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Metabolic Engineering IX
Proceedings
Summer 6-6-2012
Synthetic Control of Transcription: From Hybrid
Promoters to Promoter Engineering to Synthetic
Operon Design
Hal Alper
The University of Texas at Austin
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Recommended Citation
Hal 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
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Synthetic control of transcription:
From hybrid promoters to promoter engineering
to synthetic operon design
Hal Alper
Department of Chemical Engineering
The University of Texas at Austin
1
[email protected]
September 7, 2005
http://www.che.utexas.edu/alper_group/
Metabolic Engineering has opened up
possibilities…
• Metabolic
engineering
advances have
expanded the breath
of chemicals
produced by cells
– Pharmaceuticals and
Nutraceuticals
– Fuels
– Commodity/Specialty
Chemicals
– Polymers and
Precursors
Laboratory for Cellular
and Metabolic Engineering
Curran & Alper, Metabolic
Engineering, 2012
Hal Alper
June 6, 2012
Slide # 2
…but, requires synthetic control elements...
Pathway Control
Circuit Design
A
B
C
D
Heterologous Expression
Probing via Graded Expression
A
HAT
promoter
HAT*
B
promoter
D
promoter
C
Laboratory for Cellular
and Metabolic Engineering
promoter
Hal Alper
June 6, 2012
Slide # 3
…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:
promoter
Affords both amplification
and knockdowns.
promoter
Holstege, et al. (1998), Cell 95:717-728.
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 4
Synthetic control of transcription occurs at
many levels
Synthetic
Transcriptional
Amplifiers
5’UTR and MCSs
Genetic Context
Elements that
Influence
Expression
Mutant Promoters
Terminators and
3’UTR
Selection Markers
and Vectors
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 5
Designing novel genetic control elements
• Promoter Engineering is an effective method for
generating a collection of genetic control elements
Prior Promoter engineering example
+
Promoter
GFP
Range of promoter
strength to control gene
expression
Promoter
Library
Alper et al. PNAS, 2005.
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 6
Development of diversified promoters
Mutant
Promoter
yECitrine
TCYC1
Nevoigt et al. AEM, 2006.
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 7
Development of novel low-strength promoters
Mutant
Promoter
yECitrine
TCYC1
Fluorescence Relative to TFC1
4
3.5
3
2.5
2
1.5
1
0.5
0
Promoter
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 8
An expanding range of yeast promoters
Native range of mRNA levels
Holstege, et al. (1998), Cell 95:717-728.
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 9
Mutant Promoter Libraries
• Promoter engineering via random
mutagenesis is effective at creating diversity
• Bias in promoter libraries toward lower
expression capacity
• Expression range nearly matches “native
expression levels”—need more sequence
diversity
• Require novel tools for increasing strength of
promoters
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 10
Synthetic control of transcription occurs at
many levels
Synthetic
Transcriptional
Amplifiers
5’UTR and MCSs
Genetic Context
Elements that
Influence
Expression
Mutant Promoters
Terminators and
3’UTR
Selection Markers
and Vectors
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 11
Promoter Engineering in Yarrowia lipolytica
Yarrowia lipolytica is a fully sequenced, oleaginous yeast
Advantages
Challenges
• Naturally accumulates
Issues with immature
fatty acids (including
genetic tools and
linoleic acid) on food
expression levels
source with high C:N ratio
• Semi-developed genetic
Low plasmid copy
tools
number
• Thrives on non Lack of genetic
conventional carbon
tools
sources
“Strong”
promoters do not
readily exist
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 12
Y. lipolytica Promoter Engineering via
tandem UAS sequences
UAS1b UAS1b UAS1b UAS1b pleum
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
We sought to evaluate potential of modulating promoter
activity by tandem copies of UAS1b sites (n = 1 to 32)
UAS1b
1
Laboratory for Cellular
and Metabolic Engineering
…
UAS1b
pleum
GFP
n
Hal Alper
June 6, 2012
Slide # 13
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
(UAS1b)26
(UAS1b)25
(UAS1b)24
(UAS1b)23
(UAS1b)22
(UAS1b)21
(UAS1b)20
(UAS1b)19
(UAS1b)18
(UAS1b)17
(UAS1b)16
(UAS1b)15
(UAS1b)14
(UAS1b)13
(UAS1b)12
(UAS1b)11
(UAS1b)10
(UAS1b)9
(UAS1b)8
(UAS1b)7
(UAS1b)6
(UAS1b)5
(UAS1b)4
(UAS1b)3
(UAS1b)2
(UAS1b)1
TEF
Cen1
Mean Fluorescence
Creation of a strong promoter set in Y. lipolytica
500
450
400
350
300
250
200
150
100
50
0
Promoter Construct
Blazeck et al., AEM, 77(22), 2011
June 6, 2012
Slide # 14
Extension of hybrid promoter strategy
UAS1b
pleum
GFP
16
UAS16 Enabled TEF Promoters Tuning
500
450
TEF promoter truncation
series
Mean Fluorescence
400
350
300
Basal Promoter
250
UAS16 Basal
Promoter
200
150
100
50
0
LEUM TEFS1 TEFS2 TEFS3 TEF TEFL1 TEFL2 TEFL3 TEFL4
Basal Promoter
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 15
Identification and use of a TEF UAS
Blazeck et al., Submitted, AMB, 2012
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 16
Expanding the hybrid promoter approach to
S. cerevisiae
25000
Core Promoters
Cit UAS
CYC
Clb UAS
Tef UAS
Gu1 UAS
X
TEF
GPD
GAL
20000
Fluorescence
UAS Elements
15000
10000
5000
0
Promoter
Blazeck et al., Accepted/In Press, Biotech Bioeng, 2012
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 17
Creating the strongest constitutive promoter
