Synthetic Control of Transcription: From Hybrid Promoters ...

Engineering Conferences InternationalECI Digital Archives

Metabolic Engineering IX Proceedings

Summer 6-6-2012

Synthetic Control of Transcription: From HybridPromoters to Promoter Engineering to SyntheticOperon DesignHal AlperThe University of Texas at Austin

Follow this and additional works at: http://dc.engconfintl.org/metabolic_ix

Part of the Biomedical Engineering and Bioengineering Commons

This Conference Proceeding is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion inMetabolic Engineering IX by an authorized administrator of ECI Digital Archives. For more information, please contact [email protected].

Recommended CitationHal Alper, "Synthetic Control of Transcription: From Hybrid Promoters to Promoter Engineering to Synthetic Operon Design" in"Metabolic Engineering IX", E. Heinzle, Saarland Univ.; P. Soucaille, INSA; G. Whited, Danisco Eds, ECI Symposium Series, (2013).http://dc.engconfintl.org/metabolic_ix/15

http://dc.engconfintl.org?utm_source=dc.engconfintl.org%2Fmetabolic_ix%2F15&utm_medium=PDF&utm_campaign=PDFCoverPages

http://dc.engconfintl.org/metabolic_ix?utm_source=dc.engconfintl.org%2Fmetabolic_ix%2F15&utm_medium=PDF&utm_campaign=PDFCoverPages

http://dc.engconfintl.org/proceedings?utm_source=dc.engconfintl.org%2Fmetabolic_ix%2F15&utm_medium=PDF&utm_campaign=PDFCoverPages

http://dc.engconfintl.org/metabolic_ix?utm_source=dc.engconfintl.org%2Fmetabolic_ix%2F15&utm_medium=PDF&utm_campaign=PDFCoverPages

http://network.bepress.com/hgg/discipline/229?utm_source=dc.engconfintl.org%2Fmetabolic_ix%2F15&utm_medium=PDF&utm_campaign=PDFCoverPages

mailto:[email protected]

Synthetic control of transcription:From hybrid promoters to promoter engineering

to synthetic operon design

September 7, 2005

Hal AlperDepartment of Chemical Engineering

The University of Texas at Austin

[email protected]://www.che.utexas.edu/alper_group/

1

Metabolic Engineering has opened up

possibilities…

• Metabolic

engineering

advances have

expanded the breath

of chemicals

produced by cells

Laboratory for Cellular

and Metabolic EngineeringJune 6, 2012

Slide # Hal Alper

produced by cells

– Pharmaceuticals and

Nutraceuticals

– Fuels

– Commodity/Specialty

Chemicals

– Polymers and

Precursors

2

Curran & Alper, Metabolic Engineering, 2012

…but, requires synthetic control elements...

Pathway Control Circuit Design

A

B

C D



Slide # Hal Alper 3

Heterologous Expression Probing via Graded Expression

promoter

promoter

promoter

promoter HAT

HAT*A

B

C

D

…with a range of expression capacities.

Native range of mRNA levels

promoter

promoter

Require a promoter series(s) that span above the highest native expression down to essentially zero expression:



Slide # Hal Alper 4

Holstege, et al. (1998), Cell 95:717-728.

promoter

promoter

Affords both amplification and knockdowns.

Synthetic control of transcription occurs at

many levelsSynthetic

Transcriptional Amplifiers

5’UTR and MCSsGenetic Context

Elements that



Slide # Hal Alper 5

Mutant Promoters

Selection Markersand Vectors

Elements that

Influence

ExpressionTerminators and

3’UTR

Designing novel genetic control elements

• Promoter Engineering is an effective method for

generating a collection of genetic control elements

Prior Promoter engineering example



Slide # Hal Alper 6

Alper et al. PNAS, 2005.

GFP+

Promoter PromoterLibrary

Range of promoter strength to control gene

expression

Development of diversified promoters

yECitrineTCYC1

Mutant

Promoter



Slide # Hal Alper 7

Nevoigt et al. AEM, 2006.

Development of novel low-strength promoters

2.5

3

3.5

4

Flu

ore

sce

nce

Re

lati

ve

to

TF

C1

yECitrineTCYC1

Mutant

Promoter



Slide # Hal Alper 8

0

0.5

1

1.5

2

2.5

Flu

ore

sce

nce

Re

lati

ve

to

TF

C1

Promoter

An expanding range of yeast promoters

Native range of mRNA levels



Slide # Hal Alper 9

Holstege, et al. (1998), Cell 95:717-728.

