Difference between revisions of "Part:BBa K4273001"

Revision as of 15:25, 12 October 2022

NOTOC__ pTDH3-DDGS-tTDH1-pPGK1-OMT-tPGK1

xylose assimilation gene encoding xylose reductase(XR) that converts xylose to xylitol.

Usage and Biology

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.

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

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

Figure 2: Insertion of Xyl 1, Xyl 2, Xyl 3 to introduce a xylose-utilizing metabolic pathway. .

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 3423
Illegal XhoI site found at 2075
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 2321
Illegal BsaI.rc site found at 3433

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 <partinfo>BBa_K4273017 short</partinfo>
-Xyl1 is a xylose assimilation gene encoding xylose reductase (XR) that can converts xylose to xylitol. Xyl1 can provides an alternative carbon source for the pentose phosphate pathway that can increase S7P production.  We selected Xyl1 genes from Scheffersomyces stipitis that 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 including the promoter, coding sequence, and terminator. Xyl1 is under the control of promoter pTDH3 which provides a high and stable transcription efficiency (Apel, et. al, 2016). Then, we used PCR amplification to obtain the DNA fragment Xyl1 from Level1 plasmid, and two homology arms (LA and RA) from yeast genome.
+xylose assimilation gene encoding xylose reductase(XR) that converts xylose to xylitol.
 ==Usage and Biology==
-We selected promoters pTDH3 and pPGK1 due to their stability expression in S. cerevisiae (Apel et. al., 2016). These promoters are shown to have stable and strong expression in YPD culture mediums. pTDH3 has highest stability and strength, followed by pPGK1. Therefore, we used pTDH3 for DDGS and pPGK1 for OMT. By introducing another copy of DDGS-OMT at position Nqm1 and inserting the genes into S. cerevisiae's genome, we were able to increase the production of shinorine and porphyra-334 to a great extent.
+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.
+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==
+[[Image:t-links-china-figure211.png|thumb|right|900px|'''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.''']]
-<b>Characterization</b>
-<br>
-For our experiment, we used the strong promoters such as pTDH3 for DDGS and pPGK1 for OMT when we tried to convert S7P to 4DG efficiently, and selected DDGS(NpR5600) and OMT(NpR5599) for homologous recombination into SC.L2 genome.  Moreover, we chosed position 308 from chromosome III (Apel et. al., 2016) for genome recombinant. By using this method, we transformed pCRCT-308 plasmid, the DNA fragments of homologous arms, DDGS, and OMT into SC.L2. The recombinant strains were identified by PCR and sequencing (Fig.4C and D), demonstrating that SC.L3 was obtained in the experiment.
-[[Image:t-links-china-figure100.png|thumb|left|900px|'''Figure 1: Insertion of DDGS and OMT at 308 position. By transforming CRISPR-308 plasmid pCRCT-308, LA, DDGS, OMT and RA, the two genes should be inserted at 308 position (A). We expanded the homogenous arms, DDGS and OMT genes through PCR and transformed them into L2 strains for it to be assembled in the genome. We performed colony PCR on the yeast colonies to determine the existence of LA-DDGS and OMT (C) and verified this result through the sequencing testing (D), obtaining the L3 strain.''']]
+==Experiment==
-<br>
+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
-<b>Optimization</b>
+[[Image:t-links-china-figure213.png|thumb|right|900px|'''Figure 2: Insertion of Xyl 1, Xyl 2, Xyl 3 to introduce a xylose-utilizing metabolic pathway. .''']]
-<br>
-To increase the quantity of MAAs production, we further optimized the SC.L3 strains. We notice that Nqm1 has similar functions as TAL1 in shunting S7P into glycolytic pathway. So, to increase the S7P pool, we decided to remove this gene and insert an extra copy of DDGS-OMT simultaneously. The result shows that gene expression can be enhanced by using multiple promoters for increasing MAAs production (Yang et. al., 2018). For modification, pTDH3 was used to express OMT and pPGK1 was used to express DDGS, making the total transcription rate of the two copie roughly equal. Then, we inserted DNA fragments of LA, OMT, DDGS, RA and the pCRCT-Nqm1 plasmid into SC.L3. After PCR, DNA sequencing, and selection of recombinant colonies, we removed the pCRCT-Nqm1 plasmid, obtaining SC.L6 in the end.
-[[Image:t-links-china-figure101.png|thumb|left|900px|'''Figure 2: Optimizing production by inserting OMT-DDGS and deleting Nqm1 gene. Nqm1 function similarly as TAL1, which reduces S7P concentrations. By transforming CRISPR-Nqm1 plasmid pCRCT-Nqm1, LA, OMT, DDGS, and RA, the two genes should be inserted at Nqm1 position; we switched the promoter of DDGS to pPGK1 and OMT to pTDH3 in order to have equal net transcription rate of DDGS and OMT genes. We expanded the homogenous arms, OMT, DDGS genes through PCR and transformed them into L5 strains for it to be assembled in the genome. We performed colony PCR on the yeast colonies to determine the existence of LA-OMT, and DDGS (C) and verified this result through the sequencing testing (D), obtaining the L6 strain.''']]
-<br>
-<b>For the production of  shinorine and porphyra-334</b>
-The production of shinorine and porphyra-334 as our main experimental goal after S7P is converted to 4DG by DDGS and OMT. Shinorine and porphyra-334 are produced by ATP-grasp ligase (AGL) and D-Ala-D-Ala ligase (ALAL) with two enzymatic steps. First, 4DG is converted to mycosporine-glycine(MG) by conjugating glycine to 4DG under the action of AGL. Then, another amino acid is attached to MG by AGL to produce shinorine or porphyra-334, and L-serine for shinorine and L-threonine for porphyra-334. However, due to the fact that there are variety types of AGL and ALAL with different efficiency and amino acid preference in the enviromnment, we selected ligases from three different marine organisms: Nostoc punctiform(Np5598 and Np5597), Nostoc linckia(NlmysC and NImysD) and Actinosynnema mirum(Am4257 and Am4256, expecting to create nine combinations of AGL-AlaL. For results, we found that NlmysD has a strong selective preference toward the amino acid Threonine. Thus, it will mainly produce porphyra-334 if both Threonine and Serine are present in the environment, marking our first successful case of producing only 334. Np5597, on the other hand, shows shinorine's absorption peak, meaning it has preference toward serine.
+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.
-[[Image:t-links-china-figure52.png|thumb|left|900px|'''Figure 3: Shinorine and porphyra-334 production after optimization. We transformed porphyra-334 producing plasmid Np5598-NlmysD and shinorine producing plasmid Np5598-Np5597 into the L3 and L6 strains (A) and compared the absorption spectrum after 72 hours of fermentation. The absorption peak at 334nm of L7 strains displayed significant improvement compared to L5 strains (B) . From OD334, we concluded that in comparison with the L5 assembly, porphyra-334 production in L6 yeast increased by 91.8%, and shinorine production increased by 70.9% (C).''']]
+[[Image:t-links-china-figure212.png|thumb|right|900px|'''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. ''']]