Intracellular accumulation of c‐di‐GMP and its regulation on self‐flocculation of the bacterial cells of Zymomonas mobilis

Abstract

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

Previously uncharacterized aliphatic amino acid positions modulate the apparent catalytic activity of the EAL domain of ZMO_1055 and other cyclic di-GMP specific EAL phosphodiesterases

“…This and previous studies had shown that the A526V mutation in ZMO1055ZM401 maintains cell aggregation and the intracellular cyclic di-GMP concentrations to a greater extent (Cao, Yang et al 2022, Li, Xia et al 2023. The conservation of alanine at position 526 in subclass ZMO1055 GGDEF-EAL domain proteins indicates that alanine is important for functionality of the EAL domain.…”

“…Leucine, isoleucine and methionine are all non-polar amino acid and have similar size of side chain. Substantial DGC activity of ZMO0919 and ZMO1365 has been observed as predicted (Li, Xia et al 2023).…”

“…Local (close association of the diguanylate cyclase with the cellulose biosynthesis complex (Kader, Simm et al 2006, Massie, Reynolds et al 2012, Abidi, Torres-Sanchez et al 2022) and global signaling events might play a role. In the case of ZMO1055, although the phosphodiesterase activity of the PAS-GGDEF-EAL domain protein is dominant in the Z. mobilis flocculation and S. typhimurium rdar morphotype and motility assay, residual DGC activity has been observed in different assays (Figure 2g and 4g) as it has been demonstrated that the ZMO1055 diguanylate cyclase activity activates cellulose biosynthesis (Li, Xia et al 2023). For example, the temporal appearance of a 'brown' morphotype might indicate an imbalance towards the diguanylate cyclase versus the phosphodiesterase activity by the activation of a novel matrix or cell membrane component alternatively indicate enhanced membrane permeability or other novel mechanisms that affect dye binding.…”

“…In order to associate their catalytic capability with sequence conservation, the conserved sequence motifs were analyzed after comparison of the domain alignment with functional DGCs/PDEs. Four proteins, ZMO0919, ZMO1365, ZMO0401 and ZMO1055, contain a GGDEF domain (Figure S1a and 1a; (Li, Xia et al 2023)). The GGDEF domains of ZMO0919 and ZMO1365, possess all signature amino acid motifs except residue L29 which was substituted by I and M for ZMO0919 and ZMO1365, respectively.…”

“…ZMO1055 A526V, subsequently designated ZMO1055ZM401, displayed a lower apparent phosphodiesterase activity and highly reduced capacity to dissolute the self-flocculation phenotype caused by elevated cellulose production in strain Z. mobilis ZMO401 (Jeon, Xun et al 2012, Cao, Yang et al 2022. Indeed, deletion and overexpression analysis in combination with mutant construction of all cyclic di-GMP turnover proteins in Z. mobilis ZM4 and ZM401 showed that ZMO1055 is a key diguanylate cyclase/phosphodiesterase regulating cellulose triggered self-flocculation (Li, Xia et al 2023). Recognizing the impact of the A526V substitution located in an amino acid stretch of the EAL domain not previously identified as functionally relevant, we were asking whether the impact of the A526V substitution is observed in other model systems and whether the amino acid position equivalent to A526 in ZMO1055 is conserved among the EAL domains and whether functional conservation of the A526V substitution occurs.…”