290 14 Sequestered: Design and Construction of Synthetic Organelles
10 genes from H. neapolitanus led to fully formed CBs with a morphology nearly
identical to those from the native host in E. coli [61]. Likewise, expression of 17
genes from S. enterica can also produce EUTs in E. coli [51, 137].
Although these early attempts have defined genetic sufficiency, more work will
be required to define essential, or necessary, elements. The ultimate goal, of
course, would be to have a minimal system for expressing empty protein shells
and targeting novel proteins to the lumen. One open question is the role of each
shell protein and how many different genes are required to synthesize well‐
formed polyhedral shells. Most regulons possess multiple copies of genes for the
hexameric and pentameric protomers. Whether this is a gene dosage mechanism
for high protein expression or there is a functional difference between paralogs
remains to be seen. It should be noted that the function of shell paralogs may be
determined by their genomic position. This issue was brought to attention by
Chowdhury and colleagues, who demonstrated that PduJ is permeable to 1,2‐
propanediol only when it is expressed from the pduA locus [77]. It is unclear why
such a location effect exists, although it is thought that nascent PduJ translated
from different genomic regions may encounter different sets of binding partners.
Following from this observation, it may be possible to alter permeability of a shell
protein by changing its gene location in lieu of the labor‐intensive site‐directed
mutagenesis.
Another factor related to biogenesis that will need to be clarified is the inher
ent stability of BMCs. It is known that CBs have a more icosahedral shape [78]
and that α‐CBs, in particular, are robust enough to be isolated from cells in a
near‐pristine form. Likewise, transgenic α‐CBs display a somewhat native‐like
structure, suggesting that either the transcriptional and translational regulation
of the operon sequence “ports” over better in E. coli or that the protein–protein
interactions of α‐CB self‐assembly are inherently more robust. Future work will
be necessary to clarify to what extent this hypothesis is true and whether it holds
if BMCs are transgenically expressed in higher organisms such as yeast and
plants. In fact, Lin and colleagues have already made the first attempt to produce
carboxysomes in plants by expressing CcmM, CcmN, and three shell proteins
(CcmK2, CcmL, and CcmO) from the β‐CB in chloroplasts of Nicotiana bentha-
miana, but the resulting empty compartments were irregularly shaped [79]. It
would be of special interest to determine whether a similar experiment with an
α‐CB would result in more morphologically normal particles.14.3.2 Alternative Protein Organelles: A Minimal System
There are also several other self‐assembling protein complexes that, in principle,
could be adapted to function as an organelle. These include viral particles, large
enzyme complexes such as lumazine synthase, the ribonucleoprotein vault com
plex, and the icosahedral encapsulin complex, all of which have been studied to
some extent in attempts to engineer novel materials for both in vivo and ex vivo
applications [80–83]. Since it is not possible to cover all such applications in an
appreciable depth here, we refer the reader to previous reviews [84, 85] and
instead focus on one particular complex – encapsulin – that is a minimal alterna
tive to the more complex BMCs discussed earlier (Figure 14.5).