De novo biosynthesis of 2'-fucosyllactose by bioengineered Corynebacterium glutamicum.

2'-Fucosyllactose (2'-FL) which is well-known human milk oligosaccharide was biotechnologically synthesized using engineered Corynebacterium glutamicum, a GRAS microbial workhorse. By construction of the complete de novo pathway for GDP-L-fucose supply and heterologous expression of Escherichia coli lactose permease and Helicobacter pylori α-1,2-fucosyltransferase, bioengineered C. glutamicum BCGW_TL successfully biosynthesized 0.25 g L-1 2'-FL from glucose. The additional genetic perturbations including the expression of a putative 2'-FL exporter and disruption of the chromosomal pfkA gene allowed C. glutamicum BCGW_cTTLEΔP to produce 2.5 g L-1 2'-FL batchwise. Finally, optimized fed-batch cultivation of the BCGW_cTTLEΔP using glucose, fructose, and lactose resulted in 21.5 g L-1 2'-FL production with a productivity of 0.12 g L-1 •h, which were more than 3.3 times higher value relative to the batch culture of the BCGW_TL. Conclusively, it would be a groundwork to adopt C. glutamicum for biotechnological production of other food additives including human milk oligosaccharides.

Prebiotic activities of dextran from Leuconostoc mesenteroides SPCL742 analyzed in the aspect of the human gut microbial ecosystem.

The aim of this study was to investigate the prebiotic activities of dextran (LM742) produced by Leuconostoc mesenteroides SPCL742 in the aspect of the human gut microbial ecosystem focusing on microbiome and metabolome changes in in vitro colonic fermentation. LM742 dextran had a medium-chain structure with the molecular weight of 1394.87 kDa (DP = 7759.22) and α-1,6 and α-1,3 linkages with a 26.11 : 1 ratio. The LM742 dextran was resistent to digestive enzymes in the human gastrointestinal conditions. The individual cultivation of 30 intestinal bacteria with LM742 dextran showed the growth of Bacteroides spp., whereas in vitro human fecal fermentation with LM742 exhibited the symbiotic growth of Bacteroides spp. and beneficial bacteria such as Bifidobacterium spp. Further co-cultivation of Bacteroides xylanisolvens and several probiotics indicated that B. xylanisolvens provides a cross-feeding of dextran to probiotics. In fecal fermentation, LM742 dextran resulted in increased concentrations of short-chain fatty acids, valerate and pantothenate, but it rarely affected the conversion of betaine to trimethylamine. Lastly, LM742 dextran inhibited the adhesion of pathogenic E. coli to human epithelial cells. Taken together, these results demonstrate the prebiotic potential of LM742 dextran as a health-beneficial polysaccharide in the human intestine.

Whole-Genome Sequence of Lactobacillus plantarum SPC-SNU 72-2 as a Probiotic Starter for Sourdough Fermentation.

We report the whole-genome sequence of Lactobacillus plantarum SPC-SNU 72-2, a probiotic starter for sourdough. Genome sequencing was completed using the Pacific Biosciences RS II and Illumina platforms. This study will facilitate the understanding of microbial characteristics of L. plantarum SPC-SNU 72-2 and its roles during sourdough fermentation.

Evaluation of 2,3-Butanediol Production from Red Seaweed Gelidium amansii Hydrolysates Using Engineered Saccharomyces cerevisiae.

Hyper-thermal (HT) acid hydrolysis of red seaweed Gelidium amansii was performed using 12% (w/v) slurry and an acid mix concentration of 180 mM at 150°C for 10 min. Enzymatic saccharification when using a combination of Celluclast 1.5 L and CTec2 at a dose of 16 U/ml led to the production of 12.0 g/l of reducing sugar with an efficiency of enzymatic saccharification of 13.2%. After the enzymatic saccharification, 2,3-butanediol (2,3-BD) fermentation was carried out using an engineered S. cerevisiae strain. The use of HT acid-hydrolyzed medium with 1.9 g/l of 5-hydroxymethylfurfural showed a reduction in the lag time from 48 to 24 h. The 2,3-BD concentration and yield coefficient at 72 h were 14.8 g/l and 0.30, respectively. Therefore, HT acid hydrolysis and the use of the engineered S. cerevisiae strain can enhance the overall 2,3-BD yields from G. amansii seaweed.

Retroperitoneal Solitary Fibrous Tumor.

