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.

A pathway independent multi-modular ordered control system based on thermosensors and CRISPRi improves bioproduction in Bacillus subtilis.

Dynamic regulation is an effective strategy for control of gene expression in microbial cell factories. In some pathway contexts, several metabolic modules must be controlled in a time dependent or ordered manner to maximize production, while the creation of genetic circuits with ordered regulation capacity still remains a great challenge. In this work, we develop a pathway independent and programmable system that enables multi-modular ordered control of metabolism in Bacillus subtilis. First, a series of thermosensors were created and engineered to expand their thresholds. Then we designed single-input-multi-output circuits for ordered control based on the use of thermosensors with different transition points. Meanwhile, a repression circuit was constructed by combining CRISPRi-based NOT gates. As a proof-of-concept, these genetic circuits were applied for multi-modular ordered control of 2'-fucosyllactose (2'-FL) biosynthesis, resulting in a production of 1839.7 mg/l in shake flask, which is 5.16-times that of the parental strain. In a 5-l bioreactor, the 2'-FL titer reached 28.2 g/l with down-regulation of autolysis. Taken together, this work provides programmable and versatile thermosensitive genetic toolkits for dynamic regulation in B. subtilis and a multi-modular ordered control framework that can be used to improve metabolic modules in other chassis cells and for other compounds.

2'-Fucosyllactose production in engineered Escherichia coli with deletion of waaF and wcaJ and overexpression of FucT2.

2'-Fucosyllactose (2'-FL), a major oligosaccharide of human breast milk, and is currently supplemented into infant formula. For the overproduction of 2'-FL via fucosylation of lactose, conventional approaches have focused on the episomal overexpression of de novo or salvage GDP-L-fucose biosynthetic pathway and α-1,2-fucosyltransferase (FucT2) through T7 RNA polymerase expression system in engineered E. coli. However, these approaches have drawbacks of metabolic burden, plasmid instability, and inclusion body formation. In this study, a deletion mutant of waaF coding for ADP-heptose:LPS heptosyltransferase II was employed for 2'-FL production. As the waaF deletion induces accumulation of colanic acid, additional deletion of wcaJ coding for UDP-glucose-1-phosphate transferase in the waaF deletion mutant resulted in enhanced accumulation of GDP-L-fucose. Besides, 2'-FL yields and titers were drastically improved when T7 promoter was replaced with Trc promoter for α-1,2 fucosyltransferase expressions in the waaF and wcaJ deleted strain. As a result, when FucT2 was expressed under Trc promoter in the E. coli JM109(DE3) ΔwaaFΔwcaJ, 14.7 g/L of 2'-FL was produced with a productivity of 0.31 g/L/h in a fed-batch fermentation. We envision that the deletion-based metabolic design and decreased promoter strength for fucosyltransferase expression can resolve the drawbacks of T7 RNA polymerase-based expression design for 2'-FL production in E. coli.

Improved production of 2'-fucosyllactose in engineered Saccharomyces cerevisiae expressing a putative α-1, 2-fucosyltransferase from Bacillus cereus.

BACKGROUND: 2'-fucosyllactose (2'-FL) is one of the most abundant oligosaccharides in human milk. It constitutes an authorized functional additive to improve infant nutrition and health in manufactured infant formulations. As a result, a cost-effective method for mass production of 2'-FL is highly desirable. RESULTS: A microbial cell factory for 2'-FL production was constructed in Saccharomyces cerevisiae by expressing a putative α-1, 2-fucosyltransferase from Bacillus cereus (FutBc) and enhancing the de novo GDP-L-fucose biosynthesis. When enabled lactose uptake, this system produced 2.54 g/L of 2'-FL with a batch flask cultivation using galactose as inducer and carbon source, representing a 1.8-fold increase compared with the commonly used α-1, 2-fucosyltransferase from Helicobacter pylori (FutC). The production of 2'-FL was further increased to 3.45 g/L by fortifying GDP-mannose synthesis. Further deleting gal80 enabled the engineered strain to produce 26.63 g/L of 2'-FL with a yield of 0.85 mol/mol from lactose with sucrose as a carbon source in a fed-batch fermentation. CONCLUSION: FutBc combined with the other reported engineering strategies holds great potential for developing commercial scale processes for economic 2'-FL production using a food-grade microbial cell factory.

Efficient Production of 2'-Fucosyllactose from l-Fucose via Self-Assembling Multienzyme Complexes in Engineered Escherichia coli.

