Biology Reference
In-Depth Information
That is, not one specific auxotroph was able to form a cooperative relationship with all
other auxotrophs. Interestingly, pairings between auxotrophs that contain mutations in the
same biosynthetic pathway grew significantly less than pairings between auxotrophs that
contain mutations in distinct metabolic pathways. Using mathematical analysis, the authors
determined that pairs of auxotrophs grow the most when their growth requirement is
fulfilled using small quantities of a metabolite that are inexpensive for their growth partner
to produce and share.
Another approach to engineering communication is to use secretion of the individual
components of an enzymatic system. Arai et al. engineered three populations of
Bacillus
subtilis
to secrete the enzymatic components of an endoglucanase or a xylanase from
Clostridium cellulovorans
.
23
One strain was constructed to secrete a HIS-tagged MiniCbpA,
which serves as a scaffold protein that binds to and localizes enzymes that contain dockerin
domains. Two additional strains were created to express and secrete two cellulosomal
enzymes: EngB, which has endoglucanase activity (i.e. hydrolysis of cellulose); or XynB,
which has xylanase activity (i.e. hydrolysis of xylose). Both of these proteins contain
dockerin domains, and as such could readily bind to the MiniCbpA protein. Initially, the
authors determined that each enzyme component could be readily expressed and secreted
into the growth medium. Next, they cocultured two
B. subtilis
populations, the MiniCbpA
and XynB-expressing populations, or the MiniCbpA and EngB-expressing populations
(
Fig. 13.1c
), to determine if functional enzyme complexes could be formed in the medium.
By isolating HIS-tagged proteins from the medium, the authors observed that coculture of
these populations led to the formation of protein complexes between MiniCbpA and XynB
or MiniCbpA and EngB, both of which retained xylanase or endoglucanase activity,
respectively.
While many synthetic consortia have used either small molecules or enzymes to enable
communication, a recent study has combined both methods to program novel dynamics in
a consortium. Goldberg et al. engineered a synthetic consortium where two populations can
detect, communicate, and chemotax in concert towards two substrates.
24
The authors
engineered two
E. coli
strains that could respond to either asparagine or phenylacetyl glycine
(PAG), both of which are nonnatural chemotaxic agents in
E. coli
. To construct an
asparagine sensing strain, the authors engineered one strain to constitutively express and
export the enzyme asparaginase II (
ansB
gene), which converts asparagine into aspartate,
into the periplasm. Furthermore, they engineered this strain to express an aspartate receptor,
Tar, that, when complexed with aspartate, results in chemotaxis towards asparagine. To
construct a PAG-sensing strain, the authors placed penicillin lyase (
pac
), which converts PAG
to produce PAA, under the regulation of a constitutive promoter. To sense the presence of
PAG, the authors created a Tar receptor variant (TarPA) that could bind to PAA and activate
chemotaxis towards PAG.
247
When the authors experimentally tested each strain individually, both strains readily
chemotaxed toward their respective substrates. However, to create a consortium, the authors
swapped the receptors (i.e. Tar and TarPA) between the cell strains, creating two novel
strains. Here, one strain contains Tar and
pac
(i.e. chemotaxes towards asparagine but
hydrolyzes PAG to PAA), and the other contains TarPA and
ansB
(i.e. chemotaxes towards
PAA but hydrolyzes asparagine into aspartate). As such, when the authors grew either strain
in monoculture, neither strain performed chemotaxis towards an attractant, as each strain
lacked the ability to both
(i.e. hydrolyze a product) and respond to an attractant.
However, when the two strains were grown together on soft agar, the consortium readily
chemotaxed towards asparagine and PAG. As such, the enzyme products produced by one
strain activated chemotaxis in the other. The authors noted that both attractants were
required in order to result in chemotaxis as the consortium would not chemotax when
only one attractant was present.
sense
'
'
