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Biosynthesis of chlorophyll a in a purple bacterial phototroph and assembly into a plant chlorophyll-protein complex Andrew Hitchcock, Philip J Jackson, Jack W Chidgey, Mark J. Dickman, C. Neil Hunter, and Daniel P Canniffe ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00069 • Publication Date (Web): 12 May 2016 Downloaded from http://pubs.acs.org on May 15, 2016

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ACS Synthetic Biology

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Biosynthesis of chlorophyll a in a purple bacterial phototroph and assembly into a plant

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chlorophyll-protein complex

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Andrew Hitchcock1, Philip J. Jackson1,2, Jack W. Chidgey1, Mark J. Dickman2, C. Neil Hunter1*

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and Daniel P. Canniffe1,3

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1

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S10 2TN, UK.

Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield

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Sheffield, Sheffield S1 3JD, UK.

ChELSI Institute, Department of Chemical and Biological Engineering, University of

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State University PA 16802, USA.

Current address: Department of Biochemistry and Molecular Biology, The Pennsylvania

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*Corresponding author:

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C. Neil Hunter ([email protected])

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ACS Paragon Plus Environment


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ABSTRACT

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Improvements to photosynthetic efficiency could be achieved by manipulating

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pigment biosynthetic pathways of photosynthetic organisms in order to increase the

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spectral coverage for light absorption. The development of organisms that can produce both

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bacteriochlorophylls and chlorophylls is one way to achieve this aim, and accordingly we

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have engineered the bacteriochlorophyll-utilizing anoxygenic phototroph Rhodobacter

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sphaeroides to make chlorophyll a. Bacteriochlorophyll and chlorophyll share a common

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biosynthetic pathway up to the precursor chlorophyllide. Deletion of genes responsible for

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the bacteriochlorophyll-specific modifications of chlorophyllide and replacement of the

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native bacteriochlorophyll synthase with a cyanobacterial chlorophyll synthase resulted in

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the production of chlorophyll a. This pigment could be assembled in vivo into the plant

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water-soluble chlorophyll protein, heterologously produced in Rhodobacter sphaeroides,

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which represents a proof-of-principle for the engineering of novel antenna complexes that

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enhance the spectral range of photosynthesis.

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KEYWORDS

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Photosynthesis / chlorophyll / bacteriochlorophyll / antenna complex / water-soluble

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chlorophyll protein / Rhodobacter sphaeroides

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The overall efficiency of photosynthesis, measured on the basis of conversion to

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biomass, is in the region of 1-7% [1]. The primary steps of solar energy harvesting and

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charge separation, although inherently extremely efficient, are still constrained by the small

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portions of the available solar spectrum that can be absorbed by the light-harvesting

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pigment-protein complexes, which have evolved to be sufficient for survival in their

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particular ecological niche. One approach hypothesized to improve the efficiency of energy

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capture is to broaden the range of wavelengths absorbed by a biological system by

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producing mixtures of pigments, or mixtures of photosystems, in the same cell [1,2]. In

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oxygenic phototrophs, where the two photosystems are in competition for the same

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wavelengths of light, it has been proposed that re-engineering PSII and PSI to absorb light

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from different regions of the solar spectrum could improve energy capture, with potential

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positive impacts on growth rates and therefore crop yields and season lengths [3].

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The major photon absorbing pigments involved in solar energy capture are the

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(bacterio)chlorophylls ((B)Chls), housed in the antenna complexes of plants, algae and

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phototrophic (cyano)bacteria. The spectral range of these complexes is altered by

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modifications to the (B)Chl macrocycle, which shifts the red-most absorption band from

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~680 nm in the case of Chl a, to 800-920 nm for BChl a and as far as 1023 nm in the case of

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antennas containing Bchl b. These pigments share a common pathway up to the

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biosynthetic intermediate chlorophyllide (Chlide), the direct precursor of Chl a (Fig. 1a). In

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the case of the true BChls, Chlide is converted to a bacteriochlorophyllide species (BChlide)

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by further modifications to rings A and B, extending the Qy absorption bands of BChl

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pigments into the near infrared [4] and allowing anoxygenic phototrophic bacteria to

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harvest light that is not absorbed by the oxygenic algae and cyanobacteria higher in the

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water column/microbial mat [5].



