VL12 Biotechnology and genetic engineering PDF

Microbial Biotechnology Mitja Remus-Emsermann KöLu 12-16 Raum 129 [email protected] Selection of strains with beneficial properties Environmental microbial diversity is immense Every microorganism has potentially interesting properties How ca...

Microbial Biotechnology Mitja Remus-Emsermann KöLu 12-16 Raum 129 [email protected] Selection of strains with beneficial properties Environmental microbial diversity is immense Every microorganism has potentially interesting properties How can we make use of these properties? Making use of the properties of interest = biotechnology Sauerkraut Classical biotechnology Environmental conditions are modified to select microorganisms from the environment Goal of process? Increase shelf life of cabbage by reducing pH Increase digestibility Sauerkraut – process Cabbage is cut into thin strips Strips are place in a pot with a rim that can be filled with water Lots of salt is added Cabbage is smashed so that some of its juice is coming out Water is added to cover the cabbage A heavy weight is added on top Water is added to the rim and a heavy lid is placed on top https://www.derkleinegarten.de/images/thumb/images/phocagallery/mehr- Infos/Rezepte/thumbs/phoca_thumb_l_02-sauerkrauttopf-gaertopf-gurkentopf-fill- 320x230.jpg So what is happening? Log (CFU / ml) pH Yoghurt Classical biotechnology Goal of the process is to increase shelf life of milk by reducing pH make more digestible improve taste Yoghurt Yoghurt production employs lactic acid bacteria Where can you find lactic acid bacteria? Why is selection of bacteria from the environment usually not used anymore? What is the advantage of choosing specific inocula? The taste of yoghurt Nr. Aroma component Nr. Aroma component Nr. Aroma component Nr. Aroma component 1 Acetaldehyde 26 2,3-pentanedione 50 1,3-dimethylbenzene (1,4?) 74 3-methyl-2-butenol 2 Dimethyl sulfide 27 Dimethyl disulfide 51 3-penten-2-one 75 2-nonanone 3 Methylcyclohexane 28 Butyl acetate 52 1,3-dimethylbenzene (1,3?) 76 2-hydroxy-3-pentanone 4 Propanal 29 Hexanal 53 1-Methylpyrrole 77 Furfural 5 2-propanone 30 2-hexanone 54 3-heptanone 78 1H-pyrrole 6 Furan 31 Dimethyl trisulfide 55 1-butanol 79 Benzaldehyde 7 Methyl acetate 32 Acetic acid 56 1-ethyl-4-methylbenzene 80 2-methylpropanoic acid 8 2-methylfurane 33 Propionic acid 57 1-penten-3-ol 81 Butyric acid 9 Butanal 34 2-methyltetrahydrothiophen-3-one 58 Limonene 82 3-methylbutanoic acid 10 Ethyl acetate 35 2-undecanone 59 1,4-dimethylbenzene (1,2?) 83 2-dodecanone 11 2-butanone 36 2-furanmethanol 60 2-heptanone 84 Benzothiazole 12 Methanol 37 Pentanoic acid 61 Propylbenzene 85 2-pentadecanone 13 3-methylbutanal 38 Hexanoic acid 62 3-methyl-2-butenal 86 Nonanoic acid 14 Dichloromethane 39 Heptanoic acid 63 2-pentylfuran 87 γ-dodecalactone 15 Benzene 40 Octanoic acid 64 Pyrazine 88 Benzoic acid 16 2-propanol 41 Decanoic acid 65 Ethenylbenzene 89 Methional 17 Ethanol 42 δ-dodecalactone 66 1-pentanol 90 (2E)-nonenal 18 2-pentanone 43 1-nonen-3-one 67 3-octanone 91 Methyl 2-methyl butanoate 19 2,3-butanedione 44 2-methyltetrahydrothiophen-3-one 68 2-methyl tetrahydrofuran-3-one 92 Ethyl hexanoate 20 Acetonitrile 45 Guaiacol 69 Trimethylbenzene 93 Hexyl acetate 21 Chloroform 46 2-methylthiophene 70 Methylpyrazine 94 Diacetyl 22 Toluene 47 2-methyl-1-propanyl alcohol 71 Octanal 95 Acetone 23 2-butanol 48 Ethylbenzene 72 3-hydroxy-2-butanone 96 Acetoin 24 S-methyl thioacetate 49 Butanoic acid 73 1-methyl ethenylbenzene 97 Formic acid 25 1-propyl alcohol Glutamine / mono sodium glutamate Glutamate is highly desired in food production (Umami) Originally, glutamate was chemically produced Screening for microorganisms that produce glutamate on cheap substrates After discovery of strains, growth conditions were optimised Beer/ Wine/ Bread Saccharomyces cerevisiae and other yeasts Often in conjunction with bacteria Traditionally, yeast are selected from the environment similar to sauerkraut and