Part:BBa_K4450006
UDP-N-acetylglucosamine synthesis
UDP-N-acetylglucosamine synthesis cluster
Profile
Name: Hexosamine biosynthesis
Base Pairs: 5881 bp
Origin: Saccharomyces Cerevisiae ATCC 201388
Properties: Gene cluster of enzymes responsible for UDP-acetylglucosamine biosynthesis
Usage and Biology
UDP-N-acetylglucosamine (UDP-GlcNAc) is a nucleotide sugar and coenzyme in metabolism. N-acetylglucosamine (GlcNAc) is the monomer forming chitin and UDP-GlcNAc is the precursor to chitin. Chitin is the second most abundant polysaccharide after cellulose [1], a structurally similar polysaccharide. UDP-GlcNAc is synthesized by the hexosamine biosynthesis pathway (HBP), a pathway consistent of conserved enzymes, albeit mediated through different intermediate compounds in prokaryotes and eukaryotes, found in most living organisms [2, 3]. Despite the enzymes existing in most bacteria, taking part in synthesis of peptidoglycan, the expression is significantly lower than in yeasts, which depend on this pathway to produce chitin, a component of its cell wall [4].
The primary motivation to upregulate the expression of hexosamine biosynthesis enzymes is to produce a more biocompatible polymer then cellulose alone with specific applications in medicine and/or applications where high crystalline cellulose has a disadvantage as outlined more clearly by Yadav, et al and Helenius, et al [5, 6]. It has been proven that Komagataeibacter can incorporate amino-sugars in its endogenous cellulose synthase, creating a lysozyme-susceptible copolymer [7, 8].
Figure 1: Overview of hexosamine biosynthesis pathway and upstream glycolysis in yeast. Reused from a publication by Raimi, et al. in Royal Society of Chemistry licensed under CC BY 3.0 [9].
Figure 1 is a overview of the hexosamine pathway. There are two entry points for substrate to enter into biosynthesis UDP-GlcNAc. The first entry point is diffusion of a Glucosamine (GlcN) across the cell membrane from the extracellular space. This entry of UDP-GlcNAc biosynthesis by extracellular Glucosamine has been studied with limited success [7, 10]. This in turn limits the incorporation of non Glucose (Glc) monomers by cellulose synthesis in Komagataeibacter as hypothesized and demonstrated by Yadav et al. [5]. They focused on this route of entry by expression of relevant enzymes from Candida albicans and observed an 18-fold increase in GlcNAc vs Glc monomers in their modified cellulose with the presence of extracellular GlcN. The other entry of substrate into UDP-GlcNAc biosynthesis is directly from glycolysis. Fructose-6-phosphate enters into biosynthesis of UDP-GlcNAc transformed by four different enzymes: GFA1, GNA1, PCM1/AGM1 and QRI1/UAP1. This is the pathway that CellulALT is modifying to improve the incorporation of GlcNAc vs Glc monomers by synthesis of UDP-GlcNAc from glucose directly. To increase the concentration of intracellular UDP-GlcNAc, genes related to hexosamine biosynthesis GFA1, GNA1, PCM1/AGM1 and QRI1/UAP1 are transformed into the mutant from Saccharomyces cerevisiae.
Figure 2: Vector map of gene cluster in pUC19 plasmid.
The genes are extracted by PCR using primers from genomic DNA in S. cerevisiae. In our implementation we choose to use the pUC19 plasmid as the vector used to assemble the gene cluster to transform Escherichia coli DH5α by heat shock transformation to create an intermediate host. This cluster is then extracted and inserted into a new vector pBBR to transform the K. Xylinus using electroporation.
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 1034
Illegal SpeI site found at 5782 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal SpeI site found at 5782 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 368
Illegal BglII site found at 1031
Illegal BglII site found at 4268
Illegal BamHI site found at 4799 - 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 1034
Illegal SpeI site found at 5782 - 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 1034
Illegal SpeI site found at 5782
Illegal AgeI site found at 173
Illegal AgeI site found at 913 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 5705
Illegal SapI site found at 3371
Illegal SapI.rc site found at 1245
Characterization
Using primers found under the design tab in the basic parts, we managed to amplify and purify (by gel extraction) the target genes from S. cerevisiae. Note here that the total amplification of GFA1 was much less then the other genes, which is apparent in figure 3.
