Figure 1.
Localization of PsaM in PSI and schematic drawing of the thylakoid in the engineered strain.
A) and B) The crystal structure of a monomer of photosystem I from the cyanobacterium Thermosynechococcus elongatus seen from the cytoplasmic side of the thylakoid membrane (A) and from the trimer-facing side of the monomer (B). RCSB Protein Data Bank ID: 1JB0. The PsaM subunit is shown in green in both panels and additionally indicated by an arrow in panel A. C) Schematic representation of the PsaM-CYP79A1 fusion protein in the thylakoid membrane.
Table 1.
Sequences of oligonucleotide primers used in this study.
Figure 2.
Gene construct and verification of transformation.
A) Schematic representation of binding sites for primers used for colony PCR (primer pair d, subpanel B) and RT-PCR (primer pair e and f, subpanel C) in the transformed Synechococcus sp. PCC 7002 genomic DNA. The black lines represent the gDNA that is not part of the cloning construct. The same forward primer is used in primer pair e and f. The primers are shown in Table 1. B) Colony PCR analysis of WT and transformed Synechococcus sp. PCC 7002 colonies performed using primer pair d. The sizes of the bands correlate well with the expected sizes of 4841 bp (PsaM-CYP79A1 strain) and 2344 bp (WT) indicating that the fusion construct has replaced the native psaM gene in the transformed strain. C) RT-PCR analysis of the transcription of the psaM-CYP79A1 fusion gene. The sizes of the fragments fit the expected 77 bp for primer pair e (expected in both WT and PsaM-CYP79A1) and 142 bp for primer pair f (only expected for PsaM-CYP79A1). D) Immunoblot of WT and transformant thylakoid (proteins corresponding to 10 µg Chl) and trimeric PSI (proteins corresponding to 50 µg Chl) samples with an anti-CYP79A1 antibody. C+: CYP79A1 expressed in chloroplasts of tobacco. The expected molecular masses of the CYP79A1 with its native membrane anchor (C+) and the PsaM-CYP79A1 are ∼62 kDa and ∼61 kDa, respectively, deduced from the corresponding DNA sequences.
Figure 3.
Localization of the PsaM-CYP79A1 fusion protein in the thylakoids.
A) Immunoblot of proteins from 80 µL of the 0.5 mL fractions collected from the PsaM-CYP79A1 sucrose gradient shown in panel B), concentrated through acetone precipitation. Antibodies against the PSI subunit PsaC (∼9 kDa), the PSII subunit PsbA (∼32 kDa) and the CYP79A1 (∼61 kDa) have been used. CYP79A1, d.e.: digitally enhanced representation of the chemiluminescence signal detected from the CYP79A1 bands (shown below). C+: CYP79A1 expressed in chloroplasts of tobacco. Fraction 1 is the bottommost and fraction 28 the topmost fraction in the gradient. B) Sucrose gradients separating the components of WT and PsaM-CYP79A1 thylakoids solubilized in 1% (w/v) β-DM. C) Separation of protein complexes of β-DM-solubilized WT and PsaM-CYP79A1 thylakoids by BN-PAGE.
Figure 4.
Verification of product formation in living cells.
A) Extracted ion chromatograms (m/z 152) from the LC-MS analysis of metabolites extracted from the growth medium of Synechococcus sp. PCC 7002 WT and PsaM-CYP79A1 cultures for detection of in vivo activity of the PsaM-CYP79A1 complex, compared to a p-hydroxyphenylacetaldoxime standard (C+). The two peaks are the E and Z isomers of the p-hydroxyphenylacetaldoxime. B) LC-MS extracted ion chromatograms as in A, from analysis of extracts of PsaM-CYP79A1 cyanobacteria or growth medium.
Figure 5.
Evidence for light-driven CYP79A1 activity.
In vitro PsaM-CYP79A1 enzyme activity assays. A) Using radiolabelled tyrosine as substrate, assays were run using WT and PsaM-CYP79A1 thylakoids incubated at 25°C for 30 min under 200 µmol·m−2·s−1 illumination or in the dark. The produced radiolabelled oxime was detected by laser scanning of a storage phosphor screen incubated with the TLC plate on which the extracted cyanobacterial metabolites were separated. The data represent averages of 5 measurements and error bars show standard deviations. B) PsaM-CYP79A1 thylakoids were assayed as in A), but with unlabelled tyrosine, and metabolite extracts were analysed by LC-MS. The result from a typical experiment is shown.