Strain Improvement Strain Improvement of Microorganismsof Microorganisms

Marlia Singgih WibowoMarlia Singgih Wibowo

Definition of Definition of Strain ImprovementStrain Improvement

•• In the field of agriculture : recovery of In the field of agriculture : recovery of plantplant’’s characterss characters

•• In the In the fileldfileld of microbiology : improve the of microbiology : improve the microbemicrobe’’s productivities or characteristicss productivities or characteristics

Purposes of Purposes of Strain ImprovementStrain Improvement

•• Increase productivitiesIncrease productivities•• To change unused coTo change unused co--metabolitesmetabolites•• To improve the use of carbon and To improve the use of carbon and

nitrogen sourcesnitrogen sources•• To improve morphology of cells to be a To improve morphology of cells to be a

better cells in order to separate the cells better cells in order to separate the cells and its productsand its products

General ProcessGeneral Process

• Induction of genetic variation in some cell population of chosen microorganism

• Fermentation Process in lab scale for some cells

• Analysis (assay) to check the productivity of mutated strain

How to optimize How to optimize miicrobemiicrobe’’ssproductivity?productivity?

•• Optimize environmental condition Optimize environmental condition •• Optimize nutrition of Optimize nutrition of microorganismmicroorganism•• Genetic Modification : Mutation, Genetic Modification : Mutation,

recombination, generecombination, gene cloningcloning

•• Modification of physical parameter Modification of physical parameter (temperature, agitation, etc)(temperature, agitation, etc)

•• Modification of chemical parameter Modification of chemical parameter (pH, O2 (pH, O2 concconc, etc), etc)

•• Modification of biological parameter Modification of biological parameter (co(co--culture, culture, enzymenzym, etc), etc)

Optimize environmental Optimize environmental condition condition

•• Carbon sourceCarbon source•• Nitrogen sourceNitrogen source•• Mineral source and other sourcesMineral source and other sources•• PrecursorPrecursor•• Enzyme Enzyme

Optimize nutrition of Optimize nutrition of microorganismmicroorganism

StageStage ProcessProcess AmountAmount11 a.Mutationa.Mutation of wild type of wild type

b.Cultureb.Culture on agaron agarc.Selectionc.Selection of coloniesof coloniesd.Fermentationd.Fermentatione.Selectione.Selection of good strain of good strain

a.8 different doses a.8 different doses

c.25 of each dosec.25 of each dosed.200 strainsd.200 strainse.40 strains e.40 strains

22 a.Rea.Re test of selected strain (5 test of selected strain (5 replication)replication)b.Selectionb.Selection of best strain of best strain

Next screeningNext screening

a.40 x 5 = 200 strainsa.40 x 5 = 200 strains

b.8 strain for primary b.8 strain for primary metabolites, 40 strains for metabolites, 40 strains for 22ndnd metabolitesmetabolites

Screening in IndustryScreening in Industry

MUTAGENESISMUTAGENESIS

•• Mutagenesis is a process of mutation in Mutagenesis is a process of mutation in strain improvement of strain improvement of microorganismmicroorganism. A . A treatment to treatment to microorganismmicroorganism will cause an will cause an improvement in their improvement in their genotipicgenotipic and and fenotipicfenotipic performancesperformances

•• Mutation can be random or direct Mutation can be random or direct

Procedure of mutagenesisProcedure of mutagenesis

•• Mutation by : physical, chemical or Mutation by : physical, chemical or combination of physical and chemical combination of physical and chemical methodmethod

•• Protoplast FusionProtoplast Fusion•• RecombinationRecombination•• Gene Cloning Gene Cloning

Physical Mutation Physical Mutation •• XX--ray ray

Chromosomal mutationChromosomal mutation

•• UV Radiation (250 UV Radiation (250 –– 270 nm)270 nm)DNA DNA absorpsabsorps uvuv padapada 260 nm260 nmPoint mutationPoint mutationDimerizationDimerization of thymine of thymine PhotoPhoto--repair : gain improvement of some repair : gain improvement of some abilitiesabilities

•• RadioisotopRadioisotop

Stages in Physical mutation Stages in Physical mutation

•• WildWild--type of microbes is suspended in medium type of microbes is suspended in medium •• Counting number of cells in medium and Counting number of cells in medium and

concentration of cells is determined for concentration of cells is determined for mutation process mutation process

•• Radiation is conducted in dark area with Radiation is conducted in dark area with certain distance for certain duration.certain distance for certain duration.

