Here, we have demonstrated that the addition of sugar particularly D-Glu could improve the productivity of Synechocystis cells expressing the ene-reductase YqjM from B. subtilis. This was also corroborated by NAD(P)H fluorescence measurements showing an increased fluorescence (i.e. increased supply) upon D-Glu addition. Moreover, we have also shown that the system is also applicable in larger volumes using the internally illuminated BCR. Oxidoreductases expressed in cyanobacteria represent a strong electron drain which allows us to investigate how much of the electrons (i.e. originating from the photosynthetic electron transport chain) can be deviated towards heterologous biotechnological processes, ranging from the production of high-value products to bulk chemicals and even biofuels. The stereoselective reduction of 1a by the ene-reductase YqjM has been the fastest recorded photobiotransformation so far with specific activities of over 100 U gDCW−1 in Synechocystis [12, 21]. Stopped-flow kinetics indicated that the maximal turnover rate of the oxidative half reaction of YqjM for the reduction of 1a is more than 50 times higher than the reductive half reaction (i.e. the oxidation of NADPH). This means that in the presence of 1a the enzyme will be constantly in the oxidized state. Furthermore, YqjM has a KD value of 39.8 µM towards NADPH. On the other hand, a KD value of 140.4 µM was determined for NADH. A concentration of 500 µM is required to reach saturation and any drop below 200 µM will strongly reduce the turnover rate making this enzymatic reaction NAD(P)H-limited . Recently, Tanaka et al. reported a concentration of 87.4 nM OD730−1 for the intracellular NADPH concentration in photoautotrophically grown Synechocystis which is below this benchmark value .
Nakamura et al. reported that the reduction of 2A,3A,4A,5A,6A-pentafluoroacetophenone and other prochiral ketones in Synechococcus elongatus PCC 7942 by endogenous ketoreductases proceeds not only under light but also in darkness  and in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a specific inhibitor of the water-splitting PSII, albeit both with significantly lower rates than under light. This finding shows that glycolytic pathways in cyanobacteria can sustain heterologous NAD(P)H-consuming redox reactions, albeit at lower rates than under light. In this work, the stimulating effect of D-Glu addition was observed at different cell densities and was independent from the light intensity during the cultivation and the choice of the substrate. This demonstrates that the biotransformation can be supplied with electrons originating from glycolytic pathways. Addition of D-Glu to the reaction under light led to a 50% increase of the rate at a cell dry weight of 0.6 g L−1. This effect was observed under all the tested cell densities, most significantly at lower cell densities. At low cell densities with minimal self-shading of the cells, an improvement of the electron supplies by D-Glu addition boosts product formation.
The enzymes involved in the OPPp are expressed under photoautotrophic growth, interacting with the Calvin cycle [30, 34]. It should be noted that we applied cells for biotransformations with the metabolism adapted for autotrophic growth under high light (e.g. 180–200 µmol photons m−2 s−1), where some genes from the glycolytic pathways are less expressed [28, 35, 36]. The flux level of the OPPp is higher under the light intensity of 40 µmol m−2 s−1 than 125 µmol m−2 s−1 under mixotrophic conditions. The gluconeogenic activity upstream of glycolysis, probably caused by the gap1 gene repression enhanced the flux of the OPPp . In the earlier light period, the following genes coding for OPPp enzymes have been observed as upregulated: tktA (sll1070), devB (sll1479), and cfxE (sll0807). In the later light period, the upregulation of rpiA was noted [slr0194, encoding the ribose 5-phosphate isomerase (RPI)] . Nevertheless, the stimulating effect of D-Glu was also observed after cultivation at lower light intensity. Under mixotrophic conditions, the enzymes of the pentose phosphate cycle are down-regulated, which might contribute to the lower whole-cell activity observed after cultivation under light and addition of D-Glu to the medium .
