Combinatorial Optimization Strategies for 3-Fucosyllactose Hyperproduction in Escherichia coli.

3-Fucosyllactose (3-FL), an important fucosylated human milk oligosaccharide in breast milk, offers numerous health benefits to infants. Previously, we metabolically engineered Escherichia coli BL21(DE3) for the in vivo biosynthesis of 3-FL. In this study, we initially optimized culture conditions to double 3-FL production. Competing pathway genes involved in in vivo guanosine 5'-diphosphate-fucose biosynthesis were subsequently inactivated to redirect fluxes toward 3-FL biosynthesis. Next, three promising transporters were evaluated using plasmid-based or chromosomally integrated expression to maximize extracellular 3-FL production. Additionally, through analysis of α1,3-fucosyltransferase (FutM2) structure, we identified Q126 residues as a highly mutable residue in the active site. After site-saturation mutation, the best-performing mutant, FutM2-Q126A, was obtained. Structural analysis and molecular dynamics simulations revealed that small residue replacement positively influenced helical structure generation. Finally, the best strain BD3-A produced 6.91 and 52.1 g/L of 3-FL in a shake-flask and fed-batch cultivations, respectively, highlighting its potential for large-scale industrial applications.

De Novo Biosynthesis of Difucosyllactose by Artificial Pathway Construction and α1,3/4-Fucosyltransferase Rational Design in Escherichia coli.

Difucosyllactose (DFL) is a significant and plentiful oligosaccharide found in human breast milk. In this study, an artificial metabolic pathway of DFL was designed, focusing on the de novo biosynthesis of GDP-fucose from only glycerol. This was achieved by engineering Escherichia coli to endogenously overexpress genes manB, manC, gmd, and wcaG and heterologously overexpress a pair of fucosyltransferases to produce DFL from lactose. The introduction of α-1,2-fucosyltransferase from Helicobacter pylori (FucT2) along with α-1,3/4-fucosyltransferase (HP3/4FT) addressed rate-limiting challenges in enzymatic catalysis and allowed for highly efficient conversion of lactose into DFL. Based on these results, molecular modification of HP3/4FT was performed based on computer-assisted screening and structure-based rational design. The best-performing mutant, MH5, containing a combination of five mutated sites (F49K/Y131D/Y197N/E338D/R369A) of HP3/4FT was obtained. The best strain BLC09-58 harboring MH5 yielded 45.81 g/L of extracellular DFL in 5-L fed-batch cultures, which was the highest titer reported to date.

Engineering Escherichia coli MG1655 for Highly Efficient Biosynthesis of 2'-Fucosyllactose by De Novo GDP-Fucose Pathway.

2'-Fucosyllactose (2'-FL), the most typical human milk oligosaccharide, is used as an additive in premium infant formula. Herein, we constructed two highly effective 2'-FL synthesis producers via a de novo GDP-fucose pathway from engineered Escherichia coli MG1655. First, lacZ and wcaJ, two competitive pathway genes, were disrupted to block the invalid consumption of lactose and GDP-fucose, respectively. Next, the lacY gene was strengthened by switching its native promoter to PJ23119. To enhance the supply of endogenous GDP-fucose, the promoters of gene clusters manC-manB and gmd-fcl were strengthened individually or in combination. Subsequently, chromosomal integration of a constitutive PJ23119 promoter-based BKHT expression cassette (PJ23119-BKHT) was performed in the arsB and recA loci. The most productive plasmid-based and plasmid-free strains produced 76.9 and 50.1 g/L 2'-FL by fed-batch cultivation, respectively. Neither of them generated difucosyl lactose nor 3-fucosyllactose as byproducts.

Elimination of Byproduct Generation and Enhancement of 2'-Fucosyllactose Synthesis by Expressing a Novel α1,2-Fucosyltransferase in Engineered Escherichia coli.

