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Exploiting algal NADPH oxidase for biophotovoltaic energy

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ABSTRACT

Photosynthetic microbes exhibit light‐dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light‐dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C. reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild‐type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light‐dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.

No MeSH data available.


Superoxide anion production is restored by RBO1. (a) Validation of pSL18_RBO1 insertion into sta6rbo1 (CC‐4348) genome. To verify integration of the RBO1‐containing construct, PCRs were performed using primers specific to the plasmid. The predicted amplicon size is 1979 bp. (b) Mean ± SEM O2− production in light (120 min) from cw15, RBO1‐deficient sta6, two lines of sta6rbo1(STA6) (Blaby et al., 2013) and the three RBO1‐complemented lines shown in (a), determined using XTT (n ≤ 5). Only the presence of RBO1 was effective in restoring O2− production. Asterisks denote significant difference (P < 0.05) from indicated control (Student's t‐test; n/s, no significance).
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pbi12332-fig-0005: Superoxide anion production is restored by RBO1. (a) Validation of pSL18_RBO1 insertion into sta6rbo1 (CC‐4348) genome. To verify integration of the RBO1‐containing construct, PCRs were performed using primers specific to the plasmid. The predicted amplicon size is 1979 bp. (b) Mean ± SEM O2− production in light (120 min) from cw15, RBO1‐deficient sta6, two lines of sta6rbo1(STA6) (Blaby et al., 2013) and the three RBO1‐complemented lines shown in (a), determined using XTT (n ≤ 5). Only the presence of RBO1 was effective in restoring O2− production. Asterisks denote significant difference (P < 0.05) from indicated control (Student's t‐test; n/s, no significance).

Mentions: If RBO1 were the source of extracellular O2− production then it follows that complementing the sta6rbo1 mutant with RBO1 would restore production. To test this, the sta6rbo1 mutant was complemented with a synthetic gene construct containing the complete RBO1 gene model (Table 1). Three independently transformed strains were selected by antibiotic resistance and confirmed for RBO1 by PCR (Figure 5a). These were compared for light‐dependent O2− production against two sta6rbo1 strains that had previously been transformed to express only STA6 and so retained loss of RBO1 function (Blaby et al., 2013; Table 1). Only complementation with RBO1 restored O2− production to levels comparable to (and not significantly different from) that of the cw15 STA6RBO1 parental strain (Figure 5b). Complementation with STA6 did not increase production above that of the sta6rbo1 mutant (Figure 5b). These data show that O2− production is indeed RBO1 dependent.


Exploiting algal NADPH oxidase for biophotovoltaic energy
Superoxide anion production is restored by RBO1. (a) Validation of pSL18_RBO1 insertion into sta6rbo1 (CC‐4348) genome. To verify integration of the RBO1‐containing construct, PCRs were performed using primers specific to the plasmid. The predicted amplicon size is 1979 bp. (b) Mean ± SEM O2− production in light (120 min) from cw15, RBO1‐deficient sta6, two lines of sta6rbo1(STA6) (Blaby et al., 2013) and the three RBO1‐complemented lines shown in (a), determined using XTT (n ≤ 5). Only the presence of RBO1 was effective in restoring O2− production. Asterisks denote significant difference (P < 0.05) from indicated control (Student's t‐test; n/s, no significance).
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pbi12332-fig-0005: Superoxide anion production is restored by RBO1. (a) Validation of pSL18_RBO1 insertion into sta6rbo1 (CC‐4348) genome. To verify integration of the RBO1‐containing construct, PCRs were performed using primers specific to the plasmid. The predicted amplicon size is 1979 bp. (b) Mean ± SEM O2− production in light (120 min) from cw15, RBO1‐deficient sta6, two lines of sta6rbo1(STA6) (Blaby et al., 2013) and the three RBO1‐complemented lines shown in (a), determined using XTT (n ≤ 5). Only the presence of RBO1 was effective in restoring O2− production. Asterisks denote significant difference (P < 0.05) from indicated control (Student's t‐test; n/s, no significance).
Mentions: If RBO1 were the source of extracellular O2− production then it follows that complementing the sta6rbo1 mutant with RBO1 would restore production. To test this, the sta6rbo1 mutant was complemented with a synthetic gene construct containing the complete RBO1 gene model (Table 1). Three independently transformed strains were selected by antibiotic resistance and confirmed for RBO1 by PCR (Figure 5a). These were compared for light‐dependent O2− production against two sta6rbo1 strains that had previously been transformed to express only STA6 and so retained loss of RBO1 function (Blaby et al., 2013; Table 1). Only complementation with RBO1 restored O2− production to levels comparable to (and not significantly different from) that of the cw15 STA6RBO1 parental strain (Figure 5b). Complementation with STA6 did not increase production above that of the sta6rbo1 mutant (Figure 5b). These data show that O2− production is indeed RBO1 dependent.

View Article: PubMed Central - PubMed

ABSTRACT

Photosynthetic microbes exhibit light&#8208;dependent electron export across the cell membrane, which can generate electricity in biological photovoltaic (BPV) devices. How electrons are exported remains to be determined; the identification of mechanisms would help selection or generation of photosynthetic microbes capable of enhanced electrical output. We show that plasma membrane NADPH oxidase activity is a significant component of light&#8208;dependent generation of electricity by the unicellular green alga Chlamydomonas reinhardtii. NADPH oxidases export electrons across the plasma membrane to form superoxide anion from oxygen. The C.&nbsp;reinhardtii mutant lacking the NADPH oxidase encoded by RBO1 is impaired in both extracellular superoxide anion production and current generation in a BPV device. Complementation with the wild&#8208;type gene restores both capacities, demonstrating the role of the enzyme in electron export. Monitoring light&#8208;dependent extracellular superoxide production with a colorimetric assay is shown to be an effective way of screening for electrogenic potential of candidate algal strains. The results show that algal NADPH oxidases are important for superoxide anion production and open avenues for optimizing the biological component of these devices.

No MeSH data available.