Search

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.

Soluble expression of enzymes inside the cell is a prerequisite for the successful biotransformation of valuable products. Some key enzymes involved in biotransformation processes, however, are hardly expressed in their soluble forms. Here, we propose an inducible plasmid display, which is a molecular evolution strategy coupled with a high-throughput screening and/or selection method, as a simple and powerful tool for improving the solubility of target enzymes. Specifically, the Oct-1 DNA-binding domain and intein (i.e., auto-processing domain) were employed as anchoring and protein trans-splicing motifs to develop the system, in which the probability of protein trans-splicing is dependent on the soluble property of target proteins. The applicability of inducible plasmid display was investigated using an α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori, a highly insoluble and unstable enzyme in the cytoplasmic space of Escherichia coli, as a model protein. One round of the overall inducible plasmid display process, which consists of in vivo production of FucT2 mutants and in vitro screening, enabled soluble expression of FucT2 and selection of plasmids containing the corresponding genetic information. The inducible plasmid display developed in this study will contribute to the rapid and efficient screening and/or selection of soluble proteins.

In neuronal exocytosis, SNARE assembly into a stable four-helix bundle drives membrane fusion. Previous studies have revealed that the SM protein Munc18-1 plays a critical role for precise SNARE assembly with the help of Munc13-1, but the underlying mechanism remains unclear. Here, we used single-molecule FRET assays with a nanodisc membrane reconstitution system to investigate the conformational dynamics of SNARE/Munc18-1 complexes in multiple intermediate steps towards the SNARE complex. We found that single Munc18-1 proteins induce the closed conformation of syntaxin-1 not only in the free syntaxin-1 but also in the t-SNARE (syntaxin-1/SNAP-25) complex. These results implicate that Munc18-1 may act as a gatekeeper for both binary and ternary SNARE complex formation by locking the syntaxin-1 in a cleft of Munc18-1. Furthermore, the kinetic analysis of the opening/closing transition reveals that the closed syntaxin-1 in the syntaxin-1/SNAP-25/Munc18-1 complex is less stable than that in the closed syntaxin-1/Munc18-1 complex, which is manifested by the infrequent closing transition, indicating that the conformational equilibrium of the ternary complex is biased toward the open conformation of syntaxin-1 compared with the binary complex.

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.

2'-Fucosyllactose (2'-FL) is one of the most important human milk oligosaccharides and has several health benefits for infants. The levels of 2'-FL in breast milk or samples from other sources can bequantified by high-performance liquid chromatography. However, this method cannot be used for simultaneous detection of the target compound in numerous samples. Here, we developed a simple method for quantifying 2'-FL in a microplate format. The method involves two steps: (1) release of L-fucose from 2'-FL by α-(1-2,3,4,6)-L-fucosidase and (2) measurement of NADPH formed during the oxidation of L-fucose by L-fucose dehydrogenase. This method enables measurement of up to 5 g/l 2'-FL in 50 min using a 96-well microplate. The efficiency and simplicity of the proposed method make it suitable for the analyses of a large number of samples simultaneously.