Teaching Point: The typical imaging features of a retroperitoneal solitary fibrous tumor are relatively large-sized, have a well-defined margin, an avidly heterogeneous enhancement on arterial phase, a prolonged enhancement on delayed phase, and serpentine vessels along the periphery of the tumor.

Improved production of 3-hydroxypropionic acid in engineered Escherichia coli by rebalancing heterologous and endogenous synthetic pathways.

To improve 3-hydroxypropionic acid (3-HP) production by Escherichia coli, glycerol accumulation needs to be reduced. To accomplish this, we constructed a novel E. coli strain that overexpresses the endogenous aldehyde dehydrogenase gene (puuC) under the control of a strong promoter. The fermentation performance of the engineered strain was significantly improved compared to that of the parental control strain in the presence of glucose and xylose. We also inactivated the puu operon repressor gene, puuR, which resulted in a decrease in glycerol accumulation and an increase in 3-HP production through the co-fermentation of glucose and xylose. Through fed-batch fermentation by utilizing glucose and xylose, the engineered strain, JHS_Δgypr-PT7, produced 53.7 g/L 3-HP and accumulated 1.5 g/L glycerol. This combination strategy, wherein we overexpressed the endogenous puuC gene from a strong promoter and eliminate its transcriptional repression, may be extended to rebalance another biochemical pathway.

Suitability of Lactobacillus plantarum SPC-SNU 72-2 as a Probiotic Starter for Sourdough Fermentation.

In sourdough fermentation, lactic acid bacteria perform important roles in the production of volatile and antimicrobial compounds, and exerting health-promoting effects. In this study, we report the probiotic properties and baking characteristics of Lactobacillus plantarum SPC-SNU 72-2 isolated from kimchi. This strain is safe to use in food fermentation as it does not carry genes for biogenic amine production (i.e., hdc, tdc, and ldc) and shows no ß-hemolytic activity against red blood cells. The strain is also stable under simulated human gastrointestinal conditions, showing tolerance to gastric acid and bile salt, and adheres well to colonic epithelial cells. Additionally, this strain prevents pathogen growth and activates mouse peritoneal macrophages by inducing cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and IL-12. Furthermore, the strain possesses good baking properties, providing rich aroma during dough fermentation and contributing to the enhancement of bread texture. Taken together, L. plantarum SPC-SNU 72-2 has the properties of a good starter strain based on the observation that it improves bread flavor and texture while also providing probiotic effects comparable with commercial strains.

Production of 2,3-butanediol from glucose and cassava hydrolysates by metabolically engineered industrial polyploid Saccharomyces cerevisiae.

BACKGROUND: 2,3-Butanediol (2,3-BDO) is a valuable chemical for industrial applications. Bacteria can produce 2,3-BDO with a high productivity, though most of their classification as pathogens makes them undesirable for the industrial-scale production. Though Saccharomyces cerevisiae (GRAS microorganism) was engineered to produce 2,3-BDO efficiently in the previous studies, their 2,3-BDO productivity, yield, and titer were still uncompetitive compared to those of bacteria production. Thus, we propose an industrial polyploid S. cerevisiae as a host for efficient production of 2,3-BDO with high growth rate, rapid sugar consumption rate, and resistance to harsh conditions. Genetic manipulation tools for polyploid yeast had been limited; therefore, we engineered an industrial polyploid S. cerevisiae strain based on the CRISPR-Cas9 genome-editing system to produce 2,3-BDO instead of ethanol. RESULTS: Endogenous genes coding for pyruvate decarboxylase and alcohol dehydrogenase were partially disrupted to prevent declined growth rate and C2-compound limitation. A bacterial 2,3-BDO-producing pathway was also introduced in engineered polyploid S. cerevisiae. A fatal redox imbalance was controlled through the heterologous NADH oxidase from Lactococcus lactis during the 2,3-BDO production. The resulting strain (YG01_SDBN) still retained the beneficial traits as polyploid strains for the large-scale fermentation. The combination of partially disrupted PDC (pyruvate decarboxylase) and ADH (alcohol dehydrogenase) did not cause the severe growth defects typically found in all pdc- or adh-deficient yeast. The YG01_SDBN strain produced 178 g/L of 2,3-BDO from glucose with an impressive productivity (2.64 g/L h). When a cassava hydrolysate was used as a sole carbon source, this strain produced 132 g/L of 2,3-BDO with a productivity of 1.92 g/L h. CONCLUSIONS: The microbial production of 2,3-BDO has been limited to bacteria and haploid laboratorial S. cerevisiae strains. This study suggests that an industrial polyploid S. cerevisiae (YG01_SDBN) can produce high concentration of 2,3-BDO with various advantages. Integration of metabolic engineering of the industrial yeast at the gene level with optimization of fed-batch fermentation at the process scale resulted in a remarkable achievement of 2,3-BDO production at 178 g/L of 2,3-BDO concentration and 2.64 g/L h of productivity. Furthermore, this strain could make a bioconversion of a cassava hydrolysate to 2,3-BDO with economic and environmental benefits. The engineered industrial polyploid strain could be applicable to production of biofuels and biochemicals in large-scale fermentations particularly when using modified CRISPR-Cas9 tools.

