Biology Reference
In-Depth Information
demand for nucleotides in the replication of the DNA itself. This became known as the
metabolic burden, and reduction of the metabolic burden has become essential in the
pursuit to identify an optimal metabolic pathway. 7 10 Minimizing the metabolic burden is
challenging enough, but more problems arise when often one or two enzymes in the
pathway will not express well, or will have low activity. This causes a bottleneck or a build-
up of intermediates in the pathway, thereby reducing the overall titer. All these challenges
combined have prompted the development of powerful computational and experimental
tools for pathway engineering ( Table 3.1 ).
TABLE 3.1 A List of Available Tools for Pathway Engineering
Tool
Description
Advantages
References
Pathway design tools
BNICE
The Biochemical Network
Integrated Computational
Explorer
Design pathways based on
specific functional groups via
matrix operation and evaluate
by their thermodynamic
properties
[12,17,18]
ReBiT
Retro-Biosynthesis Tool
Pathway design and evaluation,
step-by-step suggestion on
cofactors and enzymes involved
http://www.retro-
biosynthesis.com/
PPS
The Pathway Prediction System Predict biodegradation pathways
based on chemical structures
http://umbbd.msi.umn.
edu/predict/
Pathway construction tools
DNA Assembler
Uses overlapping sequences
In vivo one-step pathway
assembly tool with high
efficiency, especially feasible
in S. cerevisiae-based research
[23,99]
44
Gibson Isothermal
Assembly
Uses overlapping sequences
In vitro one-step assembly tool
[24,25]
Ordered Gene
Assembly in
B. subtilis (OGAB)
Type II restriction enzyme
digestion
One-step pathway assembly
in B. subtilis
[26,27]
BioBrick TM /BglBrick Modular restriction digestions
Utilize restriction digestion,
BioBrick TM assembly kit
commercially available
[29]
Golden Gate
Type II restriction enzyme
digestion
One-step, one-pot assembly
in short time
[33]
OE-PCR/ CPEC
PCR with overlapping sequences PCR-based tool, easy to
manipulate
[34,35]
USER
Uses overlapping sequences
USER-friendly cloning kit
commercially available
[36
38]
SLIC
Uses overlapping sequences
Exonuclease involved, overhang
length is determined by
chewing back time
[39]
Pathway optimization tools
Optimizing gene expression
Plasmid
Copy-Number
Modify copy number of plasmid
to reduce metabolic burden
and still produce high titer
Gene expression can be
easily modified
[39,41
43,86]
CiChE
Integrate the pathway into the
chromosome
Avoid allele segregation and allow
multiple copies of gene
integration
[44]
(Continued)
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