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.

Metabolic engineering of Escherichia coli to produce 2'-fucosyllactose via salvage pathway of guanosine 5'-diphosphate (GDP)-l-fucose.

2'-Fucosyllactose (2-FL) is one of the key oligosaccharides in human milk. In the present study, the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway from fucose was employed in engineered Escherichia coli BL21star(DE3) for efficient production of 2-FL. Introduction of the fkp gene coding for fucokinase/GDP-l-fucose pyrophosphorylase (Fkp) from Bacteroides fragilis and the fucT2 gene encoding α-1,2-fucosyltransferase from Helicobacter pylori allows the engineered E. coli to produce 2-FL from fucose, lactose and glycerol. To enhance the lactose flux to 2-FL production, the attenuated, and deleted mutants of ß-galactosidase were employed. Moreover, the 2-FL yield and productivity were further improved by deletion of the fucI-fucK gene cluster coding for fucose isomerase (FucI) and fuculose kinase (FucK). Finally, fed-batch fermentation of engineered E. coli BL21star(DE3) deleting lacZ and fucI-fucK, and expressing fkp and fucT2 resulted in 23.1 g/L of extracellular concentration of 2-FL and 0.39 g/L/h productivity. Biotechnol. Bioeng. 2016;113: 2443-2452. © 2016 Wiley Periodicals, Inc.

CAR1 deletion by CRISPR/Cas9 reduces formation of ethyl carbamate from ethanol fermentation by Saccharomyces cerevisiae.

Enormous advances in genome editing technology have been achieved in recent decades. Among newly born genome editing technologies, CRISPR/Cas9 is considered revolutionary because it is easy to use and highly precise for editing genes in target organisms. CRISPR/Cas9 technology has also been applied for removing unfavorable target genes. In this study, we used CRISPR/Cas9 technology to reduce ethyl carbamate (EC), a potential carcinogen, which was formed during the ethanol fermentation process by yeast. Because the yeast CAR1 gene encoding arginase is the key gene to form ethyl carbamate, we inactivated the yeast CAR1 gene by the complete deletion of the gene or the introduction of a nonsense mutation in the CAR1 locus using CRISPR/Cas9 technology. The engineered yeast strain showed a 98 % decrease in specific activity of arginase while displaying a comparable ethanol fermentation performance. In addition, the CAR1-inactivated mutants showed reduced formation of EC and urea, as compared to the parental yeast strain. Importantly, CRISPR/Cas9 technology enabled generation of a CAR1-inactivated yeast strains without leaving remnants of heterologous genes from a vector, suggesting that the engineered yeast by CRISPR/Cas9 technology might sidestep GMO regulation.

Metabolic engineering of Corynebacterium glutamicum to produce GDP-L-fucose from glucose and mannose.

Wild-type Corynebacterium glutamicum was metabolically engineered to convert glucose and mannose into guanosine 5'-diphosphate (GDP)-L-fucose, a precursor of fucosyl-oligosaccharides, which are involved in various biological and pathological functions. This was done by introducing the gmd and wcaG genes of Escherichia coli encoding GDP-D-mannose-4,6-dehydratase and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase, respectively, which are known as key enzymes in the production of GDP-L-fucose from GDP-D-mannose. Coexpression of the genes allowed the recombinant C. glutamicum cells to produce GDP-L-fucose in a minimal medium containing glucose and mannose as carbon sources. The specific product formation rate was much higher during growth on mannose than on glucose. In addition, the specific product formation rate was further increased by coexpressing the endogenous phosphomanno-mutase gene (manB) and GTP-mannose-1-phosphate guanylyl-transferase gene (manC), which are involved in the conversion of mannose-6-phosphate into GDP-D-mannose. However, the overexpression of manA encoding mannose-6-phosphate isomerase, catalyzing interconversion of mannose-6-phosphate and fructose-6-phosphate showed a negative effect on formation of the target product. Overall, coexpression of gmd, wcaG, manB and manC in C. glutamicum enabled production of GDP-L-fucose at the specific rate of 0.11 mg g cell(-1) h(-1). The specific GDP-L-fucose content reached 5.5 mg g cell(-1), which is a 2.4-fold higher than that of the recombinant E. coli overexpressing gmd, wcaG, manB and manC under comparable conditions. Well-established metabolic engineering tools may permit optimization of the carbon and cofactor metabolisms of C. glutamicum to further improve their production capacity.