2'-Fucosyllactose (2'-FL) is a kind of fucosylated human milk oligosaccharide (HMO), representing the most abundant oligosaccharide in breast milk. We conducted systematic studies on three canonical α1,2-fucosyltransferases (WbgL, FucT2, and WcfB) to quantify the byproducts in a lacZ- and wcaJ-deleted Escherichia coli BL21(DE3) basic host strain. Further, we screened a highly active α1,2-fucosyltransferase from Helicobacter sp. 11S02629-2 (BKHT), which exhibits high in vivo 2'-FL productivity without the formation of byproducts difucosyl lactose (DFL) and 3-FL. The maximum 2'-FL titer and yield reached 11.13 g/L and 0.98 mol/mol of lactose, respectively, in shake-flask cultivation, both approaching the theoretical maximum value. In a 5 L fed-batch cultivation, the maximum 2'-FL titer reached 94.7 g/L extracellularly with a yield of 0.98 mol of 2'-FL/mol of lactose and productivity of 1.14 g L-1 h-1. Our reported 2'-FL yield is the highest from lactose reported to date.

De novo biosynthesis of 2'-fucosyllactose in a metabolically engineered Escherichia coli using a novel ɑ1,2-fucosyltransferase from Azospirillum lipoferum.

Human milk oligosaccharides are complex, indigestible oligosaccharides that provide ideal nutrition for infant development. Here, 2'-fucosyllactose was efficiently produced in Escherichia coli by using a biosynthetic pathway. For this, both lacZ and wcaJ (encoding ß-galactosidase and UDP-glucose lipid carrier transferase, respectively) were deleted to enhance the 2'-fucosyllactose biosynthesis. To further enhance 2'-fucosyllactose production, SAMT from Azospirillum lipoferum was inserted into the chromosome of the engineered strain, and the native promoter was replaced with a strong constitutive promoter (PJ23119). The titer of 2'-fucosyllactose was increased to 8.03 g/L by introducing the regulators rcsA and rcsB into the recombinant strains. Compared to wbgL-based strains, only 2'-fucosyllactose was produced in SAMT-based strains without other by-products. Finally, the highest titer of 2'-fucosyllactose reached 112.56 g/L in a 5 L bioreactor by fed-batch cultivation, with a productivity of 1.10 g/L/h and a yield of 0.98 mol/mol lactose, indicating a strong potential in industrial production.

Microbial Synthesis of l-Fucose with High Productivity by a Metabolically Engineered Escherichia coli.

l-Fucose is a natural deoxy hexose found in a variety of organisms. It possesses many physiological effects and has potential applications in pharmaceutical, cosmetic, and food industries. Microbial synthesis via metabolic engineering attracts increasing attention for efficient production of important chemicals. Previously, we reported the construction of a metabolically engineered Escherichia coli strain with high 2'-fucosyllactose productivity. Herein, we further introduced Bifidobacterium bifidum α-l-fucosidase via both plasmid expression and genomic integration and blocked the l-fucose assimilation pathway by deleting fucI, fucK, and rhaA. The highest l-fucose titers reached 6.31 and 51.05 g/L in shake-flask and fed-batch cultivation, respectively. l-Fucose synthesis was little affected by lactose added, and there was almost no 2'-fucosyllactose residue throughout the cultivation processes. The l-fucose productivity reached 0.76 g/L/h, indicating significant potential for large-scale industrial applications.

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.

High-Level De Novo Biosynthesis of 2'-Fucosyllactose by Metabolically Engineered Escherichia coli.

2'-Fucosyllactose (2'-FL) is the most abundant oligosaccharide in human milk. In this study, a highly efficient biosynthetic route for 2'-FL production was designed via the de novo pathway of GDP-l-fucose using engineered Escherichia coli BL21(DE3). Specifically, plasmid-based strains with previously deleted lacZ and wcaJ were further reconstructed by introducing de novo pathway genes and α1,2-fucosyltransferase-encoding wbgL to realize 2'-FL synthesis. The 2'-FL titer was enhanced to 3.92 g/L by further introducing rcsA and rcsB. Subsequently, the additional wbgL expression cassette was chromosomally integrated into recA locus to strengthen fucosylation reaction and a strong constitutive promoter (PJ23119) was used to replace the original promoters of manC-manB and gmd-wcaG to improve 2'-FL synthesis. The maximal 2'-FL titer reached 9.06 and 79.23 g/L in shake-flask and fed-batch cultivation, respectively. The 2'-FL productivity reached 1.45 g/L/h, showing remarkable production potential in large-scale industrial application.