Mutant Promoter Libraries

• Promoter engineering via random mutagenesis is effective at creating diversity

• Bias in promoter libraries toward lower expression capacity



Slide # Hal Alper

expression capacity

• Expression range nearly matches “native expression levels”—need more sequence diversity

• Require novel tools for increasing strength of promoters

10





Elements that



Slide # Hal Alper 11

Mutant Promoters


Elements that

Influence

Expression Terminators and 3’UTR

Promoter Engineering in Yarrowia lipolytica

Yarrowia lipolytica is a fully sequenced, oleaginous yeast

• Naturally accumulates fatty acids (including linoleic acid) on food source with high C:N ratio

� Issues with immature

genetic tools and

expression levels

Advantages Challenges




linoleic acid) on food source with high C:N ratio

• Semi-developed genetic tools

• Thrives on non-conventional carbon sources

expression levels

� Low plasmid copy

number

� Lack of genetic

tools

� “Strong”

promoters do not

readily exist

Y. lipolytica Promoter Engineering via

tandem UAS sequences

Prior research suggested linking upstream

activating sequences (UAS) to a minimal promoter

can create function. Analyzed 1 to 4 UAS site.

Madzak et al., Microbiology, 1999

UAS1b UAS1b UAS1b UAS1b pleum




UAS1b pleum GFP… UAS1b1 n

We sought to evaluate potential of modulating promoter

activity by tandem copies of UAS1b sites (n = 1 to 32)

Creation of a strong promoter set in Y. lipolytica

200

250

300

350

400

450

500

Me

an

Flu

ore

sce

nce




0

50

100

150

200

Cen1

TE

F

(UA

S1b)1

(UA

S1b)2

(UA

S1b)3

(UA

S1b)4

(UA

S1b)5

(UA

S1b)6

(UA

S1b)7

(UA

S1b)8

(UA

S1b)9

(UA

S1b)1

0

(UA

S1b)1

1

(UA

S1b)1

2

(UA

S1b)1

3

(UA

S1b)1

4

(UA

S1b)1

5

(UA

S1b)1

6

(UA

S1b)1

7

(UA

S1b)1

8

(UA

S1b)1

9

(UA

S1b)2

0

(UA

S1b)2

1

(UA

S1b)2

2

(UA

S1b)2

3

(UA

S1b)2

4

(UA

S1b)2

5

(UA

S1b)2

6

Promoter Construct

Me

an

Flu

ore

sce

nce

Blazeck et al., AEM, 77(22), 2011

Extension of hybrid promoter strategy

pleum GFPUAS1b16

UAS16 Enabled TEF Promoters Tuning

450

500




TEF promoter truncation

series

0

50

100

150

200

250

300

350

400

LEUM TEFS1 TEFS2 TEFS3 TEF TEFL1 TEFL2 TEFL3 TEFL4

Basal Promoter

Me

an

Flu

ore

sce

nce

Basal Promoter

UAS16 BasalPromoter

Identification and use of a TEF UAS




Blazeck et al., Submitted, AMB, 2012

Expanding the hybrid promoter approach to

S. cerevisiae

15000

20000

25000

Flu

ore

scen

ce

Cit UAS

Clb UAS

CYC

TEF

X

UAS Elements Core Promoters




0

5000

10000Flu

ore

scen

ce

Promoter

Tef UAS

Gu1 UAS

GPD

GAL

X

Blazeck et al., Accepted/In Press, Biotech Bioeng, 2012

Creating the strongest constitutive promoter

15000

20000

25000

Flu

ore

scen

ce

Hybrid promoter approach

created a promoter 60%

higher on fluorescence and

2.5 fold higher in mRNA




0

5000

10000

p416MCS Gpd GpdClbCittefsc3

Flu

ore

scen

ce

Promoter

2.5 fold higher in mRNA

than the strongest

constitutive promoter by

mixing disparate UAS

elements


Creating a range of inducible hybrid promoters

Galactose –Inducible Hybrid Promoters

Gal4pBS1-PLEUMGal4pBS1 PLEUM+

+

Gal4p Binding Sites PLEUM Core

CGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCG

Gal4pBS1 Gal4pBS3 Gal4pBS4

Gal4pBS2




PLEUM

PLEUM

PLEUM

PLEUM

Gal4pBS2-PLEUM

Gal4pBS3-PLEUM

Gal4pBS4-PLEUM

Gal4pBS12-PLEUM

PLEUM Gal4pBS13-PLEUM

Gal4pBS24-PLEUM

Gal4pBS34-PLEUM

Gal4pBS134-PLEUM

+

+

+

+

+

+

+

+

Gal4pBS2

PLEUM

PLEUM

PLEUM

Gal4pBS1

Gal4pBS3

Gal4pBS4

Gal4pBS2

Gal4pBS1 Gal4pBS3

Gal4pBS2 Gal4pBS4

Gal4pBS3 Gal4pBS4

Gal4pBS3 Gal4pBS4Gal4pBS1

Creating a range of inducible hybrid promoters

30000

40000

50000

60000

Me

an

Flu

ore

sce

nce

(R

FU

)