Degradation of Bisphenol A by Bacillus subtilis P74 Isolated from Traditional Fermented Soybean Foods.

Bisphenol A (BPA), one of the most widely used plasticizers, is an endocrine-disrupting chemical that is released from plastic products. The aim of this study was to screen and characterize bacteria with excellent BPA-degrading abilities for application in foods. BPA degradation ability was confirmed in 127 of 129 bacterial strains that were isolated from fermented soybean foods. Among the strains, B. subtilis P74, which showed the highest BPA degradation performance, degraded 97.2% of 10 mg/L of BPA within 9 h. This strain not only showed a fairly stable degradation performance (min > 88.2%) over a wide range of temperatures (30-45 °C) and pH (5.0-9.0) but also exhibited a degradation of 63% against high concentrations of BPA (80 mg/L). The metabolites generated during the degradation were analyzed using high-performance liquid chromatography-mass spectrometry, and predicted degradation pathways are tentatively proposed. Finally, the application of this strain to soybean fermentation was conducted to confirm its applicability in food.

Inverse metabolic engineering for improving protein content in Saccharomyces cerevisiae.

Production of Saccharomyces cerevisiae-based single cell protein (SCP) has recently received great attention due to the steady increase in the world's population and environmental issues. In this study, an inverse metabolic engineering approach was applied to improve the production of yeast SCP. Specifically, an S. cerevisiae mutant library, generated using UV-random mutagenesis, was screened for three rounds to isolate mutants with improved protein content and/or concentration. The #1021 mutant strain exhibited a respective 31% and 23% higher amino acid content and concentration than the parental S. cerevisiae D452-2 strain. Notably, the content, concentration, and composition of amino acids produced by the PAN2* strain, with a single nucleotide polymorphism in PAN2 coding for a catalytic subunit of the poly(A)-nuclease (PAN) deadenylation complex, were virtually identical to those produced by the #1021 mutant strain. In a glucose-limited fed-batch fermentation, the PAN2* strain produced 19.5 g L-1 amino acids in 89 h, which was 16% higher than that produced by the parental D452-2 strain. This study highlights the benefits of inverse metabolic engineering for enhancing the production titer and yield of target molecules without prior knowledge of rate-limiting steps involved in their biosynthetic pathways.

Consumer Preference of Traditional Korean Soy Sauce (Ganjang) and Its Relationship with Sensory Attributes and Physicochemical Properties.

This study aimed to investigate the physicochemical characteristics, sensory attributes, and consumer acceptance of the Certification of Quality of Traditional Food (CQT) ganjang samples produced in different provinces of Korea. Wide variations in physicochemical properties were found among the samples, especially in lipids, total nitrogen, acidity, and reducing sugar. Traditional fermented foods are known to be closely tied to regional features, but the composition and characteristics of CQT ganjangs might be influenced much more by individual ganjang producers than by region. Preference mapping was performed to understand consumer behavior towards ganjang, and most consumers tended to have similar preferences, implying shared a common sensory ideal. The results of the partial least squares regression revealed drivers of liking for ganjang among sensory attributes, free amino acids, and organic acids. Overall, sensory attributes such as sweetness and umami were positively associated with acceptability, while the terms related to fermentation were negatively associated. In addition, amino acids, such as threonine, serine, proline, glutamate, aspartate, and lysine, and organic acids, such as lactate and malate, were positively associated with consumer acceptance. The important implications of the findings of this study for the food industry can be utilized to develop and optimize traditional foods.

