Part:BBa_K3753000

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_K4297067) 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.