yoghurt Yeast are natural colonisers of fruit (grapes) and leaves Today, often, yeast isolates are used https://www.vinovest.co/blog/wine-making Impact of yeast on taste Bokulich and Bamforth Downloaded from htt Bokulich FIG 2 Overview of Saccharomyces metabolic activities influencing beer quality. This simplified schematic summarizes the main metabolic andlinked pathways Bamford to 2013 Plant production Plants live in intimate relationships with microorganisms some are a beneficial (mutualistic) some are pathogenic and some are commensal Nitrogen fixing rhizobacteria Brock Figure 1.14 Mycorrhorizal fungi https://koenigliche- gartenakademie.online/collections/pflanzenstarkung/products/inoq- mykorrhiza Brock Figure 23.23 Plant protection “FAO estimates that annually between 20 to 40 percent of global crop production are lost to pests. Each year, plant diseases cost the global economy around $220 billion, and invasive insects around US$70 billion.” The main cause of crop loss are fungi followed by oomycetes and bacteria  Microorganisms in agriculture Microorganisms are devastating pathogens in agriculture Bacterial canker Fireblight on Myrtle rust on on kiwifruit apples pohutukawa  Microorganisms in agriculture Microorganisms are devastating pathogens in agriculture Potato late blight Corn smut Biocontrol agents - properties Survive in the same location as the pathogen/herbivore Produce inhibiting compounds What could those be? Compete for resources What could those be? Induce plant resistance Disease progression and biological control of “Fire Blight” Red = Erwinia amylovora = bad Green = Pantoea agglomerans = good Bioremediation https://commons.wikimedia.org/wiki/File:Steps_in_a_typical_wastewater_treatment_process.png Synthetic microbiology RuBisCO RuBisCO is the most abundant protein on Earth Not because it is so successful, but because it is inefficient Maybe: Nature did not change it, because it works https://de.khanacademy.org/science/biology/photosynthesis-in-plants/photorespiration--c3-c4-cam- plants/a/c3-c4-cam-plants CETCH Cycle 1. crotonyl-CoA carboxylase/reductase 2. ethylmalonyl-CoA epimerase & mutase; 3. methylsuccinyl-CoA oxidase; 4. methylmalyl-CoA dehydratase 5. methylmalyl-CoA lyase 6. propionyl-CoA oxidase 7. crotonyl-CoA carboxylase/reductase 8. methylmalonyl-CoA epimerase & mutase 9. succinate semialdehyde dehydrogenase (acetylating) 10. 4-hydroxybutyrate dehydrogenase 11. 4-hydroxybutyryl-CoA synthetase 12. 4-hydroxybutyryl-CoA dehydratase Bar-Even 2018 CO2 fixation pathways Schwander et al. 2018  Artificial chloroplasts Using the enzymes of the CETCH pathway and spinach thylakoids Actively fixing CO2 without an oxidase side reaction T. MILLER/MAX PLANCK INSTITUTE FOR TERRESTRIAL MICROBIOLOGY  Genetic engineering Mitja Remus-Emsermann KöLu 12-16 Raum 129 [email protected]  Genetic engineering = Genetic modification of (micro)organisms Plasmids Molecular cloning Restriction ligation Isothermal assembly Golden Gate cloning (MoClo) Transposons Crispr Cas9 What are plasmids? Extrachromosomal double stranded DNA Self replicating independent of chromosome > 1 copy Low copy ~ 1-2 copies High copy > 40 copies Often circular – but linear plasmids exist Size between a few thousand base pairs and several hundred thousand base pairs Some plasmids can insert into the host chromosome Plasmids Mobile genetic elements which are highly useable for genetic engineering Common elements (bold = essential) Origin of replication Selectable marker (e.g. antibiotic resistance gene) Multiple cloning site (MCS) Screening marker (e.g. beta galactosidase) Origin of transfer Inducible promoters Constitutive promoters Terminators Plasmids are molecular tools Selectable marker - Antibiotic resistance Heterologously expressed gene Repressible/inducible Origin of transfer promoter Origin of replication Multiple cloning site Repressor Selectable marker - Antibiotic resistance Origin of replication Determines the number of plasmid copies per cell