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\begin{document}

\title{Intracellular accumulation of c-di-GMP and its regulation on
self-flocculation of the bacterial cells from Zymomonas mobilis}



\author[1]{Fengwu Bai}%
\author[1]{Kai Li}%
\author[1]{Juan Xia}%
\author[1]{Chen-Guang Liu}%
\affil[1]{Shanghai Jiao Tong University State Key Laboratory of Microbial Metabolism}%


\vspace{-1em}



  
  \date{May 26, 2024}


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\selectlanguage{english}
\begin{abstract}
\emph{Zymomonas mobilis} is an emerging chassis for being engineered to
produce bulk products due to its glycolysis through the Entner-Doudoroff
pathway with less ATP produced for lower biomass accumulation and higher
yields with targeted products. When self-flocculated, the bacterial
cells are more productive and tolerant to stresses for high product
titers, but this morphology needs to be controlled properly to avoid
internal mass transfer limitation associated with strong flocculation.
Herewith we explored the regulation of cyclic diguanosine monophosphate
(c-di-GMP) on self-flocculation of the bacterial cells through cellulose
biosynthesis. While ZMO1365 and ZMO0919 with GGDEF domains for
diguanylate cyclase activities catalyze c-di-GMP biosynthesis, ZMO1487
with an EAL domain for phosphodiesterase activities catalyzes c-di-GMP
degradation, but ZMO1055 and ZMO0401 contain the dual domains with
phosphodiesterase activities predominated. Since c-di-GMP is synthesized
from GTP, the intracellular accumulation of this signal molecule through
deactivating the activity of phosphodiesterase is preferred for
activating cellulose biosynthesis to flocculate the bacterial cells,
since such a strategy exerts less perturbance on intracellular processes
regulated by GTP. These discoveries are significant not only for
engineering unicellular \emph{Z. mobilis} strains with the
self-flocculating morphology to boost production, but also for
understanding mechanism underlying c-di-GMP biosynthesis and degradation
in the bacterium.%
\end{abstract}%



\sloppy


\textbf{Intracellular accumulation of c-di-GMP and its regulation on
self-flocculation of the bacterial cells from \emph{Zymomonas mobilis}}

Kai Li, Juan Xia, Chen-Guang Liu\textsuperscript{*}, Feng-Wu
Bai11\textsuperscript{*}Corresponding authors. E-mails:
cg.liu@sjtu.edu.cn (C. G. Liu) and fwbai@sjtu.edu.cn (F. W. Bai)

State Key Laboratory of Microbial Metabolism, Joint International
Research Laboratory of Metabolic \& Developmental Sciences, School of
Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai
200240, China.

\textbf{Abstract:}\emph{Zymomonas mobilis} ~is an emerging chassis
engineered to produce bulk products because of its glycolysis through
the Entner-Doudoroff pathway, with less ATP produced for lower biomass
accumulation and higher yields with targeted products. When
self-flocculated, the bacterial cells are more productive and tolerant
to stresses for high product titers, but this morphology needs to be
controlled properly to avoid internal mass transfer limitations
associated with strong flocculation. In this study, we explored the
regulation of cyclic diguanosine monophosphate (c-di-GMP) on
self-flocculation of bacterial cells through cellulose biosynthesis.
While ZMO1365 and ZMO0919, with GGDEF domains for diguanylate cyclase
activities, catalyze c-di-GMP biosynthesis, ZMO1487, with an EAL domain
for phosphodiesterase activity, catalyzes c-di-GMP degradation, but
ZMO1055 and ZMO0401 contain dual domains with phosphodiesterase
activities. Since c-di-GMP is synthesized from GTP, the intracellular
accumulation of this signal molecule by deactivating the activity of
phosphodiesterase is preferred for activating cellulose biosynthesis to
flocculate bacterial cells, since such a strategy exerts less
perturbation on intracellular processes regulated by GTP. These
discoveries are significant not only for engineering
unicellular~\emph{Z. mobilis} ~strains with a self-flocculating
morphology to boost production but also for understanding the mechanism
underlying c-di-GMP biosynthesis and degradation in the bacterium.