2'-Fucosyllactose (2'-FL) has been widely used as a nutritional additive in infant formula due to its multifarious nutraceutical and pharmaceutical functions in neonate health. As such, it is essential to develop an efficient and extensive microbial fermentation platform to cater to the needs of the 2'-FL market. In this study, a spatial synthetic biology strategy was employed to promote 2'-FL biosynthesis in recombinant Escherichia coli. First, the salvage pathway for 2'-FL production from l-fucose and lactose was constructed by introducing a bifunctional enzyme l-fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) derived from Bacteroides fragilis and an α-1,2-fucosyltransferase (FutC) derived from Helicobacter pylori into engineered E. coli BL21(DE3). Next, the endogenous genes involved in the degradation and shunting of the substrate and key intermediate were inactivated to improve the availability of precursors for 2'-FL biosynthesis. Moreover, to further improve the yield and titer of 2'-FL, a short peptide pair (RIAD-RIDD) was used to form self-assembling multienzyme complexes in vivo. The spatial localization of peptides and stoichiometry of enzyme assemblies were subsequently optimized to further improve 2'-FL production. Finally, cofactor regeneration was also considered to alleviate the potential cofactor deficiency and redox flux imbalance in the biocatalysis process. Fed-batch fermentation of the final WLS20 strain accumulated 30.5 g/L extracellular 2'-FL with the yield and productivity of 0.661 mol/mol fucose and 0.48 g/L/h, respectively. This research has demonstrated that the application of spatial synthetic biology and metabolic engineering strategies can dramatically enlarge the titer and yield of 2'-FL biosynthesis in engineered E. coli.

A Regulator Based "Semi-Targeted" Approach to Activate Silent Biosynthetic Gene Clusters.

By culturing microorganisms under standard laboratory conditions, most biosynthetic gene clusters (BGCs) are not expressed, and thus, the products are not produced. To explore this biosynthetic potential, we developed a novel "semi-targeted" approach focusing on activating "silent" BGCs by concurrently introducing a group of regulator genes into streptomycetes of the Tübingen strain collection. We constructed integrative plasmids containing two classes of regulatory genes under the control of the constitutive promoter ermE*p (cluster situated regulators (CSR) and Streptomyces antibiotic regulatory proteins (SARPs)). These plasmids were introduced into Streptomyces sp. TÜ17, Streptomyces sp. TÜ10 and Streptomyces sp. TÜ102. Introduction of the CSRs-plasmid into strain S. sp. TÜ17 activated the production of mayamycin A. By using the individual regulator genes, we proved that Aur1P, was responsible for the activation. In strain S. sp. TÜ102, the introduction of the SARP-plasmid triggered the production of a chartreusin-like compound. Insertion of the CSRs-plasmid into strain S. sp. TÜ10 resulted in activating the warkmycin-BGC. In both recombinants, activation of the BGCs was only possible through the simultaneous expression of aur1PR3 and griR in S. sp. TÜ102 and aur1P and pntR in of S. sp. TÜ10.

Fructooligosaccharides production by immobilized Pichia pastoris cells expressing Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase.

Fructooligosaccharides (FOSs)-fructose-based oligosaccharides-are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.

Biotechnological Production of 2'-Fucosyllactose: A Prevalent Fucosylated Human Milk Oligosaccharide.

Human milk oligosaccharide (HMO) is a key component of human milk carbohydrates and is closely related to the nutrition and health benefits of breastfeeding in infants. 2'-Fucosyllactose (2'-FL) is the most abundant fucosylated HMO, which has remarkable value in nutrition and medicine, such as suppressing pathogen infection, regulating intestinal flora, and boosting immunity. However, 2'-FL production via the method of extraction or chemical synthesis cannot meet its large demand, and as a result, environmentally friendly and efficient biotechnological approaches, including in vitro enzymatic synthesis and microbial cell factory production, have been developed and applied to its commercialized production. This review introduces, summarizes, and discusses the recent advances in the biotechnological production of 2'-FL. Furthermore, future research directions for the biotechnological production of 2'-FL as well as the strategies to further improve its concentration are highlighted and discussed.

Pathway Optimization of 2'-Fucosyllactose Production in Engineered Escherichia coli.

