Spatial organization of pathway enzymes via self-assembly to improve 2'-fucosyllactose biosynthesis in engineered Escherichia coli.

As one of the most abundant components in human milk oligosaccharides, 2'-fucosyllactose (2'-FL) possesses versatile beneficial health effects. Although most studies focused on overexpressing or fine-tuning the expression of pathway enzymes and achieved a striking increase of 2'-FL production, directly facilitating the metabolic flux toward the key intermediate GDP-l-fucose seems to be ignored. Here, multienzyme complexes consisting of sequential pathway enzymes were constructed by using specific peptide interaction motifs in recombinant Escherichia coli to achieve a higher titer of 2'-FL. Specifically, we first fine-tuned the expression level of pathway enzymes and balanced the metabolic flux toward 2'-FL synthesis. Then, two key enzymes (GDP-mannose 4,6-dehydratase and GDP- l-fucose synthase) were self-assembled into enzyme complexes in vivo via a short peptide interaction pair RIAD-RIDD (RI anchoring disruptor-RI dimer D/D domains), resulting in noticeable improvement of 2'-FL production. Next, to further strengthen the metabolic flux toward 2'-FL, three pathway enzymes were further aggregated into multienzyme assemblies by using another orthogonal protein interaction motif (Spycatcher-SpyTag or PDZ-PDZlig). Intracellular multienzyme assemblies remarkably enlarged the flux toward 2'-FL biosynthesis and showed a 2.1-fold increase of 2'-FL production compared with a strain expressing free-floating and unassembled enzymes. The optimally engineered strain EZJ23 accumulated 4.8 g/L 2'-FL in shake flask fermentation and was capable of producing 25.1 g/L 2'-FL by fed-batch cultivation. This work provides novel approaches for further improvement and large-scale production of 2'-FL and demonstrates the effectiveness of spatial assembly of pathway enzymes to improve the production of valuable products in the engineered host strain.

Interaction between the Anchoring Domain of A-Kinase Anchoring Proteins and the Dimerization and Docking Domain of Protein Kinase A: A Potent Tool for Synthetic Biology.

Nature is enriched with specific interactions between receptor proteins and their cognate ligands. These interacting pairs can be exploited and applied for the construction of well-ordered multicomponent assemblies with multivalency and multifunctionality. One of the research hotspots of this area is the formation of multienzyme complexes with stable and tunable architectures, which may bear the potential to facilitate cascade biocatalysis and/or strengthen metabolic fluxes. Here we focus on a special interacting pair, the anchoring domain (AD) derived from A-kinase anchoring protein and its interacting dimerization and docking domain (DDD) derived from cyclic AMP-dependent protein kinase, which has potential to be an effective and powerful synthetic biology tool for the construction of multienzyme assemblies. We review the origin and interaction mechanism of AD-DDD, followed by the application of this so-called dock-and-lock pair to form various bioconjugates with multivalency and multispecificity. Then several recent studies related to the construction of multienzyme complexes using AD-DDD, and more specifically, the RIAD-RIDD interacting pair, are presented. Finally, we also discuss the great biotechnology potential and perspectives of AD-DDD as a potent synthetic biology tool for post-translational modifications.