Description
1. Background
Breast milk oligosaccharides, also known as human milk oligosaccharides (HMOs), are the third abundant solid
component in breast milk after lactose and fat. Over 200 sugar compounds were identified in HMOs, among which
2′-Fucosyllactose (2’-FL) has the highest proportion in HMOs (more than 30%). Studies have proved that 2'-FL not
only promotes the development of immune system and a gut microbiota, but also helps to prevent allergic disease and
promotes brain function and cognitive development. Moreover, 2'-FL can be used as an additive in the infant formula
which is closer to breast milk in nutrients. Thus, producing the high yield of 2'-FL draws more and more
attention.Breast milk oligosaccharides, also known as human milk oligosaccharides (HMOs), are the third abundant
solid component in breast milk after lactose and fat. Over 200 sugar compounds were identified in HMOs, among which
2′-Fucosyllactose (2’-FL) has the highest proportion in HMOs (more than 30%). Studies have proved that 2'-FL not
only promotes the development of immune system and a gut microbiota, but also helps to prevent allergic disease and
promotes brain function and cognitive development. Moreover, 2'-FL can be used as an additive in the infant formula
which is closer to breast milk in nutrients. Thus, producing the high yield of 2'-FL draws more and more attention.
At present, there are three methods for synthesizing 2'-FL, including chemical synthesis, enzymatic synthesis, and
whole-cell biosynthesis. However, there are some limiting factors for the chemical synthesis and enzymatic synthesis
including the toxic reagents and cost of precursor. For instance, using the enzyme-catalyzed synthesis route, the
yield of enzyme-catalyzed synthesis of 2'-FL is not high due to the expensive substrate guanosine
5’-diphosphate-L-fucose (GDP-L-fucose). The substrate GDP-fucose is extracted from cellular which is not suitable to
reuse it for large-scale production. In contrast, the whole-cell biosynthesis method is relatively low-cost and
mainly performed in engineered E.coli to produce 2'-FL. But, for large-scale fermentation, there is possible that
endotoxin contamination. For sake of safety, we use the S. cerevisiae to produce 2'-FL. In addition, sweet potato
residues contain abundant glucose with the relative content of 56% and can be used as substrate for large-scale
fermentation.
Thus, in this project, we designed and synthesized an engineered S.
cerevisiae cellular factory producing 2'-FL
from sweet potato residues by fermentation. On one hand, producing 2'-FL in S. cerevisiae is a relatively safe
cellular factory to allow the large-scale fermentation. On the other hand, it is promising that using sweet potato
residues to further extend industrial chain, improve the utilization value of inferior biomass resources and cut the
cost. Thus, in this project, we designed and synthesized an engineered S. cerevisiae cellular factory producing
2'-FL from sweet potato residues by fermentation. On one hand, producing 2'-FL in S. cerevisiae is a relatively safe
cellular factory to allow the large-scale fermentation. On the other hand, it is promising that using sweet potato
residues to further extend industrial chain, improve the utilization value of inferior biomass resources and cut the
cost.
2. General concept
Figure1. Cellular Factory ConceptFigure1. Cellular Factory Concept
Our team aims to produce 2'-FL in S. cerevisiae for providing the economic strategy. In terms of raw material, there
are two pathways to produce GDP-L-fucose from GDP-D-mannose (called de novo pathway) or L-fucose (called salvage
pathway). In the de novo pathway, the GDP-D-mannose converts 2'-FL and secreted into medium involving three enzymes
gmd, wcaG, and wbgL, as well as transporter lac12. Our team aims to produce 2'-FL in S. cerevisiae for providing the
economic strategy. In terms of raw material, there are two pathways to produce GDP-L-fucose from GDP-D-mannose
(called de novo pathway) or L-fucose (called salvage pathway). In the de novo pathway, the GDP-D-mannose converts
2'-FL and secreted into medium involving three enzymes gmd, wcaG, and wbgL, as well as transporter lac12.
In order to obtain the high yield of 2'-FL, we introduced the four
exogenous gene including Lac12, gmd, wcaG, and Wbgl into S. cerevisiae genome using CRISPER-cas9. As shown in
Figure1, expression of Lac12 (lactose permease) from Kluyveromyces lactis is a transporter to facilitate lactose
transporting out of the cytoplasm. Other three genes, gmd, wcaG and Wbgl, are encoding the important enzymes
involving the 2'-FL synthesis. Amongst these enzymes, gmd and wcaG are converted the abundant intracellular
GDP-mannose in S. cerevisiae into GDP-fucose. Further, 2'-FL can be produced from GDP-fucose by expression of Wbgl
and metabolization. In this work, we used sweet potato residues as main carbon source.
Figure2. The metabolic pathway of biosynthesis 2’-FL in S. cerevisiae.
The purple part represents the heterologous pathway gene
3. General experiment procedure
pHCas9-Nours plasmid serves as a DNA template and amplified PCR to obtain the XI-2 and X-3 site target gene
integration plasmid. The gmd, lac12, wbgl and wcaG gene fragments were amplified by PCR using the X-3-gmd-wcaG
plasmid and the XI-2-wbgL-lac12 plasmid as template.
