FL Production Research Articles

Enhanced 2′-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate

2′-Fucosyllactose (2′-FL), a human milk oligosaccharide with confirmed benefits for infant health, is a promising infant formula ingredient. Although Escherichia coli, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Bacillus subtilis have been engineered to produce 2′-FL, their titers and productivities need be improved for economic production. Glucose along with lactose have been used as substrates for producing 2′-FL, but accumulation of by-products due to overflow metabolism of glucose hampered efficient production of 2′-FL regardless of a host strain. To circumvent this problem, we used xylose, which is the second most abundant sugar in plant cell wall hydrolysates and is metabolized through oxidative metabolism, for the production of 2′-FL by engineered yeast. Specifically, we modified an engineered S. cerevisiae strain capable of assimilating xylose to produce 2′-FL from a mixture of xylose and lactose. First, a lactose transporter (Lac12) from Kluyveromyces lactis was introduced. Second, a heterologous 2′-FL biosynthetic pathway consisting of enzymes Gmd, WcaG, and WbgL from Escherichia coli was introduced. Third, we adjusted expression levels of the heterologous genes to maximize 2′-FL production. The resulting engineered yeast produced 25.5 g/L of 2′-FL with a volumetric productivity of 0.35 g/L∙h in a fed-batch fermentation with lactose and xylose feeding to mitigate the glucose repression. Interestingly, the major location of produced 2′-FL by the engineered yeast can be changed using different culture media. While 72% of the produced 2′-FL was secreted when a complex medium was used, 82% of the produced 2′-FL remained inside the cells when a minimal medium was used. As yeast extract is already used as food and animal feed ingredients, 2′-FL enriched yeast extract can be produced cost-effectively using the 2′-FL-accumulating yeast cells.

Engineering the Substrate Transport and Cofactor Regeneration Systems for Enhancing 2'-Fucosyllactose Synthesis in Bacillus subtilis.

Human milk oligosaccharides (HMOs) have been proven to be beneficial to infants' intestinal health and immune systems. 2'-Fucosyllactose (2'-FL) is the most abundant and thoroughly studied HMO and has been approved to be an additive of infant formula. How to construct efficient and safe microbial cell factories for the production of 2'-FL attracts increasing attention. In this work, we engineered the Bacillus subtilis as an efficient 2'-FL producer by engineering the substrate transport and cofactor guanosine 5'-triphosphate (GTP) regeneration systems. First, we constructed a synthesis pathway for the 2'-FL precursor guanosine 5'-diphosphate-l-fucose (GDP-l-fucose) by introducing the salvage pathway gene fkp from Bacteriodes fragilis and improved the fucose importation by overexpressing the transporters. Then, the complete synthesis pathway of 2'-FL was constructed by introducing the heterologous fucosyltransferases from different sources, and it was found that the gene from Helicobacter pylori was the best one for 2'-FL synthesis. We also improved the substrate lactose importation by introducing heterologous lactose permeases and eliminated endogenous β-galactosidase (yesZ) to block the lactose degradation. Next, the production of 2'-FL and GDP-l-fucose was improved by fine-tuning the expression of cofactor guanosine 5'-triphosphate regeneration module genes gmd, ndk, guaA, guaC, ykfN, deoD, and xpt. Finally, a 3 L fed-batch fermentation was performed, and the highest 2'-FL titer reached 5.01 g/L with a yield up to 0.85 mol/mol fucose. We optimized the synthesis modules of 2'-FL in B. subtilis, and this provides a good starting point for metabolic engineering to further improve 2'-FL production in the future.