Effects of deletion of glycerol-3-phosphate dehydrogenase and glutamate dehydrogenase genes on glycerol and ethanol metabolism in recombinant Saccharomyces cerevisiae.

Bioethanol is currently used as an alternative fuel for gasoline worldwide. For economic production of bioethanol by Saccharomyces cerevisiae, formation of a main by-product, glycerol, should be prevented or minimized in order to reduce a separation cost of ethanol from fermentation broth. In this study, S. cerevisiae was engineered to investigate the effects of the sole and double disruption of NADH-dependent glycerol-3-phosphate dehydrogenase 1 (GPD1) and NADPH-requiring glutamate dehydrogenase 1 (GDH1) on the production of glycerol and ethanol from glucose. Even though sole deletion of GPD1 or GDH1 reduced glycerol production, double deletion of GPD1 and GDH1 resulted in the lowest glycerol concentration of 2.31 g/L, which was 46.4% lower than the wild-type strain. Interestingly, the recombinant S. cerevisiae ∆GPD1∆GDH1 strain showed a slight improvement in ethanol yield (0.414 g/g) compared with the wild-type strain (0.406 g/g). Genetic engineering of the glycerol and glutamate metabolic pathways modified NAD(P)H-requiring metabolic pathways and exerted a positive effect on glycerol reduction without affecting ethanol production.

By-product of Korean liquor fermented by Saccharomyces cerevisiae exhibits skin whitening activity.

Herein, the skin whitening effect of the fermentation residue of Saccharomyces cerevisiae was investigated. The fermentation residue showed radical scavenging activity and attenuated tyrosinase activity. Furthermore, the fermentation residue of S. cerevisiae significantly suppressed melanin generation in B16F10 cells. Interestingly, the sample-containing formulation exhibited increased skin whitening activity compared with that by the control formulation in a clinical study. Notably, the endogenous tyrosinase expression was not altered by the fermentation residue of S. cerevisiae; however, the enzymatic activity of tyrosinase was inhibited. Furthermore, the sample did not change TRP1 and TRP2 expression in B16F10 cells. Thus, the fermentation residue of S. cerevisiae was assumed to directly suppress the tyrosinase enzyme. It was confirmed that the fermentation residue of S. cerevisiae was a competitive inhibitor of tyrosinase. Taken together, the fermentation residue of S. cerevisiae could be a novel skin whitening agent originating from the traditional Korean liquor production process. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01062-7.

Simultaneous production of 2'-fucosyllactose and difucosyllactose by engineered Escherichia coli with high secretion efficiency.

BACKGROUND AND AIM: Difucosyllactose (Di-FL) has strong antimicrobial activity against various pathogens, including group B Streptococcus, identified as the leading cause of neonatal sepsis. In this study, we sought to develop Escherichia coli as a microbial cell factory for efficiently producing Di-FL as well as 2'-fucosyllactose (2'-FL), the most abundant fucosylated oligosaccharide in human milk, by utilizing the salvage guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic pathway. MAIN METHODS AND MAJOR RESULTS: The biosynthetic pathway for producing fucosylated oligosaccharides via the salvage pathway requires two enzymes, l-fucokinase/GDP-l-fucose phosphorylase (FKP) from Bacteroides fragilis and α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori. To decrease the intracellular accumulation of 2'-FL while increasing substrate accessibility to FKP and FucT2, we evaluated whether extracellular secretion of FKP and FucT2 would enhance the production of fucosylated oligosaccharides. Among various engineered strains constructed in this study, the ΔLFAR-YA/FF+P-PLA2 strain expressing phospholipase A2 (PLA2 ) from Streptomyces violaceoruber, whose native signal peptide was replaced with the PelB signal peptide (P-PLA2 ), could secrete both FKP and FucT2 into the culture medium. Notably, it was observed that FKP and FucT2 present in the extracellular fraction could catalyze the synthesis of Di-FL from lactose and fucose. As a result, a batch fermentation with the ΔLFAR-YA/FF+P-PLA2 strain resulted in the production of 1.22 ± 0.01 g L-1 Di-FL and 0.47 ± 0.01 g L-1 2'-FL, whereas the control strain could only produce 0.65 ± 0.01 g L-1 2'-FL. CONCLUSIONS AND IMPLICATIONS: This study highlights the benefits of extracellular secretion of enzymes to improve biotransformation efficiency, as the transport of substrates and/or products across the cell membrane is limited.

