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An enzyme-coupled biosensor enables (S)-reticuline production in
yeast from glucoseWilliam C. Deloache, Zachary N. Russ, Lauren Narcross,
Andrew M Gonzales, Vincent J. J. Martin, & John E. Dueber
Presented by: Emma Price and Leah Johnston
Benzylisoquinoline alkaloids (BIAs)
Family of L-tyrosine derived metabolites with a variety of therapeutic uses
WHY IS THIS STUDY IMPORTANT?
Plant Metabolic Engineering
Structures
Tools
Resources
http://labmed.ascpjournals.org/content/41/8/457/F2.expansion.html
https://bioweb.uwlax.edu/bio203/s2012/engebos_meag/nutrition.htm
Instead of plants?
http://yourweeklymicrobe.blogspot.ca/2011/04/yeast-leader-in-libations-king-of.html Shirley Owens, Center for Electron Optics, MSU, 1996
MICROBES!The use of S. cerevisiae in the production of artemisinic acid has already been shown to revolutionize pharmaceutical manufacturing
FIRST PART OF THE BIA PATHWAY
This part of the BIA pathway occurs efficiently in engineered E. coli
Naturally occurring pathway
http://www.nature.com/ncomms/journal/v2/n5/full/ncomms1327.html
BIA PathwayL-Tyrosine
L-DOPA
Dopamine 4-Hydroxyphenyl-acetaldehyde
(s)-Norcoclaurine
(s)-Reticuline
Codeine Morphine Etc.
+
S-Reticuline to Morphine(S)-Reticuline
Salutaridinol-7-O-acetateCodeinoneCodeine
Morphine
(R)-Reticuline Salutaridinol
SalAT
COR1
Many of these steps have been successfully demonstrated in S. cerevisiae
E. coli
https://en.wikipedia.org/wiki/Carl_Woesehttp://pubs.acs.org/cen/news/8229/print/8229notw6.html
CYTOCHROME P450s (CYPs)
• 21,000 distinct CYPs• Role in the generation of new backbones
WHY DO THEY NEED THE FIRST HALF OF THE PATHWAY IN YEAST?
Small-scale Large-scale
✔
= E. coli= S. cerevisiae
TYROSINE HYDROXYLASES• Tyrosine 3-monooxygenases• Copper-containing tyrosinases
Glucose L-Tyrosine L-DOPA
L-Dopaquinone Melanin
1
2
YEAST DON’T HAVE TYROSINE HYDROXLASES
WHAT IS THE ISSUE?
Glucose L-Tyrosine L-DOPA
Dopamine BIAs(e.g., morphine)
L-Dopaquinone Melanin
BetaxanthinDOD
DODC
#1: Tyrosine hydroxylase
1
2
✔
Glucose L-Tyrosine L-DOPA
Dopamine BIAs(e.g., morphine)
L-Dopaquinone Melanin
BetaxanthinDOD
DODC
Cellular fluorescence was measured to determine whether the biosensor activity related accurately to L-DOPA levels produced
SOLVING PROBLEM #1: A BIOSENSOR
DOD = DOPA dioxygenase
ISSUE # 2: Norcoclaurine Synthase (NCS)
Solution: Found a suitable, newly identified, NCS in the opium poppy Papaver somniferum
DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.
BIA PathwayL-Tyrosine
L-DOPA
Dopamine 4-Hydroxyphenyl-acetaldehyde
(s)-Norcoclaurine
(s)-Reticuline
Codeine Morphine Etc.
+
BIA PathwayL-Tyrosine
L-DOPA
Dopamine 4-Hydroxyphenyl-acetaldehyde
(s)-Norcoclaurine
(s)-Reticuline
Codeine Morphine Etc.
+
*Problem 1
*Problem 2
Problem 1
L-Tyrosine
L-DOPA
Tyrosine Hydroxylase
Problem 1Finding a Yeast-active Tyrosine Hydroxylase
SolutionDevelop an enzyme-coupled biosensor
L-Tyrosine
L-DOPABetaxanthin
Problem 1Finding a Yeast-active Tyrosine Hydroxylase
Yeast active tyrosine hydroxylase
DOPA Dioxygenase
Problem 1Finding a Yeast-active Tyrosine Hydroxylase
L-Tyrosine
L-DOPABetaxanthin
Yeast active tyrosine hydroxylase
DOPA Dioxygenase
Candidate 1: AbPPO2 Candidate 2: CYP76AD1
Problem 1Finding a Yeast-active Tyrosine Hydroxylase
DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.
Problem 1Improving the Tyrosine Hydroxylase Activity of CYP76AD1
CYP76AD1
Error prone PCR
Visual Selection of 17 Highest Betaxanthin producers
Top 6 Strains
DNA shuffling library
Problem 1Improving the Tyrosine Hydroxylase Activity of CYP76AD1
Double mutant strain of CYP76AD1
DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.
Problem 1Finding a Yeast-active Tyrosine Hydroxylase
L-Tyrosine
L-DOPABetaxanthin L-Dopaquinone
Dopamine
Unwanted DOPA oxidase activity
Problem 1Finding a Yeast-active Tyrosine Hydroxylase
Tyrosine Hydroxylase with HIGH DOPA oxidase activity
Tyrosine Hydroxylase with LOW DOPA oxidase activity
Violet Yellow
Problem 1Finding a Yeast-active Tyrosine Hydroxylase
DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.
BIA PathwayL-Tyrosine
L-DOPA
Dopamine 4-Hydroxyphenyl-acetaldehyde
(s)-Norcoclaurine
(s)-Reticuline
Codeine Morphine Etc.
+
*Problem 1
*Problem 2
Problem 2Putting it all together
Dopamine 4-Hydroxyphenyl-acetaldehyde
(s)-Norcoclaurine
(s)-Reticuline
Codeine Morphine Etc.
+
6OMTCNMT
NMCH
4’OMT
NCS
Problem 2Putting it all together
DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.
The Big Picture
Glucose
L-Tyrosine
(s)-Reticuline
S. Cerevisiae
NEXT STEPS
(S)-Reticuline titers
Identify dopamine transporter
Balancing 4-HPAA and dopamine production
Tyrosine (S)-Reticuline
(S)-Reticuline Morphine, etc
Put the 2 different pathways intoone yeast strain to produce morphine!
+
tyrosol + 4-HPA
DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). Nature Chemical Biology.
REFERENCES
DeLoache W. C., Russ Z. N., Narcross L., Gonzales A. M., Martin V. J. J., Dueber J. E. (2015). An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose. Nature Chemical Biology, 11, 465-471.
