Standardized biosynthesis of flavan-3-ols with effects on pancreatic beta-cell insulin secretion

Transcription

1 Standardized biosynthesis of flavan-3-ols with effects on pancreatic beta-cell insulin secretion Joseph A. Chemler, Lye T. Lock, Mattheos A. G. Koffas, Emmanuel S. Tzanakakis Applied Microbiology and Biotechnology 77(4): , Speaker: I-Lun Chiang Adviser: Ching-Tsan Huang, PhD Date:

2 Symptoms of Diabetes Eat a lot!! Drink a lot!! Pee a lot!! 2

3 Diabetes: A Global Problem and Epidemic +44% +102% +160% +180% +130% 3 (from WHO)

4 Diabetes Mellitus Type 1 Diabetes - Cannot produce insulin at all - insulin injection Type 2 Diabetes - Lack of insulin production - Insulin resistance -Oral medication 4

5 Diabetes Oral Medications 6 Classes : Sulfonylureas Biguanides Sulfonylureas and biguanide combination drugs Thiazolidinediones Alpha-glycosidase inhibitors Meglitinides 5

6 Sulfonylureas Stimulate pancreatic ß-cell to secrete insulin Side effects Long time hypoglycemia Water retention (edema) Worsening myocardial function Gains in body weight New drugs discovery!! 6

7 Plant-derived Flavonoids Antioxidant Flavonoids Flavan-3-ols protect islets from oxidative stress slow progressive ß-cell dysfunction may be effective against diabetes Natural hypoglycemic compounds Catechins 7

8 The Flavonoids Metabolic Pathway Plant secondary metabolites Flavan-3-ols 8

9 Strategy of Production Metabolic Engineering -The The practice of optimizing genetic and regulatory processes within cells to increase the production of a certain substance. precursor Metabolically Engineered cells Product 9

10 Metabolic Engineering of Flavan-3-ols Advantages Easy to scale up Less expensive starting materials Challenges Involves multiple enzymes in the pathway The target genes are from different plants The vectors must be designed specially The problem of plasmids incompatibility Need to optimize the production yield 10

11 Framework Precursors Metabolic engineering flavan-3-ols Insulin secretion assay Type-2 diabetes drug candidate 11

12 Metabolic engineering Identify the enzymes (b.c) C c= D =d E (e.f) b= =e B F a= A =f (a.d) 12

13 Metabolic engineering Find the genes from plants Petroselinum crispum Pc4CL-2 4CL (a) (a) Petunia hybrida CHS-A CHS (b) (b) Medicago sativa CHI (c) CHI (c) Malus domestica MdF3H (d) FHT (d) flamingo flower DFR DFR (e) (e) Desmodium uncinatum LAR LAR (f) (f) 13

14 Metabolic engineering Construct expression plasmids CHS-A (b) Md3FH (d) DFR (e) (b) (d) (e) CHI (c) Pc4CL (a) LAR (f) (b.c) (a.d) (e.f) 14

15 Metabolic engineering Transform into E. coli cells BL21Star E. coli lack of some proteases reduces degradation of heterologous proteins (e.f) (a.d) (b.c) Antibiotic Kanamycin marker: streptomycin Ampicillin 15

16 Metabolic engineering Supply of precursors c d b C D E e (e.f) (a.d) (b.c) a B A F f f e d Catechins F E Product D c b a C B Precursor A 16

17 Metabolic engineering Production yield control D eriodictyol naringenin d e f E F Catechins (+)-catechin (+)-afzelechin To increase the yield: growth medium oxygen availability enzyme s cofactor vector copy number 17

18 Production yield control Growth medium: M9 vs. LB M9 wins!! Because in LB: D E F (+)-catechin 18

19 Production yield control Production (mg/l) Oxygen availability: 20 ml liquid medium: oxygen-rich cultures 80 ml liquid medium: oxygen-limited cultures (+)-catechin from eriodictyol 20 ml Production (mg/l) (+)-afzelechin from naringenin 80 ml 80 ml 20 ml 20 ml 80 ml 0 20 ml 80 ml Volume (ml) inhibited by O ml 80 ml Volume (ml) Oxygen-limited culture wins!! 19

20 Production yield control Enzyme s cofactor: Fe(II) 80 ml Increased Decreased 20

21 Production yield control Enzyme s cofactor: Fe(II) D Fe(II) eriodictyol naringenin d e f E F Catechins Chelating effect (+)-catechin (+)-afzelechin No chelating effect Because Fe(II) chelating with the catechol groups of eriodictyol, the yield decrease Produce (+)-catechin without FeSO 4 Fe(II) can increase the yield of (+)-afzelechin Produce (+)-afzelechin with FeSO 4 21

22 Production yield control Vector copy number: Hypothesis: replace by yield? (e.f) (e.f) Medium-copy High-copy (d) (e.f) (d) (e.f) BL-FDL BL-FDLb 22

23 Production yield control Vector copy number: Results: did not get higher yield, why? 4 (+)-catechin 0.25 (+)-afzelechin Production (mg/l) Medium-copy High-copy Production (mg/l) Medium-copy High-copy BL-FDL BL-FDLb d might be limiting factor BL-FDL BL-FDLb 23

24 Metabolic engineering Production yield control D eriodictyol naringenin d e f E F Catechins (+)-catechin (+)-afzelechin To increase the yield: growth medium: M9 oxygen availability: oxygen-limited culture enzyme s cofactor: (+)-catechin without FeSO 4 (+)-afzelechin with FeSO 4 vector copy number: unclear 24

25 Results- Standardized Biosynthesis of Flavan-3-ols caffeic acid courmaric acid A B C D E F CatechinsCatechins (+)-catechin (+)-afzelechin Successful! A (+)-catechin Standardized conditions (+)-afzelechin 25

26 Framework Precursors Metabolic engineering flavan-3-ols Insulin secretion assay Type-2 diabetes drug candidate MIN6 ß-cell incubation 2.5 mm glucose 25 mm glucose KRB KRB add (+)-catechin or (+)-afzelechin Insulin secretion Quantify by ELISA 26

27 Results- Insulin secretion assay (+)-catechin can increase the glucose-stimulated insulin secretion of MIN6 β-cells. glucose-stimulated level Base-line level the same Control group KRB (Krebs-Ringer bicarbonate buffer) 27

28 Results- Insulin secretion assay (+)-afzelechin can increase both base-line level and glucose-stimulated level of insulin secretion. glucose-stimulated level Base-line level increased Control group KRB (Krebs-Ringer bicarbonate buffer) 28

29 Summary Oral medication Old drugs have side effects Discovery of new drugs Treating diabetes Precursors Metabolic engineering flavan-3-ols Insulin secretion assay Insulin injection Type-1 diabetes treatment (+)-catechin Type-2 diabetes drug candidate (+)-afzelechin Type-2 diabetes treatment 29

30 Thanks for your attention! 30