Enhanced production of GDP-L-fucose by overexpression of NADPH regenerator in recombinant Escherichia coli.

Biosynthesis of guanosine 5'-diphosphate-L-fucose (GDP-L-fucose) requires NADPH as a reducing cofactor. In this study, endogenous NADPH regenerating enzymes such as glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (Icd), and NADP(+)-dependent malate dehydrogenase (MaeB) were overexpressed to increase GDP-L-fucose production in recombinant Escherichia coli. The effects of overexpression of each NADPH regenerating enzyme on GDP-L-fucose production were investigated in a series of batch and fed-batch fermentations. Batch fermentations showed that overexpression of G6PDH was the most effective for GDP-L-fucose production. However, GDP-L-fucose production was not enhanced by overexpression of G6PDH in the glucose-limited fed-batch fermentation. Hence, a glucose feeding strategy was optimized to enhance GDP-L-fucose production. Fed-batch fermentation with a pH-stat feeding mode for sufficient supply of glucose significantly enhanced GDP-L-fucose production compared with glucose-limited fed-batch fermentation. A maximum GDP-L-fucose concentration of 235.2 ± 3.3 mg l(-1), corresponding to a 21% enhancement in the GDP-L-fucose production compared with the control strain overexpressing GDP-L-fucose biosynthetic enzymes only, was achieved in the pH-stat fed-batch fermentation of the recombinant E. coli overexpressing G6PDH. It was concluded that sufficient glucose supply and efficient NADPH regeneration are crucial for NADPH-dependent GDP-L-fucose production in recombinant E. coli.

Application of Natural Preservatives for Meat and Meat Products against Food-Borne Pathogens and Spoilage Bacteria: A Review.

Meat and meat products are excellent sources of nutrients for humans; however, they also provide a favorable environment for microbial growth. To prevent the microbiological contamination of livestock foods, synthetic preservatives, including nitrites, nitrates, and sorbates, have been widely used in the food industry due to their low cost and strong antibacterial activity. Use of synthetic chemical preservatives is recently being considered by customers due to concerns related to negative health issues. Therefore, the demand for natural substances as food preservatives has increased with the use of plant-derived and animal-derived products, and microbial metabolites. These natural preservatives inhibit the growth of spoilage microorganisms or food-borne pathogens by increasing the permeability of microbial cell membranes, interruption of protein synthesis, and cell metabolism. Natural preservatives can extend the shelf-life and inhibit the growth of microorganisms. However, they can also influence food sensory properties, including the flavor, taste, color, texture, and acceptability of food. To increase the applicability of natural preservatives, a number of strategies, including combinations of different preservatives or food preservation methods, such as active packaging systems and encapsulation, have been explored. This review summarizes the current applications of natural preservatives for meat and meat products.

Combinatorial Effects of Protective Agents on Survival Rate of the Yeast Starter, Saccharomyces cerevisiae 88-4, after Freeze-Drying.

A yeast starter is formulated for commercial practices, including storage and distribution. The cell viability of the yeast starter is one of the most important factors for manufacturing alcoholic beverages to ensure their properties during the fermentation and formulation processes. In this study, 64 potential protective agents were evaluated to enhance the survival rate of the brewing yeast Saccharomyces cerevisiae 88-4 after freeze-drying. In addition, the optimized combination of protective agents was assessed for long-term storage. Finally, response surface methodology was applied to investigate the optimal concentration of each protectant. Twenty of the 64 additives led to an increase in the survival rate of freeze-dried S. cerevisiae 88-4. Among the various combinations of protectants, four had a survival rate >95%. The combination of skim milk, maltose, and maltitol exhibited the best survival rate of 61% after 42 weeks in refrigerated storage, and the composition of protectants optimized by response surface methodology was 6.5-10% skim milk, 1.8-4.5% maltose, and 16.5-18.2% maltitol. These results demonstrated that the combination of multiple protectants could alleviate damage to yeasts during freeze-drying and could be applied to the manufacturing starters for fermented foods.