Membrane scaffold protein (MSP) is a versatile protein that can be used to study diverse membrane proteins. MSP is strongly expressed in E. coli; however, applications of MSP in in vivo studies remain limited because of contamination with large amounts of endotoxins. Endotoxins cannot be easily removed from MSP following standard purification protocols for His6-tagged proteins, washing with detergents, or Q-Sepharose anion exchange chromatography, regardless of whether the expression host is E. coli BL21(DE3) or ClearColi BL21(DE3). Furthermore, the concentrations of MSP-bound endotoxins were not reduced during nanodisc formation, such that the assembled nanodiscs still contained significant amounts of endotoxins. We hypothesized that the structural properties of MSP that are responsible for membrane scaffolding mediated the strong binding between MSP and the endotoxins. We showed that partial denaturation of MSP with 2 M urea effectively disrupted MSP-endotoxin interactions. MSP-bound endotoxins were successfully removed via Q-Sepharose chromatography following urea treatment. The combined treatment with urea and Q-Sepharose resulted in ∼80-fold reduction in the specific endotoxin level relative to that of conventional Ni-NTA chromatography combined with detergent treatment. The low endotoxin level of 2.0 EU/mg MSP obtained in this study makes it suitable for applications in animal studies.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes. Currently available in vitro assay systems for observing fusion pore opening have several weaknesses such as cargo-bleeding, incomplete removal of unencapsulated dyes, and inadequate information regarding the size of the fusion pore, limiting measurements of the final stage of membrane fusion. In the present study, we used a biotinylated green fluorescence protein and streptavidin conjugated with Dylight 594 (DyStrp) as a Föster resonance energy transfer (FRET) donor and acceptor, respectively. This FRET pair encapsulated in each v-vesicle containing synaptobrevin and t-vesicle containing a binary acceptor complex of syntaxin 1a and synaptosomal-associated protein 25 revealed the opening of a large fusion pore of more than 5 nm, without the unwanted signals from unencapsulated dyes or leakage. This system enabled determination of the stoichiometry of the merging vesicles because the FRET efficiency of the FRET pair depended on the molar ratio between dyes. Here, we report a robust and informative assay for SNARE-mediated fusion pore opening.

Lactic acid (LA) is a versatile compound used in the food, pharmaceutical, textile, leather, and chemical industries. Biological production of LA is possible by yeast strains expressing a bacterial gene encoding l-lactate dehydrogenase (LDH). Kluyveromyces marxianus is an emerging non-conventional yeast with various phenotypes of industrial interest. However, it has not been extensively studied for LA production. In this study, K. marxianus was engineered to express and co-express various heterologous LDH enzymes that were reported to have different pH optimums. Specifically, three LDH enzymes originating from Staphylococcus epidermidis (SeLDH; optimal at pH 5.6), Lactobacillus acidophilus (LaLDH; optimal at pH 5.3), and Bos taurus (BtLDH; optimal at pH 9.8) were functionally expressed individually and in combination in K. marxianus, and the resulting strains were compared in terms of LA production. A strain co-expressing SeLDH and LaLDH (KM5 La+SeLDH) produced 16.0g/L LA, whereas the strains expressing those enzymes individually produced only 8.4 and 6.8g/L, respectively. This co-expressing strain produced 24.0g/L LA with a yield of 0.48g/g glucose in the presence of CaCO3. Our results suggest that co-expression of LDH enzymes with different pH optimums provides sufficient LDH activity under dynamic intracellular pH conditions, leading to enhanced production of LA compared to individual expression of the LDH enzymes.

Fucosyllactoses (FLs), present in human breast milk, have been reported to benefit human health immensely. Especially, 3-fucosyllactose (3-FL) has numerous benefits associated with a healthy gut ecosystem. Metabolic engineering of microorganisms is thought to be currently the only option to provide an economically feasible route for large-scale production of 3-FL. However, engineering principles for α-1,3-fucosyltransferases (1,3-FTs) are not well-known, resulting in the lower productivity of 3-FL than that of 2'-fucosyllactose (2'-FL), although both 2'-FL and 3-FL follow a common pathway to produce GDP-L-fucose. The C-terminus of 1,3-FTs is composed of heptad repeats, responsible for dimerization of the enzymes, and a peripheral membrane anchoring region. It has long been thought that truncation of most heptad repeats, retaining just 1 or 2, helps the soluble expression of 1,3-FTs. However, whether the introduction of truncated version of 1,3-FTs enhances the production of 3-FL in a metabolically engineered strain, is yet to be tested. In this study, the effect of these structural components on the production of 3-FL in Escherichia coli was evaluated through systematic truncation and elongation of the C-terminal regions of three 1,3-FTs from Helicobacter pylori. Although these three 1,3-FTs contained heptad repeats and membrane-anchoring regions of varying lengths, they commonly exhibited an optimal performance when the number of heptad repeats was increased, and membrane-binding region was removed. The production of 3-FL could be increased 10-20-fold through this simple strategy.

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca(2+) stimulates fusion-pore formation in a millisecond time scale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca(2+). When 10 μM Ca(2+) was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.