Overexpression of PAD1 and FDC1 results in significant cinnamic acid decarboxylase activity in Saccharomyces cerevisiae
© Richard et al.; licensee Springer. 2015
Received: 7 January 2015
Accepted: 9 February 2015
Published: 18 February 2015
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.
Enzymes for the decarboxylation of hydroxycinnamic acids such as p-coumaric acid, caffeic acid or ferulic acid have been described. A ferulic acid decarboxylase and the corresponding gene, fdc, was identified from Bacillus pumilus (Zago et al. 1995). A ferulic acid decarboxylase was also identified from an Enterobacter species (Gu et al. 2011a) and a crystal structure obtained (Gu et al. 2011b). A p-coumaric acid decarboxylase was purified from Lactobacillus plantarum. The enzyme was inducible and the purified enzyme had a KM of 1.4 mM and a Kcat of 103 s−1(Cavin et al. 1997b). The corresponding gene, pdc, was cloned and overexpressed in E. coli (Cavin et al. 1997a). This protein was crystallised and a structure obtained (Rodríguez et al. 2010). Based on the homology to the fdc and pdc a hydroxycinnamic acid (phenolic acid) decarboxylase, pad, was identified in Bacillus subtilis (Cavin et al. 1998). These hydroxycinnamic acid decarboxylases were shown to be active in vivo and in vitro and the purified enzymes did not require cofactors and were not part of multi subunit enzyme complexes. All these enzymes have in common that they are of bacterial origin and not active with cinnamic acid.
The PAD1 and the FDC1 are clustered, i.e. they are located next to each other on the chromosomes. Such clusters with PAD1 and FDC1 homologues are widespread in yeast and filamentous fungi. In filamentous fungi the cluster contains also a transcription factor (Plumridge et al. 2010).
Cinnamic acid decarboxylase is required for the genetic engineering of styrene producing strains. Styrene can be produced from endogenous L-phenylalanine in two steps, by a phenylalanine ammonia lyase to produce cinnamic acid and a cinnamic acid decarboxylase producing styrene (McKenna and Nielsen 2011, McKenna et al. 2014). An efficient cinnamic acid decarboxylase would be desirable for the construction of strains for effective styrene production from biomass.
Materials and methods
Strains and plasmids
Strains: The yeast strain CEN.PK2-1C was obtained from Prof. Friedrich K. Zimmermann (Frankfurt) and used if not otherwise specified.
Table of plasmids used
PAD1 without targeting (MVAITG…)
PAD1 without targeting (MVVAITG…)
FDC1 with mitochondrial targeting
Enzyme activity measurement
To prepare a cell free yeast extract yeast cells were collected by centrifugation, washed with water and 1 ml of fresh cell cake was suspended in 1 ml 20 mM sodium phosphate buffer pH 7.0. 1 ml of glass beads (0.4 mm diameter, Sigma) and protease inhibitor (Complete, Roche) were added and the cells were disrupted in two 40s sessions in the Fast Prep (Bio101). The cell extract was centrifuged and the supernatant analysed for cinnamic acid decarboxylase activity. The protein concentration was measured using the Biorad Protein Assay and BSA as a standard. The extract was added to a solution of cinnamic acid in 20 mM sodium phosphate, pH 7.0, and incubated at room temperature. The reaction was quenched by heating the sample to 96°C for 10 minutes.
Cinnamic acid and hydroxy-cinnamic acids were determined by using an analytical UPLC method. The separation of analytes was carried out on an Acquity UPLC BEH C18 1.7 μm column (2.1 × 100 mm) with a Waters Acquity UPLC system including sample manager-FTN, Quaternary solvent manager and PDA eλ detector. Detector was operated on 210-400 nm. The solvents used in gradient elution 0.43 mL/min were A) 5% formic acid and B) acetonitrile. Gradient system was as followed: 0 min 95% A and 5% B; 1.13 min 90% A and 10% B; 5.67 min 60% A and 40% B; 9.00 min 10% A and 90% B; and 10.00 min 90% A and 10% B. On the basis of corresponding standards (0-1000 μM) compounds were quantified.
Analysis of styrene by SPME-GC/MS
Samples (300 μl) were analyzed by using SPME (solid phase micro extraction)-GC/MS. Extraction of styrene was done at 80°C for 30 min with preconditioned (300°C, 60 min) 75 μm Carboxen-PDMS fibre (Sulpelco, USA). After extraction the analytes were desorbed during 5 min at 250°C in the splitless injector (flow 14.9 ml/min) of the gas chromatography (Agilent 6890 Series; Palo Alto, CA, USA) combined with a MS detector (Agilent 5973Network MSD, USA) and SPME autosampler (Combipal, Varian Inc., USA). Analytes were separated on BPX5 capillary column of 60 m × 0.25 mm with a phase thickness 1.0 μm (SGE Analytical Science Pty Ltd, Australia). α-Pinene was used as internal standard. The temperature programme started at 60°C with 1 min holding time, then increased 7°C/min up to 100°C, followed by 10°C/min increase up to final temperature 200°C, where the temperature was kept for 4 min. MSD was operated in electron-impact mode at 70 eV, in the full scan m/z 40–550. The ion source temperature was 230°C and the interface was 280°C. Styrene was identified with corresponding standard and by comparing the mass spectra on Palisade Complete 600 K Mass Spectral Library (Palisade Mass Spectrometry, USA).