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In the purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides, the major

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BChl absorption bands are in the 375-400 nm , 580-600 nm and 800-900 nm spectral

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regions, with carotenoids providing additional absorption between 400 and 550 nm. Thus,

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there is a spectral region between 600 and 800 nm with no major absorption features that

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could be occupied by a pigment such as Chl a. BChl a pigments within the peripheral (LH2)

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and core (LH1) antenna effectively funnel absorbed solar energy to the photosynthetic

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reaction center (RC), where charge separation occurs [6,7]. In order to establish a

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photosystem capable of harvesting a wider range of wavelengths, it will ultimately be

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necessary to engineer an organism capable of making a mixture of Chl and BChl pigments.

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BChl biosynthesis can be viewed as an extension of the Chl pathway, so the production of

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Chl a in a photosynthetic bacterium can be achieved by truncation of native BChl

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biosynthesis, along with modifications to introduce Chl-specific enzymes that attach and

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reduce the isoprenoid alcohol moiety (Fig. 1a), which decisively increases the

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hydrophobicity of the pigment. Here we report the implementation of this strategy using

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Rba. sphaeroides as a chassis; this anoxygenic phototroph has a versatile metabolism

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permitting viability under oxic conditions in the dark, allowing for the deletion, replacement

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and addition of genes involved in pigment biosynthesis and photosynthesis [8,9]. The

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introduction of cyanobacterial Chl-specific enzymes into a Chlide-accumulating mutant of

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Rba. sphaeroides resulted in successful re-routing of BChl biosynthesis and the production

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of Chl a, the first time this ubiquitous oxygenic photopigment has been synthesized in a

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purple phototrophic bacterium. As a proof-of-principle for incorporating this engineered

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pigment into a chlorophyll-protein complex we further demonstrate that this non-native

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pigment can be sequestered by the eukaryotic water-soluble chlorophyll protein [10] from


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Brussels sprout (Brassica oleracea var. gemmifera), BoWSCP, also heterologously produced

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in the Chl a accumulating strain of Rba. sphaeroides.

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The assembly of a plant chlorophyll-protein complex in a photosynthetic bacterium

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represents an initial proof of concept step in the design of a novel, high-energy Chl a-

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containing antenna that could be able to funnel excitation energy towards the native, lower

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energy, BChl a-containing light-harvesting antenna (LH2 and LH1) and RCs. In order to

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provide the Chlide substrate for the production of Chl a in Rba. sphaeroides, a mutant

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(ΔCXF) blocked at the steps exclusive to BChl biosynthesis (ΔbchC, ΔbchX and ΔbchF) was

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used as a host (Fig. 1b) [11]. This mutant accumulates Chlide, demonstrating that the BChl

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synthase (BchG) encoded by the native bchG gene is unable to esterify this pigment to

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produce a Chl species, as reported in vitro [12], and therefore cannot grow prototrophically.

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In order to attach an alcohol moiety to Chlide in this strain, the native bchG gene was

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replaced with the gene encoding the Chl synthase enzyme (ChlG) from the cyanobacterium

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Synechocystis sp. PCC 6803 (hereafter Synechocystis), codon-optimized for expression in

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Rba. sphaeroides, generating strain ΔCXFG::chlG (Figs. 1b & 1c). Similarly, the native bchP

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gene encoding the geranylgeranyl reductase was also replaced with a codon-optimized

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version of Synechocystis chlP, generating strain ΔCXFGP::chlGP (Figs. 1b & 1c). The

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sequences of the codon-optimized genes are given in Table S1.

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The pigments that accumulated in these strains, along with those from the WT and

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ΔCXF, were extracted from pellets of microoxically-grown cells and analyzed by HPLC (Figs.

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1d & 2). As expected, the WT accumulated BChl a (Fig. 2a), while ΔCXF was unable to

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synthesize (B)Chl pigments (Fig. 2b). With our engineered strains, both the ΔCXFG::chlG and

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ΔCXFGP::chlGP strains synthesize four species of Chl a, esterified with GG, dihydroGG-

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(DHGG), tetrahydroGG- (THGG) or phytol moieties (Fig. 1a inset panel), with each detected



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by fluorescence emission at 670 nm (Fig. 2c & 2d). To confirm the identity of these

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pigments, Chls synthesised by WT and ΔchlP (Fig. S1) strains of Synechocystis were used as

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HPLC standards for Chl a (Fig. 2e) and geranylgeranyl-(GG) Chl a (Fig. 2f) respectively. In all

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cases the detected pigments were confirmed to be species of Bchl a or Chl a by their

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absorption spectrum (Fig. 1d).

