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.