Search for bacterial α1,2-fucosyltransferases for whole-cell biosynthesis of 2′-fucosyllactose in recombinant Escherichia coli

2′-Fucosyllactose (2′-FL) is the most abundant human milk oligosaccharide and is important for infant nutrition and health. Because 2′-FL has potential as a functional ingredient in advanced infant formula and as a prebiotic in various foods, a cost-effective method for 2′-FL production is desirable. α1,2-Fucosyltransferase (α1,2-FT) is one of the key enzymes enabling the microbial biosynthesis of this complex sugar. However, the α1,2-FTs reported so far for the whole-cell biosynthesis of 2′-FL originate from pathogens, posing a potential hurdle for approval as a food production method depending on countries. In this study, 10 α1,2-FT genes from bacteria of biosafety level one were identified, and the main features of the deduced amino acid sequences were characterized. Four codon-optimized α1,2-FT genes were synthesized and introduced into Escherichia coli ΔL M15 strain containing the plasmid pBCGW encoding guanosine 5′-diphosphate-l-fucose biosynthetic enzymes. Among the four genes, 2′-FL was produced only by the α1,2-FT from Thermosynechococcus elongatus (Te2FT). Bifidobacterium thermacidophilum α1,2-FT (Bt2FT) showed high expression but was not active in E. coli ΔL M15. The other two α1,2-FTs were not expressed to a detectable level. During batch flask fermentation of Te2FT-expressing E. coli ΔL M15 cells, 0.49 g/L 2′-FL was obtained after 72 h of induction. This is comparable to the values previously reported for α1,2-FTs from Helicobacter pylori and Bacteroides fragilis.

Engineering two species of yeast as cell factories for 2′-fucosyllactose

Oligosaccharides present in human breast milk have been linked to beneficial effects on infant health. Inclusion of these human milk oligosaccharides (HMOs) in infant formula can recapitulate these health benefits. As a result, there is substantial commercial interest in a cost-effective source of HMOs as infant formula ingredients. Here we demonstrate that the yeast species Saccharomyces cerevisiae and Yarrowia lipolytica both can be engineered to produce 2′-fucosyllactose (2′FL), which is the most abundant oligosaccharide in human breast milk, at high titer and productivity. Both yeast species were modified to enable uptake of lactose and synthesis of GDP-fucose – the two precursors of 2′FL – by installing a lactose transporter and enzymes that convert GDP-mannose to GDP-fucose. Production of 2′FL was then enabled by expression of α-1,2-fucosyltransferases from various organisms. By screening candidate transporters from a variety of sources, we identified transporters capable of exporting 2′FL from yeast, which is a key consideration for any biocatalyst for 2′FL production. In particular, we identified CDT2 from Neurospora crassa as a promising target for further engineering to improve 2′FL efflux. Finally, we demonstrated production of 2′FL in fermenters at rates and titers that indicate the potential of engineered S. cerevisiae and Y. lipolytica strains for commercial 2′FL production.

Construction of Escherichia coli strains with chromosomally integrated expression cassettes for the synthesis of 2′-fucosyllactose

BackgroundThe trisaccharide 2′-fucosyllactose (2′-FL) is one of the most abundant oligosaccharides found in human milk. Due to its prebiotic and anti-infective properties, 2′-FL is discussed as nutritional additive for infant formula. Besides chemical synthesis and extraction from human milk, 2′-FL can be produced enzymatically in vitro and in vivo. The most promising approach for a large-scale formation of 2′-FL is the whole cell biosynthesis in Escherichia coli by intracellular synthesis of GDP-L-fucose and subsequent fucosylation of lactose with an appropriate α1,2-fucosyltransferase. Even though whole cell approaches have been demonstrated for the synthesis of 2′-FL, further improvements of the engineered E. coli host are required to increase product yields. Furthermore, an antibiotic-free method of whole cell synthesis of 2′-FL is desirable to simplify product purification and to avoid traces of antibiotics in a product with nutritional purpose.ResultsHere we report the construction of the first selection marker-free E. coli strain that produces 2′-FL from lactose and glycerol. To construct this strain, recombinant genes of the de novo synthesis pathway for GDP-L-fucose as well as the gene for the H. pylori fucosyltransferase futC were integrated into the chromosome of E. coli JM109 by using the λ-Red recombineering technique. Strains carrying additional copies of the futC gene and/or the gene fkp (from Bacteroides fragilis) for an additional salvage pathway for GDP-L-fucose production were used and shown to further improve production of 2′-FL in shake flask experiments. An increase of the intracellular GDP-L-fucose concentration by expression of fkp gene as well as an additional copy of the futC gene lead to an enhanced formation of 2′-FL. Using an improved production strain, feasibility of large scale 2′-FL production was demonstrated in an antibiotic-free fed-batch fermentation (13 l) with a final 2′-FL concentration of 20.28 ± 0.83 g l-1 and a space-time-yield of 0.57 g l-1 h-1.ConclusionsBy chromosomal integration of recombinant genes, altering the copy number of these genes and analysis of 2′-FL and intracellular GDP-L-fucose levels, we were able to construct and improve the first selection marker-free E. coli strain which is capable to produce 2′-FL without the use of expression plasmids. Analysis of intracellular GDP-L-fucose levels identified the de novo synthesis pathway of GDP-L-fucose as one bottleneck in 2′-FL production. In antibiotic-free fed-batch fermentation with an improved strain, scale-up of 2′-FL could be demonstrated.