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The most prominent pigment extracted from the ΔCXFG::chlG strain was Chl a, with

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only extremely low amounts of GG-Chl a and intermediate levels of DHGG- and THGG-Chl a,

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showing that, unexpectedly, the native BchP is able to perform three reductions of GG-Chl

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a, albeit at a lowered efficiency when contrasted with the accumulation of a single fully-

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reduced BChl a species in the WT (Fig. 2a). We hypothesized that this lower efficiency may

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be due to BchP acting on an unnatural substrate and that replacement of the native bchP

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gene with the Synechocystis homolog, which has previously been shown to be active in Rba.

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sphaeroides [13], may result in more efficient tail reduction such that only Chl a with a fully

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reduced isoprenoid tail would accumulate. However, strain ΔCXFGP::chlGP shows no

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improvement over ΔCXFG::chlG in this respect and the proportion of fully reduced phytol-

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Chl a is actually lower than when the native BchP is used (Fig. 2d). This aspect of the

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engineered Chl a pathway in Rba. sphaeroides requires further study of the expression and

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subunit composition of BchP/ChlP enzymes.

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In order to demonstrate that we could express a protein to sequester the engineered

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Chl pigments we expressed the water-soluble chlorophyll-binding protein (WSCP) from

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plants. Mature BoWSCP lacking its 19 residue amino(N)-terminal endoplasmic reticulum

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signal peptide [14] and post-translationally cleaved 21 residue carboxy(C)-terminal

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extension peptide [15] (Fig. 3a) was codon optimized (Table S1) and expressed with a C-

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terminal 10xHis-tag in the ΔCXFGP::chlGP strain of Rba. sphaeroides. Recombinant WSCP-His


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was purified from cell free extracts by immobilized metal affinity chromatography (IMAC)

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and protein fractions that contained Chl (Fig. 3b), as judged by absorbance at 280 nm and

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665 nm, were pooled, concentrated and analyzed by SDS-PAGE and immunoblotting (Fig.

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3c). The major Coomassie stained band corresponds to a protein of the predicted MW of

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WSCP-His (20.8 kDa) (Fig. 3c lane 1) and cross-reacts with an anti-6-His antibody (Fig. 3c

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lane 2). WSCP-His was identified as the predominant protein species following affinity

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purification by mass spectrometry analysis (Table S2, data uploaded to the

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ProteomeXchange Consortium [16] via the PRIDE partner repository, identifier

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10.6019/PXD002638). The absorption spectrum of the purified pigment-protein complex

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(Fig. 3d, solid green line) has 6 peaks (summarized in Table 1) and a small shoulder at ~584

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nm, in agreement with the spectra previously reported for native and thylakoid or Chl a

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reconstituted BoWSCP [14, 17]; excitation at 431 nm resulted in fluorescence emission at

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~670 nm, as expected for Chl a (Fig. 3d, dashed blue line). The pigment bound to WSCP was

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isolated by solid phase extraction and identified by mass spectrometry. GG-, DHGG- and

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THGG-Chl a were identified, but not Chl a or Chlide (Figs. 3e, 3f & S2). The reason we did

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not detect Chl a may be that it is too tightly bound to WSCP to be extracted [12] or the level

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extracted was below the detection limit, or WSCP preferentially binds Chl a species without

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a fully reduced phytol tail. It is interesting that Chlide was not detected, showing that WSCP

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selectively binds esterified Chl a over an excess of the less hydrophobic precursor, which

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accumulates in this strain [11].

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The production of Chl a does not restore photoautotrophic growth to the ΔCXF

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mutant as Rba. sphaeroides cannot use this pigment for light harvesting or photochemistry.

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Furthermore, only a small amount of Chl a is produced compared to BChl a in the wildtype

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organism (approximately 0.1%), which is not surprising as a mutant of Rba. sphaeroides



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harbouring deletions in genes encoding photosystem apoproteins accumulates no Bchl

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despite having an intact pigment biosynthesis pathway [18]. This observation shows that, in

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the absence of the appropriate binding proteins, even the native BChl pathway shuts down,

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no doubt to avoid the production of potentially phototoxic pigments. Further optimization

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of the introduced Chl pathway, coupled with repurposing the native photosynthetic

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apparatus to incorporate Chl a or introducing foreign Chl a-utilizing photosystem

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apoproteins, can now be attempted. These ambitious aims require heterologous expression

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of multiple membrane proteins and further engineering of both pigment biosynthesis and

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assembly pathways.