2'-Fucosyllactose (2'-FL), one of the most valuable oligosaccharides in human milk, is used as an emerging food ingredient in the nutraceutical and food industries due to its numerous health benefits. Herein, the de novo and salvage pathways for GDP-fucose synthesis were engineered and optimized in Escherichia coli BL21 (DE3) to improve the production of 2'-FL. The de novo pathway genes encoding phosphomannomutase (ManB), mannose-1-phosphate guanyltransferase (ManC), GDP-d-mannose-4,6-dehydratase (Gmd), and GDP-l-fucose synthase (WcaG) combined with the gene from the salvage pathway encoding fucose kinase/fucose-1-phosphate guanylyltransferase (Fkp) were reconstructed in two vectors to evaluate the GDP-fucose biosynthesis. Then, the fucT2 gene, encoding α1,2-fucosyltransferase, was introduced into the GDP-fucose-overproducing strains to realize 2'-FL biosynthesis. Furthermore, the genes in bypass pathways, including lacZ, fucI, fucK, and wcaJ, were inactivated to improve 2'-FL production. In addition, the two GDP-fucose synthesis pathways, along with fucT2, were transcriptionally fine-tuned to efficiently increase 2'-FL production. The final metabolically engineered E. coli produced 2.62 and 14.1 g/L in shake-flask and fed-batch cultivations, respectively.

High-Titer De Novo Biosynthesis of the Predominant Human Milk Oligosaccharide 2'-Fucosyllactose from Sucrose in Escherichia coli.

Human milk oligosaccharides (HMOs) are unique components of human breast milk. Their large-scale production by fermentation allows infant formulas to be fortified with HMOs, but current fermentation processes require lactose as a starting material, increasing the costs, bioburden, and environmental impact of manufacturing. Here we report the development of an Escherichia coli strain that produces 2'-fucosyllactose (2'-FL), the most abundant HMO, de novo using sucrose as the sole carbon source. Strain engineering required the expression of a novel glucose-accepting galactosyltransferase, overexpression of the de novo UDP-d-galactose and GDP-l-fucose pathways, the engineering of an intracellular pool of free glucose, and overexpression of a suitable α(1,2)-fucosyltransferase. The export of 2'-FL was facilitated using a sugar efflux transporter. The final production strain achieved 2'-FL yields exceeding 60 g/L after fermentation for 84 h. This efficient strategy facilitates the lactose-independent production of HMOs by fermentation, which will improve product quality and reduce the costs of manufacturing.

Efficient sequential synthesis of lacto-N-triose II and lacto-N-neotetraose by a novel ß-N-acetylhexosaminidase from Tyzzerella nexilis.

ß-N-acetylhexosaminidases have attracted much attention in recent years due to their potential application in oligosaccharide production, in particular lacto-N-triose II (LNT2) and lacto-N-neotetraose (LNnT) synthesis, which can be further used as backbone precursors for human milk oligosaccharides. A novel ß-N-acetylhexosaminidase gene from Tyzzerella nexilis (TnHex189) was heterologously expressed in Bacillus subtilis. The highest ß-N-acetylhexosaminidase activity of 14.5 U mL-1 was obtained in a 5-L fermentor by fed-batch fermentation for 27 h. TnHex189 was optimally active at pH 5.0 and 45 °C. It efficiently synthesized LNT2 with a conversion ratio of 57.2% (4.7 g L-1). The synthesized LNT2 was further converted to LNnT by a reported ß-galactosidase (BgaD-D) in 8 h, with a conversion ratio of 17.3% (6.1 g L-1). These unique synthesis activities may make this enzyme a good candidate for the food industry.

Biochemical characterization of a novel α-L-fucosidase from Pedobacter sp. and its application in synthesis of 3'-fucosyllactose and 2'-fucosyllactose.