Deletion of glycerol-3-phosphate dehydrogenase genes improved 2,3-butanediol production by reducing glycerol production in pyruvate decarboxylase-deficient Saccharomyces cerevisiae.

2,3-Butanediol (2,3-BD) can be produced at high titers by engineered Saccharomyces cerevisiae by abolishing the ethanol biosynthetic pathway and introducing the bacterial butanediol-producing pathway. However, production of 2,3-BD instead of ethanol by engineered S. cerevisiae has resulted in glycerol production because of surplus NADH accumulation caused by a lower degree of reduction (γ = 5.5) of 2,3-BD than that (γ = 6) of ethanol. In order to eliminate glycerol production and resolve redox imbalance during 2,3-BD production, both GPD1 and GPD2 coding for glycerol-3-phosphate dehydrogenases were disrupted after overexpressing NADH oxidase from Lactococcus lactis. As disruption of the GPD genes caused growth defects due to limited supply of C2 compounds, Candida tropicalis PDC1 was additionally introduced to provide a necessary amount of C2 compounds while minimizing ethanol production. The resulting strain (BD5_T2 nox_dGPD1,2_CtPDC1) produced 99.4 g/L of 2,3-BD with 0.5 g/L glycerol accumulation in a batch culture. The fed-batch fermentation led to production of 108.6 g/L 2,3-BD with a negligible amount of glycerol production, resulting in a high BD yield (0.462 g2,3-BD/gglucose) corresponding to 92.4 % of the theoretical yield. These results demonstrate that glycerol-free production of 2,3-BD by engineered yeast is feasible.

Edgeworthia papyrifera Regulates Osteoblast and Osteoclast Differentiation In Vitro and Exhibits Anti-osteoporosis Activity in Animal Models of Osteoporosis.

Osteoporosis is a clinical condition characterized by low bone strength that leads to an increased risk of fracture. Strategies for the treatment of osteoporosis involve inhibition of bone resorption by osteoclasts and an increase of bone formation by osteoblasts. Here, we identified the extract derived from the stem part of Edgeworthia papyrifera that enhanced differentiation of MC3T3-E1 cells to osteoblast-like cells and inhibited osteoclast differentiation of RAW 264.7 cells in vitro. In support of our observation, rutin and daphnoretin, which were previously reported to inhibit osteoclast differentiation, were identified in E. papyrifera extract. In an animal model of osteoporosis, the ovariectomy-induced increases in bone resorption biomarkers such as pyridinoline and tartrate-resistant acid phosphatase were significantly reduced by E. papyrifera extract administration at 25.6 and 48.1%, respectively. Furthermore, the ovariectomy-induced bone loss in animal models of osteoporosis was significantly prevented by the administration of E. papyrifera in our study. Taking these observations into account, we suggest that E. papyrifera is an interesting candidate for further exploration as an anti-osteoporotic agent.

Overexpression of Endogenous Xylose Reductase Enhanced Xylitol Productivity at 40 °C by Thermotolerant Yeast Kluyveromyces marxianus.

Xylitol is a valuable substance utilized by food and biochemical industries. NAD(P)H-dependent xylose reductase (XR)-encoded by the yeast KmXYL1 gene-is the key enzyme which facilitates reduction of xylose to xylitol. Multi-copy integration of a mutant KmXYL1 (mKmXYL1) gene was carried out using thermotolerant yeast Kluyveromyces marxianus KCTC17555ΔURA3, in order to enhance xylitol production. After multi-copy integration, the highest xylitol producing strain was isolated and named K. marxianus 17555-JBP2. This strain exhibited 440% higher xylitol production than the parental strain at 30 °C. Due to a multi-copy integration of the mKmXYL1 gene, various additional differences between K. marxianus 17555-JBP2 and the parental strain were observed, including a 66% increase in NAD(P)H-dependent XR activity at high temperature (45 °C). Quantitative real-time PCR and transcriptome analysis demonstrated that, relative to the parent strain, K. marxianus 17555-JBP2 exhibited two more copies of mKmXY1 genes and a 9.63-fold elevation in transcription of NAD(P)H-dependent XR. After optimization of bioreactor fermentation conditions (agitation speed), high-temperature (40 °C) xylitol productivity of K. marxianus 17555-JBP2 exhibited an 81% improvement relative to the parental strain. In this study, we demonstrated that the overexpression of endogenous XR could enhance xylitol productivity at 40 °C by thermotolerant K. marxianus.