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METHODS

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Growth conditions

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Rba. sphaeroides strains were grown microoxically in the dark in a rotary shaker (150 rpm) at 34°C in liquid M22+ medium [19] supplemented with 0.1% casaminoacids.

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Escherichia coli strains JM109 [20] and S17-1 [21] transformed with pK18mobsacB or

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pIND4 plasmids were grown shaking (250 rpm) at 37°C in LB medium supplemented with 30

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µg·ml-1 kanamycin. For strains and plasmids used in this study see Supplementary Table 3.

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Construction of mutants of Rba. sphaeroides

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The Rba. sphaeroides bchG and bchP genes were replaced with their counterparts

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from Synechocystis (chlG and chlP, respectively) using the allelic exchange vector

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pK18mobsacB [22]. Briefly, the chl genes were codon-optimized for expression in Rba.

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sphaeroides using J-Cat [23], and these sequences, flanked by the up- and downstream

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regions of the target for replacement, were synthesized by Bio Basic (Ontario, Canada). The


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fragments were subcloned into pK18mobsacB cut with EcoRI/HindIII. Sequenced clones

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were conjugated into Rba. sphaeroides from E. coli S17-1, and transconjugants in which the

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clone had integrated into the genome by homologous recombination were selected on

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M22+ medium supplemented with 30 µg·ml-1 kanamycin. Transconjugants that had

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undergone a second recombination were subsequently selected on M22+ supplemented

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with 10% (w/v) sucrose and lacking kanamycin. Sucrose-resistant kanamycin-sensitive

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colonies had excised the allelic exchange vector through the second recombination event

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[24]. Gene replacements were confirmed by sequencing at the relevant loci. Sequences of

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primers used in the present study can be found in Supplementary Table 4.

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Strains harbouring the gene encoding WSCP from Brassica oleracea var. gemmifera

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(BoWSCP) were created as follows. The gene (designed and synthesized as above) encoding

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C-terminally His10-tagged holo-BoWSCP, codon-optimized for expression in Rba. sphaeroides

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and flanked by NcoI and HindIII restriction sites. The fragment was digested and cloned into

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the NcoI/HindIII sites of pIND4 [25]. The resulting sequenced plasmid was conjugated into

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relevant strains of Rba. sphaeroides from E. coli S17-1 and transconjugants were selected

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for and maintained on M22+ medium supplemented with 30 µg·ml-1 kanamycin.

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Pigment analysis

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Pigments were extracted from cell pellets after washing in 20 mM HEPES pH 7.2 by

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adding 9 pellet volumes of 0.2% (v/v) ammonia in methanol, vortex-mixing for 30 s and

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incubating on ice for 20 min. The extracts were clarified by centrifugation (15000 g for 5 min

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at 4°C) and the supernatants were immediately analyzed on an Agilent 1200 HPLC system.

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(B)Chl a species were separated on a Phenomenex Aqua C18 reverse-phase column (5 μm

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particle size, 125 Å pore size, 250 mm × 4.6 mm) using a method modified from that of

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Addlesee & Hunter [26]. Solvents A and B were 64:16:20 (v/v) methanol/acetone/H2O and



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80:20 (v/v) methanol/acetone respectively. The buffer composition changed from 0-100%

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solvent B over 10 min, and was maintained at 100% for 15 min, during which time (B)Chl

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species were eluted. Elution of Chl a species were monitored by checking absorbance at

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665 nm or fluorescence emission at 670 nm with excitation at 431 nm, while elution of

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BChl a species were monitored by checking absorbance at 770 nm. To compare how much

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Chl a is produced in engineered strains compared to BChl a in the wildtype, the areas of

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HPLC peaks (detected by absorbance at 665 nm for Chl a species or 770 nm for BChl a) were

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integrated and the molar stoichiometry estimated using literature reported extinction

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coefficients for Chl a [27] and BChl a [28].

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Pigment from ~500 µg of WSCP in 200 µL IMAC elution buffer (see below) was

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extracted as above and isolated by solid phase extraction as previously described [29]. The

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sample was dried under nitrogen, dissolved in 20 µL methanol and infused at 3 µL·min-1 via

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an atmospheric pressure chemical ionisation source into a Maxis UHR-TOF mass

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spectrometer (Bruker Daltonics) operating with the following parameters: capillary

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potential: 4 kV, corona current: 4 µA, dry gas: 3.5 L·min-1 at 220 °C, nebuliser gas: 2 bar,

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vaporiser temperature 350 °C, ion cooler transfer time: 60 µs. All other parameters were as

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per the manufacturer’s default settings.

