CEN 551: Biochemical Engineering

1. CEN 551: Biochemical Engineering Instructor: Dr. Christine Kelly Chapter 8

2. Schedule • Exam 2. Thursday, March 4, before spring break. • Exam 2: Take home on chapter 8 material, in-class on Chapter 9, 10 and 11. • Take home exam - Genetic engineering. • In-class - operation of bioreactors, scale-up and control of bioreactors, recovery and purification. • 3 weeks from Thursday.

3. Homework • By Friday at 5:00 pm email ckelly@syr.edu a 1 paragraph description of your project topic. • Chapter 8 • Problems 2, 6, 7, and 8. • Due Thursday, February 19.

4. Chapter 8: How cellular information is altered • Mutation and Selection • Natural Mechanisms for Gene Transfer and Rearrangement • Genetically Engineering Cells • Genomics

5. We can alter cells by using mutation or genetic engineering. Mutation is subjecting the cells to stress causing changes in the genetic make-up. Genetic engineering is the purposeful transfer of DNA from one type of organism to another.

6. Mutations and Selection • Mutations = mistakes in the genetic code (can arise from replication and/or damage) • Mutant = organism with a genetic mutation • Wild type = the organism without the genetic organism • Genotype = genetic construction of an organism • Phenotype = characteristics expressed by an organisms. • Expression = usually refers to transcription+translation+posttranslation processing

7. Examples • Strain A has the tol operon for toluene degradation, and is in a reactor growing on glucose. • Strain B has the tol operon for toluene degradation, and is in a reactor growing on toluene. • These strains have the same genotype, but different phenotypes.

8. Point mutation: single base change Consequences – base change may or may not result in an amino acid change. If the amino acid is the same as before the mutation there is no consequence. If the amino acid is different, but not in the region of the active site, there may be no consequences. If the mutation is in the active site, there may be some enzyme activity consequence. If the mutation changes the amino acid to a stop codon, the resulting protein will be truncated and probably not active.

9. Selection Selectable mutation: confers upon the mutant an advantage for growth, survival or detection under a set of environmental conditions that the wild type does not have. Examples Antibiotic resistance Ability to grow on toluene Inability to produce lysine Ability to produce bioluminescence Ability to produce more of an enzyme Inability to grow at higher temperatures

10. Serial Dilution Plating

11. Natural Mutation Rates • 10-3-10-9 mutations per cell conversion • 10-6 = 1 mutation/1,000,000 divisions • How do we increase mutation rates? • Why do we want to increase mutation rates?

12. Increase Mutation Rates Mutagens: chemicals, radiation Lots of growth (i.e. lots of divisions)

13. Why do we want to increase mutations? We want a cell to develop specific characteristics that are advantages for us. For example, removing feed back inhibition of lysine to increase lysine production

14. Natural Gene Transfer/Rearrangement • Transformation: uptake of free DNA by a cell. The cell membrane has to be permeable to DNA. • Transduction: DNA is carried into the call in a phage. • Conjugation: Cell to cell transfer of DNA. Also called mating. Once the DNA is inside the cell it can remain separate from the chromosome in self replicating plasmid, or integrate into the chromosome. To integrate, the DNA must be complementary to the chromosomal DNA on the ends.

15. Mutation and Selection Using mutation and selection engineers and microbiologists were able to increase penicillin from 0.001 g/L to 50 g/L.

16. Genetic Engineering Using natural mechanisms to purposefully manipulate DNA. The DNA is manipulated outside of the cell, and then sent into the cell.

17. Genetic Engineering Tools • Restriction enzymes: enzymes that cut DNA at specific sequences. Different enzymes will cut at different sequences. • Gel electrophoresis (Southern Blot): A method to detect what sizes of DNA a sample contains. • Polymerase chain reaction (PCR): A process used to make many copies of a piece of DNA. • Plasmid: self replicating, circular piece of DNA that can survive in a cell.

18. http://www.accessexcellence.org/AB/GG/nucleic.html

20. Enzyme Organism from which derived Target sequence(cut at *)5' -->3' Ava I Anabaena variabilis C* C/T C G A/G G Bam HI Bacillus amyloliquefaciens G* G A T C C Bgl II Bacillus globigii A* G A T C T Eco RI Escherichia coli RY 13 G* A A T T C Eco RII Escherichia coli R245 * C C A/T G G Hae III Haemophilus aegyptius G G * C C Hha I Haemophilus haemolyticus G C G * C Hind III Haemophilus inflenzae Rd A* A G C T T Hpa I Haemophilus parainflenzae G T T * A A C Kpn I Klebsiella pneumoniae G G T A C * C Mbo I Moraxella bovis *G A T C Mbo I Moraxella bovis *G A T C Pst I Providencia stuartii C T G C A * G Sma I Serratia marcescens C C C * G G G SstI Streptomyces stanford G A G C T * C Sal I Streptomyces albus G G * T C G A C Taq I Thermophilus aquaticus T * C G A Xma I Xanthamonas malvacearum C * C C G G G

