Mathematical modeling of 1,2-propanediol utilization bacterial microcompartments in vivo activity

TL;DR

A revised in vivo model of WT growth on 1,2-PD in which PduCDE is fully encapsulated, while much of the downstream Pdu activity occurs in the cytosol is proposed, suggesting that MCP-localized enzymes contribute little to in vivo metabolic flux downstream of PduCDE.

Abstract

On exposure to 1,2-propanediol (1,2-PD), Salmonella enterica serovar Typhimurium LT2 produces 1,2-PD utilization (Pdu) microcompartments (MCPs), nanoscale protein-bound shells that encapsulate metabolic enzymes. MCPs serve as a bioengineering platform to study reaction organization and enhance flux through specific pathways. However, a recently published assay of purified wild-type (WT) MCPs reported metabolic activity that differed markedly from that observed in vivo. Using kinetic modeling, we attribute these discrepancies to in vivo cell growth and to the cytosolic presence of MCP-associated enzymes and promiscuous alcohol dehydrogenases, which are not present in the purified MCPs. Assays of purified MCPs in E. coli lysate, together with an LT2 growth assay in which the native Pdu MCP-associated alcohol dehydrogenase, PduQ, was knocked out, support the conclusion that exogenous Pdu cytosolic enzyme activity can narrow the gap between in vitro and in vivo experiments. Our modeling further suggests that MCP-localized enzymes contribute little to in vivo metabolic flux downstream of PduCDE. We therefore propose a revised in vivo model of WT growth on 1,2-PD in which PduCDE is fully encapsulated, while much of the downstream Pdu activity occurs in the cytosol.

Figures (25)

• Figure 1: Proposed reaction model of the Pdu MCPs with complete encapsulation of PduCDE and partial encapsulation of PduP, PduQ, PduL, and PduW. In this model, 1,2-propanediol is converted to propionyl-CoA, propionate, and 1-propanol. Propionate and 1-propanol act as shunts for excess carbon flux and are reconverted to propionyl-CoA. Propionyl-CoA is further metabolized to generate ATP via the 2-methylcitrate cycle, the tricarboxylic acid (TCA) cycle, and the electron transport chain Horswill1999aHorswill2001bHorswill1999aPalacios2003Dolan2018Noster2019Deshpande2022. The findings presented in this study support partial encapsulation of PduP, PduQ, PduL, and PduW.
• Figure 2: Model simulations of A) in vitro and B) in vivo metabolite dynamics for i) 1,2-propanediol, ii) propionaldehyde, iii) propionate, and iv) 1-propanol. Experimental time-series data were compared with three model configurations: in vitro calibrated parameters (short red dashed lines, Ai–iv), in vitro calibrated parameters with in vivo priors (long red dashed lines, Ai–iv), and jointly calibrated in vitro + WT parameters (solid blue lines, Ai–iv and Bi–vi).
• Figure 3: Panel of plots showing MCP and cytosolic enzyme flux contribution to metabolite dynamics. A) Contribution of MCP PduQ, cytosolic PduQ, and cytosolic alcohol dehydrogenase to the total cytosolic 1-propanol. B) Comparison of MCP PduP and cytosolic PduP Propionyl-CoA flux. C) Comparison of MCP PduL and cytosolic PduL Propionyl-Phosphate flux. D) Contribution of MCP PduW, cytosolic PduW, and cytosolic PrpE to the total cytosolic propionate flux.
• Figure 4: Metabolic activity of WT MCPs assayed with 1,2-propanediol in buffer or E. coli lysate, compared to MCPs deficient in the PduP/PduQ catalytic proteins assayed with 1,2-propanediol in E. coli lysate. A) 1,2-propanediol, B) propionaldehyde, C) propionate, and D) 1-propanol concentrations were measured over a 20–24 h period. Propionate and 1-propanol profiles are consistent with the hypothesis that exogenous enzymes are required to elevate metabolite dynamics to in vivo levels.
• Figure 5: Metabolic activity of $\Delta$pduQ. A–B) Growth curves (A) and 1-propanol activity (B) of WT and $\Delta$pduQ in vivo. C) PduCDE–GFP fluorescence in $\Delta$PocR, WT, and $\Delta$pduQ strains. D) Comparison of in silico predictions of $\Delta$pduQ 1,2-propanediol dynamics, generated using in vitro and in vivo calibrated model parameters, with experimental measurements.