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Purification of BoWSCP from Rba. sphaeroides and identification by nanoflow LC-MS/MS

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Expression of WSCP from pIND4 was induced in Rba. sphaeroides cultures at OD710

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0.8 by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of

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125 µM. Cultures were harvested by centrifugation (6300 g for 15 min at 4oC) 12 h after

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induction. Cell pellets were resuspended in binding buffer (25 mM HEPES pH 7.5, 500 mM

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NaCl, 5mM imidazole) and broken by sonication on ice (6 × 30 s bursts separated by 30 s

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gaps). The clarified cell-free extract was isolated as the supernatant following centrifugation


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(43000 g for 20 min at 4oC), passed through a 0.4 µm filter and fractionated by immobilized

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Ni-affinity chromatography. Following washes with at least 10 column volumes each of

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binding buffer containing 5, 20, 50 and 100 mM imidazole, remaining bound protein was

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eluted with 400 mM imidazole in 25 mM HEPES pH 7.5 with 100 mM NaCl. Protein fractions

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were analyzed by SDS polyacrylamide gel electrophoresis (PAGE) and stained with

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Coomassie Brilliant Blue, or transferred onto a polyvinylidene fluoride membrane for

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immunodetection. Membranes were incubated with an anti-6-His primary antibody (Bethyl)

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followed by a secondary antibody conjugated with horseradish peroxidase (Sigma-Aldrich).

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To identify WSCP by nanoflow LC-MS/MS, 50 µg protein (3 µL) was diluted to 10 µL

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with 100 mM Tris-HCl pH 8.5 containing 8 M urea and 5 mM DTT and incubated at 37°C for

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30 min. S-alkylation was carried out by addition of 1 µL 100 mM iodoacetic acid followed by

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incubation in the dark at room temperature for 30 min. 2 µg trypsin/endoproteinase LysC

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mix (Promega) was added and the protein digested at 37 °C for 3 h. After dilution with 75 µL

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50 mM Tris-HCl pH 8.5, 10 mM CaCl2, digestion was continued overnight. The digest was

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desalted using a C18 spin column (Thermo Scientific) following the manufacturer’s

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instructions and 500 ng analyzed by nanoflow liquid chromatography (Dionex UltiMate 3000

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RSLCnano system) coupled on-line to a Q Exactive HF mass spectrometer (Thermo

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Scientific). Mass spectra were processed using Mascot Distiller v. 2.5.1.0 for database

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searching with Mascot Server v. 2.5.1 (Matrix Science) against NCBInr (Viridiplantae).

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SUPPORTING INFORMATION

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Supplementary Method Growth of Synechocystis and construction of a ΔchlP mutant;

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Supplementary Figure 1 Construction of a chlP (sll1091) mutant in Synechocystis sp. PCC6803;

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Supplementary Figure 2 Observed and theoretical isotopic patterns for mono-oxidized



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geranylgeranyl-chlorophyll a; Supplementary Table 1 Nucleotide sequences of codon optimized

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genes used in this study; Supplementary Table 2 Identification of WSCP by nanoflow LC-MS/MS;

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Supplementary Table 3 Strains and plasmids described in this study; Supplementary Table 4 Primers

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used in this study.

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ABBREVIATIONS

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(B)Chl = (bacterio)chlorophyll; Rba = Rhodobacter; Chlide = Chlorophllide a; IMAC =

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immobilised metal affinity chromatography; LH = light harvesting complex; RC = reaction

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center; WSCP = water-soluble chlorophyll protein; GG = geranylgeranyl; DHGG =

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dihydrogeranylgeranyl; THGG = tetrahydrogeranylgeranyl.

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AUTHOR INFORMATION

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Corresponding author

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*C. Neil Hunter. Email: [email protected]. Current address: 1Department of

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Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.

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Author contributions

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C.N.H. and D.P.C. conceived the study. A.H., P.J.J., J.W.C. and D.P.C performed the

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experiments. All authors designed the experiments and analyzed and interpreted the data.

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A.H., P.J.J., J.W.C., C.N.H. and D.P.C. wrote the manuscript.

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Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGEMENTS


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A.H., P.J.J., M.J.D., C.N.H. and D.P.C. were supported by grant BB/M000265/1 from the

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Biotechnology and Biological Sciences Research Council (BBSRC), UK. A.H., J.W.C. and C.N.H.

