Nanoparticle Mediated NADPH Regeneration for Enhanced Ethanol Production by Engineered Synechocystis sp. PCC 6803

The ethanol synthesis pathway engineered Synechocystis sp. PCC 6803 was used to investigate the influence of metal oxide mediated NADPH regeneration on ethanol synthesis. Among the metal oxides, Fe2O3 and MgO showed considerable improvement in growth, chlorophyll a content and ethanol synthesis. The in-vitro studies proved that the selected metal oxides have the potential to regenerate the NADPH under light illumination. The results clearly indicate that the light energy is the key factor for activation of metal oxides and to a less extent light itself has the possibility for direct regeneration of NADPH. Under optimized light intensity and NADP addition, the maximum MgO mediated ethanol production of 5100mg/L, about a 2-fold increase compared to the control, was obtained after 20 days cultivation at 5L level. This study indicates that the efficient NADPH regeneration aided by metal oxide is crucial to improve ethanol productivity in Synechocystis sp. PCC 6803. IMPORTANCE Cyanobacteria are efficient ethanol producing organisms from atmospheric CO2 upon engineering of pathway. In cyanobacterial ethanol synthesis pathway, NADPH plays an important role acetaldehyde to ethanol conversion. Here we elucidated the NADPH regeneration through extracellular addition of metal oxides. The metal oxide mediated NADPH regeneration study allows us to dissect the importance of metal oxides in enhancing ethanol production through NADPH regeneration while also providing insight into the regulatory functions of metal oxides in growth, photosynthetic apparatus and various carbon metabolisms.

(200 μL) was collected, and filtered through a 0.45 μm Millipore filter before the analysis of 114 acetate, ethanol, pyruvate and succinate by HPLC. 115 In-vitro studies on NADPH regeneration 116 The effects of NADP, metal oxides, and light intensity on NADPH regeneration were studied by  In-situ studies on NADPH regeneration 123 The effect of metal oxide (Fe 2 O 3 :20 µM and MgO: 200 µM) mediated NADPH regeneration on 124 biomass, chlorophyll a and ethanol production was analysed by adding the NADP (100 µM). 125 The metal oxides such as Fe 2 O 3 and MgO were used as NADPH activating materials. The effects 126 of light/dark cycles were analysed by growing the cells in Erlenmeyer flask (250 mL) containing 127 Intracellular pigments of Synechocystis cell suspension were extracted by dimethylformamide. 137 Chlorophyll awas determined according to the method of Moran (33). The polyhydroxybutyrate 138 (PHB) content was determined as described by Monshupanee and Incharoensakdi (34) using 139 HPLC (Shimadzu, Japan) equipped with InertSustain 3-µm C18 column (GL Sciences, Japan) 140 and UV/Vis detector. The estimation of lipid content was performed using the method described 141 by Monshupanee and Incharoensakdi (34). The estimation of glycogen was performed by acid 142 hydrolysis followed by sugar analysis by HPLC and the theoretical factor 1.111 was used for the 143 glycogen to glucose conversion. The sugar and ethanol contents were quantified using HPLC 144 system equipped with refractive index detector (RID 10A, Shimadzu, Japan). Metabolic 145 intermediates such as acetate, pyruvate and succinate were quantified using HPLC equipped with 146 UV/Vis detector (SPD-20A, Shimadzu, Japan). The components were separated in Phenomenex,

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Rezex ROA-Organic acid column (150 × 7.8 mm) with 5 mM H 2 SO 4 as a mobile phase at a flow 148 rate of 0.6 mL/min. The regeneration of NADPH was determined by the method described in 149 Sigma Aldrich protocol (MAK038). Briefly, the samples were centrifuged at 10000 × g for 10 150 min and filtered to remove the protein. inhibitory effect on growth (Fig. 1a). The oxidation states of Cu(I) and Cu(II) make the Cu an 165 ideal factor for oxidoreduction reaction. However, they generate reactive oxygen species through 166 Fenton and Haber-Weis reaction, thereby affecting the lipids, proteins and DNA (35). This 167 nature of Cu affects the Synechocystis, thereby reducing the biomass concentration (Fig. 1a).

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Another metal, Mn is also acting as a cofactor for superoxide dismutase and catalase, butit   (46). The compatibility of metal oxide is a major 222 concern to perform in-situ process development. In this study, the type and concentration of 223 metal oxides were chosen based on the metals present in the cyanobacterial medium (29) and the 224 influences of metal oxide concentration were studied. As can be seen in Fig. 3a showed that an increase in light intensity constantly increased the NADPH regeneration ( Fig.   229 3b). However, when comparing two metal oxides, the MgO always showed higher regeneration.

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The control experiment in the absence of light also regenerated the NADPH upto a certain level, 231 which might be due to the reactivity of metal oxides to regenerate the NADPH. Factors such as 232 metal oxide and light illumination may also cause the harmful effect on cyanobacteria, thus, it is 233 important to analyse its compatibility in in-situ experiments.  (Table 1).

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The control experiments (without metal oxides) showed higher extracellular acetate, succinate  improved NADPH level intracellularly, the biomass and ethanol concentrations were increased (6). In the present study, the increased NADPH regeneration mediated by metal oxides has been 251 achieved without engineering other natural pathways of Synechocystis and the production of 252 ethanol is also higher than that reported by Choi and Park (6).

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Effect of light intensity on biomass, chlorophyll a and ethanol production 254 The light acts as a strong metabolic valve for the photosynthetic organisms. In this context, we 255 examined the influence of continuous light at various intensities on biomass, chlorophyll a and 256 ethanol production (Fig. 5). The continuous light highly influencesbiomass, chlorophyll a and 257 ethanol production, which signifies that the light illumination is an indispensable factor to sustain 258 the growth rate and to regenerate the NADPH (9). It should be noted that even in the presence of 259 externally added metal oxides and NADP, the continuous light does not affect the growth upto 260 normal light illumination (100 µE/m 2 /s), which indicates the compatibility of the integrated 261 treatment in cyanobacterial system (Fig. 5a). When comparing the metal oxides, the Fe 2 O 3 262 showed higher biomass and chlorophyll a content, whereas the MgO produced higher ethanol 263 production ( Fig. 5 and Table 2). When comparing the light intensities, the light intensity higher The intracellular glycogen, PHB and lipid contents in Synechocystis were analysed to monitor 288 the changes in metabolism of engineered strain under NADPH regenerationat 5 L level (Fig. 6b).

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After 20 days of growth, the engineered strain produced 23.8% (g/g DCW) glycogen in BG11 however, it is not upto considerable level. As observed with glycogen content, the lipid content Fe 2 O 3 . The control values were always higher than that of metal oxide treatment without NADP, 296 which indicates the NADP also influences glycogen, PHB and lipid contents ( Fig. 2 and 6b). The 297 1.5-fold increase in lipid content upon NADPH regeneration was already reported by Osada et al.

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(48). The results further confirm that the presence of metal oxides regenerated the NADPH and 299 improved the lipid accumulation.

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The analysis of intracellular metabolites such as acetate, succinate and pyruvate showed the 301 significant improvement in concentration upon NADPH regeneration (Fig. 6b). The highest 302 intracellular acetate, succinate and pyruvate concentration of about 8.9, 7.0 and 12.2 mg/g,