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Overexpression of PAD1 and FDC1 results in significant cinnamic acid decarboxylase activity in Saccharomyces cerevisiae.

Richard P, Viljanen K, Penttilä M - AMB Express (2015)

Bottom Line: This was suggested for the reason that the over-expression of PAD1 resulted in increased tolerance toward cinnamic acid, up to 0.6 mM.The overexpression of PAD1 and FDC1 resulted also in increased activity with the hydroxycinnamic acids ferulic acid, p-coumaric acid and caffeinic acid.Styrene can be produced from endogenously produced L-phenylalanine which is converted by a phenylalanine ammonia lyase to cinnamic acid and then by a decarboxylase to styrene.

View Article: PubMed Central - PubMed

Affiliation: VTT Technical Research Centre of Finland Ltd, Tietotie 2, P.O. box 1000, 02044 VTT Espoo, Finland.

ABSTRACT
The S. cerevisiae PAD1 gene had been suggested to code for a cinnamic acid decarboxylase, converting trans-cinnamic acid to styrene. This was suggested for the reason that the over-expression of PAD1 resulted in increased tolerance toward cinnamic acid, up to 0.6 mM. We show that by over-expression of the PAD1 together with the FDC1 the cinnamic acid decarboxylase activity can be increased significantly. The strain over-expressing PAD1 and FDC1 tolerated cinnamic acid concentrations up to 10 mM. The cooperation of Pad1p and Fdc1p is surprising since the PAD1 has a mitochondrial targeting sequence and the FDC1 codes for a cytosolic protein. The cinnamic acid decarboxylase activity was also seen in the cell free extract. The activity was 0.019 μmol per minute and mg of extracted protein. The overexpression of PAD1 and FDC1 resulted also in increased activity with the hydroxycinnamic acids ferulic acid, p-coumaric acid and caffeinic acid. This activity was not seen when FDC1 was overexpressed alone. An efficient cinnamic acid decarboxylase is valuable for the genetic engineering of yeast strains producing styrene. Styrene can be produced from endogenously produced L-phenylalanine which is converted by a phenylalanine ammonia lyase to cinnamic acid and then by a decarboxylase to styrene.

No MeSH data available.


Related in: MedlinePlus

Cinnamic acid decarboxylation with a cell free extract of a yeast strain over-expressing PAD1 and FDC. The protein concentration is 0.32g/l.
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Fig2: Cinnamic acid decarboxylation with a cell free extract of a yeast strain over-expressing PAD1 and FDC. The protein concentration is 0.32g/l.

Mentions: It was previously shown that the PAD1 is responsible for cinnamic acid dehydratase activity and that the overexpression of PAD1 results in increased cinnamic acid dehydratase activity (Clausen et al. 1994, Larsson et al. 2001) using intact cells of S. cerevisiae. In order to test if this activity could be also seen in cell free extracts we analysed the cell free extracts of the CEN.PK strains which were not modified or expressed the PAD1 gene. The PAD1 gene was amplified from the S. cerevisiae strain S288C since the CEN.PK has a mutation in the PAD1 gene resulting in a stop codon instead of the tyrosine in position 98. The cell extracts were incubated with 500 μM cinnamic acid at neutral pH and the disappearance of cinnamic acid was followed by UPLC, however no decarboxylase activity was detected. We followed the decrease in cinnamic acid over a period of 5 hours at a protein concentration of 2 g/l but could not detect a decrease. The detection limit for the decrease in concentration was estimated 15 μM. We also tested if the overexpression of FDC1 would result cinnamic acid decarboxylase activity in the cell free extract however no activity was detected. However when the two cell-free extracts with PAD1 and the extracts with FDC1 were mixed, cinnamic acid decarboxylase activity was detected. After 5 hours the cinnamic acid concentration was decreased by 250 μM. This suggested that both enzymes, Pad1p and Fdc1p, are required. We then expressed PAD1 and FDC1 in the same yeast strain using two multicopy plasmids. This extract had a much higher cinnamic acid decarboxylase activity. This is summarized in the Figure 2. The initial rate was about 0,019 μmol per minute and mg of extracted protein. We also confirmed that the reaction product was styrene.Figure 2


Overexpression of PAD1 and FDC1 results in significant cinnamic acid decarboxylase activity in Saccharomyces cerevisiae.