The DNA fragments gmd-wcaG and wbgL-lac12, as well as XI-2 and X-3 integration plasmids were digested with NotI and
XhoI to form the cohesive ends, respectively. Then, the DNA fragments and vector were joined together by the ligase.
The recombinant plasmid transformed into the competent cells and verified by colony PCR and Sanger sequencing. After
that, the corrected gmd-wcaG and wbgL-lac12 DNA fragments, which were digested with NotI, and gRNA were introduced
into the yeast that already contains the Cas9 expression plasmid using lithium acetate transformation method. These
colonies were used colony PCR to verify whether the colony’s genome carried recombinant DNA fragments, and the
positive transformants further confirmed through Sanger sequencing.
Finally, the positive colony incubated in the shaker, enabling produce 2'-FL in YPD30L2 medium and sweet potato
mash. Finally, we could obtain high yield of 2'-FL in the engineered yeast.
4. Expected results
1. Successfully obtain two constructs: X-3-gmd-wcaG plasmid and the XI-2-wbgL-lac12 plasmid.
2. Produce 2'-FL in S. cerevisiae strain using the sweet potato as substrates.
2. Produce 2'-FL in S. cerevisiae strain using the sweet potato as substrates.
Reference
1. Chaturvedi, P., C.D. Warren, M. Altaye, et al., Fucosylated human milk oligosaccharides vary between individuals
and over the course of lactation.[J] Glycobiology, 2001. 11(5):365-372.
2. Mei, X., T.-H. Mu, and J.-J. Han, Composition and Physicochemical Properties of Dietary Fiber Extracted from Residues of 10 Varieties of Sweet Potato by a Sieving Method.[J] Journal of Agricultural and Food Chemistry, 2010. 58(12):7305-7310.
3. Conze, D.B., C.L. Kruger, J.M. Symonds, et al., Weighted analysis of 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 3′-sialyllactose, and 6′-sialyllactose concentrations in human milk.[J] Food and Chemical Toxicology, 2022. 163:112877.
4. Jung, S.-M., Y.-W. Chin, Y.-G. Lee, et al., Enhanced production of 2’-fucosyllactose from fucose by elimination of rhamnose isomerase and arabinose isomerase in engineered Escherichia coli.[J] Biotechnology and Bioengineering, 2019. 116(9):2412-2417.
5. Xu, M., X. Meng, W. Zhang, et al., Improved production of 2′-fucosyllactose in engineered Saccharomyces cerevisiae expressing a putative α-1, 2-fucosyltransferase from Bacillus cereus.[J] Microbial Cell Factories, 2021. 20(1):165.
6. Lee, J.W., S. Kwak, J.-J. Liu, et al., Enhanced 2′-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate.[J] Metabolic Engineering, 2020. 62:322-329.
7. Zhu, Y., L. Wan, W. Li, et al., Recent advances on 2′-fucosyllactose: physiological properties, applications, and production approaches.[J] Critical Reviews in Food Science and Nutrition, 2022. 62(8):2083-2092.
8. Lu, M., I. Mosleh, and A. Abbaspourrad, Engineered Microbial Routes for Human Milk Oligosaccharides Synthesis.[J] ACS Synthetic Biology, 2021. 10(5):923-938.
2. Mei, X., T.-H. Mu, and J.-J. Han, Composition and Physicochemical Properties of Dietary Fiber Extracted from Residues of 10 Varieties of Sweet Potato by a Sieving Method.[J] Journal of Agricultural and Food Chemistry, 2010. 58(12):7305-7310.
3. Conze, D.B., C.L. Kruger, J.M. Symonds, et al., Weighted analysis of 2′-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 3′-sialyllactose, and 6′-sialyllactose concentrations in human milk.[J] Food and Chemical Toxicology, 2022. 163:112877.
4. Jung, S.-M., Y.-W. Chin, Y.-G. Lee, et al., Enhanced production of 2’-fucosyllactose from fucose by elimination of rhamnose isomerase and arabinose isomerase in engineered Escherichia coli.[J] Biotechnology and Bioengineering, 2019. 116(9):2412-2417.
5. Xu, M., X. Meng, W. Zhang, et al., Improved production of 2′-fucosyllactose in engineered Saccharomyces cerevisiae expressing a putative α-1, 2-fucosyltransferase from Bacillus cereus.[J] Microbial Cell Factories, 2021. 20(1):165.
6. Lee, J.W., S. Kwak, J.-J. Liu, et al., Enhanced 2′-Fucosyllactose production by engineered Saccharomyces cerevisiae using xylose as a co-substrate.[J] Metabolic Engineering, 2020. 62:322-329.
7. Zhu, Y., L. Wan, W. Li, et al., Recent advances on 2′-fucosyllactose: physiological properties, applications, and production approaches.[J] Critical Reviews in Food Science and Nutrition, 2022. 62(8):2083-2092.
8. Lu, M., I. Mosleh, and A. Abbaspourrad, Engineered Microbial Routes for Human Milk Oligosaccharides Synthesis.[J] ACS Synthetic Biology, 2021. 10(5):923-938.