Translational Collocational Strategies of Arab Learners of English

Arab learners of English encounter a serious problem with collocational sequences. The present study purports to determine the extent to which university English language majors can use English collocations properly. A two-form translation test of 16 Arabic collocations was administered to both graduate and undergraduate students of English. The first form included the English translation in a multiple-choice format whereas the other was given as a free translation task. The findings confirmed the writers’ hypothesis that Arab learners of English at all levels face difficulty with English collocations. Moreover, the study aimed at the characterization of the communicative strategies imple-mented by the subjects in their attempts to convey the English meaning. Twelve such strategies have been identified, exemplified and described. The findings have substanti-ated the role of the NL in FL production as well as the need for explicit instructional focus on collocation in school and university.

Relaties Tussen VT-Grammaticaliteitsoordelen en VT-Productie

Teacher training institutes in the Netherlands submit to their students tests of grammaticality judgments concerning FL senten-ces, in order to prepare them for their future task. Comparison of the results of these tests with results of FL production tests of the same students suggested that the former task was more difficult that the latter. The aim of this study was to examine two questions: (1) Is the production of FL grammatical structures different from, that is, more difficult than giving a judgment of grammatical acceptability concerning the same structures? (2) How do students proceed when judging the grammaticality of a given FL sentence? Existing studies on FL grammaticality judgments do not give a clear-cut onswer to these questions. Therefore we decided to carry out an experimental study, concentrating on the characteristics of grammaticality judgment tasks. 30 French syntactic structures were selected which often give rise to errors. These structures were incorporated in a grammaticality judgment test and a production test. The tests were administerd to matched groups of Dutch students of French. The hypothesis was that the two tasks were of a different degree of difficulty. The results did not confirm this hypothesis: although differences were found between the three conditions explored (judgment of a correct sentence, of an incorrect one, and production), these differences were not significant. The grammaticality judgment test was also administered to two groups of French subjects in order to compare their behaviour to that of the Dutch group. The French subjects were found to behave more homogeneously than the Dutch ones. Finally, the grammaticality judgment test was administered to a small group of Dutch subjects in a thinking-aloud-setting. The results of the pilot study suggest that the two tasks are not essentially different. This can be explained in the framework of the literature discussed. An article reporting this study will appear in ITL Review of Applied Linguistics (1986 or 1987)

Relaxions Between fl Grammaticality Judgments and fl Production

Teacher training institutes in the Netherlands submit to their students tests of grammaticality judgments concerning FL sentences, in order to prepare them for their future task. Comparison of the results of these tests with results of FL production tests of the same students suggested that the former task was more difficult than the latter. The purposes of this study were to examine two questions : (1) Is production of FL grammatical structures different from, that is, more difficult than giving a judgment of grammatical acceptability concerning the same structures? (2) How do students proceed when judging the grammaticality of a given FL sentence? To this end, an experiment was designed. 30 French syntactic structures were selected which often give rise to errors. These structures were incorporated in a grammaticality judgment test and a production test. The tests were administered to matched groups of Dutch students of French of the level mentioned. The hypothesis was that the two tasks were of a different degree of difficulty. The results did not confirm this hypothesis : although differences were found between the three conditions explored (judgment of a correct sentence, of an incorrect one, and production), these differences were not significant. The grammatically judgment test was also administered to two groups of French subjects in order to compare their behaviour to that of the Dutch group. The French subjects were found to behave more homogeneously than the Dutch ones. The results of the pilot study suggest that the two tasks are not essentially different.