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were also supported by an Advanced Award (338895) from the European Research Council.

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D.P.C. acknowledges funding from a European Commission Marie Skłodowska-Curie Global

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Fellowship (660652). M.J.D acknowledges further support from BBSRC (BB/M012166/1).

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REFERENCES

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Gunner, M.R., Junge, W., Kramer, D.M., Melis, A., Moore, T.A., Moser, C.C., Nocera, D.G.,

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Nozik, A.J., Ort, D.R., Parson, W.W., Prince, R.C. and Sayre, R.T. (2011) Comparing

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photosynthetic and photovoltaic efficiencies and recognizing the potential for improvement.

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Science 332, 805-809.

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8. Canniffe, D. P. and Hunter, C. N. (2014) Engineered biosynthesis of bacteriochlorophyll b

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9. Chi, S. C., Mothersole, D. J., Dilbeck, P., Niedzwiedzki, D. M., Zhang, H., Qian, P., Vasilev,

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Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating

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carotenoids from the spirilloxanthin pathway. Biochim. Biophys. Acta. 1847, 189-201.

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soluble chlorophyll protein in

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binding protein. Plant Cell Physiol. 42, 906-911.

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of C8-vinyl reduction in chlorophyll and bacteriochlorophyll biosynthesis. Biochem. J. 462,

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433-440.

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12. Kim, E.J. and Lee, J.K. (2010) Competitive inhibitions of the chlorophyll synthase

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of Synechocystis sp. strain PCC 6803 by bacteriochlorophyllide a and the bacteriochlorophyll

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synthase of Rhodobacter sphaeroides by chlorophyllide a. J. Bacteriol. 192, 198-207.

Satoh,

H.,

Ucihida,

A.,

Nakayame,

Brassicaceae

K. plants

and is

Okada, a


M.

(2001)

Water-

stress-induced chlorophyll-

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13. Addlesee, H.A., Gibson, L.C., Jensen, P.E. and Hunter, C.N. (1996) Cloning, sequencing

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and functional assignment of the chlorophyll biosynthesis gene, chlP, of Synechocystis sp.

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PCC 6803. FEBS Lett. 389, 126-130.

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14. Takahashi, S., Yanai, H., Nakamaru, Y., Uchida, A., Nakayama, K. and Satoh H. (2012)

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Molecular cloning, characterization and analysis of the intracellular localization of a water-

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soluble Chl-binding protein from Brussels sprouts (Brassica oleracea var. gemmifera).Plant

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Cell Physiol. 53, 879-891.

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15. Takahashi, S., Uchida, A., Nakayama, K. and Satoh H. (2014) The C-terminal extension

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peptide of non-photoconvertible water-soluble chlorophyll-binding proteins (Class II WSCPs)

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affects their solubility and stability: comparative analyses of the biochemical

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and chlorophyll-binding properties of recombinant Brassica, Raphanus and Lepidium WSCPs

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with or without their C-terminal extension peptides. Protein J. 33, 75-84.

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16. Vizcaíno, J. A., Deutsch, E.W., Wang, R., Csordas, A., Reisinger, F., Ríos, D., Dianes,

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J.A., Sun, Z., Farrah, T., Bandeira, N., Binz, P.A., Xenarios, I., Eisenacher, M., Mayer,

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G., Gatto, L., Campos,

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S., Apweiler, R., Omenn, G.S., Martens, L., Jones, A.R. and Hermjakob, H. (2014)

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ProteomeXchange provides globally co-ordinated proteomics data submission and

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dissemination. Nat. Biotechnol. 30, 223-226.

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17. Takahashi, S., Ono, M., Uchida, A., Nakayama, K. and Satoh H. (2013) Molecular cloning

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and functional expression of a water-soluble chlorophyll-binding protein from Japanese wild

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radish. J Plant Physiol. 170, 406-412.

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18. Jones, M.R., Fowler, G.J.S., Gibson, L.C.D., Grief, G.G., Olsen, J.D., Crielaard, W. and

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Hunter, C.N. (1992) Mutants of Rhodobacter sphaeroides lacking one or more pigment-

A., Chalkley,

R.J., Kraus, H.J., Albar,


J.P., Martinez-Bartolomé,


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protein complexes and complementation with reaction centre, LH1, and LH2 genes. Mol.

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Microbiol. 6, 1173-84.

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19. Hunter, C. N. and Turner, G. (1988) Transfer of genes coding for apoproteins of reaction

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centre and light-harvesting LH1 complexes to Rhodobacter sphaeroides. J. Gen. Microbiol.

