Part:BBa_K4772001
ChlF: A photo-oxidoreductase that uses light to oxidize chlorophyll a to produce chlorophyll f.
Chlamydomonas reinhardtii are organisms that convert visible photons into chemical energy via oxygenic photosynthesis. And chlorophylls(Chls) are the essential pigments of photosynthesis, for which they both harvest light and transduce it into chemical energy. Chlamydomonas reinhardtii usually uses chlorophyll a as photochemically active pigment. The absorption wavelength of Chl a is 680nm in photosystem Ⅱ (PSⅡ)and 700nm in photosystem Ⅰ(PSⅠ). Therefore, 680nm in PSⅡand 700nm in PSⅠwere assumed to be the longest wavelength able to power photosynthesis. [1]. However, recently, a red-shifted pigment, chlorophyll f (Chl f), has been discovered.[2]. It was found in terrestrial cyanobacteria that live in an environment under near-infrared light(720nm) because of shading by plants or because of their associations with soil crusts, benthic mat communities, or dense cyanobacterial blooms. For evolution pressure, these cyanobacteria have evolved a novel far-red light photoacclimation (FaRLiP) that enable them to use far-red light (FRL) for photosynthesis. Chlorophyll f (Chl f) is the key pigment in FaRLiP whose maximal absoption is at around 707nm.[3].And Chlf is the enzyme that produce chlorophyll f by oxidizing chlorophyll a. Similar to the living environment of terrestrial cyanobacteria, in runway ponds for industrial production and cultivation of Chlamydomonas, Chlamydomonas in the upper layer uses near-red light for photosynthesis. Therefore, a serious problem of near-red light attenuation in the depth of runway ponds exists, which leads to serious decrease of the photosynthetic efficiency of Chlamydomonas in the depth of runway ponds. In order to solve this problem, we adopted the means of synthetic biology.
Sequence and Features
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Contents
Usage and Biology
Biology: Far-red light photoacclimation (FaRLiP)
Chlamydomonas reinhardtii are organisms that convert visible photons into chemical energy via oxygenic photosynthesis. And chlorophylls (Chls) are the essential pigments of photosynthesis, for which they both harvest light and transduce it into chemical energy. Chlamydomonas reinhardtii usually uses chlorophyll a as photochemically active pigment. The absorption wavelength of Chl a is 680nm in photosystem Ⅱ (PSⅡ)and 700nm in photosystem Ⅰ(PSⅠ). Therefore, 680nm in PSⅡand 700nm in PSⅠ were assumed to be the longest wavelength able to power photosynthesis[4]. However, recently, a red-shifted pigment, chlorophyll f (Chl f), has been discovered.[5] It was found in terrestrial cyanobacteria that live in an environment under near-infrared light (720nm) because of shading by plants or because of their associations with soil crusts, benthic mat communities, or dense cyanobacterial blooms. For evolution pressure, these cyanobacteria have evolved a novel far-red light photoacclimation (FaRLiP) that enable them to use far-red light (FRL) for photosynthesis. Chlorophyll f (Chl f) is the key pigment in FaRLiP whose maximal absoption is at around 707nm[6]. And ChlF is the enzyme that produce chlorophyll f by oxidizing chlorophyll a.
Usage: Our Design
Similar to the living environment of terrestrial cyanobacteria, in runway ponds for industrial production and cultivation of Chlamydomonas, Chlamydomonas in the upper layer uses near-red light for photosynthesis. Therefore, a serious problem of near-red light attenuation in the depth of runway ponds exists, which leads to serious decrease of the photosynthetic efficiency of Chlamydomonas in the depth of runway ponds. In order to solve this problem, we adopted the means of synthetic biology. We try to express enzyme ChlF from cyanobacteria to introduce FaRLiP into Chlamydomonas, thereby extending their light harvesting into far red (700 to 800 nm) and improving their photosynthetic light use efficiency.
Background verification
Efficacy of FaRLiP: Light absorption and energy output of photosynthetic units under a dense plant canopy
The performance of oxygenic photosynthesis in the presence of Chl f might differ markedly from that in Chl a-only photosystems. Therefore,it is vital to gain detailed and comprehensive investigation of the effects of FaRLiP on the energy equilibration and trapping dynamics in FRL photosystems, especially under physiological conditions. To quantify the gain in photosynthetic capacity achieved with FaRLiP, Roberta Croce calculated the wavelength distribution of the photons absorbed by the white-light and FRL units at the bottom of a dense plant canopy[7]. The gain in absorption is particularly evident for PSII (Fig.1), as FRL PSII harvests about 3.6 times more photons than its white-light counterpart (as Chl a-only PSII does not absorb above 700 nm).
Figure.1 Light absorption and energy output of photosynthetic units under a dense plant canopy.