•• Exposed colonies are inoculated in growth Exposed colonies are inoculated in growth medium for several days medium for several days

•• Check the growth and select some colonies for Check the growth and select some colonies for further process further process

Chemical Mutation Chemical Mutation •• Base Base analoganalog : : substance that similar to substance that similar to purinepurine or or

pirimidinepirimidine, so transition will , so transition will occuredoccured , example : , example : 55--bromouracyl : substitute C (cytosine) with T bromouracyl : substitute C (cytosine) with T ((thyminthymin) ) 22--aminopurine : substitute A (adenine) with G aminopurine : substitute A (adenine) with G (guanine)(guanine)

•• De amine and De amine and alkylatingalkylating agentagent : : transition, transition, example:example:Nitrite (NaNONitrite (NaNO22) can cause de) can cause de--aminedamined A and C so A and C so replication of AT will change to GC, or replication of AT will change to GC, or viceversaviceversaEMS (EMS (EtilEtil metilmetil SulfonatSulfonat), MNNG (), MNNG (MetilMetil--nitronitro--nitrosonitroso--guanidineguanidine))

•• IntercalationIntercalationDye Dye acridineacridine ((proflavineproflavine) inserted between ) inserted between base pair, will cause a framebase pair, will cause a frame--shift and will shift and will misread its genetic informationmisread its genetic information

Mutation of Mutation of MonascusMonascus purpureuspurpureususing EMSusing EMS

Albino mutant of Albino mutant of MonascusMonascus purpureuspurpureusstable after 5 generationstable after 5 generation

PROTOPLAST FUSIONPROTOPLAST FUSION

• Protoplast fusion is a general technic to induce genetic recombination in some prokariot and eukariotic microorganisms

• Protoplast : whole cell without cell wall

• Fusion has some benefits to improve productivity of industrial microorganisms, e.g. Actinomycetes.

• The Method is easy, no transduction phage needed, plasmid or competention development, the only things is how to get the stable protoplast

• In protoplast fusion will produce hybrid cells

Method of Protoplast FusionMethod of Protoplast Fusion•• Protoplast is prepared by removing the cell wall Protoplast is prepared by removing the cell wall

using using lyticlytic enzyme in a osmotic solution. enzyme in a osmotic solution. •• With the help of PEG (With the help of PEG (polyetilenglicolpolyetilenglicol) at certain ) at certain

conc. (conc. (fusogenicfusogenic agent), the fusion can be done agent), the fusion can be done and hybrid cells will produced (diploid) and hybrid cells will produced (diploid)

•• During this stage , genome (or During this stage , genome (or chromosomchromosom) ) from both cells will be fused and mixed . The from both cells will be fused and mixed . The genetic recombination will genetic recombination will occuredoccured

Protoplast Fusion for microorganismProtoplast Fusion for microorganism

• For prokariot Bacilli and Streptomycetes , recombination will occur in a very high frequency because its chromosomes is free in the cytoplasm

• For eukariot microorganism, protoplast fusion should be followed by fusion of nucleus to obtain the real recombination

• The important step in protoplast fusion is REGENERATION of the cells after fusion

• Protoplast fusion mostly done for the same species to obtain the best strain with high productivity

ExampeExampe of Protoplast Fusion : of Protoplast Fusion : AspergillusAspergillus terreusterreus