Galactose

Glucose




0

10000

20000

Me

an

Flu

ore

sce

nce

(R

FU

)

Promoter

Glucose


Hybrid Promoter Engineering Examples

Largest library of promoters in Y. lipolytica Strongest constitutive promoter in S. cerevisiae




Largest range of synthetic inducible promoters in S. cerevisiae

Blazeck et al., AEM, 77(22), 2011 & Blazeck et al., Accepted/In Press, Biotech Bioeng, 2012

Synthetic, hybrid promoters

• UAS elements serve as synthetic transcriptional amplifiers

• UAS elements and core promoters can serve as modular synthetic components



Slide # Hal Alper

• Hybrid promoter engineering can amplify expression and create highly-functional libraries

22





Elements that




Mutant Promoters


Elements that

Influence


3’UTR

GOI

Multicloning sites influence 5’UTR sequences




Promoter RS1 RS2 RS3 RS4 RS5 RS6RS1 RS2 RS3 RS6

Crook et al., Nucleic Acids Research, 39(14), e92, 2011

Model System

• Yeast model system (S. cerevisiae)

• pBluescript SK II Multicloning Site

• CYC1, TEF, or GPD promoters



Slide # Hal Alper

• CYC1, TEF, or GPD promoters

• yECitrine

– Codon-optimized

25

>80%

3-fold

Restriction site affects protein expression




absolute

variationrelative

variation

Effect is most pronounced with short, codon-

optimized genes

No

rmali

zed

Rep

ort

er

Exp

ressio

n




No

rmali

zed

Rep

ort

er

Exp

ressio

n

Modeling and re-designing MCSs

• Goal: Use model to re-design better MCSs not susceptible to position effect by mitigating 5’UTR secondary structure




NNNNN

Conceptual model of inhibition

• Region(s) of secondary structure in 5’UTR can impede ribosome progression




Modeling and re-designing MCSs:

GPD/TDH3-based Promoter




Crook et al., Nucleic Acids Research, 39(14), e92, 2011

5’UTR Influence

• Choice of site in MCS can influence 5’UTR regions via secondary structure

• Most pronounced by short, codon-optimized genes (i.e. when transcription



Slide # Hal Alper

optimized genes (i.e. when transcription and translation rates are not limiting)

• Can use model-based approach to improve MCS design (esp. with GPD)

• Genetic context is important for characterizing parts

31





Elements that




Mutant Promoters


Elements that

Influence


Importance of genetic context




Genetic Context

• Results highlight importance of genetic context of synthetic parts

– Plasmids vs. genomic integration site

• Highlight need for insulating elements



Slide # Hal Alper

• Highlight need for insulating elements

36





Elements that




Mutant Promoters


Elements that

Influence


Terminator choice can influence performance

Test impact of changing

terminator region




>5 fold difference in

expression output by

changing terminator





Elements that




Mutant Promoters


Elements that

Influence


3’UTR

Final Thoughts

• Synthetic control elements are a critical tool for

implementing M.E. strategy

• “Promoters” cannot be thought of as singular,

isolated elements

• Currently require slightly more range, but



Slide # Hal Alper

• Currently require slightly more range, but

significantly more sequence diversity

• Starting to understanding fundamental design

principles for this critical components

• These elements are greatly enhancing our

capacity to metabolically engineer pathways

40

Acknowledgements

Students

Graduate

John Blazeck

Joseph Cheng

Nathan Crook

Kate Curran

Amanda Lanza

John Leavitt

Sun-mi Lee

Leqian Liu

Undergraduate

Vaibhav Agarwala

Heming Bai

Alex Bailey

Austin Comer

Tim Dyess

Rishi Garg

Rachelle Gerstner

Akash Gupta




Leqian Liu

Heidi Redden

Eric Young

Akash Gupta

Daniel Huang

Taylor Jellison

Ashty Karim

Do Soon Kim

Peter Otoupal

Ashley Poucher

Annie Pan

Heidi Redden

Ben Reed

Lindsey Rey

Andrea Zuzack

Synthetic Control of Transcription: From Hybrid Promoters ...

Documents

Transcript of Synthetic Control of Transcription: From Hybrid Promoters ...