25000
20000
Fluorescence
Hybrid promoter approach
created a promoter 60%
higher on fluorescence and
2.5 fold higher in mRNA
than the strongest
constitutive promoter by
mixing disparate UAS
elements
15000
10000
5000
0
p416MCS
Laboratory for Cellular
and Metabolic Engineering
Gpd
Promoter
GpdClbCittefsc3
Blazeck et al., Accepted/In Press, Biotech Bioeng, 2012
Hal Alper
June 6, 2012
Slide # 18
Creating a range of inducible hybrid promoters
Gal4pBS1
Gal4pBS3
Gal4pBS4
CGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCG
Gal4pBS2
Gal4p Binding Sites
Gal4pBS1
Gal4pBS2
Gal4pBS3
Gal4pBS4
Gal4pBS1 Gal4pBS2
Gal4pBS1 Gal4pBS3
Gal4pBS2 Gal4pBS4
Gal4pBS3 Gal4pBS4
Gal4pBS1 Gal4pBS3 Gal4pBS4
Laboratory for Cellular
and Metabolic Engineering
PLEUM Core
+
+
+
+
+
+
+
+
+
Galactose –Inducible Hybrid Promoters
PLEUM
Gal4pBS1-PLEUM
PLEUM
Gal4pBS2-PLEUM
PLEUM
Gal4pBS3-PLEUM
PLEUM
Gal4pBS4-PLEUM
PLEUM
Gal4pBS12-PLEUM
PLEUM
Gal4pBS13-PLEUM
PLEUM
Gal4pBS24-PLEUM
PLEUM
Gal4pBS34-PLEUM
PLEUM
Gal4pBS134-PLEUM
Hal Alper
June 6, 2012
Slide # 19
Creating a range of inducible hybrid promoters
60000
Mean Fluorescence (RFU)
50000
40000
30000
Galactose
Glucose
20000
10000
0
Promoter
Blazeck et al., Accepted/In Press, Biotech Bioeng, 2012
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 20
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
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 21
Synthetic, hybrid promoters
• UAS elements serve as synthetic
transcriptional amplifiers
• UAS elements and core promoters can serve
as modular synthetic components
• Hybrid promoter engineering can amplify
expression and create highly-functional
libraries
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 22
Synthetic control of transcription occurs at
many levels
Synthetic
Transcriptional
Amplifiers
5’UTR and MCSs
Genetic Context
Elements that
Influence
Expression
Mutant Promoters
Terminators and
3’UTR
Selection Markers
and Vectors
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 23
Multicloning sites influence 5’UTR sequences
GOI
Promoter
RS1 RS2 RS3
RS4
RS5
RS6
Crook et al., Nucleic Acids Research, 39(14), e92, 2011
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 24
Model System
• Yeast model system (S. cerevisiae)
• pBluescript SK II Multicloning Site
• CYC1, TEF, or GPD promoters
• yECitrine
– Codon-optimized
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 25
Restriction site affects protein expression
>80%
3-fold
absolute
variation
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
relative
variation
June 6, 2012
Slide # 26
Normalized Reporter Expression
Effect is most pronounced with short, codonoptimized genes
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 27
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
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 28
Conceptual model of inhibition
• Region(s) of secondary structure in 5’UTR
can impede ribosome progression
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 29
Modeling and re-designing MCSs:
GPD/TDH3-based Promoter
Crook et al., Nucleic Acids Research, 39(14), e92, 2011
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 30
5’UTR Influence
• Choice of site in MCS can influence 5’UTR
regions via secondary structure
• Most pronounced by short, codonoptimized 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
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 31
Synthetic control of transcription occurs at
many levels
Synthetic
Transcriptional
Amplifiers
5’UTR and MCSs
Genetic Context
Elements that
Influence
Expression
Mutant Promoters
Terminators and
3’UTR
Selection Markers
and Vectors
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 32
Importance of genetic context
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 33
Importance of genetic context
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 34
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 35
Genetic Context
• Results highlight importance of genetic
context of synthetic parts
– Plasmids vs. genomic integration site
• Highlight need for insulating elements
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 36
Synthetic control of transcription occurs at
many levels
Synthetic
Transcriptional
Amplifiers
5’UTR and MCSs
Genetic Context
Elements that
Influence
Expression
Mutant Promoters
Terminators and
3’UTR
Selection Markers
and Vectors
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 37
Terminator choice can influence performance
Test impact of changing
terminator region
>5 fold difference in
expression output by
changing terminator
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 38
Synthetic control of transcription occurs at
many levels
Synthetic
Transcriptional
Amplifiers
5’UTR and MCSs
Genetic Context
Elements that
Influence
Expression
Mutant Promoters
Terminators and
3’UTR
Selection Markers
and Vectors
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 39
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
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
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
June 6, 2012
Slide # 40
Acknowledgements
Students
Graduate
John Blazeck
Joseph Cheng
Nathan Crook
Kate Curran
Amanda Lanza
John Leavitt
Sun-mi Lee
Leqian Liu
Heidi Redden
Eric Young
Laboratory for Cellular
and Metabolic Engineering
Hal Alper
Undergraduate
Vaibhav Agarwala
Heming Bai
Alex Bailey
Austin Comer
Tim Dyess
Rishi Garg
Rachelle Gerstner
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
June 6, 2012
Slide # 41