate formate lyase

Acetyldehydrogenase

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 dissolvedand 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 labelledwith 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

Genediscovery

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)