1. Metabolic engineering of bacteria Increasing biological production of small molecules Random screening for overproducing strains (genome shuffling) Rational engineering of pathways

2. 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

3. 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(and other small molecules)--traditional methods Obtain organism that produces a specific compound-- Penicillium mold originally made micrograms per liter of culture Randomly mutagenize the organism and screen for increased production, repeat using top producing organism Outcome: grams of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!

4. An alternative to simple random mutagenesis: genome shuffling

5. The shuffling advantage: simultaneous recombination of entire genomes (breeding) with multiple parents (old way) (new way)

6. 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

7. 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 StrainDescription S. coelicolor 2684 12 proA1 argA1 uraA1 S. coelicolor 2685 12 proA1 cysD18 uraA1 S. coelicolor 2686 12 argA1 cysD18 uraA1 S. coelicolor M124 12 proA1 argA1 cysD18 4 parental strains

8. Indicates increased efficiency of recombination

9. 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)

10. Comparing CSI to genome shuffling

11. 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

12. 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

13. 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]

14. Introduce isatin hydrolase (from a soil microbe) to prevent production of indirubin (color) from isatin blue burgundy

15. 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

16. How to overproduce phenylalanine? 1) Remove feedback inhibition (select strains resistant to phenylalanine analogues) 2) Avoid repression (place genes under control of non- phe controlled promoters) 3) Remove pathway competition (delete tyr and trp specific genes) 4) Overexpress phe-specific genes 5) Increase E4P and PEP synthesis

17. 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

18. Non-E.coli Bacterial Cloning Homologous recombination

19. 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

20. 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)

21. 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!

22. 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)

23. 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”

24. 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)

25. Homologous recombination: portrait of a single cross-over

26. Recombination (single crossover) Transfer plasmid (or linear piece of DNA) into host in which it cannot replicate Select for antibiotic marker

27. Recombination in genome engineering: gene flank (genome) (PCR product) recombination (engineered genome) Cell is Tet r, and red gene is knocked out Tet r

28. Things that can be easily done with PCR products, transformation, and recombination…. Gene deletions (with or without the antibiotic resistance gene) Addition of tags to chromosomal proteins Gene replacement (targeted mutagenesis)

29. Recap Non-E.coli bacteria can be useful for recombinant DNA studies, though not as versatile as E. coli Natural transformation is an important feature of some species Shuttle vectors: hybrid plasmids with more than one type of replicon to increase host range Recombination is an important tool for maintaining recombinant DNA and for manipulating the genome