Fucosyllactoses have gained much attention owing to their multiple functions, including prebiotic, immune, gut, and cognition benefits. In this study, human milk oligosaccharide (HMO) 2'-fucosyllactose (α-L-Fuc-(1,2)-D-Galß-1,4-Glu, 2'FL) and its isomer 3'-fucosyllactose (α-L-Fuc-(1,3)-D-Galß-1,4-Glu, 3'FL) with potential prebiotic effect were synthesized efficiently by a novel recombinant α-L-fucosidase. An α-L-fucosidase gene (PbFuc) from Pedobacter sp. CAU209 was successfully cloned and expressed in Escherichia coli (E. coli). The deduced amino acid sequence shared the highest identity of 36.8% with the amino sequences of other reported α-L-fucosidases. The purified α-L-fucosidase (PbFuc) had a molecular mass of 50 kDa. The enzyme exhibited specific activity (26.3 U/mg) towards 4-nitrophenyl-α-L-fucopyranoside (pNP-FUC), 3'FL (8.9 U/mg), and 2'FL (3.4 U/mg). It showed the highest activity at pH 5.0 and 35 °C, respectively. PbFuc catalyzed the synthesis of 3'FL and 2'FL through a transglycosylation reaction using pNP-FUC as donor and lactose as acceptor, and total conversion ratio was up to 85% at the optimized reaction conditions. The synthesized mixture of 2'FL and 3'FL promoted the growth of Lactobacillus delbrueckii subsp. bulgaricus NRRL B-548, L. casei subsp. casei NRRL B-1922, L. casei subsp. casei AS 1.2435, and Bifidobacterium longum NRRL B-41409. However, the growths of E. coli ATCC 11775, S. enterica AS 1.1552, L. monocytogenes CICC 21635, and S. aureus AS 1.1861 were not stimulated by the mixture of 2'FL and 3'FL. Overall, our findings suggest that PbFuc possesses a great potential for the specific synthesis of fucosylated compounds.Key Points• A novel α-L-fucosidase (PbFuc) from Pedobacter sp. was cloned and expressed.• PbFuc showed the highest hydrolysis activity at pH 5.0 and 35 °C, respectively.• It was used for synthesis of 3'-fucosyllactose (3'FL) and 2'-fucosyllactose (2'FL).• The mixture of 3'FL and 2'FL promoted the growth of some Lactobacillus sp. and Bifidobacteria sp.

Chemoenzymatic synthesis of 3-deoxy-3-fluoro-l-fucose and its enzymatic incorporation into glycoconjugates.

The first synthesis of 3-deoxy-3-fluoro-l-fucose is presented, which employs a d- to l-sugar translation strategy, and involves an enzymatic oxidation of 3-deoxy-3-fluoro-l-fucitol. Enzymatic activation (FKP) and glycosylation using an α-1,2 and an α-1,3 fucosyltransferase to obtain two fluorinated trisaccharides demonstrates its potential as a novel versatile chemical probe in glycobiology.

An endoxylanase rapidly hydrolyzes xylan into major product xylobiose via transglycosylation of xylose to xylotriose or xylotetraose.

Here, we proposed an effective strategy to enhance a novel endoxylanase (Taxy11) activity and elucidated an efficient catalysis mechanism to produce xylooligosaccharides (XOSs). Codon optimization and recruitment of natural propeptide in Pichia pastoris resulted in achievement of Taxy11 activity to 1405.65 ±â€¯51.24 U/mL. Analysis of action mode reveals that Taxy11 requires at least three xylose (xylotriose) residues for hydrolysis to yield xylobiose. Results of site-directed mutagenesis indicate that residues Glu119, Glu210, and Asp53 of Taxy11 are key catalytic sites, while Asp203 plays an auxiliary role. The novel mechanism whereby Taxy11 catalyzes conversion of xylan or XOSs into major product xylobiose involves transglycosylation of xylose to xylotriose or xylotetraose as substrate, to form xylotetraose or xylopentaose intermediate, respectively. Taxy11 displayed highly hydrolytic activity toward corncob xylan, producing 50.44 % of xylobiose within 0.5 h. This work provides a cost-effective and sustainable way to produce value-added biomolecules XOSs (xylobiose-enriched) from agricultural waste.

Biosynthesis of the anti-diabetic metabolite montbretin A: glucosylation of the central intermediate mini-MbA.

Type 2 diabetes (T2D) affects over 320 million people worldwide. Healthy lifestyles, improved drugs and effective nutraceuticals are different components of a response against the growing T2D epidemic. The specialized metabolite montbretin A (MbA) is being developed for treatment of T2D and obesity due to its unique pharmacological activity as a highly effective and selective inhibitor of the human pancreatic α-amylase. MbA is an acylated flavonol glycoside found in small amounts in montbretia (Crocosmia × crocosmiiflora) corms. MbA cannot be obtained in sufficient quantities for drug development from its natural source or by chemical synthesis. To overcome these limitations through metabolic engineering, we are investigating the genes and enzymes of MbA biosynthesis. We previously reported the first three steps of MbA biosynthesis from myricetin to myricetin 3-O-(6'-O-caffeoyl)-glucosyl rhamnoside (mini-MbA). Here, we describe the sequence of reactions from mini-MbA to MbA, and the discovery and characterization of the gene and enzyme responsible for the glucosylation of mini-MbA. The UDP-dependent glucosyltransferase CcUGT3 (UGT703E1) catalyzes the 1,2-glucosylation of mini-MbA to produce myricetin 3-O-(glucosyl-6'-O-caffeoyl)-glucosyl rhamnoside. Co-expression of CcUGT3 with genes for myricetin and mini-MbA biosynthesis in Nicotiana benthamiana validated its biological function and expanded the set of genes available for metabolic engineering of MbA.