\textbf{Keywords:} \emph{Zymomonas mobilis} ; chassis;
self-flocculation; c-di-GMP; microbial cell factories; industrial
production

\textbf{1 Introduction}

Compared with the Embden-Meyerhof-Parnas (EMP) pathway, which is
commonly used by other microorganisms, the ethanologenic bacterium
\emph{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
\emph{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, \emph{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 \emph{S. cerevisiae} with
the redox pathway for the same purpose (Gopinarayanan et al., 2019).
These merits make \emph{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
of\emph{Z. 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 \emph{Z.
mobilis}(Carre\selectlanguage{ngerman}ón-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 \emph{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 \emph{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 \emph{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 \emph{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 from\emph{Z. 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.

\textbf{2 Materials and Methods}

\textbf{2.1 Strains, media and culture}

All strains used in this study are listed in Table S1. \emph{Z.
mobilis}stocks were inoculated into the rich medium (RM) composed of 10
g/L yeast extract, 20 g/L glucose, and 2 g/L
KH\textsubscript{2}PO\textsubscript{4}, grown statically at 30 °C until
their exponential phase, and then transferred into 250 mL flasks, each
containing 100 mL RM medium with 10\% inoculation for subculture to
increase OD\textsubscript{600} to \textasciitilde{}1.0. When needed, 20
\selectlanguage{greek}µ\selectlanguage{english}g/mL tetracycline was added to the RM medium. Congo red staining was
used to visualize the cellulose produced by \emph{Z. mobili} s, and 2 \selectlanguage{greek}µ\selectlanguage{english}L
of the subculture was inoculated onto RM agar plates containing 70 \selectlanguage{greek}µ
µ\selectlanguage{english}g/mL Congo red, which were incubated for 12-24 h at 30 \selectlanguage{ngerman}°C for visual
inspection (Trivedi et al., 2016; Thongsomboon et al., 2020).

\textbf{2.2 Development of recombinant strains}

Primers and plasmids used in this study are listed in Tables S2 and S3.
The suicide vector, pEX18Tc, was used (Hoang et al., 1998). Fragments of
500\selectlanguage{english}-1000 bp for flanking genes to be deleted were amplified from\emph{Z.
mobilis} , which were cloned into \emph{E. coli} DH5\selectlanguage{greek}α \selectlanguage{english}for amplification.
The recovered plasmids were fused with enzymatically digested pEX18Tc
and confirmed by sequencing. Subsequently, the plasmids were transformed
into \emph{E. coli} JM110 for demethylation for efficient transformation
into \emph{Z. mobilis} ZM4 or ZM401 through electroporation. RM medium
supplemented with tetracycline was used to select colonies harboring the
target plasmids, and 5\% sucrose was added to the RM medium for
counter-selection of homologous colonies (Li et al., 2013). The selected
mutants were verified using tetracycline sensitivity and Colony PCR. The
shuttle vector pHW20a was used to carry target genes (Dong et al.,
2011). The overexpressed genes were colonized from \emph{Z. mobilis} ,
which, together with the promoter of the glyceraldehyde-3-phosphate
dehydrogenase gene (Pgap), were amplified by PCR for fusion with pHW20a.
The expression plasmids were demethylated in\emph{E. coli} JM110 and
then transformed into \emph{Z. mobilis} as previously described (Xia et
al., 2018). When engineered with the empty vector pHW20a, no significant
difference was observed compared with the wild-type strains ZM4 and
ZM401 (Fig. S2). Therefore, ZM4/pHW20a and ZM401/pHW20a were used as
controls for the comparative analysis. All the engineered strains were
verified by PCR.

