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2'-Fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, has multiple beneficial effects on human health. However, its biosynthesis by metabolically engineered

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineeredSaccharomyces cerevisiaeoffers an advance towards more cost-effective second-generation (2G) ethanol production. As xylan is one of the most abundant polysaccharides present in lignocellulosic residues, the transport and breakdown of XOS in an intracellular environment might bring a competitive advantage for recombinant strains in competition with contaminating microbes, which are always present in fermentation tanks; furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. In the present work, the previously engineeredS. cerevisiaestrain, SR8A6S3, expressing NADPH-linked xylose reductase (XR), NAD+-linked xylitol dehydrogenase (XDH) (for xylose assimilation), as well as NADH-linked acetylating acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthetase (ACS) (for an NADH-dependent acetate reduction pathway), was used as the host for expressing of two β-xylosidases,GH43-2andGH43-7, and a xylodextrin transporter,CDT-2, fromNeurospora crassa, yielding the engineered strain SR8A6S3-CDT2-GH432/7. Both β-xylosidases and the transporter were introduced by replacing two endogenous genes,GRE3andSOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, which catalyse steps in xylitol production. Xylitol accumulation during xylose fermentation is a problem for 2G ethanol production since it reduces final ethanol yield. The engineered strain, SR8A6S3-CDT2-GH432/7, produced ethanol through simultaneous co-utilization of XOS, xylose, and acetate. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan compared with the parental strain. The consumption of XOS, xylose, and acetate expands the capabilities ofS. cerevisiaefor utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production.HighlightsIntegration of XOS pathway in an acetate-xylose-consumingS. cerevisiaestrain;Intracellular fermentation of XOS, acetate and xylose improved ethanol production;Deletion of bothsor1Δ andgre3Δ reduced xylitol production.

Escherichia coli-based whole-cell biocatalysts are widely used for the sustainable production of value-added chemicals. However, weak acids present as substrates and/or products obstruct the growth and fermentation capability of E. coli. Here, we show that a viroporin consisting of the influenza A matrix-2 (M2) protein, is activated by low pH and has proton channel activity in E. coli. The heterologous expression of the M2 protein in E. coli resulted in a significant increase in the intracellular pH and cell viability in the presence of various weak acids with different lengths of carbon chains. In addition, the feasibility of developing a robust and efficient E. coli-based whole-cell biocatalyst via introduction of the proton-selective viroporin was explored by employing (Z)-11-(heptanolyoxy)undec-9-enoic acid (ester) and 2-fucosyllactose (2'-FL) as model products, whose production is hampered by cytosolic acidification. The engineered E. coli strains containing the proton-selective viroporin exhibited approximately 80% and 230% higher concentrations of the ester and 2'-FL, respectively, than the control strains without the M2 protein. The simple and powerful strategy developed in this study can be applied to produce other valuable chemicals whose production involves substrates and/or products that cause cytosolic acidification.

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.

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.

Directed evolution is widely used to optimize protein folding and solubility in cells. Although the screening and selection of desired mutants is an essential step in directed evolution, it generally requires laborious optimization and/or specialized equipment. With a view toward designing a more practical procedure, we previously developed an inducible plasmid display system, in which the intein (auto-processing) and Oct-1 DNA-binding (DBD) domains were used as the protein trans-splicing domain and DNA-binding module, respectively. Specifically, the N-terminal (CfaN) and C-terminal (CfaC) domains of intein were fused to the C-terminal end of the His-tag and the N-terminal end of Oct-1 DBD to generate His6-CfaN and CfaC-Oct-1, respectively. For such a system to be viable, the efficiency of protein trans-splicing without the protein of interest (POI) should be maximized, such that the probability of occurrence is solely dependent on the solubility of the POI. To this end, we initially prevented the degradation of l -arabinose (the inducer of the PBAD promoter) by employing an Escherichia coli host strain deficient in the metabolism of l -arabinose. Given that a low expression of His6-CfaN, compared with that of CfaC-Oct-1, was found to be conducive to the generation to a soluble product of the protein trans-splicing event, we designed the expression of His6-CfaN and CfaC-Oct-1 to be inducible from the PBAD and PT7 promoters, respectively. The optimized system thus obtained enabled in vitro selection of the plasmid–protein complex with high yield. We believe that the inducible plasmid display system developed in this study would be applicable to high-throughput screening and/or selection of protein variants with enhanced solubility.

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.

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.

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 Ni 2+ 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.

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.

Caveolae are membrane-budding structures that exist in many vertebrate cells. One of the important functions of caveolae is to form membrane curvature and endocytic vesicles. Recently, it was shown that caveolae-like structures were formed in Escherichia coli through the expression of caveolin-1. This interesting structure seems to be versatile for a variety of biotechnological applications. Targeting of heterologous proteins in the caveolae-like structure should be the first question to be addressed for this purpose. Here we show that membrane proteins co-expressed with caveolin-1 are embedded into the heterologous caveolae (h-caveolae), the cavaolae-like structures formed inside the cell. Two transmembrane SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, Syntaxin 1a and vesicle-associated membrane protein 2 (VAMP2), were displayed on the h-caveolae surface. The size of the h-caveolae harboring the transmembrane proteins was ∼100 nm in diameter. The proteins were functional and faced outward on the h-caveolae. Multi-spanning transmembrane proteins FtsH and FeoB could be included in the h-caveolae, too. Furthermore, the recombinant E. coli cells were shown to endocytose substrate supplemented in the medium. These results provide a basis for exploiting the h-caveolae formed inside E. coli cells for future biotechnological applications.

Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins generate energy required for membrane fusion. They form a parallelly aligned four-helix bundle called the SNARE complex, whose formation is initiated from the N terminus and proceeds toward the membrane-proximal C terminus. Previously, we have shown that this zippering-like process can be controlled by several flavonoids that bind to the intermediate structures formed during the SNARE zippering. Here, our aim was to test whether the fluorescence resonance energy transfer signals that are observed during the inner leaflet mixing assay indeed represent the hemifused vesicles. We show that changes in vesicle size accompanying the merging of bilayers is a good measure of progression of the membrane fusion. Two merging vesicles with the same size D in diameter exhibited their hydrodynamic diameters 2D + d (d, intermembrane distance), 2D and 2D as membrane fusion progressed from vesicle docking to hemifusion and full fusion, respectively. A dynamic light scattering assay of membrane fusion suggested that myricetin stopped membrane fusion at the hemifusion state, whereas delphinidin and cyanidin prevented the docking of the vesicles. These results are consistent with our previous findings in fluorescence resonance energy transfer assays.

Epothilone B (EpoB) is a paclitaxel (PTX)-like microtubule-stabilizing agent that induces mitotic arrest and apoptosis in cells. EpoB is a promising anti-tumor agent, and is thought to have the potential to overcome well-known PTX resistance. However, EpoB is barely soluble in water, and is fatal to normal cells due to extremely potent cytotoxicity. To reduce the unwanted cytotoxicity of EpoB to normal cells, a reconstituted highdensity lipoprotein (rHDL)-like nanoparticle of EpoB (EpoB-rHDL) was assembled using apolipoprotein A-I (apoA-I). The EpoB-rHDL, as well as PTX-rHDL (a HDLlike nanoparticle of paclitaxel), were indeed mild (nontoxic) to certain cell lines such as MCF7, MDA-MB-231, and SK-OV-3, while free EpoB and PTX were very toxic to those same cells. In contrast, the EpoB-rHDL and PTXrHDL were very effective in killing the Caco-2 and ZR-75-1 while free drugs were less toxic to those cells. The susceptibility of cell lines to rHDLs was dependent on the expression of scavenger receptor class B type I (SR-BI), indicating that EpoB-rHDL selectively and efficiently kills only SR-BI-overexpressing cells. Furthermore, the EpoBrHDL released EpoB only at acidic pH, which may facilitate the escape of drugs from acidic endosome. Thus, EpoB-rHDL shown in this study enables safe and targeted delivery of the potent EpoB to cancer cells in SR-BIdependent manner.

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.

Fusion of synaptic vesicles with the presynaptic plasma membrane in the neuron is mediated by soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor (SNARE) proteins. SNARE complex formation is a zippering-like process which initiates at the N-terminus and proceeds to the C-terminal membrane-proximal region. Previously, we showed that this zippering-like process is regulated by several polyphenols, leading to the arrest of membrane fusion and the inhibition of neuroexocytosis. In vitro studies using purified SNARE proteins reconstituted in liposomes revealed that each polyphenol uniquely regulates SNARE zippering. However, the unique regulatory effect of each polyphenol in cells has not yet been examined. In the present study, we observed SNARE zippering in neuronal PC12 cells by measuring the fluorescence resonance energy transfer (FRET) changes of a cyan fluorescence protein (CFP) and a yellow fluorescence protein (YFP) fused to the N-termini or C-termini of SNARE proteins. We show that delphinidin and cyanidin inhibit the initial N-terminal nucleation of SNARE complex formation in a Ca(2+)-independent manner, while myricetin inhibits Ca(2+)-dependent transmembrane domain association of the SNARE complex in the cell. This result explains how polyphenols exhibit botulinum neurotoxin-like activity in vivo.

Anti-allergic effects of dietary polyphenols were extensively studied in numerous allergic disease models, but the molecular mechanisms of anti-allergic effects by polyphenols remain poorly understood. In the present study, we show that the release of granular cargo molecules, contained in distinct subsets of granules of mast cells, is specifically mediated by two sets of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, and that various polyphenols differentially inhibit the formation of those SNARE complexes. Expression analysis of RBL-2H3 cells for 11 SNARE genes and a lipid mixing assay of 24 possible combinations of reconstituted SNAREs indicated that the only two active SNARE complexes involved in mast cell degranulation are Syn (syntaxin) 4/SNAP (23 kDa synaptosome-associated protein)-23/VAMP (vesicle-associated membrane protein) 2 and Syn4/SNAP-23/VAMP8. Various polyphenols selectively or commonly interfered with ternary complex formation of these two SNARE complexes, thereby stopping membrane fusion between granules and plasma membrane. This led to the differential effect of polyphenols on degranulation of three distinct subsets of granules. These results suggest the possibility that formation of a variety of SNARE complexes in numerous cell types is controlled by polyphenols which, in turn, might regulate corresponding membrane trafficking.