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134, 1471-1480.

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20. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved M13 phage cloning vectors

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and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103-

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21. Simon, R., Priefer, U. & Pühler, A. (1983) A broad host range mobilization system for in

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vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Nat.

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Biotechnol. 1, 784-791.

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22. Schäfer, A., Tauch, A., Jäger, W., Kalinowski, J., Thierbach, G. and Pühler, A. (1994) Small

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mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18

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and pK19: selection of defined deletions in the chromosome of Corynebacterium

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glutamicum. Gene 145, 69-73.

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23. Grote, A., Hiller, K., Scheer, M., Münch, R., Nörtemann, B., Hempel, D. C. and Jahn, D.

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(2005) JCat: a novel tool to adapt codon usage of a target gene to its potential expression

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host. Nucleic Acids Res. 33, W526-W531.

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24. Hamblin, P. A., Bourne, N. A. and Armitage, J. P. (1997) Characterization of the

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chemotaxis protein CheW from Rhodobacter sphaeroides and its effect on the behaviour of

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Escherichia coli. Mol. Microbiol. 24, 41-51.

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25. Ind, A. C., Porter, S. L., Brown, M. T., Byles, E. D., de Beyer, J. A., Godfrey, S. A. and

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Armitage, J. P. (2009) Inducible-expression plasmid for Rhodobacter sphaeroides and

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Paracoccus denitrificans. Appl. Environ. Microbiol. 75, 6613-6615.


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26. Addlesee, H. A. and Hunter, C. N. (1999) Physical mapping and functional assignment of

379

the geranylgeranyl-bacteriochlorophyll reductase gene, bchP, of Rhodobacter sphaeroides. J.

380

Bacteriol. 181, 7248-7255.

381

27. Porra, R.K., Thompson, W.A. and Kriedemann, P.E. (1989) Determination of accurate

382

extinction coefficients and simultaneous equations for assaying chlorophylls a and b

383

extracted with four different solvents:verification of the concentration of chlorophyll

384

standardsby atomic absorption spectroscopy. Biochim. Biophys. Acta. 975, 384-94.

385

28. Permentier, H.P., Schmidt, K.A., Kobayashi, M., Akiyama, M., Hager-Braun, C., Neerken,

386

S., Miller, M. and Amesz, J. (2000) Composition and optical properties of reaction centre

387

core complexes from the green sulfur bacteria Prosthecochloris aestuarii and Chlorobium

388

tepidum. Phtosynth. Res. 64, 27-39.

389

29. Canniffe, D.P., Jackson, P.J., Hollingshead, S., Dickman, M.J., and Hunter, C.N. (2013)

390

Identification of an 8-vinyl reductase involved in bacteriochlorophyll biosynthesis in

391

Rhodobacter sphaeroides and evidence for the existence of a third distinct class of the

392

enzyme. Biochem. J. 450, 397-405.

393



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

394

Table 1 Absorption peaks (nm) of purified WSCP-His in 200 mM Tris-HCl (pH 8.0) at 25 oC Protein

Soret

WSCP-His expressed in Rba. sphaeroides ΔCXFGP::chlGP WSCP+ a Native BoWSCP

b

BoWSCP-His expressed in E. coli (thylakoid membrane reconstituted) BoWSCP-His expressed in E. coli (Chl a reconstituted) b,c 395

Page 18 of 24

a

b

Q

342

384

418

436

628

673

343

384

423

437

629

673

342

383

422

437

629

673

417/421

436/437

629/630 673/674

342/343 384

this study; b [14]; c [17].

396


Page 19 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

397


FIGURE LEGENDS

398 399

Figure 1 Concept for constructing a Chl a biosynthesis pathway in the purple phototroph

400

Rba. sphaeroides. a, The native BChl a biosynthetic pathway (blue arrows). Removal of

401

BchF, BchX and BchC (red cross) from the native pathway halts the production of BChl at the

402

level of Chlide, and ChlG and ChlP from Synechocystis (green arrows) are introduced to re-

403

route the pathway to Chl a. Dashed arrows represent multiple enzymatic steps. Chemical

404

structures of Chl a (IUPAC numbered) and BChl a are shown. 3V, C3-vinyl; 3HE, C3-

405

hydroxyethyl. R denotes the attached alcohol moiety. Inset: Alcohol moieties of (B)Chls,

406

denoted by R. Highlighted bonds are those sequentially reduced by the gene product of

407

chlP/bchP.

