Part:BBa_K4273016
pTDH3-xyl1-tTDH1-pPGK1-xyl2-tPGK1-pTEF2-xyl3-tSSA1
xylose assimilation gene encoding xylose reductase(XR), xylitol dehydrogenase(XDH), xylulokinase(XK) that converts xylose to xylulose-5-phosphate . The genes Xyl1, Xyl2, and Xyl3 introduces a xylose-utilizing metabolic pathway to provide additional carbon source for the production of S7P. Xyl3 encodes for xylulosekinase which converts xylulose into xylulose-5-phosphate. Xylulose-5-phosphate could then be converted into S7P through the endogenous penta-phosphate pathway.
We create a collection including BBa_K4273016, BBa_K4273017, BBa_K4273018, BBa_K4273019, BBa_K4273020,
BBa_K4273021 and BBa_K4273000. This collection can provide inspiration and efficient methods to utilize the penta phosphate pathway or to produce other types of MAAs in S. cerevisiae for other teams.
Usage and Biology
Xyl1, Xyl2, and Xyl3 genes from Scheffersomyces stipitis proved to be successfully heterologous expressed for efficiently xylose-fermenting in S. Cerevisiae. We used yeast toolkit (Lee, 2015) to assemble the Level 1 plasmid concluding the promoter, coding sequence, and terminator. Xyl1, Xyl2, and Xyl3 are under the control of promoters pTDH3, pPGK1, and pTEF2 respectively which have a high and stable transcription efficiency (Apel, et. al, 2016).Then we used PCR amplification to obtain the DNA fragment Xyl1, Xly2, Xly3 from Level1 plasmid, and two homology arm(LA and RA)from yeast genome(Fig.3B). The five fragments are transformed into the SC.L1 strain along with a pCRCT-His3 plasmid that will cut open the yeast genome at the His 3 position. The recombinant strains were identified by pcr and sequencing(Fig.3C and D), and the pCRCT plasmid was discarded using URA nutrient deficient medium. We name the strain with Xyl 1, 2, 3 inserted at His3 position SC.L2.
Experiment
Insertion of Xyl 1, Xyl 2, Xyl 3 to introduce a xylose-utilizing metabolic pathway. By transforming CRISPR-His3 plasmid pCRCT-His3, His3 Left Arm (LA), xyl1, xyl2, xyl3, and His3 Right Arm (RA), the three genes are inserted at His3 loci (A). We expanded the homogenous arms, xyl1, xyl2, and xyl3 genes through PCR and transformed them into L1 strains for it to be assembled in the genome. We performed colony PCR on the yeast colonies to determine the existence of LA-xyl1, xyl2, and xyl3-RA (C) and verified this result through the sequencing testing (D), obtaining the L2 strain
To verify the previous engineering on yeast strains works, four strains(Wild Type, SC.L1, SC.L2, and SC.L3) were cultivated in four different media containing a different ratio of glucose to xylose. We found that in the medium containing only glucose(2%, 20 g/L), the growth curves of the 4 strains are roughly identical(Fig.5A), implying that our modification had no impact on the ability of cells to use glucose for growth when glucose is abundant. In the medium containing 1% glucose and 1% xylose, SC. L2 and SC.L3 containing Xyl1/2/3 showed a slight growth advantage compared with WT and SC.L1, which suggests xylose may be used as a potential carbon source for cell growth in yeast. As the concentration of glucose decreased and xylose increased further, the cell growth of all four strains is inhibited, especially SC.L3 and SC.L4(Fig. 5C and D). This result indicates most xylose is converted into S7P for MAAs production instead of being used for growth when xylose is abound and the xylose-utilizing pathway is introduced. Furthermore, the conversion of xylose into S7P requires the usage of ATP and NADPH, which might also affect growth. Therefore, we concluded that we need to supply xylose to generate S7P and maintain certain levels of glucose as the carbon source for cell growth. We chose the media containing 1% glucose and 1% xylose for later yeast fermentation.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 770
Illegal BglII site found at 1159
Illegal BglII site found at 2994
Illegal BglII site found at 5119
Illegal XhoI site found at 1646
Illegal XhoI site found at 6620 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 790
Illegal AgeI site found at 3123
Illegal AgeI site found at 3699
Illegal AgeI site found at 5167 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1892
Illegal BsaI site found at 6606
Illegal BsaI.rc site found at 3004
Illegal BsaI.rc site found at 6856
Illegal SapI site found at 4764
Illegal SapI.rc site found at 5434
Reference
Park SH, Lee K, Jang JW, Hahn JS. Metabolic Engineering of Saccharomyces cerevisiae for Production of Shinorine, a Sunscreen Material, from Xylose. ACS Synth Biol. 2019;8(2):346-357.
Jin C, Kim S, Moon S, Jin H, Hahn JS. Efficient production of shinorine, a natural sunscreen material, from glucose and xylose by deleting HXK2 encoding hexokinase in Saccharomyces cerevisiae. FEMS Yeast Res. 2021;21(7):foab053.
Chen M, Rubin GM, Jiang G, Raad Z, Ding Y. Biosynthesis and Heterologous Production of Mycosporine-Like Amino Acid Palythines. J Org Chem. 2021 Aug 20;86(16):11160-11168.
Osborn AR, Almabruk KH, Holzwarth G, Asamizu S, LaDu J, Kean KM, Karplus PA, Tanguay RL, Bakalinsky AT, Mahmud T. De novo synthesis of a sunscreen compound in vertebrates. Elife. 2015 May 12;4:e05919.
Reider Apel A, d'Espaux L, Wehrs M, Sachs D, Li RA, Tong GJ, Garber M, Nnadi O, Zhuang W, Hillson NJ, Keasling JD, Mukhopadhyay A. A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae. Nucleic Acids Res. 2017 Jan 9;45(1):496-508.
Zhang H, Jiang Y, Zhou C, Chen Y, Yu G, Zheng L, Guan H, Li R. Occurrence of Mycosporine-like Amino Acids (MAAs) from the Bloom-Forming Cyanobacteria Aphanizomenon Strains. Molecules. 2022 Mar 7;27(5):1734.
Cress BF, Toparlak ÖD, Guleria S, et al. CRISPathBrick: Modular Combinatorial Assembly of Type II-A CRISPR Arrays for dCas9-Mediated Multiplex Transcriptional Repression in E. coli. ACS Synth Biol. 2015;4(9):987-1000.
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