Difference between revisions of "Part:BBa K3753000"
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K3753000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3753000 SequenceAndFeatures</partinfo>
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==Contribution From NNU-China 2021==
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==Contribution From NJXDF-CHN 2022==
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'''Group''': [https://2022.igem.wiki/njxdf-chn/contribution]
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'''Group''': NJXDF-CHN
  
 
'''Author''': Yang Gu
 
'''Author''': Yang Gu
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'''Summary''': Identification of the capacity of to Y.lipolytica produce 2-PE in lipolytic yeast
 
'''Summary''': Identification of the capacity of to Y.lipolytica produce 2-PE in lipolytic yeast
  
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===Characterization from iGEM21-NNU-China===
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===Characterization from iGEM22-NJXDF-CHN===
  
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA, consisting of dSpCas9, CDA, and UGI. It was first registered in 2020. In order to test the editing efficiency of this composite part, we construct the dual plasmid system based on the (<partinfo>BBa_K3645011</partinfo>). We selected the cadA, maeA, and maeB genes as the testing sites, and the related pTarget plasmids were constructed. Results showed that the (<partinfo>BBa_K3645011</partinfo>) can successfully work in the BL21 (DE3), and the editing efficiency of single gene editing, double genes editing and triple genes editing can reach 85%, 56% and 25%, respectively (Fig. 1). These results provide references for future iGEM teams to choose gene-editing tools in E.coli.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Microbially produced 2‑Phenylethanol (2-PE) is mainly obtained by two routes, containing the de novo pathway from glucose and bioconversion from L-phenylalanine by the Ehrlich pathway. Compared to the multi-step pathway, the Ehrlich pathway provides more efficient synthesis of 2-PE In the Ehrlich pathway, the generation of L-phenylalanine by oxidative decarboxylation to phenylethylaldehyde is a key step in the high yield of 2-PE (Farhi et al. 2010) (Fig. 1). Petunia hybrida hybrid phenylacetaldehyde synthase (PAAS) was first registered in 2021, which had been shown to have functional activity in Saccharomyces cerevisiae BY4741. We introduced this gene into the genome of Y. lipolytica polf and tested it in shake flask fermentation with 4 g/L of L-phe. The experimental result showed that overexpression of PhPAAS <partinfo>BBa_K3753000</partinfo> does not affect the normal growth of Y. lipolytica. Simultaneously, there was a significant increase in the yield of 2-PE, reaching 957.35 mg/L (Fig. 1). These results provide references for future iGEM team to select suitable sources of PAAS.
 
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:'''Fig.1 The gene editing efficiency of the part of dCas9-CDA-UGI.'''  
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:'''Fig. 1. A. The key synthetic steps of the Ehrlich pathway. B. Overexpression of PhPAAS enhances 2-PE yield.'''  
 
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<p><b><h2>Reference</h2></b></p>
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<p>1. Farhi M, Lavie O, Masci T, Hendel-Rahmanim K, Weiss D, Abeliovich H, Vainstein A (2010) Identification of rose phenylacetaldehyde synthase by functional complementation in yeast. Plant Mol Biol 72(3):235-245.</p>
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3753000 parameters</partinfo>
 
<partinfo>BBa_K3753000 parameters</partinfo>
 
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Latest revision as of 01:36, 14 October 2022


PAAS-Petunia

Petunia hybrida hybrid phenylacetaldehyde synthase (PAAS) is capable of transforming L-phenylalanine (L-phe) into phenylacetaldehyde by oxidative decarboxylation. Subsequently, phenylacetaldehyde can be reducted into phenylethanol, which possesses elegant, fascinating and long-lasting fragrance.

Characterization


Fig.1 The growth curve of recombinant strains.


The figure above shows the changes of biomass concentration in WT, BY4741-pRS426-Petunia, BY4741-pRS426-Vanda, BY4741-pRS426-Rosa yeast over time. The OD600 of different strain cultures is measured at the designated time points (0h, 24h,48h,72h). The result shows that the growth trend of the recombinant strain is basically the same as the wild-type strain, indicating that the introduction of heterogeneous gene has no significant effect on the growth of yeast.

Fig.2 The 2-PE production of the recombinant strains.
After 72 hours of fermentation, 2-PE production of the wild-type of BY4741, pRS426-Petunia, pRS426-Vanda and pRS426-Rosa was tested by HPLC. Data represent means of triplicate culture±standard error.


Saccharomyces cerevisiae BY4741 contains Ehrlich pathway and other metabolic pathways to operate simultaneously to produce 2-PE, so the wild-type of BY4741 has a certain amount of 2-PE production (1.205g/L). After the introduction of heterogeneous paas gene, the 2-PE production has remarkablely increased. Among them, the production of 2-PE produced by the yeast strain which was introduced petunia-paas increased the most(1.570g/L), followed by the strain which was introduced vanda-paas (1.514g/L) BBa_K3753001 and rosa-paas (1.341g/L) BBa_K3753002.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 60
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 60
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 60
  • 1000
    COMPATIBLE WITH RFC[1000]

Contribution From NJXDF-CHN 2022

Group: NJXDF-CHN

Author: Yang Gu

Summary: Identification of the capacity of to Y.lipolytica produce 2-PE in lipolytic yeast

Characterization from iGEM22-NJXDF-CHN

        Microbially produced 2‑Phenylethanol (2-PE) is mainly obtained by two routes, containing the de novo pathway from glucose and bioconversion from L-phenylalanine by the Ehrlich pathway. Compared to the multi-step pathway, the Ehrlich pathway provides more efficient synthesis of 2-PE In the Ehrlich pathway, the generation of L-phenylalanine by oxidative decarboxylation to phenylethylaldehyde is a key step in the high yield of 2-PE (Farhi et al. 2010) (Fig. 1). Petunia hybrida hybrid phenylacetaldehyde synthase (PAAS) was first registered in 2021, which had been shown to have functional activity in Saccharomyces cerevisiae BY4741. We introduced this gene into the genome of Y. lipolytica polf and tested it in shake flask fermentation with 4 g/L of L-phe. The experimental result showed that overexpression of PhPAAS BBa_K3753000 does not affect the normal growth of Y. lipolytica. Simultaneously, there was a significant increase in the yield of 2-PE, reaching 957.35 mg/L (Fig. 1). These results provide references for future iGEM team to select suitable sources of PAAS.

Fig. 1. A. The key synthetic steps of the Ehrlich pathway. B. Overexpression of PhPAAS enhances 2-PE yield.

Reference

1. Farhi M, Lavie O, Masci T, Hendel-Rahmanim K, Weiss D, Abeliovich H, Vainstein A (2010) Identification of rose phenylacetaldehyde synthase by functional complementation in yeast. Plant Mol Biol 72(3):235-245.