CRISPR/Cas9-mediated Inactivation of arginase in a yeast strain isolated from Nuruk and its impact on the whole genome.

Despite the advantages of CRISPR/Cas9 technology in the food industry, controversy over its off-target effects exists. We engineered an industrial Saccharomyces cerevisiae strain isolated from a Korean rice wine starter, Nuruk, using CRISPR/Cas9 to decrease ethyl carbamate (EC) formation. We disrupted the CAR1 gene encoding arginase, which plays a key role in EC formation. Subsequently, we compared the whole genome of the engineered strain to that of the wild type by analyzing heterozygous and homozygous mutations through variant calling. Homozygous mutations in the genome of the engineered strains were identified as the target mutations in CAR1 induced by CRISPR/Cas9, and no other off-target effects were observed. Our findings have critical implications for the use of CRISRP/Cas9 technology in yeasts in the food industry.

Analysis of Volatile Compounds in Soju, a Korean Distilled Spirit, by SPME-Arrow-GC/MS.

The SPME Arrow technology-a novel solid phase micro-extraction technique-was used to analyze Soju, a traditional Korean distilled liquor, in barrels made of Quercus spp. The volatile compounds detected when the barrels were toasted were analyzed. Five types of sorbents-carbon wide range/polydimethylsiloxane, divinylbenzene/carbon wide range/polydimethylsiloxane, divinylbenzene/polydimethylsiloxane, polydimethylsiloxane, and polyacrylate-were used for this investigation. Fifty-four volatile compounds were detected in Soju using gas chromatography/mass spectrometry. A high extraction efficiency was obtained using carbon wide range/polydimethylsiloxane. Nineteen samples were analyzed using barrels made of six species of carbonated oak (Q. aliena, Q. variabilis, Q. dentate, Q. acutissima, Q. mongolica, and Q. serrata) and control groups in three ways: noncharring, medium charring, and heavy charring. Ethanol, 1-propanol, isoamyl acetate, and isoamyl alcohol can be used as indicator volatile components for Soju and other such traditional Korean distilled liquors. We believe our study results can be used to design better analysis methods for Soju and other distilled liquors.

Heterologous expression of Deinococcus geothermalis amylosucrase in Corynebacterium glutamicum for luteolin glucoside production.

Amylosucrase (ASase) has great industrial potential owing to its multifunctional activities, including transglucosylation, polymerization, and isomerization. In the present study, the properties of Deinococcus geothermalis ASase (DGAS) expressed in Corynebacterium glutamicum (cDGAS) and purified via Ni-NTA affinity chromatography were compared to those of DGAS expressed in Escherichia coli (eDGAS). The pH profile of cDGAS was similar to that of eDGAS, whereas the temperature profile of cDGAS was lower than that of eDGAS. The melting temperature of both enzymes did not differ significantly. Interestingly, polymerization activity was slightly lower in cDGAS than in eDGAS, whereas luteolin (an acceptor molecule) transglucosylation activity in cDGAS was 10 % higher than that in eDGAS. Analysis of protein secondary structure via circular dichroism spectroscopy revealed that cDGAS had a lower strand/helix ratio than eDGAS. The present results indicate that cDGAS is of greater industrial significance than eDGAS.

Genome sequence of the potential probiotic eukaryote Saccharomyces cerevisiae KCCM 51299.

