Difference between revisions of "Part:BBa K4273001"
 
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<partinfo>BBa_K4274000 short</partinfo>
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<partinfo>BBa_K4273001 short</partinfo>
  
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<i>Cl</i>SS_S533A is an optimized biobrick part encoding the gene for alpha-santalene synthase from <i>Clausena lansium</i>. The enzyme catalyzes the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the alpha-santelene in a single step. It is reported that <i>Cl</i>SS's basic amino acid residue S533’s mutation to typical nonpolar amino acid Ala could result in a 1.7-fold increase in production of alpha-santalene compared to the nonmutated strain (Jia Z.et al., 2022) .
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xylose assimilation gene encoding xylose reductase(XR) that converts xylose to xylitol.
  
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<br>This year, we are using <i>Cl</i>SS_S533A to construct composite parts ptac-RiboJ-B0034-Cl</i>SS_S533A-B0034-ERG20-B0015 (part: BBa_K4274020), ptac-RiboJ-B0034-<i>Cl</i>SS_S533A-B0034-ERG20_F96W-B0015 (part: BBa_K4274021) and ptac-RiboJ-B0034-<i>Cl</i>SS_S533A-FL-ERG20_F96W-B0015 (part: BBa_K4274023). This will allow engineering <i>E.coli</i> DH5a (tnaA-) to heterologously express <i>Cl</i>SS_S533A. Other teams can utilize this part for <i>E. coli</i> alpha-santalene production.
 
  
 
==Usage and Biology==
 
==Usage and Biology==
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<i>Cl</i>SS_S533A is an optimized biobrick part encoding the gene for alpha-santalene synthase from Clausena lansium. It was firstly characterized by Jia Z.in 2022, who mutated <i>Cl</i>SS's basic amino acid residue S533 to increase the production of alpha-santalene in <i>E. coli</i> (Jia Z.et al., 2022).
 
  
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xylose assimilation gene encoding xylose reductase(XR), xylitol dehydrogenase(XDH), xylulokinase(XK) that converts xylose to xylulose-5-phosphate .                       
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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.
  
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Natural <i>Cl</i>SS is an alpha-santalene synthase which could catalyze the conversion of the common isoprenoid intermediate farnesyl pyrophosphate (FPP) into the alpha-santelene in a single step. It has been successfully heterologously expressed to produce functional terpene product in both yeast (Wenlong Z.et al., 2020) and <i>E. coli</i> (Jia Z.et al., 2022). But the mutation of residue S533 to Ala led to the addition of two hydrogen bonds near this site (<4 Å), which resulted in a high α-santalene production <i>E.coli</i> strain.
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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.
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In our study, <i>Cl</i>SS_S533A could be heterologously expressed in <i>E. coli</i>, and allows for fused proteins to be bound to ERG20 (Part: BBa_K849001) and ERG20_F96W (Part: BBa_K4274002)
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[[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.''']]
  
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==Source==
 
  
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<i>Scheffersomyces stipitis</i>
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==Experiment==
  
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==Characterization==
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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
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[[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. .''']]
  
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After engineering, <i>E. coli</i> could utilize both MEP pathway and MVA pathway for the universal precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), then synthesize santalene with the help of FPP Synthase (FPPS) and santalene synthase (SS). Except heterologously expressed MVA pathway and ERG20 of <i>Saccharomyces cerevisiae</i> and santalene synthase of <i>Clausena lansium</i> (<i>Cl</i>SS), several modifications upon ERG20 or <i>Cl</i>SS by amino acid mutation, binding to a hydrophillic tag and the construction of fusion protein were tested for the higher yield of santalene. Therefore, with the help of the co-transformation of pMVA plasmid with various pW1 plasmids, including pW1_CE, pW1_CEM, pW1_TCEM and pW1_CEM_FL, different strains like CE, CEM, TCEM, CEM_FL were successfully constructed (Figure 1). The complete pathway we designed for producing santalene in <i>E. coli</i> is illustrated in Figure 1.
 
  
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[[Image:Parts-keystone-santalene1.jpeg|thumbnail|750px|center|'''Figure 1:'''
 
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Construction and expression of santalene. (a) Enzymes and some of the reaction intermediates necessary for the production of santalene through the MEP pathway and MVA pathway. (b) Schematic representing the structure of pMVA, pW1_CE, pW1_CEM, pW1_TCEM and pW1_CEM_FL transformed into <i>E.coli</i> DH5α ∆TnaA. ]]
 
  
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<br>Afterwards, the various engineering of <i>E.coli</i> DH5α ∆TnaA mentioned above were used for santalene production. After rapid centrifugation, the supernatant of dodecane was spiked with with 0.475 g/L a-humulene as an internal standard, and then injected into GC/MS for verification of α-santalene production. It turned out that all samples from four strains appeared a significant peak at the retention time of 26-27 min, and various peak area of different samples exhibited santalene production with differing levels, indicating the general success of <i>E. coli</i> engineering. It can be concluded that the <i>E. coli</i> strain CEM (with pW1_CEM plasmid) produces the maximal level of α-santalene compared to other strains (73.93 mg/L). Furthermore, our study elucidates that the mutation of 96th amino acid into tryptophan could increase the yield of α-santalene by about 20%, substantiating the prominent performance of ERG20_F96W in enhancing the supply of FPP and α-santalene production in E. coli (Figure 2).  
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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.
  
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[[Image:]t-links-china-figure1.png|thumbnail|750px|center|'''Figure 1:'''
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[[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. ''']]
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Quantification analysis of α-santalene production. (a) Measurement santalene production of different strains by GC/MS results. (b) Quantification of α-santalene is analyzed with 0.475 g/L α-humulene as an internal standard. And the GC/MS results demonstrate that the peaks at the retention time of 26-27 min and 28-29 min respectively were α-santalene and α-humulene. (c) GC/MS results of samples originated from CE, CEM, TCEM and CEM-FL strains. ]]
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==Sequence and Features==
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<partinfo>BBa_K4274000 SequenceAndFeatures</partinfo>
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4273001 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K4274000 parameters</partinfo>
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<partinfo>BBa_K4273001 parameters</partinfo>
 
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==Reference==
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[1] Wenlong Z., Tianyue A., Ting L., et al. Reconstruction of the Biosynthetic Pathway of Santalols under Control of the GAL Regulatory System in Yeast. ACS Synth. Biol. 9 (2), 449-456 (2020). https://doi.org/10.1021/acssynbio.9b00479
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<b>Reference</b>
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<br>[2] Jia Z., Xun W., Xinyi Z., et al. Sesquiterpene Synthase Engineering and Targeted Engineering of α-Santalene Overproduction in Escherichia coli. J. Agric. Food Chem. 70 (17), 5377-5385 (2022). https://doi.org/10.1021/acs.jafc.2c00754
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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.  
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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.  
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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.  
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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.
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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.
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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.  
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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.

Latest revision as of 09:40, 13 October 2022

xyl1

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 89
    Illegal BglII site found at 478
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 109
  • 1000
    COMPATIBLE WITH RFC[1000]



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.