1. Introductions to Lysine in E.coli Metabolism Pathways. Group 9 張智宏 楊翊文 王翔昱 2005.1.14

2. Outline  Describe the usage of Lysine.  Process  Find the metabolic pathways involved in the synthesis of the Lysine.  Find the enzymes and genes involved in these pathway reactions.  Is E. Coli suitable for biosynthesis of Lysine ?

3. Introductions to Lysin Query interface... Query interface... Symbol : lys k, 離胺酸 Molecular formula : C6H14N2O2 Molecular weight : 146.19 Isoelectric point (pH) : 9.59 pKa values : 2.20, 8.90, 10.28 CAS Registry Number : 56-87-1 Type : lonic Other Forms L-lysine acetylsalicylate (LAS) Lysine clonixinate (LC) L-lysine monohydrochlorine (LMH)

4. Introductions to Lysine 3D Molecular Model H2N-(CH2)4-CH(NH2)-COOH Lysine under the microscope

5. Introductions to Lysine Discovered in 1989. Lysine is incorporated into proteins at the rate of 7 percent on a molar basis compared to the other amino acids. The side chain of Lysine has three methylene groups, so that even though the terminal amino group will be charged under physiological conditions, the side chain does have significant hydrophobic character. Lysine is an essential amino acid that you must get from food because your body cannot make enough of it. Lysine helps your body process fatty acids, and it is particularly important for proper growth. Lysine helps your body absorb calcium, and it plays an important role in the formation of collagen, a substance important to your bones and tissues.

6. Medical Information of Lysine Lysine is used to treat herpes infections caused by both herpes simplex and herpes zoster viruses. Taking lysine supplements can speed your recovery time and reduce chances of reinfection. Some studies have found lysine helpful in treating cardiovascular disease, osteoporosis, asthma, migraine, nasal polyps, and postepisiotomy pain. If you get too little lysine in your diet, your body may develop a poor nitrogen balance, and you may ultimately develop kidney stones. Signs of getting too little lysine include fatigue, nausea, dizziness, appetite loss, emotional agitation, bloodshot eyes, decreased immunity, slow growth, anemia, enzyme deterioration, reproductive disorders, pneumonia, and acidosis (a pH imbalance in the body). The most concentrated sources of lysine are torula yeast, dried and salted cod, Parmesan cheese, pork loin (excluding fat), dried and frozen tofu.

7. Outline  Describe the usage of Lysine.  Process  Find the metabolic pathways involved in the synthesis of the Lysine.  Find the enzymes and genes involved in these pathway reactions.  Is E. Coli suitable for biosynthesis of Lysine ?

8. Process, What we have done http://www.genome.jp/kegg/ Click “ Ligand ” to enter query interface Query “Lysine” with “ name” function

9. Process, What we have done Select the entry that match our desire: entry C00047 We are interested in Lysine biosynthesis, select the path map00300

10. Process, What we have done

11. Show the enzymes exist in E.Coli with green color

13. Outline  Describe the usage of Lysine.  Process  Find the metabolic pathways involved in the synthesis of the Lysine.  Find the enzymes and genes involved in these pathway reactions.  Is E. Coli suitable for biosynthesis of Lysine ?

14. Find the metabolic pathways involved in the synthesis of the Lysine.

15. Lysine Biosynthesis (Alanine and Aspartate Metabolism)  L-Asparate  2.7.2.4  L-4-Aspartyl Phosphate  1.2.1.11  L-Aspartate 4-Semialdehyde  4.2.1.52  2,3-Dihydrodipicolinate  1.3.1.26   2,3,4,5,-Tetrahydro-dipicolinate  2.3.1.117   N-Succinyl-2-amino-6-oxopimelate  2.6.1.17   N-Succinyl-L-2,6-diaminopimelate  3.5.1.18  2,6-diominopimelate  5.1.1.7  meso-2,6-Diaminopimelate  4.1.1.20  L-Lysine

16. Alanine and Aspartate Metabolism (Glycosis)  Pyruvate  Acetyl-Coa  Cirate  2-Oxoglutarate  Succinate  Fumarate  4.3.1.1  L-Aspartate (Lysine Biosynthesis)

17. Glycolysis D-Glucose  2.7.1.69  α-D-Glucose-6p  5.3.1.9  β-D-Fructose-6p  2.7.1.11  β-D-Fructose-1,6p2  4.1.2.13  Glyceraldehyde-3p  1.2.1.12  Glycerate-1,3p2  3.6.1.7 (2.7.2.3)  Glycerate-3p  5.4.2.1  Glycerate-2p  4.2.1.11  Phosphoenolpyruvate  2.7.1.40  (Alanine and Aspartate Metabolism)

18. Outline  Describe the usage of Lysine.  Process  Find the metabolic pathways involved in the synthesis of the Lysine.  Find the enzymes and genes involved in these pathway reactions.  Is E. Coli suitable for biosynthesis of Lysine ?