408

pyrophosphate; THGG, tetrahydrogeranylgeranyl pyrophosphate. b, The organization of

409

native (upper) and modified (lower) partial photosynthesis gene clusters of Rba.

410

sphaeroides. Blue, bch genes; magenta, crt genes; peach, regulatory genes; grey, unassigned

411

genes. Deletions of bchF, C and X (red cross) in the native pathway are shown as well as the

412

sites where the native bchG and bchP were replaced by chlG and chlP from Synechocystis

413

(green). c, Agarose gel of PCR products confirming the replacement of bchG with chlG in

414

ΔCXFG::chlG and bchG and bchP with chlG and chlP in ΔCXFGP::chlGP. In each PCR reaction a

415

bchG/P or chlG/P specific forward primer was used with a reverse primer in the downstream

416

rsp_0278 (for bchG/chlG) or rsp_0276 gene (for bchP/chlP). Lanes 1, 5, 9 = bchG specific

417

primers; lanes 2, 6, 10 = chlG specific primers; lanes 3, 7, 11 = bchP specific primers; lanes 4,

418

8, 12 = chlP specific primers; M = HyperladderTM 1kb (Bioline). d, Absorption spectra of

419

methanol extracted (B)Chl pigments.

GGPP,

geranylgeranyl

pyrophosphate;

DHGG,

420


dihydrogeranylgeranyl


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

421

Figure 2 HPLC analysis of (B)Chls extracted from described strains of Rba. sphaeroides.

422

Elution profiles of pigments extracted from pellets of a, WT, b, ΔCXF, c, ΔCXFG::chlG,

423

d, ΔCXFGP::chlGP, e, standard of Chl a from WT Synechocystis, f, standard of GG-Chl a from

424

a ΔchlP strain of Synechocystis. Elution of BChls and Chls was monitored by absorbance at

425

770 nm (grey) or by fluorescence emission at 670 nm with excitation at 431 nm (black),

426

respectively. Traces are normalized to major peak height for clarity.

427 428

Figure 3 Purification of pigment-bound WSCP produced in Chl a synthesizing Rba.

429

sphaeroides. a, Sequence alignment of apo BoWSCP and recombinant WSCP-His used in this

430

study. Positions of the Kunitz trypsin inhibitor (KTI) and [F/Y]DPLGL chlorophyll binding

431

motifs are indicated by black bars. The 19 residue N-terminal endoplasmic reticulum signal

432

peptide (underlined) and 21 residue C-terminal extension peptide (grey box) that are post-

433

translationally cleaved in holo BoWSCP are absent in WSCP-His, which has a 10xHis tag

434

(shown in bold). b, Photograph of purified WSCP-His. c, SDS-PAGE and anti-6-His

435

immunoblot analysis of purified WSCP-His. Lanes 1 and 2 contain 20 µg and 5 µg of protein

436

respectively. Lane M = Precision Plus ProteinTM Standards (Biorad). d, Absorption (Abs, solid

437

green line) and fluorescence (Flu, dotted blue line) spectra of pigment bound WSCP-His. e,

438

Mass spectrum showing [M + H]+ ions for geranylgeranyl-chlorophyll a (GG-Chl a) and

439

dihydrogeranylgeranyl-chlorophyll a (DHGG-Chl a). Also detectable are the mono- and di-

440

oxidation products of GG-Chl a, DHGG-Chl a and tetrahydrogeranylgeranyl-chlorophyll a

441

(THGG-Chl a) formed by exposure to air during isolation and storage. Mass assignments

442

were obtained at an accuracy of < 3 ppm with external calibration and identifications were

443

confirmed by isotope pattern (see Fig. S2). f, Mass spectrum showing the expected positions

444

of [M + H]+ ions for Chlide a and its mono-oxidation product, neither of which are detected.


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445


FIGURES

446 447

Figure 1

448 449

450 451



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

452

Figure 2

453 454

455 456 457 458 459 460 461 462


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463


Figure 3

464

465 466 467 468 469 470 471



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Page 24 of 24

For Table of Contents Use Only Manuscript title: Biosynthesis of chlorophyll a in a purple bacterial phototroph and assembly

into a plant chlorophyll‐protein complex Authors: Hitchcock, Andrew; Jackson, Philip; Chidgey, Jack; Dickman, Mark; Hunter, C. Neil; Canniffe, Daniel


Biosynthesis of Chlorophyll a in a ... - ACS Publications

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