Figure 3: Gel electrophoresis of PCR from S. cerevisiae genomic DNA with GFA1, GNA1, PCM1/AGM1, and QRI1/UAP1 primer pairs.
References
[1] Elieh-Ali-Komi, & Hamblin, M. R. (2016). Chitin and Chitosan: Production and Application of Versatile Biomedical Nanomaterials. International Journal of Advanced Research (Indore), 4(3), 411–427.
[2] Lockhart, Stanley, M., Raimi, O. G., Robinson, D. A., Boldovjakova, D., Squair, D. R., Ferenbach, A. T., Fang, W., & van Aalten, D. M. F. (2020). Targeting a critical step in fungal hexosamine biosynthesis. The Journal of Biological Chemistry, 295(26), 8678–8691. https://doi.org/10.1074/jbc.RA120.012985
[3] Li, Kang, J., Yu, W., Zhou, Y., Zhang, W., Xin, Y., & Ma, Y. (2012). Identification of M. tuberculosis Rv3441c and M. smegmatis MSMEG_1556 and essentiality of M. smegmatis MSMEG_1556. PloS One, 7(8), e42769–e42769. https://doi.org/10.1371/journal.pone.0042769
[4] Brown, Esher, S. K., & Alspaugh, J. A. (2019). Chitin: A “Hidden Figure” in the Fungal Cell Wall. Current Topics in Microbiology and Immunology, 425, 83–111. https://doi.org/10.1007/82_2019_184
[5] Yadav, Paniliatis, B. J., Shi, H., Lee, K., Cebe, P., & Kaplan, D. L. (2010). Novel In Vivo-Degradable Cellulose-Chitin Copolymer from Metabolically Engineered Gluconacetobacter xylinus. Applied and Environmental Microbiology, 76(18), 6257–6265. https://doi.org/10.1128/AEM.00698-10
[6] Helenius, Bäckdahl, H., Bodin, A., Nannmark, U., Gatenholm, P., & Risberg, B. (2006). In vivo biocompatibility of bacterial cellulose. Journal of Biomedical Materials Research. Part A, 76A(2), 431–438. https://doi.org/10.1002/jbm.a.30570
[7] Shirai, Takahashi, M., Kaneko, H., Nishimura, S.-I., Ogawa, M., Nishi, N., & Tokura, S. (1994). Biosynthesis of a novel polysaccharide by Acetobacter xylinum. International Journal of Biological Macromolecules, 16(6), 297–300. https://doi.org/10.1016/0141-8130(94)90059-0
[8] Ogawa, Miura, Y., Tokura, S., & Koriyama, T. (1992). Susceptibilities of bacterial cellulose containing N-acetylglucosamine residues for cellulolytic and chitinolytic enzymes. International Journal of Biological Macromolecules, 14(6), 343–347. https://doi.org/10.1016/S0141-8130(05)80076-5
[9] Raimi, Hurtado-Guerrero, R., Borodkin, V., Ferenbach, A., Urbaniak, M. D., Ferguson, M. A. J., & van Aalten, D. M. F. (2020). A mechanism-inspired UDP- N -acetylglucosamine pyrophosphorylase inhibitor. RSC Chemical Biology, 1(1), 13–25. https://doi.org/10.1039/C9CB00017H
[10] Jin W. Lee, Fang Deng, Walter G. Yeomans, Alfred L. Allen, Richard A. Gross, & David L. Kaplan. (2001). Direct Incorporation of Glucosamine andN-Acetylglucosamine into Exopolymers byGluconacetobacter xylinus (=Acetobacter xylinum) ATCC 10245: Production of Chitosan-Cellulose and Chitin-Cellulose Exopolymers. Applied and Environmental Microbiology, 67(9), 3970. https://doi.org/10.1128/AEM.67.9.3970-3975.2001
Functional Parameters
| None |