In vitro activity
In vivo activity
Growth on increasing cinnamic acid concentrations
Cinnamic acid concentration
The combination of PAD1 and FDC1 seemed to result in cinnamic acid decarboxylase activity although the proteins are targeted to different cellular compartments. We tested if the activity could be increased by targeting the proteins to the same cellular compartment. In one case we targeted both proteins to the mitochondria. For that purpose we added the mitochondrial targeting sequence of the PAD1 to the FDC1. In the other case we targeted both to the cytosol. For that we removed the targeting signal from the PAD1. In both cases the cell free extract of the yeast strain over-expressing both genes, PAD1 and FDC1 with added targeting sequence or FDC1 and PAD1 with removed targeting sequence, showed no cinnamic acid decarboxylase activity. Removing or adding mitochondrial targeting signals might affect the protein folding, the lack of activity is therefore not necessarily related to the targeting.
Activity with hydroxy-cinnamic acids
Although it is known that yeast and other microorganisms have the ability to convert cinnamic acid to styrene, the enzyme to catalyse this reaction has not been identified. This is mainly because most of the studies were carried in vivo and only a few used a cell free environment (Mukai et al. 2010, Stratford et al. 2012).
In S. cerevisiae the PAD1 was suggested to code for the cinnamic acid decarboxylase. A PAD1 mutant showed increased sensitivity toward cinnamic acid and expression of PAD1 repaired the phenotype (Clausen et al. 1994). In the same study permeabilised cells were used to measure cinnamic acid decarboxylase activity. The parent strain showed an activity of 0–3.9 nM/mg dry mass per h and after expression of PAD1 29.4 nM/mg dry mass per h (Clausen et al. 1994). Assuming that 40% of the dry mass is extractable protein this would correspond to about 75 nM per mg extracted protein and hour. When we overexpressed FDC1 and PAD1 and analysed the cell extract we estimated the decarboxylase activity to be about 1 mM per mg extracted protein and hour. This corresponds to an increase of activity of about 4 orders of magnitude.
The Pad1p and the Fdc1p are located in different compartments in S. cerevisiae. The Pad1p is in mitochondria and the Fdc1p in the cytosol. The fact that the overexpression of both proteins results in an increased resistance toward cinnamic acid can be interpreted in different ways. One interpretation is that cinnamic acid is converted to styrene in two steps with an intermediate that is passing through the mitochondrial membrane. Another interpretation is that the proteins are not confined to their compartment. However for the Pad1p it was shown that after tagging with the green fluorescence protein the enzyme was exclusively located in the mitochondria (Huh et al. 2003).
No information is available about a possible protein complex including Pad1p and Fdc1p. In an genome wide survey to identify protein complexes by systematic tagging of open reading frames to identify protein complexes, no protein complexes with Pad1p or Fdc1p were identified (Gavin et al. 2006).
The physiological role of cinnamic acid decarboxylation is detoxification. Cinnamic acid or cinnamate is more toxic than the reaction product styrene. Cinnamic acid decarboxylation is also of potential biotechnological relevance. L-Phenylalanine can be efficiently converted to cinnamic acid by the action of an L-phenylalanine ammonia lyase (PAL). PAL and cinnamic acid decarboxylase constitute a pathway for styrene production from L-phenylalanine. This had been practiced by McKenna et al. in E. coli to produce styrene from D-glucose (McKenna and Nielsen, 2011). In this work the PAD1 and the FDC1 of S. cerevisiae were expressed in E. coli but it was concluded that functional cinnamic acid decarboxylase activity in E. coli depends solely upon FDC1 over-expression and is not dependent on co-expression of PAD1. The expression of the S.cerevisiae PAD1 in E. coli did not result in cinnamic acid decarboxylation activity; however the combined expression of PAD1 and FDC1 resulted in such activity. The expression of FDC1 alone also resulted in activity suggesting that the PAD1 is not required for activity; however it cannot be excluded that an E. coli protein homologous to PAD1 would be functional (McKenna and Nielsen, 2011). There are indeed proteins in E. coli with homologies to the PAD1. One is the UbiX that has a role in coenzyme Q biosynthesis (Gulmezian et al. 2007). Another one is a close homologue, Pad1, that has been crystallized and a structure determined (Rangarajan et al. 2004), however no function was assigned.
This work was supported by the Academy of Finland through the Sustainable Energy (SusEn) programme (Grant 271025).
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