An enzyme-coupled biosensor enables (S)-reticuline production in yeast from glucose William C....

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.