(a) Absorption spectra of PSII of Synechococcus eleongatus (from Kuhl, H. et al. 2000)[8] (designated as white-light PSII (WL PSII), black) and FRL PSII of C. fritschii (red) normalized to the total Qy absorbance area (λ = 600–800 nm) and relative spectral photon irradiance of unshaded sunlight (daylight) and light filtered by a dense leaf canopy (canopy) (from Gan, F. & Bryant, D. A. et al. 2015)[9].
(b) Wavelength distribution of photons absorbed under a dense plant canopy by the white-light and FRL PSII complexes, obtained by multiplying the absorption spectra of the complexes by the spectral irradiance under the canopy shown in a at each wavelength. The coloured areas represent the wavelength-dependent energy output of each PSII obtained by multiplying the wavelength distribution of absorbed photons by the PSII photochemical yield calculated from the in vivo Fm/Fo ratios as ΦWL PSII = 1 − 1/4.6 = 78% and ΦFRL PSII = 1 – 1/1.6 = 37%. The total light absorption is estimated as the area below the curve, and the total output is the total absorption times the photochemical yield.
Since the efficiency of Chl f-containing PSII in vivo is roughly halved relative to white-light PSII (on the basis of the fluorescence-lifetime data), the total energy output of FRL PSII (calculated as the product of total light absorption and photochemi- cal yield) is still 70% larger than for white-light PSII in this specific environment. In the case of PSI, both the total absorption and total energy output are approximately doubled when Chl f is inserted[10].
Optical power meter data of environment of Chlamydomonas Cultivation
To verify our background for Chlamydomonas, we designed an experimental device to simulate the light environment of Chlamydomonas. Respectively, we use a red laser light source and a white light source to irradiate the Chlamydomonas reinhardtii culture solution contained in a container of the same diameter. The control group was Chlamydomonas reinhardtii culture medium(TAP) , and the experimental group was Chlamydomonas reinhardtii culture solution with algae concentrations of 106, 107, and 108 cells/ml, respectively.
We used an optical power meter to measure the light intensity of red and white light after passing through the culture medium. The instruments are as follows.
Figure 2 Our optical power meter instrument.
A. Red light laser；
B. Support frame;
C. Clear aperture;
D. Optical power meter.
After taking the average of multiple measurements, the following data was obtained:
| Light Intensity (w) | Control | 106 cells/ml | 107 cells/ml | 108 cells/ml |
| Red Light | 24.2 | 20.5 | 6.1 | 0.0158 |
| White Light | 6.05 | 5.5 | 2.9 | 0.00606 |
| Transmittance | Control | 106 cells/ml | 107 cells/ml | 108 cells/ml |
| Red Light | 1 | 0.85 | 0.25 | 0.00065 |
| White Light | 1 | 0.91 | 0.48 | 0.001 |
It can be seen that after passing through the same length of Chlamydomonas reinhardtii culture solution, the transmittance of red light is lower than that of white light, indicating that the proportion of red light decreases when light passes through the Chlamydomonas reinhardtii culture solution, for some of the red light is absorbed by the chlorophyll of Chlamydomonas reinhardtii. This can simulate the light absorption of Chlamydomonas reinhardtii in the runway pond. The proportion of red light received by Chlamydomonas reinhardtii in the depth of the runway pond decreases, while the proportion of far-red light increases. However, the original chlorophyll a and b in Chlamydomonas reinhardtii cannot utilize far-red light, resulting in a relative decrease in photosynthetic efficiency. This illustrates the necessity of introducing chlorophyll f into Chlamydomonas.
Experiment Results
Construction of ChlF expression plasmid
First, we obtained the sequence fragment of ChlF through company synthesis. To express and verify expression of ChlF in Chlamydomonas, we performed homologous recombination to fuse the sequence of ChlF to a plasmid backbone containing a YFP tag that can be expressed in Chlamydomonas. Based on the sequencing results, we successfully constructed the ChlF plasmid that can be expressed in Chlamydomonas reinhardtii. And the YFP tag made sure that the expression of ChlF can be verified by performing Western Blot.
Figure 3. Successful Construction of ChlF expression plasmid with YFP tag.
Successful expression of ChlF in Chlamydomonas
Fluorescence intensity detection
To verify whether ChlF was successfully expressed in Chlamydomonas reinhardtii, we detected the fluorescence intensity of Chlamydomonas reinhardtii, as ChlF was tagged with YFP.
Figure 4. Fluorescence intensity detection heatmap.
Excitation light wavelength: 488nm
Absorption light wavelength range: 500-520nm
The first and second lines are two repeats. Samples in the same columns but different lines come from one strains of algae. Scale of the heat map is shown on the right. NC: Chlamy that do not express YFP; PC: Chlamy that express YFP; ChlF-YFP: Chlamy that express ChlF-YFP plasmid. ChlF-YFP-1 and ChlF-YFP-2 are two different strains of Chlamy.
From fluorescence intensity, we can propose that two strains of Chlamydomonas reinhardtii successfully expressed ChlF.