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.

Enhanced production of 3-hydroxypropionic acid from glucose and xylose by alleviation of metabolic congestion due to glycerol flux in engineered Escherichia coli.

Among platform chemicals obtained from renewable biomass, 3-hydroxypropionic acid (3-HP) has attracted considerable attention. A GC/TOF-MS study revealed that the intracellular metabolites of the TCA cycle and fatty acid synthesis increased in JHS01302, a galP-overexpressing strain of Escherichia coli, during glucose and xylose co-fermentation. Decreased intracellular glycerol levels and increased intracellular biosynthesis of 3-HP were also detected in the strain. Based on these results, the yeast GPD1 gene was replaced with the endogenous gpsA gene to modulate the rate of glycerol metabolism. In flask cultures, JHS01304 containing the gpsA gene displayed 43% lower glycerol accumulation and 52% higher 3-HP production than the control. JHS01304 produced 37.6 g/L 3-HP with a productivity rate of 0.63 g/L/h and yield of 0.17 g/g in the fed-batch fermentation. The metabolome analysis provided valuable information for alleviating the metabolic burden of glycerol flux to improve the production of 3-HP during glucose and xylose co-fermentation.

Production of 3-Fucosyllactose in Engineered Escherichia coli with α-1,3-Fucosyltransferase from Helicobacter pylori.

3-Fucosyllactose (3-FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α-1,3-fucosyltransferase, three bacterial α-1,3-fucosyltransferases are expressed in engineered E. coli deficient in ß-galactosidase activity and expressing the essential enzymes for the production of guanosine 5'-diphosphate-l-fucose, the donor of fucose for 3-FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3-FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ∆LP-YA+FT shows a much lower performance of 3-FL production (4.50 g L-1 ) than the ∆L-YA+FT strain grown in a glycerol medium (10.7 g L-1 ), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ∆LW-YA+FT expressing the essential genes for 3-FL production and blocking the colanic acid biosynthetic pathway (∆wcaJ) exhibits the highest concentration (11.5 g L-1 ), yield (0.39 mol mol-1 ), and productivity (0.22 g L-1 h) of 3-FL in glycerol-limited fed-batch fermentation.

L-Fucose production by engineered Escherichia coli.

L-Fucose (6-deoxy-L-galactose) is a major constituent of glycans and glycolipids in mammals. Fucosylation of glycans can confer unique functional properties and may be an economical way to manufacture L-fucose. Research can extract L-fucose directly from brown algae, or by enzymatic hydrolysis of L-fucose-rich microbial exopolysaccharides. However, these L-fucose production methods are not economical or scalable for various applications. We engineered an Escherichia coli strain to produce L-fucose. Specifically, we modified the strain genome to eliminate endogenous L-fucose and lactose metabolism, produce 2'-fucosyllactose (2'-FL), and to liberate L-fucose from 2'-FL. This E. coli strain produced 16.7 g/L of L-fucose with productivity of 0.1 g·L-1 ·h-1 in a fed-batch fermentation. This study presents an efficient one-pot biosynthesis strategy to produce a monomeric form of L-fucose by microbial fermentation, making large-scale industrial production of L-fucose feasible.

Production of biofuels and chemicals from xylose using native and engineered yeast strains.

