Development of fluorescent Escherichia coli for a whole-cell sensor of 2'-fucosyllactose.

2'-Fucosyllactose (2'-FL), a major component of fucosylated human milk oligosaccharides, is beneficial to human health in various ways like prebiotic effect, protection from pathogens, anti-inflammatory activity and reduction of the risk of neurodegeneration. Here, a whole-cell fluorescence biosensor for 2'-FL was developed. Escherichia coli (E. coli) was engineered to catalyse the cleavage of 2'-FL into L-fucose and lactose by constitutively expressing α-L-fucosidase. Escherichia coli ∆L YA, in which lacZ is deleted and lacY is retained, was employed to disable lactose consumption. E. coli ∆L YA constitutively co-expressing α-L-fucosidase and a red fluorescence protein (RFP) exhibited increased fluorescence intensity in media containing 2'-FL. However, the presence of 50 g/L lactose reduced the RFP intensity due to lactose-induced cytotoxicity. Preadaptation of bacterial strains to fucose alleviated growth hindrance by lactose and partially recovered the fluorescence intensity. The fluorescence intensity of the cell was linearly proportional to 1-5 g/L 2'-FL. The whole-cell sensor will be versatile in developing a 2'-FL detection system.

Development of a quantitative assay for 2´-fucosyllactose via one-pot reaction with α1,2-fucosidase and l-fucose dehydrogenase.

2'-Fucosyllactose (2'-FL) is the most abundant milk oligosaccharide in human breast milk and it has several benefits for infant health. The quantification of 2'-FL in breast milk or in samples from other sources generally requires lengthy analyses. These methods cannot be used to simultaneously detect 2'-FL in numerous samples, which would be more time-efficient. In this study, two genes, namely α1,2-fucosidase from Xanthomonas manihotis and l-fucose dehydrogenase from Pseudomonas sp. no. 1143, were identified, cloned and overexpressed in E. coli. The recombinant enzymes were produced as 6 × His-tagged proteins and were purified to homogeneity using Ni2+ affinity chromatography. The purified α1,2-fucosidase and l-fucose dehydrogenase are monomers with molecular masses of 63 kDa and 36 kDa, respectively. Both enzymes have sufficiently high activities in phosphate-buffered saline (pH 7.0) at 37 °C, making it possible to develop a coupled enzyme reaction in a single buffer system for the quantitative determination of 2'-FL in a large number of samples simultaneously. This method can be used to quantify 2'-FL in infant formulas and in samples collected from different phases of the biotechnological production of this oligosaccharide. Furthermore, the method is applicable for the rapid screening of active variants during the development of microbial strains producing 2'-FL.

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.

Endocytosing Escherichia coli as a Whole-Cell Biocatalyst of Fatty Acids.

Whole cell biocatalysts can be used to convert fatty acids into various value-added products. However, fatty acid transport across cellular membranes into the cytosol of microbial cells limits substrate availability and impairs membrane integrity, which in turn decreases cell viability and bioconversion activity. Because these problems are associated with the mechanism of fatty acid transport through membranes, a whole-cell biocatalyst that can form caveolae-like structures was generated to promote substrate endocytosis. Caveolin-1 ( CAV1) expression in Escherichia coli increased both the fatty acid transport rate and intracellular fatty acid concentrations via endocytosis of the supplemented substrate. Furthermore, fatty-acid endocytosis alleviated substrate cytotoxicity in E. coli. These traits attributed to bacterial endocytosis resulted in dramatically elevated biotransformation efficiencies in fed-batch and cell-recycle reaction systems when caveolae-forming E. coli was used for the bioconversion of ricinoleic acid (12-hydroxyoctadec-9-enoic acid) to ( Z)-11-(heptanoyloxy) undec-9-enoic acid. We propose that CAV1-mediated endocytosing E. coli represents a versatile tool for the biotransformation of hydrophobic substrates.

Plasmid Display for Stabilization of Enzymes Inside the Cell to Improve Whole-Cell Biotransformation Efficiency.

Recombinant whole-cell biocatalysts are widely used for biotransformation of valuable products. However, some key enzymes involved in biotransformation processes are unstable and cannot be easily expressed in the functional form. In this study, we describe a versatile platform for enzyme stabilization inside the cell: Intracellularly Immobilized Enzyme System (IIES). A 1,2-fucosyltransferase from Pedobactor saltans (PsFL) and a 1,3-fucosyltransferase from Helicobacter pylori (HpFL), chosen as model proteins, were fused with Oct-1 DNA-binding domain, which mediated the formation of a plasmid-protein complex. Oct-1 fusion enabled both soluble and stable expression of recombinant proteins in the cytoplasm because the fusion proteins were stabilized on the plasmid like immobilized enzymes bound to solid surface. As a result, Oct-1-fusion proteins exhibited significantly greater product titer and yield than non-fusion proteins. Use of fusion proteins PsFL-Oct-1 with C-terminal Oct-1 and Oct-1-PsFL with N-terminal Oct-1 resulted in ~3- and ~2-fold higher 2'-fucosyllactose titers, respectively, than with the use of PsFL alone. When Oct-1 was fused to HpFL, which requires dimerization through heptad repeats, almost two times more 3-fucosyllactose was produced. Fucosyllactose has been used as a food additive because it has various beneficial effects on human health. We anticipate that IIES using Oct-1 fusion protein developed in this study can be applied to stabilize other unstable enzymes.