Richard P, Viljanen K, Penttilä M - AMB Express (2015)

Cinnamic acid decarboxylation with a cell free extract of a yeast strain over-expressing PAD1 and FDC. The protein concentration is 0.32g/l.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4384992&req=5

Fig2: Cinnamic acid decarboxylation with a cell free extract of a yeast strain over-expressing PAD1 and FDC. The protein concentration is 0.32g/l.
Mentions: It was previously shown that the PAD1 is responsible for cinnamic acid dehydratase activity and that the overexpression of PAD1 results in increased cinnamic acid dehydratase activity (Clausen et al. 1994, Larsson et al. 2001) using intact cells of S. cerevisiae. In order to test if this activity could be also seen in cell free extracts we analysed the cell free extracts of the CEN.PK strains which were not modified or expressed the PAD1 gene. The PAD1 gene was amplified from the S. cerevisiae strain S288C since the CEN.PK has a mutation in the PAD1 gene resulting in a stop codon instead of the tyrosine in position 98. The cell extracts were incubated with 500 μM cinnamic acid at neutral pH and the disappearance of cinnamic acid was followed by UPLC, however no decarboxylase activity was detected. We followed the decrease in cinnamic acid over a period of 5 hours at a protein concentration of 2 g/l but could not detect a decrease. The detection limit for the decrease in concentration was estimated 15 μM. We also tested if the overexpression of FDC1 would result cinnamic acid decarboxylase activity in the cell free extract however no activity was detected. However when the two cell-free extracts with PAD1 and the extracts with FDC1 were mixed, cinnamic acid decarboxylase activity was detected. After 5 hours the cinnamic acid concentration was decreased by 250 μM. This suggested that both enzymes, Pad1p and Fdc1p, are required. We then expressed PAD1 and FDC1 in the same yeast strain using two multicopy plasmids. This extract had a much higher cinnamic acid decarboxylase activity. This is summarized in the Figure 2. The initial rate was about 0,019 μmol per minute and mg of extracted protein. We also confirmed that the reaction product was styrene.Figure 2

Bottom Line: This was suggested for the reason that the over-expression of PAD1 resulted in increased tolerance toward cinnamic acid, up to 0.6 mM.The overexpression of PAD1 and FDC1 resulted also in increased activity with the hydroxycinnamic acids ferulic acid, p-coumaric acid and caffeinic acid.Styrene can be produced from endogenously produced L-phenylalanine which is converted by a phenylalanine ammonia lyase to cinnamic acid and then by a decarboxylase to styrene.

View Article: PubMed Central - PubMed

Affiliation: VTT Technical Research Centre of Finland Ltd, Tietotie 2, P.O. box 1000, 02044 VTT Espoo, Finland.

ABSTRACT
The S. cerevisiae PAD1 gene had been suggested to code for a cinnamic acid decarboxylase, converting trans-cinnamic acid to styrene. This was suggested for the reason that the over-expression of PAD1 resulted in increased tolerance toward cinnamic acid, up to 0.6 mM. We show that by over-expression of the PAD1 together with the FDC1 the cinnamic acid decarboxylase activity can be increased significantly. The strain over-expressing PAD1 and FDC1 tolerated cinnamic acid concentrations up to 10 mM. The cooperation of Pad1p and Fdc1p is surprising since the PAD1 has a mitochondrial targeting sequence and the FDC1 codes for a cytosolic protein. The cinnamic acid decarboxylase activity was also seen in the cell free extract. The activity was 0.019 μmol per minute and mg of extracted protein. The overexpression of PAD1 and FDC1 resulted also in increased activity with the hydroxycinnamic acids ferulic acid, p-coumaric acid and caffeinic acid. This activity was not seen when FDC1 was overexpressed alone. An efficient cinnamic acid decarboxylase is valuable for the genetic engineering of yeast strains producing styrene. Styrene can be produced from endogenously produced L-phenylalanine which is converted by a phenylalanine ammonia lyase to cinnamic acid and then by a decarboxylase to styrene.

No MeSH data available.


Related in: MedlinePlus