Enhanced production of 2'-fucosyllactose from fucose by elimination of rhamnose isomerase and arabinose isomerase in engineered Escherichia coli.

2'-Fucosyllactose (2-FL), one of the most abundant oligosaccharides in human milk, has been spotlighted for its neutraceutical and pharmaceutical potentials. Microbial production of 2-FL is promising since it is efficient as compared to other production methods. In 2-FL microbial production via the salvage pathway for biosynthesis of guanosine 5'-diphosphate (GDP)-l-fucose from fucose, the conversion yield from fucose is important because of the high price of fucose. In this study, deletion of the genes (araA and rhaA) coding for arabinose isomerase (AraA) and rhamnose isomerase (RhaA) was attempted in engineered Escherichia coli for improving 2-FL production by using fucose, lactose, and glycerol. The engineered E. coli constructed previously is able to express fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori and deficient in ß-galactosidase (LacZ), fucose isomerase (FucI), and fuculose kinase (FucK). The additional double-deletion of the araA and rhaA genes in the engineered E. coli enhanced the product yield of 2-FL to 0.52 mole 2-FL/mole fucose, and hence the concentration of 2-FL reached to 47.0 g/L, which are 44% and two-fold higher than those (23.1 g/L and 0.36 mole 2-FL/mole fucose) of the control strain in fed-batch fermentation. Elimination of sugar isomerases exhibiting promiscuous activities with fucose might be critical in the microbial production of 2-FL through the salvage pathway of GDP-l-fucose.

Search for bacterial α1,2-fucosyltransferases for whole-cell biosynthesis of 2'-fucosyllactose in recombinant Escherichia coli.

2'-Fucosyllactose (2'-FL) is the most abundant human milk oligosaccharide and is important for infant nutrition and health. Because 2'-FL has potential as a functional ingredient in advanced infant formula and as a prebiotic in various foods, a cost-effective method for 2'-FL production is desirable. α1,2-Fucosyltransferase (α1,2-FT) is one of the key enzymes enabling the microbial biosynthesis of this complex sugar. However, the α1,2-FTs reported so far for the whole-cell biosynthesis of 2'-FL originate from pathogens, posing a potential hurdle for approval as a food production method depending on countries. In this study, 10 α1,2-FT genes from bacteria of biosafety level one were identified, and the main features of the deduced amino acid sequences were characterized. Four codon-optimized α1,2-FT genes were synthesized and introduced into Escherichia coli ΔL M15 strain containing the plasmid pBCGW encoding guanosine 5'-diphosphate-l-fucose biosynthetic enzymes. Among the four genes, 2'-FL was produced only by the α1,2-FT from Thermosynechococcus elongatus (Te2FT). Bifidobacterium thermacidophilum α1,2-FT (Bt2FT) showed high expression but was not active in E. coli ΔL M15. The other two α1,2-FTs were not expressed to a detectable level. During batch flask fermentation of Te2FT-expressing E. coli ΔL M15 cells, 0.49 g/L 2'-FL was obtained after 72 h of induction. This is comparable to the values previously reported for α1,2-FTs from Helicobacter pylori and Bacteroides fragilis.

Flavonol Biosynthesis Genes and Their Use in Engineering the Plant Antidiabetic Metabolite Montbretin A.

The plant metabolite montbretin A (MbA) and its precursor mini-MbA are potential new drugs for treating type 2 diabetes. These complex acylated flavonol glycosides only occur in small amounts in the corms of the ornamental plant montbretia (Crocosmia × crocosmiiflora). Our goal is to metabolically engineer Nicotiana benthamiana using montbretia genes to achieve increased production of mini-MbA and MbA. Two montbretia UDP-dependent glycosyltransferases (UGTs), CcUGT1 and CcUGT2, catalyze the formation of the first two pathway-specific intermediates in MbA biosynthesis, myricetin 3-O-rhamnoside and myricetin 3-O-glucosyl rhamnoside. In previous work, expression of these UGTs in N. benthamiana resulted in small amounts of kaempferol glycosides but not myricetin glycosides, suggesting that myricetin was limiting. Here, we investigated montbretia genes and enzymes of flavonol biosynthesis to enhance myricetin formation in N. benthamiana We characterized two flavanone hydroxylases, a flavonol synthase, a flavonoid 3'-hydroxylase (F3'H), and a flavonoid 3'5'-hydroxylase (F3'5'H). Montbretia flavonol synthase converted dihydromyricetin into myricetin. Unexpectedly, montbretia F3'5'H shared higher sequence relatedness with F3'Hs in the CYP75B subfamily of cytochromes P450 than with those with known F3'5'H activity. Transient expression of combinations of montbretia flavonol biosynthesis genes and a montbretia MYB transcription factor in N. benthamiana resulted in availability of myricetin for MbA biosynthesis. Transient coexpression of montbretia flavonol biosynthesis genes combined with CcUGT1 and CcUGT2 in N. benthamiana resulted in 2 mg g-1 fresh weight of the MbA pathway-specific compound myricetin 3-O-glucosyl rhamnoside. Additional expression of the montbretia acyltransferase CcAT1 led to detectable levels of mini-MbA in N. benthamiana.