\textbf{2.3 Characterization for the self-flocculation of \emph{Z.
mobilis}}

The culture (4 mL) was sampled and de-flocculated by cellulases
following a previously established protocol for measuring
OD\textsubscript{600} using a Microplate Reader (Thermo Fisher,
Multiskan GO, USA) (Xia et al., 2018), which was used as the basis (A)
for quantifying the flocculation efficiency of \emph{Z. mobilis} . The
culture collected simultaneously was rested statically for 5 min, and
then 400 \selectlanguage{greek}μ\selectlanguage{english}L supernatant was sampled, which was also treated with
cellulases to de-flocculate any suspended small flocs and mixed
vigorously to obtain a homogenous suspension to measure
OD\textsubscript{600} (B). The flocculation efficiency (F) of \emph{Z.
mobilis} was calculated using the following equation:

F = (1 -- B/A) \selectlanguage{ngerman}× 100\%.

\textbf{2.4 Extraction and quantification of intracellular c-di-GMP}

c-di-GMP was extracted as previously reported, with some modifications
(Xu et al., 2013). Briefly, 15 mL of \emph{Z. mobilis} culture was grown
to the exponential stage and centrifuged at 5, 000 g for 3 min. The
pellet was immediately suspended in 1000 \selectlanguage{greek}μ\selectlanguage{english}L buffer (40\% methanol: 40\%
acetonitrile: 20\% dH\textsubscript{2}O) by vigorous vortexing at -20\selectlanguage{ngerman}°C
for 30 min, which was by centrifuged at 12, 000 g and 4 °C for 5 min,
and the supernatant was collected into a tube cooled with ice. Cell
debris was extracted twice and the supernatant was collected. All
supernatants were combined for vacuum evaporation to condense c-di-GMP,
which was dissolved in 80 \selectlanguage{greek}µ\selectlanguage{english}L of buffer (50\% acetonitrile and 50\%
dH\textsubscript{2}O) for analysis.

c-Di-GMP quantification was performed using a Waters I-Class Acquity
UPLC (Waters, UK) coupled with a Vion IMS QToF mass spectrometer
(Waters, UK). The separation of c-di-GMP was performed using a SeQuant
ZIC-HILIC column (100 mm \selectlanguage{ngerman}× 2.1 mm) packed with 3.5 \selectlanguage{greek}µ\selectlanguage{english}m
polyetheretherketone and operated at 45\selectlanguage{ngerman}°C; and the mobile phase composed
of 50 mM ammonium formate in water (A), and acetonitrile (B) was pumped
at 0.4 mL/min under the following gradient elution conditions: 0--10
min, 90--50\% B; 10--12 min, 50--90\% B; 12--15 min, 90\% B. c-di-GMP
was detected through electrospray ionization operated in the
negative-ion mode. The software UNIFI 1.8.1 was used for data
processing.

c-Di-GMP with a purity of 98\% (Biolog, Germany) was used as the
standard to calibrate the analysis.



\textbf{3 Results}

\textbf{3.1 Role of c-di-GMP in self-flocculation of the bacterial
cells}

To explore the role of intracellular accumulation of c-di-GMP in the
self-flocculation of \emph{Z. mobilis} , genes \emph{wspR}
and\emph{yhjH} encoding DGC and PDE in \emph{Pseudomonas aeruginosa}
and\emph{Escherichia coli} for c-di-GMP biosynthesis and degradation
(Güvener et al., 2007; Lindenberg et al., 2013) were overexpressed in
ZM4 (ZM4/\emph{wspR} ) and ZM401 (ZM401/\emph{yhjH} ), respectively.
These manipulations would enhance or compromise the intracellular
accumulation of c-di-GMPs in bacterial cells.

While ZM4/\emph{wspR} self-flocculated incompletely for a loose and
flake-like morphology (Fig. 1A) with a flocculating efficiency of 30.9\%
compared to 6.1\% observed in the control ZM4/pHW20a, the
self-flocculating phenotype of the control ZM401/pHW20a was disrupted
completely, and the flocculation efficiency decreased to 5.5\% (Fig.
1B). On the other hand, the intracellular accumulation of c-di-GMP
increased drastically to 72.7 pg/mg protein in ZM4/\emph{wspR} from 10.0
pg/mg proteins detected for the control, but only 1.4 pg (c-di-GMP)/mg
protein was detected in ZM401/\emph{yhjH} , compared to that of 14.3
pg/mg protein detected in the control (Fig. 1B).