19. Lysine Biosynthesis Pathway EntryEC 2.3.1.117 ECO: b0166(dapD)b0166 EntryEC 2.6.1.17 ECO: b2379(yfdZ)b2379 EntryEC 3.5.1.18 ECO: b2472(dapE)b2472 EntryEC 5.1.1.7 ECO: b3809(dapF)b3809 EntryEC 4.1.1.20 ECO: b2838(lysA)b2838 EntryEC 2.7.2.4 ECO: b0002(thrA)b0002 b3940(metL)b3940 b4024(lysC)b4024 EntryEC 1.2.1.11 ECO: b3433(asd)b3433 EntryEC 4.2.1.52 ECO: b2478(dapA)b2478 EntryEC 1.3.1.26 ECO: b0031(dapB)b0031

20. Alanine and Aspartate Metabolism Pathway EntryEC 4.3.1.1 ECO: b4139(aspA)b4139 Name : aspartate ammonia-lyase aspartase fumaric aminase L-aspartase Definition : aspartate ammonia-lyase (aspartase)

21. Glycosis Pathway EntryEC 1.2.1.12 ECO: b1416(gapC_2) b1417(gapC) b1779(gapA) EntryEC 2.7.2.3 ECO: b2926(pgk) EntryEC 5.4.2.1 ECO: b0755(gpmA) b3612(yibO) EntryEC 4.2.1.11 ECO: b2779(eno) EntryEC 2.7.1.40 ECO: b1676(pykF) b1854(pykA) EntryEC 2.7.1.69 ECO: b0129(yadI) b0679(nagE) b0731(hrsA) b1101(ptsG) b1621(malX) b1736(celC) b1738(celA) ….. EntryEC 5.3.1.9 ECO: b4025(pgi) EntryEC 2.7.1.11 ECO: b1723(pfkB) b3916(pfkA) EntryEC 4.1.2.13 ECO: b2097(dhnA) b2925(fba)

22. Outline  Describe the usage of Lysine.  Process  Find the metabolic pathways involved in the synthesis of the Lysine.  Find the enzymes and genes involved in these pathway reactions.  Is E. Coli suitable for biosynthesis of Lysine ?

23. Is E. Coli suitable for biosynthesis of Lysine ? Methylotrophs, microorganisms that use reduced one- carbon compounds, are diverse and ubiquitous. Even though many methylotrophs have been described, only a few are gram positive. The industrial advantages of using methanol as a substrate for large-scale production of fermentation products have been outlined by other groups. Methanol is relatively inexpensive, prices are relatively stable, production exceeds demand, and it is easily stored and transported. In addition, methanol is available in pure form, it is highly soluable in water, methanol solutions are not explosive, and residual methanol can be easily removed from products after fermentation is completed. Bacillus species have been used extensively in industrial fermentation processes

24. Second Part Using Linear program

25. Our goal is to maximize the quantity of Lysine by using Linear program. 1129 biochemical reactions and 1177 compounds of E.Coli are parsed to obtain the coefficient matrix In the solution X each xi denotes the percentage of each reaction.

32. Resource 1129 biochemical reactions and 1177 compounds of E.Coli are parsed to obtain the coefficient matrix We fail to figure out the result if all biochemical reactions and compounds are considered.

33. Alternative resource But it works when we ignore all reactions and compounds. The relationship of compounds related to Lysine is represented as follow :

34. subject to R1=100 R1-R2=0 R2-R3=0 R3-R4=0 R4-R5=0 R5-R6=0 R6-R7=0 R7-R8=0 R8-R9-R16=0 R9-R10=0 R10-R11=0 R11-R12=0 R12-R13-R17=0 R13-R14=0 R14-R15=0 R16+R28-R29=0 R17-R18=0 R18-R19=0 R19-R20=0 R20-R21=0 R21-R22=0 R22-R23=0 R23-R24=0 R24-R25=0 R25-R26=0 R26-R27=0 R27-R28=0 R29-R30=0 R30-R31=0 R31-R39-R32=0 R32-R33=0 R33-R34=0 R34-R35=0 R35-R36=0 R36-R37=0 R37-R38=0 R40-R41=0 R39-R40-R42=0 R42-R43=0 R43-R44=0 -100<=Ri<=100 max C00047(lysine)

35. Result Input data: E.coli reactions (KEGG) Result Methionine=0, Threonine=0, Lysine=47.5

36. Describe the usage of Lysine. Find the metabolic pathways involved in the synthesis of the following compound. Find the enzymes and genes involved in these pathway reactions. Is E. Coli suitable for biosynthesis of Lysine ? Thank You ! Group9, 2004.11.19