Strain 1 Strain 2

Fusant after fusion

Genetic EngineeringGenetic Engineering

Genetic EngineeringGenetic Engineering

•• RekayasaRekayasa genetikgenetik adalahadalah salahsalah satusatu caracara pembuatanpembuatanDNA DNA barubaru, , biasanyabiasanya melaluimelalui rekombinasirekombinasi DNA DNA daridariorganismeorganisme yang yang berbedaberbeda dandan meproduksimeproduksi banyakbanyakkopi kopi daridari DNA DNA rekombinanrekombinan tersebuttersebut melaluimelalui prosesprosesyang yang disebutdisebut KLONINGKLONING

•• KloningKloning adalahadalah suatusuatu prosesproses dimanadimana suatusuatu urutanurutanDNA DNA tertentutertentu disisipkandisisipkan keke dalamdalam suatusuatu vektorvektor((berupaberupa plasmid plasmid atauatau kromosomkromosom fagafaga) ) dandanselanjutnyaselanjutnya direplikasidireplikasi sebanyaksebanyak mungkinmungkin. . ReplikasiReplikasiterjaditerjadi didi dalamdalam suatusuatu inanginang (host) yang (host) yang memungkinkanmemungkinkan replikasireplikasi ituitu terjaditerjadi

PrinsipPrinsip mutasimutasi dengandenganrekayasarekayasa genetikgenetik

•• InformasiInformasi genetikgenetik yang yang ditujudituju, , diisolasidiisolasi daridariorganismeorganisme donor donor dandan dipotongdipotong menjadimenjadi bagianbagiantunggaltunggal menggunakanmenggunakan enzimenzim restriksirestriksi

•• PotonganPotongan iniini digabungkandigabungkan didi dalamdalam suatusuatu DNA DNA pembawapembawa ((vektorvektor) ) dandan selanjutnyaselanjutnya bersamabersamavektorvektor tersebuttersebut ditransferditransfer keke dalamdalam suatusuatu selselinanginang (host)(host)

•• SelSel inanginang akanakan bereplikasibereplikasi dandan mensekresimensekresimetabolitmetabolit yang yang sesuaisesuai dengandengan informasiinformasi genetikgenetikyang yang ditransferditransfer sebelumnyasebelumnya..

SyaratSyarat untukuntuk vektorvektor

•• MemilikiMemiliki Selectable markers Selectable markers ((penandapenanda selektifselektif))

•• MemilikiMemiliki Restriction sites Restriction sites ((situssitus restriksirestriksi))

•• BeratBerat molekulmolekul serendahserendah mungkinmungkin

PlasmidPlasmid•• Plasmid Plasmid adalahadalah suatusuatu molekulmolekul DNA DNA sirkularsirkular

(BM 10(BM 1066 –– 2 x 102 x 1088) ) daridari bakteribakteri, yang , yang membawamembawa 11--3% 3% genomgenom selsel dandan mengkodemengkodesuatusuatu sifatsifat genetikgenetik pentingpenting yang yang tidaktidak dikodedikodesecarasecara normal normal oleholeh kromosomkromosom bakteribakteritersebuttersebut

•• SifatSifat yang yang banyakbanyak digunakandigunakan : : resistensiresistensiantibiotikantibiotik, , produksiproduksi antibiotikantibiotik, , degradasidegradasisenyawasenyawa aromatikaromatik, , dlldll..

FagaFaga ((bakteriofagabakteriofaga))•• suatusuatu subsub--kelompokkelompok virus yang virus yang menginfeksimenginfeksi

bakteribakteri dengandengan caracara menyisipkanmenyisipkan asamasamnukleatnukleat nyanya keke dalamdalam bakteribakteri host host nyanya..