May lead to incompatibility Plasmids featuring similar origin of replications are incompatible and one plasmid will maintained on a long run  Incompatibility example Plasmid 1 Bacterial cell Plasmid 2 Origin determines 3 plasmid copies By chance only one kind is Cell division event transmitted to the next generation Plasmid is lost to future generations Plasmids are molecular tools Selectable marker - Antibiotic resistance Heterologously expressed gene Repressible/inducible Origin of transfer promoter Origin of replication Multiple cloning site Repressor Selectable marker - Antibiotic resistance Change bacterial genetics using plasmids Heterologous expression Fluorescent proteins Overexpression of genes to characterise protein Directed knockout of genes using homologuous recombination Schlechter et al. 2018 Biosensors Shuttle plasmids e.g. to transform other bacteria, yeast, plants, etc. General process cloning https://blog.addgene.org/plasmids-101-gibson-assembly?_ga=2.172299107.1996653639.1652623766-1857694467.1639342506 Cloning using restriction/ ligation Restriction enzymes molecular scissors often highly specific and dependend on DNA sequence if not this is called star activity Restriction enzymes derive from bacterial “immune systems” against viruses DNA ligases covalently connect DNA strands Cloning using Situation: two cut sites available restriction/ ligation https://blog.addgene.org/plasmids-101-restriction-cloning?_ga=2.172299107.1996653639.1652623766-1857694467.1639342506 Cloning using Situation: one cut sites available restriction/ ligation Single stranded nicks remain but are repaired in vivo https://blog.addgene.org/plasmids-101-restriction-cloning?_ga=2.172299107.1996653639.1652623766-1857694467.1639342506  BioBrick tools Isocaudomeric restriction enzymes generate compatible overhangs Example: XbaI and PstI Schada von Borzyskowski, Remus-Emsermann et al. 2014 PCR polymerase chain reaction Amplicon modification: Isothermal (Gibson) assembly https://blog.addgene.org/plasmids-101-gibson-assembly?_ga=2.172299107.1996653639.1652623766-1857694467.1639342506 Golden Gate cloning The restriction enzyme BsaI cuts outside of its recognition site BsaI is active in ligation reaction mixes and can be added to recleave unwanted religated vectors https://international.neb.com/-/media/nebus/files/application-notes/technote_breaking_through_the_limits_of_golden_gate_assembly.pdf?rev=d8ac2ce3ef664c6c875e126d86153ec1 Transformation/ Conjugation/ Transduction Please refer to the lectures of Haike Antelmann All these phenomena have a natural background and are used as methods for genomic engineering Transposons as molecular tools What are transposons? Mobile genetic elements Inverted repeat Transposon machinery Inverted repeat One or more genes Discovered by Barbara McClintock Insertions sites of transposons can: disrupt genes change levels of expressions of genes -> change the phenotype of an organism Mechanisms of transposition Transposase recognizes, cuts, ligates DNA. Conservative: Transposon is excised from one location and reinserted at a second location (e.g., Tn5) Copy number is constant = one. DNA transposon Replicative: A new copy of transposon is produced and inserted at a second location. Number of transposons present doubles. retrotransposons “cut-and-paste” “copy-and-paste” Lodish et al., Molecular Cell Biology, 7th ed. Fig 10-8 Crispr Cas9 a bacterial foreign DNA defense system https://international.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology CRISPR Cas9 https://international.neb.com/tools-and-resources/feature-articles/crispr-cas9-and-targeted-genome-editing-a-new-era-in-molecular-biology Take home points Most biotechnological approaches rely on natural phenomena Often, environmental conditions select of the organisms of interest Isolates are used alternatively Biotechnology allows to reconstruct pathways Techniques of genetic engineering rely on enzymes and tools that were discovered in all kingdoms of life