Co-Localizing Key Pathway Enzymes By Protein Scaffold To Enhance Geraniol Production In Escherichia Coli

Geraniol exhibits extensive applications in various fields,such as food,medicine,cosmetics,energy,and others,and possesses enormous market potential.However,the utilization of geraniol extracted from plants is restricted by the issue of raw material supply,while chemical synthesis faces challenges such as high energy consumption and environmental pollution.Microbial biosynthesis of geraniol has emerged as a promising and eco-friendly alternative method with significant development potential.Nevertheless,the large gap between microbial synthesis of geraniol and commercial application remains a challenge,mainly due to the toxicity of metabolic intermediates in the geraniol synthesis pathway,which impedes the further increase of geraniol yield.The protein scaffold system represents the latest strategy for regulating metabolic flux and influencing metabolic intermediates.By utilizing the protein scaffold co-localization approach to key enzymes in the geraniol synthesis pathway,several advantages can be attained,including the enhancement of substrate,enzyme,and reaction intermediate concentrations,the provision of a more suitable microenvironment for enzymes,the concentration of metabolic pathway enzymes to reduce reaction competition loss,the reduction of side reactions to yield a purer product,and the mitigation of the toxic effect of reaction intermediates on host cells to enhance the conversion rate of metabolites.This study focused on investigating the metabolic pathway of geraniol biosynthesis in Escherichia coli and introducing key pathway enzymes of geraniol synthesis co-localized by protein scaffolds.The co-localization of metabolic enzymes was aimed at bringing them closer together to accelerate cascade reactions and ultimately decrease the toxic effects of metabolic intermediates on host cells.The main research results included:（1）Plasmids encoding the three pathway enzymes were linked to distinct ligand proteins,and the resulting recombinant strains were expressed in E.coli to screen for an optimal ligand protein partition protocol.The effect of different ligand proteins on pathway enzymes was evaluated by measuring geraniol production.Ligand proteins were fused directly with pathway enzymes via a SEG linker,and the optimal ligand protein partition scheme was determined to be IDI-SH3lig,GES-Ccdockerin,and GPPS-Ctdockerin.Simultaneous expression of these three ligand proteins in the recombinant strain led to a geraniol yield of 597.2 mg/L,which was14.6%higher than the yield obtained with unligated ligand protein（521 mg/L）.（2）Plasmids expressing scaffold proteins were constructed,and the proteins were purified after culture expression to verify the binding effect of ligand proteins and scaffold proteins.A linker was used to link the binding domain in the scaffold protein,and a histidine tag was attached at the C-terminus of the protein for nickel column purification.The interaction between ligand proteins and scaffold proteins was verified by in vitro immunoprecipitation pull-down experiments.Our results showed that the scaffold SH3-Ctcohesin-Cccohesin（STC）and ligand proteins IDI-SH3lig,GES-Ccdocherin,and GPPS-Ctdocherin)underwent distinct protein-protein interactions outside the cell and could form related complexes.（3）The scaffold system was expressed in recombinant strain WS,while recombinant strain ns was used as a control to contrast the effects of colocalization of pathway enzymes by monitoring the cultivation process.Our results showed that recombinant strain WS produced more geraniol on the first day of cultivation,but subsequent geraniol production decreased and the final yield was lower than that of recombinant strain NS.This was attributed to the competition between scaffold expression and geraniol production for substrate,and the absence of glycerol led to a decrease in the final yield.This conclusion was also verified by supplementing the substrates during the cultivation process.The geraniol yield of the final recombinant strain WS reached 677.1 mg/L,which was slightly higher than that of recombinant strain NS（641 mg/L）.（4）Optimization of the incubation conditions（incubation temperature,initial glycerol content,starting concentration of induction,concentration of inducer）for geraniol production by recombinant strain WS was performed.The optimal culture conditions for scaffold constructs were found to be incubated at 25°C in shake flasks with an initial glycerol concentration of 30 g/L and an inducer at an initial cell concentration OD₆₀₀ of 1.5supplemented with 0.5 mm.Incubation under optimal conditions for 24 h yielded 777.8 mg/L geraniol,a 2.2-fold increase over 341.2 mg/L before optimization.To further improve the geraniol yield,simultaneous batch fed fermentation was performed on recombinant strains WS and NS.The geraniol yield reached 2102.5 mg/L at 112 h under the optimal condition for recombinant strain WS,while it was 1460.6 mg/L under the optimal condition for recombinant strain NS.Our results showed a 42.9%increase in the yield of geraniol by the scaffold co-localization system over the free system.