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)