Saccharomyces cerevisiae KCCM 51299, a potential probiotic yeast overproducing glutathione, has been isolated from among 272 yeast strains from the relatively safe Nuruk. The genome sequence of S. cerevisiae KCCM 51299 was analyzed and a near-complete genome (12 Mb) with 19 contigs was assembled after PacBio single-molecule real-time (SMRT) sequencing. The genome of S. cerevisiae KCCM 51299 was compared to the S. cerevisiae s288c reference genome. Additionally, genes involved in glutathione biosynthesis were identified, and the glutathione biosynthesis pathway was constructed in silico based on these genes. Furthermore, S. cerevisiae KCCM 51299 genes were compared with those in other yeast genomes. Finally, genome-scale in silico flux analysis was carried out, and a metabolic engineering strategy for glutathione biosynthesis was generated. These results provide useful information to further develop eukaryotic probiotics to overproduce glutathione.

Improved production of 2'-fucosyllactose in engineered Escherichia coli by expressing putative α-1,2-fucosyltransferase, WcfB from Bacteroides fragilis.

2'-Fucosyllactose (2-FL) is one of most abundant oligosaccharides in human milk, which is involved in many biological functions for infant health. Since 2-FL has a great potential in application to functional food materials and pharmaceuticals, several microbial systems for mass production of 2-FL have been developed in recent years. Microbial production of 2-FL was suggested to be influenced by a number of factors including fucosylation activity of α-1,2-fucosyltransferase. In the present study, the wcfB gene coding for α-1,2-fucosyltransferase from Bacteroides fragilis was screened from eleven candidates of putative α-1,2-fucosyltransferase. Introduction of the wcfB gene allows the lacZ-deleted strain of E. coli expressing the genes for guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic enzymes to produce 2-FL. As a result of fed-batch fermentation, 15.4g/L extracellular concentration of 2-FL with 2-FL yield of 0.858g/g lactose and productivity of 0.530g/L/h were obtained. In addition, the feasibility of industrial production of 2-FL using this microbial system was demonstrated by performing fed-batch fermentation in a 75L bioreactor.

Complete genome sequence of Leuconostoc garlicum KCCM 43211 producing exopolysaccharide.

Leuconostoc garlicum KCCM 43211 isolated from traditional Korean fermented food is an intensive producer of exopolysaccharide (EPS). Here we report the first complete genome sequence of L. garlicum KCCM 43211. The genome sequence displayed that this strain contains genes involved in production of EPS possibly composed of glucose monomers. An uncharacterized EPS from the L. garlicum KCCM 43211 strains was also produced during fermentation in the sucrose medium. The MALDI-TOF results displayed the typical mass spectrometry pattern of dextran. This uncharacterized EPS may have use in commercial prebiotics, food additives, and medical purposes. The complete genome sequence of L. garlicum KCCM 43211 will provide valuable information for strain engineering based on the genetic information.

Enhanced production of 2'-fucosyllactose in engineered Escherichia coli BL21star(DE3) by modulation of lactose metabolism and fucosyltransferase.

2'-Fucosyllactose (2-FL) is one of most abundant functional oligosaccharides in human milk, which is involved in many biological functions for human health. To date, most microbial systems for 2-FL production have been limited to use Escherichia coli JM strains since they cannot metabolize lactose. In this study, E. coli BL21star(DE3) was engineered through deletion of the whole endogenous lactose operon and introduction of the modified lactose operon containing lacZ△M15 from E. coli K-12. Expression of genes for guanosine 5'-diphosphate (GDP)-l-fucose biosynthetic enzymes and heterologous α-1,2-fucosyltransferase (FucT2) from Helicobacter pylori allowed the engineered E. coli BL21star(DE3) to produce 2-FL with 3-times enhanced yield than the non-engineered E. coli BL21star(DE3). In addition, the titer and yield of 2-FL were further improved by adding the three aspartate molecules at the N-terminal of FucT2. Overall, 6.4 g/L 2-FL with the yield of 0.225 g 2-FL/g lactose was obtained in fed-batch fermentation of the engineered E. coli BL21star(DE3) expressing GDP-l-fucose biosynthetic enzymes and three aspartate tagged FucT2.