Metabolic engineering of bacteria课件.ppt

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(and other small molecules)--traditional methods Obtain organism that produces a specific compound--Penicillium mold originally made micrograms per liter of culture Randomly mutagenize the organism and screen for increased production, repeat using top producing organism Outcome: grams of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 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 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 don’t 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 Potential routes for overproducing biological compounds Remove rate-limiting transcriptional controls Remove rate-limiting enzy