Western Blot
To verify whether ChlF was successfully expressed in the two strains of Chlamydomonas reinhardtii, we extracted the protein from Chlamydomonas and performed Western Blot experiment. According to the Western Blot results, YFP was successfully expressed in the two strains of Chlamydomonas reinhardtii.
It is worth noting that the size of the band in the Figure 5 is smaller than the theoretical size of ChlF-YFP and larger than the size of YFP alone. Considering that ChlF and YFP share the same start codon, ChlF should also be successfully expressed, and the incorrect band size may be due to some degree of degradation of ChlF-YFP during the protein extraction process or other processes.
Figure 5 Western Blot for YFP.
The lane of ChlF-YFP and YFP is indicated. Lane 1: ChlF-YFP-1; Lane 9: ChlF-YFP-2; Lane 2-8,10: Chlamy that failed to express ChlF-YFP plasmid. NC: Chlamy that do not express YFP; PC: Chlamy that express YFP;
* The smaller lane of PC might result from degradation of YFP or other unknown reasons.
Introduction of FaRLiP in Chlamydomonas Improve their Performance under Far-red Light
Introduction of FaRLiP in Chlamydomonas Improve their Performance under Far-red Light
As mentioned in the Background Verification section, Roberta Croce's calculations showed that under special conditions of high far-red light, the total energy output (calculated as the product of total light absorption and photochemical heat production) of FaRLiP PSII is 70% greater than that of white light PSII.
To verify whether the introduction of FaRLiP is truly beneficial for the growth of Chlamydomonas reinhardtii under far-red light, we cultured Chlamydomonas under far-red light and tried to measure the growth curves of Chlamydomonas with FaRLiP and WT for comparison. If the growth rate of Chlamydomonas reinhardtii with FaRLiP under far-red light is higher than that of WT, this may be due to the higher total energy output of Chlamydomonas reinhardtii with the introduction of FaRLiP under far-red light conditions, resulting in faster growth rates. If so, we can see that introducing FaRLiP to solve the shade problem of Chlamydomonas reinhardtii in industrial production runway pond is a promising and feasible approach.
Result
Firstly, in order to achieve far-red light culture, we purchased far-red light source as the only light source during the culture process of Chlamydomonas. According to the light source spectrum test report of the far-red light source, the peak wavelength of the far-red light source is at 740nm, which meets our requirements.
Figure 6 Spectroscopic data of far-red light source
Color parameters:
Color coordinate: x=0.4907 y=0.2927 / u'= 0.3549 v'=0.4763
Related color temperature: CCT=1516K (color difference Duv=0.0429)
Main wavelength: Ad=700.0nm; Color purity: Purity=39.9%; Color ratio: R=39.6% G=55.0% B=5.4%
Peak wavelength: λp=735.8nm; Half-peak bandwidth: FWHM=20.3nm
Color rendering index: Ra=21.8
Unfortunately, due to time constraints, we were unable to obtain detailed growth curve data of Chlamydomonas’s performance under far-red light. However, if other iGEM teams want to use this Part, they can consider using our far-red light culture methods that is mentioned above to further perfect the functional verification of this Part.
- ↑ Björn, L. O., Papageorgiou, G. C., Blankenship, R. E. & Govindjee A viewpoint: why chlorophyll a? Photosynth. Res. 99, 85–98 (2009)
- ↑ Chen, M. et al. A red-shifted chlorophyll. Science 329, 1318–1320 (2010)
- ↑ Y. Li, Z.-L. Cai, M. Chen, Spectroscopic properties of chlorophyll f. J. Phys. Chem. B 117, 11309–11317 (2013)
- ↑ Björn, L. O., Papageorgiou, G. C., Blankenship, R. E. & Govindjee A viewpoint: why chlorophyll a? Photosynth. Res. 99, 85–98 (2009).
- ↑ Chen, M. et al. A red-shifted chlorophyll. Science 329, 1318–1320 (2010).
- ↑ Y. Li, Z.-L. Cai, M. Chen, Spectroscopic properties of chlorophyll f. J. Phys. Chem. B 117, 11309–11317 (2013).
- ↑ Gan, F. & Bryant, D. A. Adaptive and acclimative responses of cyanobacteria to far-red light. Environ. Microbiol. 17, 3450–3465 (2015).
- ↑ Kuhl, H. et al. Towards structural determination of the water-splitting enzyme: purifcation, crystallization, and preliminary crystallographic studies of photosystem II from a thermophilic cyanobacterium. J. Biol. Chem. 275, 20652–20659 (2000).
- ↑ Gan, F. & Bryant, D. A. Adaptive and acclimative responses of cyanobacteria to far-red light. Environ. Microbiol. 17, 3450–3465 (2015).
- ↑ Mascoli, V., Bersanini, L. & Croce, R. Far-red absorption and light-use efficiency trade-offs in chlorophyll f photosynthesis. Nat. Plants 6, 1044–1053 (2020).
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