Elsevier

Metabolic Engineering

Volume 35, May 2016, Pages 83-94
Metabolic Engineering

Original Research Article
Expression of holo-proteorhodopsin in Synechocystis sp. PCC 6803

https://doi.org/10.1016/j.ymben.2016.02.001Get rights and content

Highlights

  • Here we show that proteo-opsin can be expressed up to levels of 105 molecules per cell in membranes of Synechocystis sp. PCC6803.

  • The heterologously expressed protein is equally distributed (based on amount of protein) over thylakoid- and cytoplasmic membranes.

  • Synechocystis sp. PCC6803 is able to synthesize all-trans retinal and generate proteorhodopsin without requiring to add external retinal.

  • Proteorhodopsin expressed in Synechocystis sp. PCC6803 can make a measurable contribution to light-energy conversion in this cyanobacterium.

Abstract

Retinal-based photosynthesis may contribute to the free energy conversion needed for growth of an organism carrying out oxygenic photosynthesis, like a cyanobacterium. After optimization, this may even enhance the overall efficiency of phototrophic growth of such organisms in sustainability applications. As a first step towards this, we here report on functional expression of the archetype proteorhodopsin in Synechocystis sp. PCC 6803. Upon use of the moderate-strength psbA2 promoter, holo-proteorhodopsin is expressed in this cyanobacterium, at a level of up to 105 molecules per cell, presumably in a hexameric quaternary structure, and with approximately equal distribution (on a protein-content basis) over the thylakoid and the cytoplasmic membrane fraction. These results also demonstrate that Synechocystis sp. PCC 6803 has the capacity to synthesize all-trans-retinal. Expressing a substantial amount of a heterologous opsin membrane protein causes a substantial growth retardation Synechocystis, as is clear from a strain expressing PROPS, a non-pumping mutant derivative of proteorhodopsin. Relative to this latter strain, proteorhodopsin expression, however, measurably stimulates its growth.

Introduction

Concerns about global warming and the depletion of fossil fuels have led to an increasing need for the development of alternative, more sustainable, methods to produce biofuel and commodity chemicals. Cyanobacteria, like all phototrophs, can utilize the energy from sunlight to fix CO2 and produce a range of valuable carbon-based products via ‘direct conversion’ (Angermayr et al., 2009, Ducat et al., 2011, Machado and Atsumi, 2012). The relatively high growth rate of these organisms, the use of water as the source of electrons and their genetic accessibility, has made them the preferred organisms for such applications.

Rhodopsins are light-sensitive seven-helix transmembrane proteins that bind a retinal molecule as their chromophore. This family has members with either a sensory or a chemiosmotic function in energy transduction. Bacteriorhodopsin from Halobacterium salinarum is the archetype of the chemiosmotic-energy-transducing rhodopsins (Lanyi, 1978). It pumps protons, driven by light, and hence is able to generate a proton motive force (pmf). Proteorhodopsins (PRs) form a subgroup of the rhodopsins from the Domain of the Bacteria, which utilize light energy to translocate protons over a membrane against an electrochemical proton gradient. The gene encoding the first discovered member of this group was detected in the genome sequence of an uncultured γ-proteobacterium from oceanic waters (Beja et al., 2000). Since then, PRs have turned out to be highly abundant in the oceans (Palovaara et al., 2014, Rusch et al., 2007, Finkel et al., 2013, Campbell et al., 2008), and organisms containing them, including a cyanobacterium (Miranda et al., 2009) can be found in many other habitats as well (Rusch et al., 2007, Atamna-Ismaeel et al., 2008, Atamna-Ismaeel et al., 2012, Koh et al., 2010). In vivo experiments have shown that pumping of protons by PR can lead to increases in growth rate under nutrient-limited conditions (Palovaara et al., 2014, Gomez-Consarnau et al., 2007, Kimura et al., 2011), production of ATP (Steindler et al., 2011), increased fixation of CO2 (Palovaara et al., 2014; Gonzalez et al., 2008), and/or survival under starvation or stress conditions (Steindler et al., 2011, Gomez-Consarnau et al., 2010, Akram et al., 2013, Wang et al., 2012). However, these enhancements generally require nutrient-limitation or stress conditions before they exceed experimental error.