•• FagaFaga terkecilterkecil hanyahanya mengandungmengandung 3 3 gengenpengkodepengkode ssss RNARNA

•• T4, T4, suatusuatu fagafaga bakteribakteri yang yang mengandungmengandung lebihlebihkurangkurang 60 60 gengen pengkodepengkode dsds genomgenom DNA DNA fagafagatersebuttersebut

CosmidCosmid•• CosmidCosmid adalahadalah partikelpartikel sintetiksintetik yang yang dapatdapat bereplikasibereplikasi

sendirisendiri•• BerasalBerasal daridari plasmid yang plasmid yang mengandungmengandung fragmenfragmen

lambdalambda--DNA (DNADNA (DNA--λλ) yang ) yang mengkodemengkode urutanurutan coscos sitesite((situssitus pengenalanpengenalan) ) untukuntuk sistemsistem λλ yang yang akanakanmembentukmembentuk strukturstruktur sirkularsirkular..

•• DNA DNA λλ fagafaga iniini bereplikasibereplikasi melaluimelalui suatusuatu mekanismemekanismekhususkhusus yaituyaitu rolling circlerolling circle yang yang yangyang selanjutnyaselanjutnyamembentukmembentuk strukturstruktur concatemerconcatemer ((berulangberulang secarasecara linear) linear) setelahsetelah disisipkandisisipkan DNA DNA asingasing..

•• BentukBentuk linear linear iniini merupakanmerupakan substratsubstrat untukuntuk reaksireaksipackaging packaging secarasecara inin--vitro vitro membentukmembentuk partikelpartikel. . SelanjutnyaSelanjutnya partikelpartikel tersebuttersebut digunakandigunakan untukuntukmenginfeksimenginfeksi selsel inanginang, , dandan bentukbentuk linear linear cosmidcosmid akanakanmembentukmembentuk sirkularsirkular kembalikembali padapada sisisisi coscos sitesite

PrinsipPrinsip kloningkloning gengen menggunakanmenggunakanplasmid plasmid E.coliE.coli pBRpBR 322322

Plasmid yang mengandung gen resistensithp ampisilin dan tetrasiklin dipotong padasisi tetrasiklin dengan enzim restriksi

DNA asing di sisipkan pada plasmid denganenzim ligase

Plasmid yang telah mengandung DNA asing tsb ditransfer ke dalam sel bakteri

Seleksi sel yang hanyamengandung genresistensi ampisilindan DNA asing

Pembiakan

JenisJenis MutanMutan

•• MutanMutan AukosotrofAukosotrof ((AuxotrophicAuxotrophic mutanmutan))•• MutanMutan ResistenResisten (Resistant)(Resistant)•• MutanMutan yang yang sensitifsensitif terhadapterhadap temperaturtemperatur•• MutanMutan KonstitutifKonstitutif katabolikkatabolik•• MutanMutan KonstitutifKonstitutif anabolikanabolik

•• MutanMutan aukosotrofaukosotrof adalahadalah mutanmutan yang yang tidaktidak dapatdapatmensintesismensintesis suatusuatu molekulmolekul organikorganik yang yang sebenarnyasebenarnyasangatsangat diperlukandiperlukan untukuntuk salahsalah satusatu jalurjalur biosintesisbiosintesismetabolitmetabolit nyanya. . MisalnyaMisalnya : : nitratnitrat, , antibiotikantibiotik

•• MutanMutan resistenresisten adalahadalah mutanmutan daridari organismeorganisme yang yang sebelumnyasebelumnya sensitifsensitif terhadapterhadap suatusuatu senyawasenyawa

•• MutanMutan yang yang sensitifsensitif terhadapterhadap temperaturtemperaturadalahadalah mutanmutan daridari oraganismeoraganisme yang yang semulasemula tidaktidak sensitifsensitifterhadapterhadap temperaturtemperatur

•• MutanMutan yang yang memilikimemiliki enzimenzim konstitutifkonstitutifkatabolikkatabolik : : antiinduksiantiinduksi, limiting factor, , limiting factor, dlldll..

•• MutanMutan yang yang memilikimemiliki enzimenzim konstitutifkonstitutifanabolikanabolik : : antimetabolitantimetabolit