1 Introduction
Compared with the
Embden-Meyerhof-Parnas (EMP) pathway, which is commonly used by other
microorganisms, the ethanologenic
bacterium Zymomonas
mobilis employs the Entner-Doudoroff (ED) pathway for glycolysis with
less ATP produced for lower biomass accumulation, as
ATP is dissipated predominantly
through biosynthesis, particularly cell growth of cells (Xia et al.,
2019). From the viewpoint of mass balance, more sugar can be directed to
ethanol production with improved yield, which is most important for
producing ethanol as a biofuel with a major cost from sugar consumption
(Gombert et al., 2015). On the other hand, the bacterial cells are
smaller than the brewing yeast Saccharomyces cerevisiae due to a
high specific surface to assimilate sugar faster, which, together with
the low energy-coupling ED pathway, forms a catabolic pathway for carbon
metabolism to produce ethanol faster (Rutkis et al., 2016).
Moreover, Z. mobilis can
be engineered with pentose metabolism through the isomerase pathway
without cofactor imbalance for intermediate accumulation (Zhang et al.,
1995), which is an intrinsic drawback for engineering S.
cerevisiae with the redox pathway for the same purpose (Gopinarayanan
et al., 2019). These merits make Z. mobilis suitable for
engineering to produce not only cellulosic ethanol but also other bulk
products from lignocellulosic biomass (He et al., 2014).
ZM401, a mutant developed from ZM4, a unicellular model strain ofZ. mobilis , self-flocculates with advantages for industrial
production (Cao et al., 2022). When self-flocculated, bacteria can be
conveniently immobilized within bioreactors for high cell density to
improve productivity, as highlighted previously in ethanol fermentation
with self-flocculating yeast (Zhao et al., 2019). In addition, bacterial
flocs can be recovered through cost-effective gravity sedimentation
instead of centrifugation, a regular practice for harvesting unicellular
cells with high capital investment in centrifuges, as well as intensive
energy consumption during their operation, needless to say cost with
frequent maintenance.
Tolerance to environmental stresses is a prerequisite for the robust
production of microbial strains because various stresses are present
under industrial conditions (Gong et al., 2017). Product inhibition is
one of these stresses, because a high product titer has been pursued
endlessly in industry to save energy consumption on product recovery and
reduce the discharge of wastewater, which has been highlighted in
high-gravity ethanol fermentation (Puligundla et al., 2019). Toxicity
from by-products is another stress, one of the biggest challenges for
lignocellulose biorefineries to produce biofuels and bio-based
chemicals, as toxic byproducts, including
furfural,
5-hydroxymethylfurfural, and acetic acid, are inevitably generated
during the pretreatment of lignocellulosic biomass (Ling et al., 2014).
Although various technologies, such as physical adsorption, chemical
treatment, and biological degradation, have been developed for
detoxifying the hydrolysate of lignocellulosic biomass, none of them is
economically feasible for industrial applications (Nogueira et al.,
2021). Meanwhile, tolerance to individual stresses such as ethanol,
acetic acid, and high temperature has been studied for Z. mobilis(Carreón-Rodríguez et al., 2019; Yang et al., 2020; Li et al., 2021),
but the progress is less significant because multiple stresses always
co-exist under industrial production conditions, and general stress
responses are preferred (Guan et al., 2017). Self-flocculation with Z.
mobilis bacterial cells of Z. mobilis can make them more tolerant
to elevated ethanol and inhibitors present in the hydrolysate of
lignocellulosic biomass (Zhao et al., 2014).
Microbes can develop multicellular morphologies, such as biofilms and
activated sludge, under stressful conditions (Ciofu et al., 2022; Wilén
et al., 2018). However, self-flocculation with Z. mobilis bacterial
cells of Z. mobilis presents a unique morphology. Compared to
amorphous biofilms, which generally require abiotic surfaces for
development with a life cycle (Rumbaugh et al., 2020), no surface is
needed for the bacterial cells to
self-flocculate, since the process is mediated by cellulose fibers that
are self-synthesized (Xia et al., 2018). Furthermore, a dynamic balance
can develop between the breakup of large flocs and the re-flocculation
of small flocs under specific hydrodynamic conditions that are developed
within bioreactors, which can renew the inside time for the bacterial
flocs to sustain viability and perform production efficiently. Unlike
activated sludge, which is formed naturally during mixed cultures with
abundant microbes as a core community for more efficient syntrophy, such
as bacteria for the degradation of short-chain fatty acids and
methanogens for methane production in anaerobic digestion (Saunders et
al., 2016; Hao et al., 2020), self-flocculation of bacterial cells
occurs under pure culture conditions.
The chemical basis for self-flocculation with the bacterial cells of
ZM401 was experimentally validated to be cellulose fibrils (Xia et al.,
2018), which are synthesized in the mutant more efficiently by the
bacterial cellulose synthase (Bcs) complex due to single nucleotide
polymorphism (SNP) mutations occurring in genes ZMO1082 and ZMO1055 (Cao
et al., 2022). As a second messenger, cyclic diguanosine monophosphate
(c-di-GMP) regulates intracellular processes through a dynamic balance
between its biosynthesis and degradation, which are catalyzed by
diguanylate cyclases (DGC) and phosphodiesterases (PDE), respectively
(Ute et al., 2006; Jenal et al., 2012). Since bacterial cellulose
biosynthesis is regulated by c-di-GMP (Ross et al., 1987; Morgan et al.,
2014), we hypothesized that the intracellular accumulation of c-di-GMP
in Z. mobilis could impact the self-flocculation of the bacterial
cells through its regulation of the biosynthesis of cellulose fibrils.
In this study, we explored genes
encoding enzymes related to c-di-GMP metabolism in Z. mobilis and
studied the intracellular accumulation of this signal molecule as well
as its impact on the self-flocculation of bacterial cells. This progress
is significant not only for engineering unicellular strains fromZ. mobilis with such a multicellular morphology for robust
production but also for understanding the mechanism underlying c-di-GMP
metabolism through intracellular
biosynthesis and degradation in
the bacterium.