Previous studies have confirmed that cellulose fibrils are the chemical
basis for the development of the self-flocculating phenotype in
ZM401(Xia et al., 2018). Congo red binds to 1,4-\selectlanguage{greek}β-\selectlanguage{english}D-glucopyranosyl units
with a strong affinity, which can be used to qualitatively characterize
cellulose production qualitatively (Trivedi et al., 2016). When the
cultures were inoculated onto agar plates containing the rich medium
(RM) supplemented with Congo red and incubated properly, dark red was
observed for ZM401/pHW20a due to cellulose production, but a weak
staining reaction occurred with ZM4/\emph{wspR} , ZM4/pHW20a, and
ZM401/\emph{yhjH} , indicating that less cellulose was produced (Fig.
1C). Therefore, intracellular accumulation of c-di-GMP regulates the
development of the self-flocculating phenotype in \emph{Z. mobilis} ,
but the synthesis of sufficient cellulose by the bacterial cells would
be a prerequisite for them to self-flocculate, with significance in
industrial applications.

Fig. 1

In the genome of ZM4, ZMO1055, ZMO0401, ZMO1487, ZMO1365 and ZMO0919 are
predicted to encode proteins with conserved domains of GGDEF and/or EAL
for DGC and/or EAL activities, catalyzing the biosynthesis and
degradation of c-di-GMP, respectively (Jones-Burrage et al., 2019).
While ZMO1055 and ZMO0401 contain both GGDEF and EAL domains, ZMO1487
contains an EAL domain only, and both ZMO1365 and ZMO0919 contains GGDEF
domains (Table 1).

Table 1

\textbf{3.2 Endogenous c-di-GMP metabolism in \emph{Z. mobilis}}

Comparative genome analysis between ZM401 and ZM4 detected the SNP
mutation in ZMO1055: thymine was replaced by cytosine for the amino acid
substitution Ala526Val, and its role in the degradation of c-di-GMP and
development of the self-flocculating phenotype in ZM401 has been studied
recently (Cao et al., 2022). However, little attention has been paid to
the role of wild-type ZMO1055 (ZMO1055\textsuperscript{+}) from ZM4 in
the biosynthesis and degradation of c-di-GMP.

When ZMO1055\textsuperscript{+} was overexpressed in ZM401, its
self-flocculating phenotype was disrupted with the flocculating
efficiency decreased to 5.0\%, and the intracellular accumulation of
c-di-GMP decreased drastically to 0.44 pg/mg protein, compared to that
of 92.5\% and 14.25 pg/mg protein, respectively, observed in the control
(Fig. 2). However when ZMO1055 with the SNP mutation from ZM401
(ZMO1055\textsuperscript{-}) was overexpressed in ZM401, its
self-flocculating phenotype was compromised slightly with the
flocculating efficiency decreased to 85.9\%, and the intracellular
accumulation of c-di-GMP compromised less to 6.07 pg/mg protein,
indicating that the SNP mutation substantially mitigated the PDE
activity of ZMO1055\textsuperscript{+} for c-di-GMP degradation, which
was supported by the deletion of ZMO1055\textsuperscript{-} from ZM401
for the signal molecule to further increase to 16.68 pg/mg protein (Fig.
2). Manipulation of ZMO1055\textsuperscript{+} and
ZMO1055\textsuperscript{-} in ZM4 through their overexpression and
deletion also indicated a strong PDE activity of the wild-type protein
for c-di-GMP degradation.

