Revision as of 15:13, 12 October 2022

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.

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.

Figure 1: The designed metabolic pathways in S. cerevisiae in order to produce MAAs. We introduced the genes Xyl 1, Xyl 2, Xyl 3 into S. cerevisiae to increase the production of S7P. We knocked out the downstream genes TAL1 and Nqm1 to prevent turnover of S7P into undesired F6P.

Experiment

Figure 2: 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.

Figure 3: Growth curves of wild type S. cerevisiae, L1, L2, and L3 under 2% glucose + 0% xylose (A), 1% glucose + 1% xylose (B), 0.4% glucose + 1.6% xylose (C), 0% glucose + 2% xylose (D). WT represents wild type CEN.PK2 strain. L1 represents S. cerevisiae with TAL1 gene removed. L2 represents strains with Xyl 1, Xyl 2, Xyl 3 inserted into L1. L3 represents L2 strains with DDGS-OMT inserted.

Sequence and Features