metabolicengineeringofbacteria课件

Metabolic engineering of bacteria,Increasing biological production of small molecules Random screening for overproducing strains (genome shuffling) Rational engineering of pathways,Many biological small molecules are useful,Antibiotics Vitamins Amino acids and derivatives (indigo, aspartame) “secondary metabolites” from plants-alkaloids (caffeine, theobromine, etc.) Etc. Synthesis often requires multiple steps and enzymes,Increasing production of antibiotics (and other small molecules)-traditional methods,Obtain organism that produces a specific compound-Penicillium mold originally made micrograms per liter of culture Randomly mutagenize the organism and screen for increased production, repeat using top producing organism Outcome: grams of penicillum per liter of culture (1000-fold increase in production) Time consuming and expensive process!,An alternative to simple random mutagenesis: genome shuffling,The shuffling advantage: simultaneous recombination of entire genomes (breeding) with multiple parents,(old way),(new way),The set-up,Compare classical strain improvement (CSI) to genome shuffling Streptomyces sp.: produce polyketide antibiotics Induce recombination by recursive protoplast fusion: Fuse protoplasts Regenerate cell walls, grow as a population (F1) Make protoplasts with F1, repeat until F4 Test with 4 auxotrophy markers (next page) Test for increased antibiotic production,Supplements required: pro, arg, ura (not cys) pro, cys, ura (not arg) arg, cys, ura (not pro) pro, arg, cys (not ura),Can strains be isolated that can grow in the absence of pro, arg, ura, and cys (indicating progeny with all 4 genes wild type)? YES.,Test of recursive shuffling,4 parental strains,Indicates increased efficiency of recombination,Test case: increase tylosin production by S. fradiae?,SF1 was treated with NTG, 11 strains selected (22000 screened), those 11 strains were shuffled once (GS1) and then again (GS2),Comparing CSI to genome shuffling,Genome shuffling,Technique has also been used to generate acid-tolerant strains of Lactobacillus (useful for production of lactic acid) Applicable to eukaryotic microbes? Still dont know the mutations that have occurred, or what the state of the genome is following several fusion events,Increasing production of a biological compound: rational design,1) Increase production of a naturally produced commercial compound Modify existing genes 2) Obtain a new organism that can convert an existing compound into a commercial compound Introduce new genes Modify existing genes,Engineering E. coli to produce indigo Mutate tryptophan synthase complex to release indole Introduce napthalene dioxygenase (from Pseudomonas putida),woad,Pict (“painted”-with woad),natural source of indigo: woad Isatis tinctoria,Introduce isatin hydrolase (from a soil microbe) to prevent production of indirubin (color) from isatin,blue,burgundy,Potential routes for overproducing biological compounds,Remove rate-limiting transcriptional controls Remove rate-limiting enzyme allostery controls Kinetically enhance rate-limiting enzymes Genetically block competing pathways Enhance commitment of carbon to the pathway of interest Enhance transport of compound out of cell,How to overproduce phenylalanine?,Remove feedback inhibition (select strains resistant to phenylalanine analogues) Avoid repression (place genes under control of non-phe controlled promoters) Remove pathway competition (delete tyr and trp specific genes) Overexpress phe-specific genes Increase E4P and PEP synthesis,Rational metabolic engineering,Requires at least some knowledge of the biochemical pathway required for compound synthesis Trial and error-try something, see if it works, or where new block is (and focus on the new block) Potentially very labor intensive But high degree of control over the organism,Non-E.coli Bacterial Cloning Homologous recombination,Cloning in bacteria other than E.coli?,Utility: Study bacterial processes and pathways that may not be correctly expressed in E. coli, eg. pathogenesis, antibiotic production properties not available in E.coli, eg. natural transformation Disadvantages: Often a poor selection of “specialized” vectors Transformation (by the usual techniques) may be difficult,Necessary components for non-E.coli cloning,Method for introducing DNA Transformation (spontaneous) Transformation (chemical, electroporation) Conjugation Method for replicating DNA Plasmid replicon Integration into chromosome (homologous recombination) Cloned gene must be expressed in the non-E. coli host (if you want to use the new host as an expression vector),Natural transformation,Spontaneous uptake of DNA from the environment (Likely to be a major route for “horizontal gene transfer”) Fairly common in bacteria- but this is one thing E. coli cannot do!,Conjugation as a method of transfer,Promiscuous plasmids-self-transmissible to many hosts (not a complete substitute for transformation, since DNA must often be manipulated in vitro, then reintroduced),Plasmid Host Range,Host-range of plasmid replicons is highly variable E. coli specialized vectors: have narrow host range But their range can be increased by creating hybrid plasmids that replicate in E. coli and in new host: “Shuttle Vectors”,Integration by recombination,If transformed DNA has homology to chromosome (or other plasmid), this DNA can be integrated by homologous recombination Two pieces of DNA with the same sequence: RecA protein guides a complex that causes strand exchange between homologous sequences Homologous recombination is rare but spontaneous (with a highly predictable frequency: 1/1000 cells will recombine),Homologous recombination: portrait of a single cross-over,Recombination (single crossover),Transfer plasmid (or linear piece of DNA) into host in which it cannot replicate Select for antibiotic marker,Recombination in genome engineering:,gene,flank,flank,(genome),(PCR product),recombinati