To further confirm the PDE activity, we constructed an expression
plasmid carrying ZMO1055\textsuperscript{+} but with a site-directed
mutation of the amino acid substitution Ala356Glu to change the
catalytic domain from EAL to AAL, which was experimentally validated to
deactivate PDE activity in \emph{P. aeruginosa} (Kuchma et al., 2007;
Nesbitt et al., 2015). When ZMO1055\textsuperscript{-} was knocked out
and the recombinant plasmid was transformed into ZM401, no significant
change was observed in the intracellular accumulation of c-di-GMP, and
the mutant ZM401\selectlanguage{greek}Δ\selectlanguage{english}ZMO1055\textsuperscript{-}/1055\textsuperscript{AAL}
maintained the self-flocculating phenotype (Fig. 2). These experimental
results indicate that the substitution of Ala526Val on
ZMO1055\textsuperscript{+} in ZM401 compromised the protein's PDE
activity for c-di-GMP degradation, as exhibited by the native gene
bearing the AAL domain. This conclusion was validated by the reverse
substitution of Val526Ala in ZM401
(ZM401\selectlanguage{greek}Δ\selectlanguage{english}ZMO1055\textsuperscript{-}/1055\textsuperscript{+}), as well as
the substitution of Ala526Val in ZM4
(ZM4\selectlanguage{greek}Δ\selectlanguage{english}ZMO1055\textsuperscript{+}/1055\textsuperscript{-}) to compromise
the protein's PDE activity for intracellular accumulation of c-di-GMP to
as high as 18.1 pg/mg protein (Fig. 2).

ZMO1055 also contains the GGDEF (GGDQF) domain, which catalyzes the
biosynthesis of c-di-GMP. When GGDQF was replaced by GGAQF through the
substitution of Asp232Ala to deactivate the protein's DGC activity in
ZM401, the intracellular accumulation of c-di-GMP decreased to 7.82
pg/mg protein, about 50\% of that detected in the control, and its
self-flocculating phenotype was disrupted completely (Fig. 2). These
results indicate the DGC activity of ZMO1055 and its contribution to the
biosynthesis of c-di-GMP for the intracellular accumulation of this
signal molecule, as well as the development of the self-flocculating
phenotype in ZM401.

Fig. 2

ZMO0401 is another protein with the dual domains speculated for the
biosynthesis and degradation of c-di-GMP in \emph{Z. mobilis} . When it
was overexpressed, ZM401 lost the self-flocculating phenotype with the
flocculating efficiency decreased to 5.6\%, and in the meantime
extremely low intracellular accumulation of c-di-GMP (0.76 pg/mg
protein) was detected, indicating that ZMO0401 might function
predominantly on c-di-GMP degradation (Fig. 3). We therefore constructed
the knockout mutants ZM401\selectlanguage{greek}Δ\selectlanguage{english}0401 and ZM4\selectlanguage{greek}Δ\selectlanguage{english}0401, respectively, to validate
such a speculation.

When ZMO0401 was deleted from ZM401, the intracellular accumulation of
c-di-GMP increased to 29.2 from 15.5 pg/mg protein, but no significant
change in the self-flocculating phenotype was observed for the mutant,
indicating that the intracellular accumulation of c-di-GMP was high
enough for activating the biosynthesis of cellulose to flocculate the
bacterial cells. As for ZM4, the intracellular accumulation of c-di-GMP
increased significantly to 28.2 from 9.8 pg/mg protein when ZMO0401 was
deleted, but the flocculating efficiency of ZM4\selectlanguage{greek}Δ\selectlanguage{english}0401 increased slightly
to 15.5\%, due to its inability to synthesize sufficient cellulose,
which we previously showed relies on a mutation in the protein ZMO1082
involved in cellulose production (Cao et al., 2022) (Fig. 3).

Fig. 3

ZMO1487 was predicted to encode a protein with an EAL domain only for
c-di-GMP degradation. When ZMO1487 was overexpressed in ZM401 and ZM4,
respectively, the intracellular accumulation of c-di-GMP decreased
drastically to 0.95 and 0.54 pg/mg protein from 15.48 and 10.04 pg/mg
protein detected in the controls, and the self-flocculating phenotype of
ZM401 was disrupted (Fig. 4). On the other hand, when ZMO1487 was
deleted from ZM4, intracellular accumulation of c-di-GMP increased to
17.62 pg/mg protein, but no significant difference was observed when
ZMO1487 was deleted from ZM401 (Fig. 4). The reason for this phenomenon
might be due to relatively weak impact of ZMO1487 on c-di-GMP metabolism
in ZM401 compared to ZMO1055 and ZMO0401, particularly when the SNP
mutation in ZMO1055 substantially compromised its PDE activity and
enhanced the intracellular accumulation of c-di-GMP. These experiments
indicate the catalytic function of the EAL domain in ZMO1487 on c-di-GMP
degradation.

