Kinetic compartmentalization by unnatural reaction for itaconate production

Abstract

Physical compartmentalization of metabolism using membranous organelles in eukaryotes is helpful for chemical biosynthesis to ensure the availability of substrates from competitive metabolic reactions. Bacterial hosts lack such a membranous system, which is one of the major limitations for efficient metabolic engineering. Here, we employ kinetic compartmentalization with the introduction of an unnatural enzymatic reaction by an engineered enzyme as an alternative strategy to enable substrate availability from competitive reactions through kinetic isolation of metabolic pathways. As a proof of concept, we kinetically isolate the itaconate synthetic pathway from the tricarboxylic acid cycle in Escherichia coli, which is natively separated by mitochondrial membranes in Aspergillus terreus. Specifically, 2-methylcitrate dehydratase is engineered to alternatively catalyze citrate and kinetically secure cis-aconitate for efficient production using a high-throughput screening system. Itaconate production can be significantly improved with kinetic compartmentalization and its strategy has the potential to be widely applicable.

Fig. 1. Schematic diagram of overall strategies for kinetic compartmentalization using the non-natural enzyme reaction.

2-Methylcitrate dehydratase (PrpD) was evolutionarily engineered to alter the substrate preference and catalytic efficiency towards citrate. The desired PrpD mutant has higher activity towards citrate to secure cis-aconitate, resulting in increased itaconate production. The non-natural enzyme exhibits a kinetically compartmentalized effect without actual spatial separation. Citrate (blue); isocitrate (orange); cis-aconitate (green); itaconate (red).

Fig. 2. Design and validation of itaconate-responsive screening system.

a Schematic diagram of itaconate-responsive screening system based on the tetracycline resistance gene. b Specific growth rates of WS strains were plotted in accordance with itaconate concentration under varied tetracycline concentrations. Data were presented as mean values and error bars indicate the standard deviations from three biological replicates. Source data are provided as a Source Data file.

Fig. 3. Fermentation profiles of itaconate production.

a WAICP, b WAICP^VTL, and c WAICP^VL strains. The left y-axis and y-offset represent the cell biomass (g DCW/L) and acetate (g/L), respectively. The right y-axis indicates the production of itaconate and citrate (g/L). The x-axis denotes time (h). Symbols: circles, cell biomass; up-triangles, acetate; squares, itaconate; down-triangles, citrate. Data were presented as mean values and error bars indicate the standard deviations from three biological replicates. Source data are provided as a Source Data file.

Fig. 4. Further optimization of itaconate production.

a Comparison of the itaconate titer of strains with varied aceA expression for 48 h. White dots indicate actual data. b Fermentation profile of WAICPG5 strain. The left y-axis and y-offset represent the cell biomass (g DCW/L) and acetate (g/L), respectively. The right y-axis indicates the production of citrate and itaconate (g/L). The x-axis denotes time (h). Data were presented as mean values and error bars indicate the standard deviations from three biological replicates. Source data are provided as a Source Data file.

Fig. 5. Fed-batch fermentation profile of WAICPG5.

The left y-axis and y-offset represent the cell biomass (g DCW/L) and consumed acetate (g/L), respectively. The right y-axis indicates the production of itaconate (g/L). The x-axis denotes time (h). The representative fermentation profiles was plotted. Source data are provided as a Source Data file.

Fig. 6. Application of kinetic compartmentalization approach for itaconate production from glucose.

Comparison of the a cell biomass and b itaconate titer with glucose-utilizing strains after 60 h. The right y-axis indicates the cell biomass and production of itaconate (g/L), respectively. The x-axis denotes strains. Data were presented as mean values and error bars indicate the standard deviations from three biological replicates. White dots indicate actual data. Source data are provided as a Source Data file.