4 Discussion
Z. mobilis has been acknowledged as a potential chassis to be
engineered for biorefinery of lignocellulosic biomass to produce bulk
products with major costs from feedstock consumption, such as ethanol as
a biofuel and 2, 3-butanediol as a building block (Xia et al., 2019;
Yang et al., 2016). However, unlike E. coli and S.
cerevisiae which have been intensively studied and delicately
engineered as chassis cells (Mienda et al., 2021; Mitsui et al., 2021),
much less is known about Z. mobilis being engineered as a
microbial cell factory for robust production of bulk products, in
particular through rational design.
As a signaling molecule, c-di-GMP regulates intracellular processes,
including cellulose biosynthesis in bacteria (Ute et al., 2006; Jenal et
al., 2012; Ross et al., 1987; Morgan et al., 2014). Therefore,
understanding the biosynthesis and degradation of c-di-GMP in Z.
mobilis is fundamental for its development as a suitable chassis. On
the one hand, this knowledge can contribute directly to developing
strategies for controlling the morphological shift from unicellular
cells to multicellular flocs with physiological and metabolic merits,
including stress tolerance, and advantages in bioprocess engineering,
such as biomass recovery and immobilization of bacterial cells within
bioreactors. However, it will be beneficial to explore internal cues
related to the replication of genetic materials for division and
differentiation to support cell growth, since c-di-GMP can act as a cell
cycle oscillator to drive chromosome replication (Lori et al., 2015).
Bacteria have evolved specialized sensory and regulatory domains for
responding to c-di-GMP, which accumulates intracellularly at different
levels through a dynamic balance between its biosynthesis and
degradation, and variants of enzymes with GGDEF and/or EAL domain(s) for
DGC or/and PDE activities can fulfill such a task (Hengge et al., 2021;
Petchiappan et al., 2020). ZMO1055, ZMO0401, ZMO1487, ZMO1365, and
ZMO0919 are involved in c-di-GMP metabolism in ZM4 for the intracellular
accumulation of c-di-GMP at different levels, but previous studies have
confirmed that only ZMO0919 exhibits DGC activity (Jones-Burrage et al.,
2015).
Our experimental results
confirmed the function of ZMO0901, as reported previously (Jones-Burrage
et al., 2015), and further validated that all four other genes are
functional for c-di-GMP metabolism in ZM4. In addition to ZMO1055, which
has been studied recently (Cao et al., 2022), the catalytic functions of
ZMO0401, ZMO1487, and ZMO1365 on c-di-GMP metabolism in Z.
mobilis were revealed for the first time. The reason for this
discrepancy may be the different culture conditions. While rich medium
was employed in our studies, minimal medium was used by Jones-Burrage et
al. (Jones-Burrage et al., 2015),
which could affect the expression
of genes and functions of encoded proteins. Therefore, these genes could
be selected as targets for engineering to explore the role of c-di-GMP
in metabolic regulation in Z. mobilis .
ZMO1365 and ZMO0919 enhanced c-di-GMP biosynthesis. Under the catalysis
of DGC, c-di-GMP is synthesized from 2 mol of
guanosine
triphosphate (GTP) with 2 mol of
diphosphate produced
(Schirmer et al., 2016). As a high-energy compound, GTP is actively
involved in multiple cellular processes, such as G-protein signaling
through RGS proteins and protein biosynthesis through GTPase switch
regulation (Wolff DW et al., 2022; Masuho et al., 2020; Cherfils et al.,
2011), and also acts as a building block for synthesizing
RNA during transcription
(Attwater et al., 2018; Akoopie et al., 2020). Therefore,
energy-intensive GTP production is finely regulated within cells to
reduce the consumption of energy in the form of ATP. As a result, the
overexpression of ZMO1365 and ZMO0919 in Z. mobilis to synthesize
more c-di-GMP from GTP would not be an economic strategy for developing
this species as a suitable chassis to be engineered as a microbial cell
factory, since such a strategy could potentially affect intracellular
processes involved with GTP.
Although ZMO1055 and ZMO0401 encode dual-functional proteins with both
DGC and PDE domains to catalyze the biosynthesis and degradation of
c-di-GMP, their PDE activities dominate the DGC activities for c-di-GMP
degradation. Moreover, ZMO1487, with PDE activity, only catalyzes the
degradation of c-di-GMP. Therefore, deactivating PDE activities by
deleting ZMO1055, ZMO0401, and ZMO1487 would be preferred to compromise
c-di-GMP degradation in Z. mobilis to enhance its intracellular
accumulation, and consequently activate cellulose biosynthesis to
flocculate bacterial cells (Xia et al., 2018; Morgan et al., 2014).
These manipulations would exert less perturbation on other intracellular
processes involved or regulated by GTP.
When engineered only with the overexpression or deletion of genes
related to the biosynthesis and degradation of c-di-GMP, ZM4 could not
develop a self-flocculating phenotype for applications from the
viewpoint of bioprocess engineering. Thus, overexpression of the wholebcs operon composed of ZMO1082-1085 is needed for bacterial cells
to synthesize sufficient amounts of cellulose under the regulation of
c-di-GMP. Therefore, we proposed a strategy for engineering unicellularZ. mobilis strains with a self-flocculating phenotype through
rational design (Fig. 6).
Fig. 6
It is worth noting that the size of the bacterial flocs needs to be
controlled properly. Large flocs benefit biomass recovery through
cost-effective gravity sedimentation, and also could enhance their
tolerance to stresses for less demand on detoxification of toxic
byproducts in the hydrolysate of lignocellulosic biomass. However, they
also present risk for internal mass transfer limitation for substrate
transport from bulk environment (outside) into the inner core of the
bacterial flocs (inside), and vice versa for transporting product from
the inside to outside. No doubt understanding of the regulation of
c-di-GMP on self-flocculation of the bacterial cells provides insights
on controlling their self-flocculating process at molecule levels,
which, together with bioprocess engineering strategies for developing
suitable hydrodynamic conditions within bioreactors, could ultimately
optimize their size for robust production.