PRs have also been heterologously expressed in non-photosynthetic hosts (Beja et al., 2000, Walter et al., 2007, Martinez et al., 2007, Johnson et al., 2010, Hunt et al., 2010, Kim et al., 2012). For example, the introduction of a PR in Shewanella oneidensis increased the pmf, which resulted in increases in electrical current generation, lactate uptake, and survival under starvation conditions (Johnson et al., 2010, Hunt et al., 2010). In Escherichia coli, the pmf generated by a PR resulted in ATP synthesis (Martinez et al., 2007), was able to drive the flagellar motor (Walter et al., 2007), and could be used to significantly increase the production of hydrogen by a co-introduced hydrogenase (Kim et al., 2012, Kuniyoshi et al., 2015). Very recently, functional expression of a proteorhodopsin in E. coli was shown to cause a minute increase the growth rate of this organism when growing fermentatively (Wang et al., 2015). This stimulation, however, may be strictly limited to anaerobic conditions: photo-activation of Gloeobacter rhodopsin under aerobic conditions led to a decrease in growth rate of the organism because of increased oxidative stress (Na et al., 2015).

Cyanobacteria and green algae have a significantly lower absorption in the green part of the visible spectrum (i.e. 450–550 nm), a range that is well covered by the absorption spectrum of many PRs (Walter et al., 2010, Claassens et al., 2013). Introduction of PRs into such a system could lead to increased CO2 fixation, which could ultimately increase production rates of interesting compounds by cyanobacterial cell factories (Angermayr et al., 2012). Hence, it has previously been suggested that PRs could supply additional free energy to oxy-phototrophic organisms (Walter et al., 2010, Claassens et al., 2013), even more so when it turns out to be possible to shift the window of absorption of this pigmented protein outside that of the PAR region (Ganapathy et al., 2015). Several proposals have been made to increase the efficiency of oxygenic photosynthesis beyond its natural limits (e.g. Blankenship et al., 2011). In the most recent proposal (Ort et al., 2015) PSI is substituted by an (infrared-light dependent) proton pump; NADPH would then have to be derived via the combined action of PSII and NAD(P)H-dehydrogenase (Ort et al., 2015). Functional expression of PR may aid in the engineering towards obtaining such strains.

As a first step towards this we have expressed the gene sequence of the proteorhodopsin from Monterey Bay ((Beja et al., 2000); also known as GPR, but hereafter referred to as PR) in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). We first quantitated the amount of expressed apo-PR and its retinal chromophore, and characterized the sub-cellular distribution of the protein. Using the assay of growth (rate), we were then able to demonstrate an improvement in growth of the transgenic Synechocystis strain expressing PR, when compared with the control strain expressing a non-pumping proteorhodopsin, PROPS (Kralj et al., 2011). To our knowledge, this is the first demonstration of a beneficial effect of PR on the growth of a cyanobacterium. By implication, we demonstrate for the first time that Synechocystis can synthesize all-trans-retinal in vivo.

Section snippets

Strains and growth conditions

Strains of E. coli were routinely grown at 37 °C, either in liquid LB medium while shaking at 200 rpm, or on solid LB plates containing 1.5% (w/v) agar. Where appropriate, antibiotics were added to a final concentration of 100 μg ml−1 for ampicillin, and 25–50 μg ml−1 for kanamycin.

All Synechocystis sp. PCC 6803 strains used in this study were derived from a single wild-type strain (a glucose tolerant strain, obtained from D. Bhaya, Stanford University, Stanford, CA). Unless specified otherwise,

Expression of PR in Synechocystis

To explore the possibility of functional expression of PR in Synechocystis, we constructed a series of expression plasmids based on the broad host-range conjugation vector pVZ321 (Zinchenko et al., 1999). Each plasmid carried a promoter, a ribosome binding site, the structural gene of PR, with an extension encoding a C-terminal poly-histidine tag (PR-His), and a bi-directional terminator (see Section 2 for further detail). We preferred a plasmid-based expression system over a genomic

Discussion

In this manuscript we report the heterologous (holo-protein) expression of a PR-based proton pump in the membranes of Synechocystis sp. PCC 6803. The heterologous protein incorporates approximately evenly (on a protein-content basis) in both types of membrane of the cells (i.e. CM and TM). It could also make a contribution to the cell׳s energy conversion in both membrane types. Furthermore, we demonstrated that this protein can make a measurable contribution to the conversion of solar energy

Authors׳ contributions

JBvdS, QC and KJH designed experiments; QC, JBvdS and HLD performed experiments; JBvdS, QC and KJH wrote the paper; and SG and WJdG contributed to the writing of the paper and the overall experimental design.

Conflict of interest

The authors declare that they have no conflict of interest. KJH is scientific advisor to the start-up company Photanol BV. This does not create a conflict of interest nor does it alter the authors’ adherence to accepted policies on sharing data and materials.

Acknowledgments

This project was carried out within the research program of BioSolar Cells (BSC core project Grant C2.9 to WJdG and KJH), co-financed by the Dutch Ministry of Economic Affairs. The authors would like to acknowledge Jos C. Arents and Louis Hartog for their help with experiments.

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