Both ZMO1365 and ZMO0919 were predicted to encode DGC domains for
c-di-GMP biosynthesis. Compared to the intracellular accumulation of
c-di-GMP at 9.8 pg/mg protein in the control, the overexpression of
ZMO1365 and ZMO0919 in ZM4 increased its intracellular accumulation of
c-di-GMP to 82.49 and 27.77 pg/mg protein, respectively (Fig. 4B). The
extremely high intracellular accumulation of the signal molecule
stimulated partial development of the self-flocculating phenotype in the
mutants, with their flocculating efficiency increasing to 46.1\% and
30.1\%, respectively, compared to only 5.5\% observed in the control
(Fig. 4B). These experimental results validated the catalytic function
of the DGC domains in ZMO1365 and ZMO0919 in c-di-GMP biosynthesis.

Fig. 4

\textbf{3.3 Engineering ZM4 with the self-flocculating phenotype}

To elucidate the functions of genes involved in the biosynthesis and
degradation of c-di-GMP, we targeted genes encoding proteins with PDE
activities for c-di-GMP degradation to explore the effect of their
combinatory knockout on the development of the self-flocculating
phenotype in ZM4.

When both ZMO1055 and ZMO0401 were deleted, the flocculation efficiency
of the bacterial cells improved to 24.4\%, and the flocculation
efficiency was further improved to 34.5\% for the double knockout mutant
with both ZMO1055 and ZMO1487 deleted (Fig. 5A). No further improvement
in the self-flocculating phenotype was observed when all three genes
were deleted in ZM4. However, such genetic manipulation is preferred for
reducing the genome of \emph{Z. mobilis} to engineer this species as a
more reliable method to accommodate heterogeneous genes more
effectively.

Cellulose fibrils have been validated as the chemical basis for
developing the self-flocculating phenotype in ZM401(Xia et al., 2018).
ZM4 also contains a bacterial cellulose synthase (\emph{bcs} ) operon
composed of ZMO1082, ZMO1083, ZMO1084, and ZMO1085. ZMO1082 was
predicted to be a putative gene encoding a short peptide composed of 67
amino acid residues only (Xia et al., 2018), which is less likely to be
functional and thus can be manipulated together with ZMO1083. Therefore,
we engineered ZM4 with the overexpression of ZMO1082-1083 and
ZMO1082-1084 and the whole \emph{bcs} operon ZMO1082-1085 to investigate
their contribution to the self-flocculation of the bacterial cells. As a
result, the development of the self-flocculating phenotype was observed
in the mutants with their flocculating efficiencies of 28.9\%, 43.1\%
and 66.7\%, respectively (Fig. 5A).

When both strategies were employed in ZM4, enhancing its intracellular
accumulation of c-di-GMP to 94.42 pg/mg protein through the deletion of
ZMO1055, ZMO0401, and ZMO1487, and the biosynthesis of cellulose fibrils
through the overexpression of the \emph{bcs} operon, a flocculating
efficiency of 97.3\% was observed for the bacterial cells, which was
higher than that of 92.5\% detected with ZM401 (Fig. 5A). Morphologies
were further shown for ZM4 strains engineered with the deletion of
ZMO1055, ZMO0401, and ZMO1487, overexpression of the \emph{bcs} operon,
and the combination of these two strategies (Fig. 5B).

Fig. 5

When industrial strains are engineered with new phenotypes, such as the
self-flocculation of microbial cells for more advantages, their
production performance should not be compromised. \emph{Z. mobilis} is
ethanologenic, and suitable for producing cellulosic ethanol. Therefore,
we compared ethanol fermentation performance between the strain
engineered with the self-flocculating phenotype and its unicellular
wild-type ZM4. As can be seen (Fig. S1), no difference was observed when
medium supplemented with 100 g/L glucose, equivalent to total sugars in
the hydrolysate of lignocellulosic biomass, was fermented to produce
ethanol.

