Biology Reference
In-Depth Information
TABLE 3.1
(Continued)
Tool
Description
Advantages
References
Codon Optimization
Express a heterologous enzyme,
change the DNA sequence to
complement host codon bias
Can be done by DNA synthesis
[50
53]
Tuning Intergenic
Regions
Improve gene expression far from
promoter, improve stability
of mRNA by introducing
secondary structures to reduce
mRNA degradation
Control of gene expression in
an operon structure
[54]
TIGR Library
Avoid rationally designing the
intergenic regions, creation
of a library
Library approach allows for
nonintuitive mutations
[55]
Promoter Engineering Engineer different promoters with
different strengths and apply
them to a pathway to
overexpress rate-limiting
enzymes
Allows fine-tuning of gene
expression for a single gene
[57]
Combinatorial
Promoter Library
Library approach to promoter
engineering
Fine tuning of multiple gene
expression simultaneously
[58]
Dynamic
Transcriptional
Regulation
Gene circuit designed to express
certain genes to redirect flux
towards desired product
Express superfluous genes
only when needed, reducing
burden
[59,60]
RBS Engineering
Improve translation efficiency by
optimizing the RBS site by
mathematical models
Reduce the library size required
to screen
[63]
Optimizing protein activity
Increasing Protein
Activity
45
In vitro enzyme engineering to
improve activity increases
product titer
Proteins with higher activity do
not need high expression
[64]
Improving Substrate
Specificity
In vitro enzyme engineering
to alter specificity to desired
substrate
Reduce side reactions and
redirect the flux towards the
product
[65]
Alter Cofactor
Specificity
In vitro enzyme engineering
to alter specificity to desired
cofactor, can create internal
regeneration mechanisms for
cofactor
Reduce the competition for
cofactors between required
metabolism reactions and the
desired pathway
[72
74]
Library of
Homologous
Enzymes
In vivo library combination of
different enzyme homologues
with different activities
Can select proteins for pathway
with best activity, most
substrate specificity, and
cofactor usage simultaneously
[75]
Protein Scaffolds
Anchor the protein in a special
manner to stimulate active site
channeling
Shuttle intermediates directly
to the next reaction, reducing
diffusion
[79,80]
DESIGN AND CONSTRUCTION OF PATHWAYS
Pathway Design Tools
In the design of metabolic pathways for synthesis of target compounds, traditional methods
simply modify existing endogenous pathways or recruit partial/entire pathways into a
heterologous host. Recent advances in bioinformatics have allowed a new approach,
wherein the computational tools can be used to design novel and specific pathways for the
desired compound. This strategy relieves the inherent regulatory restrictions of endogenous