Download Next-Generation Approaches to Overcome the Challenges of

Next-Generation Approaches to Overcome the Challenges
of Metabolic Pathway Engineering
1Department
Abstract
Ashlee Smith1, Clay Swackhamer2, Sam Krug1, Emily Sileo3, Howard Salis1,2, Tom Richard2
of Chemical Engineering, 2Department of Ag. and Biological Engineering 3Department of Biochemistry and Molecular Biology
The Pennsylvania State University
Our Construct
Engineering metabolic pathways requires answering several questions and
often performing many experiments. We sought to answer such "combinatorial
questions" using new biophysical models and experimental approaches. First,
we employed the CRISPR/Cas9 system to identify the missing enzymes of a
metabolic pathway that functions in one organism, but not in another. We
demonstrate our approach on a 5-enzyme catabolic pathway that functions in
P. putida, but not in E. coli. Once a multi-enzyme pathway functions in a
preferred host, the DNA sequence of the system must then be optimized to
achieve a desired metabolic activity. We took a forward engineering approach
to systematically investigate the differences in translation rate capacity when
using either "frequent", "fast", "rare", or "slow" synonymous codons during
codon optimization of protein coding sequences. Our research will enable
metabolic engineers to overcome two of the most commonly encountered
combinatorial questions during metabolic pathway engineering.
Codon Optimization Objectives
Sac1
Pst1
Inverse PCR
pFTV(2.8kb)
Pst1
Inverse PCRProducts
OldCodingSequence
Pst1
Cla1
Inserting GFP
pFTV(2.8kb)
Leader Sequence
Variant GFP
Leader Sequence
Xho1
Cla1
Variant GFP
Currently, we have successfully
completed all constructs except
for slow GFP (4).
Stop
Codon
pFTV with GFP
pFTV(2.8kb)
Xho1
Cla1
Start Leader Sequence
Codon
Variant GFP
Design and Methods
Stop Codon
1) Use Gibson CBA and restriction digests and ligations to
assemble plasmid containing dCas9
2) Insert dCas9 (plasmid 1) into P. putida genome using
homologous recombination
Terminator
dRBS sequence (36 nt)
Pst1
Sac1
Completed Construct
CHOSENCODON
J23100 Promoter
Chloramphenicol Resistance
Current Progress
Assembled the first plasmid and attempting to insert
into the genome via homologous recombination
HmfABCDE gene cluster
breaks
down
furan
derivatives to components
of the citric acid cycle
Stop Codon
Start
Codon
Design
Koopman et. al. identified
the 5 gene pathway that
detoxifies
furan
derivatives in C. basilensis
and P. putida – does not
function in E. coli
Cla1
Xho1
Pst1
Optimized superfolder GFP, a gene derived from GFP mut3b by
Stephan Milde and Jim Haseloff, iGEM Cambridge 2008
Substitute degenerate codons with higher or lower
translation efficiency without altering the amino acid
protein sequence to change protein expression levels
1) Validate the HMF pathway and dCas9 function in P.
putida
2) Identify the essential genes of the HMF pathway
using dCas9 combinatorial gene knockdown
3) Develop a program for automated genome
comparisons
4) Engineer the functioning pathway in E. coli
Used inverse PCR to excise the
existing GFP, amplify the backbone,
introduce new restriction sites to
insert a dRBS containing leader
sequence and variant coding
sequence
pFTV(2.8kb)
Sac1 Pst1
Goal: Determine the essential genes of the 5-(hydroxymethyl)
furfural (HMF) pathway and engineer E. coli with this pathway.
Allow for more efficient microbial biofuel production and
higher biofuel yields.
Cla1
• Search for novel optimization criteria and apply
them to a reporter gene
• Express the codon optimized
variants and draw conclusions
about the effects of non-standard
codon optimization methods on
translation elongation.
• Investigate an additional point of
control for genetic engineering
Used pFTV vector to express variant
GFPs - contained effective origin of
replication, antibiotic resistance
marker, promoter, and terminator
Forward Primer
OldCodingSequence
Sac1
Objectives
Cla1
Reverse Primer
Sac1
Biodetoxification Introduction
The kanamycin resistance gene is part of the overlap
for the next plasmid containing the HMF pathway
Cannot complete cloning until first plasmid is inserted
into genome
ColE1 Origin of Replication
Results
Average FLPC of two replicates for each strain of common
2
1
3
4000
4
3500
5
3000
DegenerateCodons
AVerage FLPC
2500
dCas9 PCR
2000
FTV backbone PCR
1500
Future Steps
1000
500
0
G2 T5
G2 T17 G2 T19
G2 T7
G2 T10
G2 T3
G2 T8
G2 T18 G2 T14 G2 T11 G2 T15
G2 T4
G2 T1
G2 T13
G2 T6
G2 T16 G2 T12
G2 T2
G2 T9
G2 T20 G2 T21
Average FLPC of two replicates for each strain of fast
4500
4000
3500
Coding sequence analysis. Blue is for rare, Green is for rare, Yellow
is for codons that do not fall under the common or rare category.
Our degenerate Ribosome
Binding Site (dRBS)was designed
C
using the Ribosome Binding Site The dRBS
Calculator (Salis, Nature Biotech (48 distinct sequences) A C A G
2009). Each GFP coding
CTGGAGGARTAGSTTMGRGVGGTC
sequence started with a leader
A GC G
A G
sequence to eliminate
discrepancies in translation
initiation rate.
Average FLPC
3000
2500
3) Construct plasmid containing HMF pathway, insert into genome
4) Test the function of dCas9 by knocking out RFP and test the
function of HMF pathway by doing a furfural consumption assay
5) Assemble CRISPR RNA library and insert into plasmid
6) Conduct gene knockdown experiments using combinatorial
dCas9
Combinatorial Gene Knockdown Using dCas9
2000
1500
1000
500
0
1
4
2
5
7
9
8
3
11
12
6
10
18
20
16
13
32
19
34
22
21
29
As of now, common codon optimization results in better expression of
superfolder GFP over varying translation initiation rate. Further studies
would be to compare and characterize rare and slow codon optimized
GFP coding sequence variants. This could lead to more effective ways to
repress gene expression for undesirable genes.
Iman Farasat identified 19
target genes that could be
related to the HMF pathway
Knock out combinations of 2
or 3 genes
Sequence the constructs
that no longer consumes
furfural
Targets: Cofactors, chaperone
proteins, ATPases, accessory
proteins
Continue attempts to insert HMF and dCas9
into P. putida genome
Validate function of dCas9 using RFP
knockout
Continue characterization of Slow, Rare GFP
variants
Acknowledgements
Dr. Howard Salis and Dr. Tom Richard for helping develop our project ideas
Graduate students Iman Farasat and Chiam Yu Ng for assisting us in wetlab work
Penn State Institutes of Energy and the Environment for funding
References:
(1) Koopman, Frank, et al. "Identification and characterization of the furfural and 5-(hydroxymethyl) furfural degradation pathways of
Cupriavidus basilensis HMF14." Proceedings of the National Academy of Sciences 107.11 (2010): 4919-4924.
(2) Graf, Nadja, and Josef Altenbuchner. "Development of a method for markerless gene deletion in Pseudomonas putida." Applied and
environmental microbiology 77.15 (2011): 5549-5552
(3) Subramaniam, Arvind R, Tao Pan, and Philippe Cluzel. “Environmental Perturbations Lift the Degeneracy of the Genetic Code to
Regulate Protein Levels in Bacteria.” Proceedings of the National Academy of Sciences of the United States of America 110.6 (2013):
2419–24. Web. 26 May 2014.