\textbf{4 Discussion}

\emph{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 \emph{E. coli} and \emph{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 \emph{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 \emph{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 \emph{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 \emph{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
\emph{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 \emph{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
whole\emph{bcs} 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
unicellular\emph{Z. 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.

\textbf{5 Conclusions}

Intracellular accumulation of c-di-GMP in \emph{Z. mobilis} through
biosynthesis and degradation catalyzed by enzymes with DGC and PDE
activities activates the biosynthesis of cellulose fibrils for the
bacterial cells to self-flocculate with significant advantages in
industrial production. Compared to the overexpression of proteins with
DGC activity for synthesizing more c-di-GMP from GTP, deactivation of
PDE activity to compromise c-di-GMP degradation is preferred for
intracellular accumulation of the signal molecule, since this strategy
would exert less perturbation on intracellular processes involved with
or regulated by GTP. This study provides insights into the regulation of
c-di-GMP on the morphological shift in \emph{Z. mobilis} from
unicellular cells to multicellular flocs, as well as guidelines for
engineering unicellular strains from this species and other bacteria
with the self-flocculating phenotype through rational design. Moreover,
this study provides a basis for exploring the mechanism underlying
c-di-GMP metabolism in \emph{Z. mobilis} and its regulation of other
intracellular processes.

\textbf{Competing interests}

The authors declare no competing interests.

\textbf{Author contributions}

K.L. drafted the manuscript. J. X. performed all the experiments and
performed a preliminary analysis of the experimental results. C.G.L. and
F.W.B. supervised this study.

\textbf{Funding}

This work was financially supported by the National Natural Science
Foundation of China (NSFC) under grant numbers 31970026 and 21536006.

\textbf{Data availability}

All data are available in the main text and supplementary information.
Upon request, all the materials developed in this work are available for
non-commercial use.

\textbf{Acknowledgements}

We thank Professors Jie Bao at the East China University of Science and
Technology, Shihui Yang at Hubei University, and Ningyi Zhou at Shanghai
Jiao Tong University for donating plasmids for our research.

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\textbf{Table 1} Protein domains predicted for c-di-GMP metabolism
in\emph{Z. mobilis} (ZM4).\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Protein ID & Domain for c-di-GMP metabolism & Domain architecture & ORF
(aa)\tabularnewline
\midrule
\endhead
ZMO1055 & Cyclase/phosphodiesterase for c-di-GMP synthesis/degradation &
& 579\tabularnewline
ZMO0401 & Cyclase/phosphodiesterase for c-di-GMP synthesis/degradation &
& 683\tabularnewline
ZMO1487 & Phosphodiesterase for c-di-GMP degradation & &
258\tabularnewline
ZMO0919 & Cyclase for c-di-GMP synthesis & & 1008\tabularnewline
ZMO1365 & Cyclase for c-di-GMP synthesis & & 1045\tabularnewline
\bottomrule
\end{longtable}

Beta-propeller signal transduction domain; Periplasmic ligand-binding
sensor domain;

GGDEF domain for cyclase activity; EAL domain for phosphodiesterase
activity;

Domain for transmembrane helices; ORF: open reading frame; aa: amino
acids.

\textbf{Figure Legends}

\textbf{Fig. 1} Overexpression of \emph{wspR} encoding diguanylate
cyclase from \emph{P. aeruginosa} in ZM4 (ZM4/wspR) and
\emph{yhjH}encoding phosphodiesterase from \emph{E. coli} in ZM401
(ZM401/yhjH). (A) Phenotypes observed on the engineered strains cultured
in the rich medium, (B) Flocculating efficiency () and intracellular
accumulation of c-di-GMP () of the bacterial cells, and (C) Qualitative
characterization of extracellular cellulose by Congo red staining.
ZM4/pHW20 and ZM401/pHW20: ZM4 and ZM401 engineered with the empty
vector pHW20a as the control.

