commercial product synthesis by recombinant microorganisms

1. Commercial Product Synthesis by Recombinant Microorganisms Historically centered on protein production Now branched to include low molecular weight compounds Vitamins Amino acids Antibiotics Used to increase quantity and/or new product variants

2. Restriction Endonuclease Systems Protect bacteria from invading foreign DNAs such as bacteriophage chromosomes Cut at specific DNA sequences Complementary methylase protects bacterium’s own DNA

3. Isolating a Gene for a Restriction Endonuclease

4. RE Gene Isolation Vector has cut site for target enzyme Make genomic library Transform Isolate plasmids Cut with enzyme Those with RE/Methylase Are not cut Retransform Screen transformants for RE activity

5. Ascorbic Acid Biosynthesis Sorbitol to sorbose microbial conversion Remainder are industrial chemical reactions Target to produce 2KLG from glucose by cofermentation

6. 2KLG Production By Cofermentation Fermentation of glucose by Erwinia produces 2,5-DKG Cofermentation with Corynebacterium converts 2,5-DKG to 2-KLG

7. 2-KLG by Recombinant Erwinia Clone 2,5-DKG reductase gene from Corynebacterium library Screen using synthetic probe from enzyme aa sequence (71% GC organism—codon usage) Transform into Erwinia E. coli not easily used due to genetic differences Complete conversion to 2-KLG by Erwinia Engineer to improve enzyme properties

8. Microbial Synthesis of Indigo Pseudomonads have many enzymes/pathways for degradation of aromatics During studies of NAH pathway for naphthalene degradation some strains turned blue on tryptophan media Made indigo E. coli with xylene oxidase and naphthalene dioxygenase genes can now make indigo

9. Microbial Synthesis of Indigo

10. Bioreactors Stable matrix Cells chemically or physically immobilized on matrix Run as a continuous flow system Substrate in and product out…

11. Biosynthesis of Amino Acids Amino acids uses in food industry Flavor enhances Antioxidants Nutritional supplements Amino acid uses in agriculture Feed additives Amino acid uses in medicine Infusion solutions Amino Acid uses in industry Starting materials for polymer and cosmetic production

12. Commercial Applications of Amino Acids

13. Tryptophan Biosynthetic Pathway Most amino acids produced by protein hydrolysis or bacterial fermentation Tryptophan biosynthetic pathway and regulation shown

14. Tryptophan Production C. glutamicum Strains Identify rate limiting step Use E. coli/Corenebacterium glutamicum shuttle vector Add second antranilate synthetase gene

15. Cysteine Biosynthesis by E. coli Serine acetyltransferase is feedback inhibited Site-directed mutagenesis Transform into E. coli strain that does not degrade cysteine Even better feedback insensitive enzyme genes isolated as cDNAs from Arabidopsis and transformed into strain

16. Lipid Removal Fatty stains are a persistent laundry problem High temperature/alkalinity required for effective removal Damages material/colors Enzyme destruction by lipase But most effective ligase difficult to produce Both required genes isolated/cloned High copy plasmid for P. alcaligenes gave 5X increase in lipase But scale up to fermentor gave less lipase activity Carbon dioxide levels needed to be controlled/reduced

17. Lipase Expression Module Two required genes isolated on separate clones and fused together to form complete module Placed in high copy broad host range vector

18. Improving Antibiotic Production 12,000 antibiotics have been identified Most from Gram-positive soil bacterium Streptomyces Some from fungi and other Gram-positive or Gram-negative bacteria 100,000 tons of antibiotics produced per year 200-300 new discoveries per year Cost to bring new one to market very high 1-2% prove useful Fleming’s fungal production 2 U/ml, now 70,000 U/ml Biotechnology…

19. Cloning Antibiotic Genes Mutate antibiotic producing strain to antibiotic negative Introduce plasmids from genomic library of wt strain Use clones to screen large insert library Pathway may require up to 20-30 steps…

20. Undecylprodigiosin Convenient for gene isolation because antibiotic producing cells are colored red

22. BAC Vectors

38. Most Common Microbially Synthesized Antibiotics

39. Antibiotics Produced by Strptomyces Strains and Those Transformed by Plasmids Pij2303 and Pij2315

40. Production of Xanthan Gum by Wild Type and Transformed X. campestris