Interestingly, the enhanced YqjM production rate in the presence of D-Glu is not caused by an increase in the photosynthetic electron transport rate. We hypothesize that under the photoautotrophic conditions, the reaction is electron-limited, which is mitigated by the metabolized D-Glu. These findings are in line with the observation that whole-cell biotransformations led to a decrease of intracellular NAD(P)H levels . Addition of D-Glu may increase the amount of NADH and NADPH available to YqjM, but the extra electrons are likely derived from glycolysis and OPPp. Still, a light-dependent component in the D-Glu-induced enhancement of YqjM-catalyzed substrate conversion was observed (Fig. 2), suggesting involvement of a light-induced regulatory mechanism. It should be mentioned that 1a is a thiol-reactive compound that potentially disturbs the thiol-based regulatory mechanisms in the cell, such as the thioredoxin and glutaredoxin systems. Indeed, glycolysis, the TCA cycle, the Calvin–Benson–Bassham cycle (CBB), and OPPp, all of which profoundly affect the NADPH/NADH metabolism, are likely subject to light-dependent redox regulation . Nevertheless, a significant activating effect of D-Glu was also observed with 4a a substrate that does not have this side-reactivity.
The possibility to boost redox reactions under light with sugar addition also implies that the initial rates observed under photoautotrophic conditions might be partially fuelled by the catabolism of storage compounds. As glycolytic pathways are usually regulated on a metabolic level by the ratio of reduced and oxidized nicotinamide cofactors, we assume that lower NAD(P)H levels increase the activity in these pathways. Several factors are involved upon the addition of D-Glu. The breakdown of storage compounds may be to some extent light dependent. Taking into account that in plant chloroplasts the degradation of starch can be activated by light especially during stressful conditions  as such as the high light utilized during our study.
The rate increase caused by D-Glu addition was also observed in a 200 mL internally illuminated photobioreactor, leading to a volumetric productivity as high as 10 mM h−1. This demonstrates that biotransformations fuelled by a combination of photosynthesis and carbohydrate catabolism can also be exploited for biotechnological production.
Our results showed that under photoautotrophic conditions, the ene-reductase is not NAD(P)H-saturated and has a higher capacity for substrate conversion. Cyanobacteria harboring heterologous enzymes such as monooxygenases and oxidoreductases have already been employed for the production of targeted chemicals and value-added compounds. Specific activities in the range of 5–6 U gDCW−1 have been reported for monooxygenases [13, 39] while higher activities have been reported for oxidoreductases ranging from 20 to over 100 U gDCW−1 [15, 21]. Moreover, biotransformations using Baeyer–Villiger monooxgenases report specific activities ranging from 25 to 60 U gDCW−1 [16, 17]. These works provide proof-of-concept, but also show that the rates obtained in photoautotrophic microorganisms are not higher than rates obtained in heterotrophs. This raises the question on how cyanobacteria might be engineered to sustain processes for the production of chemicals and biofuels with much higher electron consumption. In this context, some recent works indicate the possibility to engineer the cyanobacterial metabolism for an increased electron supply: deletion of flavodiiron proteins (FDPs) as competing electron sinks increased the specific activities of ene-reductions  and Baeyer–Villiger oxidations  in Synechocystis. By expressing heterologous metabolic sinks in cyanobacteria, an improved photosynthetic efficiency and performance was reported . Moreover, by inactivation of NDH-1, electron flow was increased by 30% to a heterologous cytochrome P450 expressed in Synechococcus PCC 7002 .
In conclusion, the addition of sugar specifically D-Glu during a photobiotransformation by an ene-reductase in Synechocystis improved the specific activity without an increase of the photosynthetic activity, indicating that electron-consuming processes at a specific activity of 100 gDCW L−1 are already electron limited. The higher activity can be attributed to an increase of NAD(P)H. As glycolytic pathways lead to the reduction of NADPH and NADH, and the enzyme accepts electrons from both nicotinamide cofactors, it is not possible to assign the activity increase to either one of them. A further increase of the yields of cyanobacterial processes thus requires a substantial improvement by cell engineering. Nevertheless, several recent works demonstrated the feasibility of such an optimization, which opens an avenue toward much higher productivity in the photosynthetic production of biofuels and bioproducts.