23. Gel Electrophoresis

26. Polymerase Chain Reaction

27. PCR Primers DNA polymerase nucleotides

29. Polymerase Chain Reaction (PCR) PCR allows scientists to extract and analyze bits of microbial DNA from samples, meaning they don’t need to find and grow whole cells. PCR is an essential element in DNA fingerprinting and in the sequencing of genes and entire genomes. Basically, it’s like a technique to photocopy pieces of DNA. In a matter of a few hours, a single DNA sequence can be amplified to millions of copies. PCR lets scientists work with samples containing even very small starting amounts of DNA. http://www.microbeworld.org/htm/aboutmicro/tools/genetic.htm

30. The technique makes use of the DNA repair enzyme polymerase. This enzyme, present in all living things, fixes breaks or mismatched nucleotides in the double-stranded DNA helix. These breaks or mismatches could cause genes to malfunction if left unfixed. http://www.microbeworld.org/htm/aboutmicro/tools/genetic.htm

31. Polymerase uses the intact half of the DNA molecule as a template and attaches the right nucleotides, which circulate constantly in the cell, to the complementary nucleotide at the site of the break. (DNA consists of two strands of nucleotide bases, which are represented as A, G, C, and T. In the laws of DNA base-pairing, A joins with T and G with C.) http://www.microbeworld.org/htm/aboutmicro/tools/genetic.htm

32. Not all polymerases are created equal, however. Many fall apart in high heat. PCR was developed in 1985 following the discovery of an unusual heat-loving bacterium called Thermus aquaticus in a hot spring in Yellowstone National Park. This bacterium’s polymerase, dubbed Taq, does its job of matching and attaching nucleotides even in the high heat generated by the successive “photocopying” cycles required during PCR. Taq made PCR possible. http://www.microbeworld.org/htm/aboutmicro/tools/genetic.htm

33. http://www.microbeworld.org/htm/aboutmicro/tools/genetic.htm

35. Plasmids and Cloning

39. Movies • 65601 missense mutation • 65701 nonsense mutation • 95301 bacterial transformation • 153301 mutation • 151401 virulence transformation • 156401 heat DNA • 92201 restriction enzyme, recombination • 112601 PCR • 165401 Sequencing

40. Example • I have two organisms: 1. A fast growing yeast that grows well in a fermentor. 2. A fungi that is difficulty to grow. • The fungi produces an enzyme that may be valuable, but I cannot grow enough fungi to produce enough enzyme to even test the enzyme. • How can I use the genetic engineering tools to get enough enzyme?

41. Genetic Engineering • PCR the enzyme DNA from the fungi, get a bunch of the DNA that encodes for the valuable enzyme. • Find a restriction enzyme that will cut the valuable enzyme DNA on ether side (but not in the middle). • Obtain a plasmid that will replicate in the yeast, that has a site that the same restriction enzyme will cut downstream of a strong promoter. • Cut the valuable enzyme DNA and the plasmid with the restriction enzyme. • But the valuable enzyme DNA and plasmid together and let them recombine. • Get the plasmid into the yeast. • At all steps, use gel electrophoresis to check and make sure you have the right DNA.

42. White-Rot Fungi • Fungi with mycelium type growth. • Able to degrade lignocellulosic materials using several enzyme systems (lignin and manganese peroxidases, laccases). • Expresses and secretes MnP under nitrogen limitation at low concentrations. • Expresses several degradative enzymes – has been widely studied for bioremediation applications. • Not suitable for conventional industrial fermentations.

43. White-rot Fungi P. chrysosporium

44. Manganese Peroxidase • Glycosylated enzyme that uses H2O2 to oxidize manganese, which in turn oxidizes lignin. • White-rot fungi produces a 41-47 kDa MnP under secondary metabolism. • Native fungal secretion signal directs secretion out of the cell. • Requires a heme cofactor for ligninolytic activity.

45. glycosylation MnP crystal structure. (Sundaramoorthy et al., 1994).

46. Pichia pastoris • Methylotrophic (methanol as a sole carbon source) yeast. • Capable of eukaryotic post translational modifications. • Higher yields, less expensive, higher cell density, and easier to scale up than mammalian and fungal systems. • Secretes only small amounts of native proteins. • Many cloning and expression vectors available.

48. Efforts to Increase Production of MnP Homologous expression • P. chrysosporium primary metabolism: low concentration Heterologous expression • Bacteria (E. coli): inactive inclusion bodies • Insect cells: active enzyme, low concentration (5 mg/L), heme addition, expensive • Fungal (Aspergillus spp.): active enzyme, higher concentration (100 mg/L), heme addition

49. Cloning of mnp 1. The white-rot fungi was grown under nitrogen limitation. 2. The total RNA was extracted from the culture. • Reverse transcriptase polymerase chain reaction (RT-PCR) was performed with oligo dT primers to create DNA complementary to the mRNA. • PCR was performed with primers specific for the MnP gene.

50. tubes Thermocycler (PCR machine)