Numerous metabolic engineering strategies have allowed yeasts to efficiently assimilate xylose, the second most abundant sugar component of lignocellulosic biomass. During the investigation of xylose utilization by yeasts, a global rewiring of metabolic networks upon xylose cultivation has been captured, as opposed to a pattern of glucose repression. A clear understanding of the xylose-induced metabolic reprogramming in yeast would shed light on the optimization of yeast-based bioprocesses to produce biofuels and chemicals using xylose. In this review, we delved into the characteristics of yeast xylose metabolism, and potential benefits of using xylose as a carbon source to produce various biochemicals with examples. Transcriptomic and metabolomic patterns of xylose-grown yeast cells were distinct from those on glucose-a conventional sugar of industrial biotechnology-and the gap might lead to opportunities to produce biochemicals efficiently. Indeed, limited glycolytic metabolic fluxes during xylose utilization could result in enhanced production of metabolites whose biosynthetic pathways compete for precursors with ethanol fermentation. Also, alleviation of glucose repression on cytosolic acetyl coenzyme A (acetyl-CoA) synthesis, and respiratory energy metabolism during xylose utilization enhanced production of acetyl-CoA derivatives. Consideration of singular properties of xylose metabolism, such as redox cofactor imbalance between xylose reductase and xylitol dehydrogenase, is necessary to maximize these positive xylose effects. This review argues the importance and benefits of xylose utilization as not only a way of expanding a substrate range, but also an effective environmental perturbation for the efficient production of advanced biofuels and chemicals in yeasts.

Isolation of lactic acid bacteria starters from Jeung-pyun for sourdough fermentation.

Lactic acid bacteria (LAB) are key for the fermentation of sourdoughs to improve the quality and nutritive value of bread. The aim of this study was to isolate the LAB starter for sourdough fermentation from Jeung-pyun, a Korean traditional rice cake. Among the twenty two LAB screened, five isolates were selected based on exo-polysaccharide production. Among them, three isolates showed cell growth greater than 8.5 Log CFU/g, maximum increase in the volume of dough, and dextran concentration up to 0.16%. During the sourdough fermentation, pH and total titratable acidity (TTA) were changed, as the three isolates synthesized lactic acid and acetic acid with fermentation quotients less than 2.0. They were identified as Leuconostoc lactis EFEL005, Lactobacillus brevis EFEL004, and Le. citreum EFEL006. They displayed good fermentation properties (growth, dextran production, pH, and TTA) in dough and they are regarded as potential starters to be used in sourdough fermentation.

Elimination of biosynthetic pathways for l-valine and l-isoleucine in mitochondria enhances isobutanol production in engineered Saccharomyces cerevisiae.

Saccharomyces cerevisiae has a natural ability to produce higher alcohols, making it a promising candidate for production of isobutanol. However, the several pathways competing with isobutanol biosynthesis lead to production of substantial amounts of l-valine and l-isoleucine in mitochondria and isobutyrate, l-leucine, and ethanol in cytosol. To increase flux to isobutanol by removing by-product formation, the genes associated with formation of l-valine (BAT1), l-isoleucine (ILV1), isobutyrate (ALD6), l-leucine (LEU1), and ethanol (ADH1) were disrupted to construct the S. cerevisiae WΔGBIALA1_2vec strain. This strain showed 8.9 and 8.6 folds increases in isobutanol concentration and yield, respectively, relative the corresponding values of the background strain on glucose medium. In a bioreactor fermentation with a gas trapping system, the WΔGBIALA1_2vec strain produced 662 mg/L isobutanol concentration with a yield of 6.71 mgisobutanol/gglucose. With elimination of the competing pathways, the WΔGBIALA1_2vec strain would serve as a platform strain for isobutanol production.

Anti-melanogenic activity of schaftoside in Rhizoma Arisaematis by increasing autophagy in B16F1 cells.

Skin pigmentation involves multiple processes, including melanin synthesis, transport, and melanosome release. Melanin content determines skin color and protects against UV radiation-induced damage. Autophagy is a cooperative process between autophagosomes and lysosomes that degrades cellular components and organelles. In the present study, B16F1 cells were treated with Rhizoma Arisaematis extract (RA) and assessed for pigmentation and autophagy regulation. RA treatment suppressed the α-MSH-stimulated increase of melanogenesis and down-regulated the expression of tyrosinase and TRP1 proteins in B16F1 cells. In addition, autophagy was activated in RA-treated cells. Inhibition of autophagy reduced the anti-melanogenic activity of RA in α-MSH-treated B16F1 cells. We identified schaftoside as an effector molecule by LC-MS analysis of RA. Consistently, treatment of schaftoside showed anti-melanogenic effect and induced autophagy activation in B16F1 cells. Inhibition of autophagy by 3 MA treatment reduced the anti-melanogenic effect of the schaftoside and recovered expression level of melanogenesis regulators in α-MSH-treated B16F1 cells. Taken together, our results suggest that schaftoside from RA inhibits skin pigmentation through modulation of autophagy.

Engineering of α-1,3-fucosyltransferases for production of 3-fucosyllactose in Escherichia coli.

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.