Biology Reference
In-Depth Information
has been met with great success, especially with multigene pathways. Other successful
examples include the mevalonate pathway and the biosynthesis of antibiotics.
39,42,43
CHROMOSOMAL INTEGRATION
As shown by Jones et al
.
comparing high- and low-copy plasmids,
41
the plasmid
expression of gratuitous proteins can be successful. However, there are disadvantages to
the plasmid-based system. Plasmid instability has been shown to be a major problem. Due
to the metabolic burden on the plasmid-bearing organism, it has a selective disadvantage
compared to a plasmid-free cell. Without selection pressure, an entire culture could be
dominated by the plasmid-free cell. The loss of the productive pathway (allele segregation)
has been described as the fundamental flaw in plasmid-based gene expression. Tyo et al.
44
developed a technique called chemical-inducible chromosomal evolution (CiChE) to
integrate the heterologous genes into the chromosome at multiple sites, with up to 40
consecutive copies. Integration into the chromosome leads to long-term stability and
circumvents the loss of productivity by allele segregation due to use of plasmid. More than
offering stability over a long period like other integration methods, this particular method
is a facile way to finely tune the gene expression by multiple insertions and having exact
copy-numbers, allowing genes with low activity to have higher expression and thereby
reducing bottlenecks.
CODON OPTIMIZATION
The degeneracy of the genetic code is exploited systematically in different ways by different
organisms.
45,46
In the early days of recombinant DNA expression, it became abundantly
clear that codon optimization would be crucial for efficient protein expression. Studies have
shown that the preferred codons correlate with the abundance of cognate tRNAs available
within the cell.
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49
Thus, if a gene from an organism with a certain codon bias is
recombinantly expressed in a host with a different codon bias, the translation of the
recombinant protein will be suboptimal. This can be overcome by codon optimization,
wherein the codon sequence of the target gene is altered to match that of the codon bias of
the host. Codon optimization is commercially available from DNA synthesis companies
and has proven to increase protein expression in multiple projects.
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50
OPERONS AND TUNING INTERGENIC REGIONS
Grouping the genes of interest for the pathway into a single polycistronic mRNA can be
beneficial to optimize the enzyme expression. This one-step gene expression allows for
simplified induction processes and improved regulatory mechanisms. However, the design
of the basic operon can greatly affect expression. For example, the genes in closest proximity
to the promoter will be expressed the greatest. To improve the expression of genes further
downstream from the promoter, the efficiency of the transcription can be controlled by
optimizing transcriptional processes. The transcription termination, mRNA stability, and
translation initiation can all affect how well the proteins are expressed. The DNA regions
which control these processes are the intergenic regions.
Smolke et al.
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showed that altering the mRNA stability by engineering the intergenic
regions would increase protein production. By introducing RNA hairpin turns into the 3
0
and
5
0
intergenic region of the gene of interest, the mRNA was less susceptible to mRNA
degradation by the RNaseE nuclease. Activity analysis of the enzyme of interest suggested that
with increasing hairpin turns, the enzyme activity increased. This was the first example of
differential control of gene expression established in an operon. This idea of independently
controlling gene expression within an operon was further explored by Pfleger et al.
55
By creating a library of tunable intergenic regions (TIGRs), the expression of several genes
within an operon was simultaneously tuned for optimal expression. The TIGRs contained
control elements that include mRNA secondary structures, RNase cleavage sites, and ribosomal
