294 14 Sequestered: Design and Construction of Synthetic Organelles
successful due to higher levels of FDP in the mitochondrion or whether sesquit
erpene synthase is simply more active in the mitochondrial matrix. Circumstantial
evidence from plants suggests that the mitochondria matrix contains both IPP
and DMAPP, agreeing with the former and alluding to a more complicated bio
synthetic picture of mitochondrial function [102].
This approach has also been used for the production of another class of
important chemicals, higher alcohols, which are potential gasoline replace
ments. Higher alcohols (aka fusel alcohols), such as isobutanol, are biosyn
thetically produced from the catabolism of amino acids via the Ehrlich pathway
[103]. Interestingly, while the initial biosynthesis of amino acids occurs in
mitochondria, the final Ehrlich decarboxylation and dehydrogenase reactions
occur in the cytosol. Avalos and colleagues hypothesized that co‐localizing the
entire isobutanol pathway (derived from leucine) together could result in
improved flux between enzymes and increased production [104]. This was
indeed the case, and a complete mitochondrial‐localized pathway resulted in a
260% increase in production. Interestingly, control experiments found that co‐
localization of the same enzymes to the cytoplasm improves yields only a 10%
increase, suggesting the mitochondria possesses an inherent biosynthetic
capability. Indeed, the mitochondrial targeting system has been used to
optimize the production of acetoin [105] and fumarate [106] in yeasts and
artemisinin in plant [107].14.4.1.2 The Vacuole
The vacuole is the central degradative structure in fungi, such as S. cerevisiae,
and is roughly the functional equivalent of the lysosome in mammals. It main
tains a low pH and possesses numerous hydrolytic enzymes involved in catabolic
processes [108]. These properties led to the classic notion of the vacuole simply
as the cell’s “trash can”. However, recent evidence suggest that the vacuole is a
highly regulated structure, which carefully maintains stores of specific free sug
ars and amino acids, and is critical to cellular pH homeostasis, mitochondrial
function, and replicative life span in yeast [109, 110]. Much of the specificity in
this process results from the numerous transporters localized to the vacuole,
which selectively transport individual sugars, amino acids, ions, and other
species. Intriguingly, although many have been identified and cloned, some are
simply hypothetical based on electrophysiology studies [111]. A better under
standing of this metabolic potential will be essential for future metabolic engi
neering efforts.
One metabolite whose vacuolar accumulation has been exploited for
metabolic engineering purposes is S‐adenosyl methionine (SAM). SAM is the
principal cellular currency for methyl transfer reactions and is a key cofactor
in numerous enzymatic reactions. The majority of cellular SAM is stored in
the vacuole [112]. Recently, Bayer and colleagues undertook a metagenomic
approach to identifying enzymes involved in the biosynthesis of methyl hal
ides, industrially relevant commodity chemicals that can be upconverted to
numerous other chemicals using zeolite catalysts [113]. During the initial
work in E. coli, it was postulated that SAM concentrations were limiting pro
duction. Switching to yeast, Bayer et al. used a well‐known targeting sequence,