280 14 Sequestered: Design and Construction of Synthetic Organelles
Co‐localization of a pathway also ensures substrate channeling of intermedi
ates between enzymatic steps to improve both kinetics and yield and reduce
host toxicity [5]. Channeling commonly occurs in multifunctional enzymes
where a labile or toxic molecule is passed from one active site to another via a
protein channel. Examples include tryptophan synthase (indole intermediate),
acetyl‐CoA synthase/carbon monoxide dehydrogenase (carbon monoxide), and
carbamoyl phosphate synthase (ammonia) [6–8]. Similar mechanisms occur in
bacterial microcompartments (BMCs), large proteinaceous shells that encapsu
late short metabolic pathways. These will be discussed in greater detail later,
but briefly, various evidence suggests these protein complexes are able to
sequester/channel both volatile substrates (CO 2 , acetaldehyde) and those
potentially toxic (propanal) to the rest of the cell [9–11]. In a related context, it
is important to note that self‐assembly of enzymes and pathways into large
complexes is more common than previously realized. It is perhaps an inevitable
outcome of the fact that metabolic enzymes are highly expressed and allosteri
cally regulated [12]. Thus, substrate channeling is often critical to metabolic
pathway function.
Recent synthetic biological efforts have leveraged these principles for improved
biocatalysis. The goal of metabolic engineering is to produce important chemi
cals, such as pharmaceuticals, materials, and biofuels, from cheap and sustain
able biomass [13, 14]. Doing so requires high productivities and yields for
engineered pathways, but this optimization is often counter to the growth and
fitness of the host organism. Drawing inspiration from nature, one promising
metabolic engineering strategy is to repurpose organelles or protein complexes
as cellular factories for improving the performance of engineered pathways – in
other words, to engineer synthetic organelles. In a striking example of this strat
egy, Dueber and colleagues have engineered scaffold proteins from the yeast
mitogen‐activating signaling cascade as an enzymatic assembly line to improve
production titers of the isoprenoid precursor pathway nearly 80‐fold while
reducing intermediate toxicity [15]. Similarly, Sachdeva et al. improved synthesis
of pentadecane from fatty acyl‐ACP by co‐localizing fatty acyl‐ACP reductase
and aldehyde‐deformylating oxygenase to an RNA scaffold, providing a strategy
for optimizing microbial biofuel production [16].
Building upon the idea of enhancing pathway flux through co‐localization,
various molecular chassis and metabolic engineering strategies have been devel
oped to facilitate catalysis. To this end, here we review recent advances and open
questions in the engineering and use of synthetic organelles for bioengineering
applications (Figure 14.1). As most research heretofore has centered on metabo
lism, our focus is largely on metabolic engineering applications. The physical
composition of an organelle – whether it is made from lipids or proteins –
profoundly shapes potential uses, so our review is conceptually broken down
into these two areas. Finally, it should be noted that the compartmentalization of
engineering metabolism spans many orders of magnitude, from metabolically
engineered cocultures of microbes down to single enzymes (Figure 14.1). We will
focus on the middle regime, from protein compartments to repurposing existing
organelles, but direct the engaged reader to previous reviews focused on the
former [17] and latter [18, 19].