\textbf{Fig. 2} Manipulations of ZMO1055 and impact on self-flocculation
of the bacterial cells of \emph{Z. mobilis} (A), and intracellular
accumulation of c-di-GMP (B). + and -: wild-type and mutated genes from
ZM4 and ZM401. / and \selectlanguage{greek}Δ: \selectlanguage{english}gene overexpression and deletion.
ZM401\selectlanguage{greek}Δ\selectlanguage{english}ZMO1055\textsuperscript{-}/1055\textsuperscript{+}(AAL) and
ZM401\selectlanguage{greek}Δ\selectlanguage{english}ZMO1055\textsuperscript{-}/1055\textsuperscript{+}(GGAQF): ZM401
manipulated with the deletion of ZMO1055\textsuperscript{-} and
overexpression of the recombinant plasmid bearing the doman AAL or GGAQF
developed through site-directed mutations in
ZMO1055\textsuperscript{+}for the amino acid substitute Ala356Glu and
Asp232Ala, respectively.
ZM401\selectlanguage{greek}Δ\selectlanguage{english}ZMO1055\textsuperscript{-}/1055\textsuperscript{-}(V526A): ZM401
engineered with the delection of ZMO1055\textsuperscript{-} and
overexpression of the recombinant plasmid bearing
ZMO1055\textsuperscript{-} with the site-directed reverse mutation for
the amino acid substitute Val526Ala.
ZM4\selectlanguage{greek}Δ\selectlanguage{english}ZMO1055\textsuperscript{+}/1055\textsuperscript{+}(A526V): ZM4
engineered with the delection of ZMO1055\textsuperscript{+} and
overexpression of the recombinant plasmid bearing
ZMO1055\textsuperscript{+} with the site-directed mutation for the amino
acid substitute Ala526Val. ZM4/pHW20 and ZM401/pHW20: ZM4 and ZM401
engineered with the empty plasmid pHW20 as the controls.

\textbf{Fig. 3} Contribution of ZMO0401 to self-flocculation of the
bacterial cells of \emph{Z. mobilis} () and intracellular accumulation
of c-di-GMP () (A), and morphologies observed in flask cultures when
shaking was stopped for 3-5 seconds (B). / and \selectlanguage{greek}Δ: \selectlanguage{english}gene overexpression
and deletion. ZM4/pHW20 and ZM401/pHW20: ZM4 and ZM401 engineered with
the empty vector pHW20a as the controls.

\textbf{Fig. 4} Impact of the deletion (\selectlanguage{greek}Δ\selectlanguage{english}) and overexpression (/) of
ZMO1487 (a), ZMO1365 and ZMO0919 (b) on the self-flocculation () of the
bacterial cells of \emph{Z. mobilis} and intracellular accumulation of
c-di-GMP (). ZM4/pHW20 and ZM401/pHW20: ZM4 and ZM401 engineered with
the empty vector pHW20a as the controls.

\textbf{Fig. 5} Engineering ZM4 (A) with the deletion (\selectlanguage{greek}Δ\selectlanguage{english}) of ZMO1055,
ZMO0401 and ZMO1487, combinationally (), overexpression (/) of
ZMO1082-1083, ZMO1082-1084 and ZMO1082-1085 () as well as the deletion
of ZMO1055, ZMO0401 and ZMO1487 (1), together with the overexpression of
the whole \emph{bcs} operon ZMO1082-1085 (2), for strategy (1) + (2) to
develop the self-flocculating phenotype (), and the morphologies
observed in flask culture of the engineered strains (B). ZM4/pHW20: ZM4
engineered with the empty vector pHW20a as the control ().

\textbf{Fig. 6} Strategies for engineering unicellular \emph{Z. mobilis}
strains with the self-flocculating phenotype through enhancing
intracellular accumulation of c-di-GMP to activate cellulose
biosynthesis (genes in bold and plain fonts are for deletion and
overexpression, respectively).\selectlanguage{english}
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\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