Engineering two species of yeast as cell factories for 2'-fucosyllactose.

Oligosaccharides present in human breast milk have been linked to beneficial effects on infant health. Inclusion of these human milk oligosaccharides (HMOs) in infant formula can recapitulate these health benefits. As a result, there is substantial commercial interest in a cost-effective source of HMOs as infant formula ingredients. Here we demonstrate that the yeast species Saccharomyces cerevisiae and Yarrowia lipolytica both can be engineered to produce 2'-fucosyllactose (2'FL), which is the most abundant oligosaccharide in human breast milk, at high titer and productivity. Both yeast species were modified to enable uptake of lactose and synthesis of GDP-fucose - the two precursors of 2'FL - by installing a lactose transporter and enzymes that convert GDP-mannose to GDP-fucose. Production of 2'FL was then enabled by expression of α-1,2-fucosyltransferases from various organisms. By screening candidate transporters from a variety of sources, we identified transporters capable of exporting 2'FL from yeast, which is a key consideration for any biocatalyst for 2'FL production. In particular, we identified CDT2 from Neurospora crassa as a promising target for further engineering to improve 2'FL efflux. Finally, we demonstrated production of 2'FL in fermenters at rates and titers that indicate the potential of engineered S. cerevisiae and Y. lipolytica strains for commercial 2'FL production.

Enzymatic synthesis of non-natural trisaccharides and galactosides; Insights of their interaction with galectins as a function of their structure.

Galectins are a family of carbohydrate-recognizing proteins that by interacting with specific glycoepitopes can mediate important biological processes, including immune cell homeostasis and activation of tolerogenic circuits. Among the different members of this family, Galectin 1 and 3 have shown pro-tumorigenic effects, being overexpressed in numerous neoplasic diseases, proving to be relevant in tumor immune escape, tumor progression and resistance to drug-induced apoptosis. Thus, generation of specific glycosides that could inhibit their pro-tumorigenic ability by blocking their carbohydrate recognition domain is one of the current major challenges in the field. Considering that galectin-ligand binding strength is closely related to the ligand structure, analysis of this relationship provides valuable information for rational design of high-affinity ligands that could work as effective galectin inhibitors. Taking profit of the ability of glycosidases to catalyze transglycosylation reactions we achieved the enzymatic synthesis of ß-d-Galp-(1 → 6)-ß-d-Galp-(1 → 4)-d-Glcp(2), a mixture of ß-d-Galp-(1 → 6)-ß-d-Glcp-(1 → 4)-d-Glcp(5) and ß-d-Galp-(1 → 3)-ß-d-Glcp-(1 → 4)-d-Glcp(6), and finally benzyl ß-d-galactopyranoside (9), with reaction yields between 16 and 27%. All the galactosides were purified, and characterized using 1H and 13C nuclear magnetic resonance spectroscopy. Docking results performed between the synthesized compounds and human Galectin 1 (hGal-1) and human Galectin 3 (hGal-3) showed that the replacement of a glucose moiety linked to the terminal galactose with a galactose moiety, decreases the affinity for these galectins. Moreover, regarding the interglycosidic bond the most favorable ß-Gal linkage seems to be ß(1 → 4) followed by ß(1 → 3) and ß(1 → 6) for hGal-1, and ß(1 → 4) followed by ß(1 → 6) and ß(1 → 3) for hGal-3. These results were in accordance with the IC50 values obtained with in vitro solid phase inhibition assays. Therefore, docking results obtained in this work proved to be a very good approximation for predicting binding affinity of novel galactosides.