The base strain of cyanobacteria we utilize for our sucrose measurements is constructed as reported in Abramson et al., 2016. Briefly, this strain contains a copy of sucrose permease (cscB) under an IPTG-inducible Ptrc promoter installed into Neutral Site 3. An additional copy of sucrose phosphate synthase (sps) cloned from Synechocystis sp. PCC 6803 is also expressed under Ptrc and is integrated into the genome at Neutral Site 2. Inducible sps allows us to bypass the requirement for external salt for sucrose production that is in many sucrose-secreting strains. For results from “wild-type” (WT) strains, we use the ATCC strain of S. elongatus PCC 7942 (ATCC #33912).

S. elongatus PCC 7942 strains were routinely grown in BG-11 media (C3061, Sigma-Aldrich, USA) supplemented with 1 g L^-1 HEPES and set to pH 8.3 with NaOH to maintain or expand cultures. Cultures were incubated in a Multitron Infors HT incubator at 32°C with 2% CO₂, 150 rpm shaking, and ~150 μmol photons^-2 s^-1 of continuous light from Sylvania 15 W Gro-Lux fluorescent bulbs. To standardize the growth history of strains between different experiments, all cultures were back-diluted 1:10 into BG-11 for three days prior to initiating any of the reported experiments. When needed, 1 mM IPTG was added to induce the expression of cscB and sps genes at time = 0 h. Chloramphenicol (Cm; 25 μg mL^-1) and kanamycin (Kn; 50 μg mL^-1) were used to maintain cscB and sps, respectively. Antibiotics were not added on the final day of back-dilution to minimize confounding effects.

The MC-1000 multi-cultivator system (Photon System Instruments, Czech Republic) was used to examine the light tolerance of S. elongatus PCC 7942; the device emits cool white light at varying light intensities (150, 250, 500, 1000, 2000, or 2500 μmol photons m^-2 s^-1). MC-1000 cultures were kept at 32°C and provided with 3% CO₂. Overnight cultures were back-diluted to an OD₇₅₀ of ~0.3 into MC-1000 vessels with fresh BG-11 media supplemented with 10 mM HCO₃ ^-, and induced with 1 mM IPTG as needed for low-starting density experiments. Samples were taken at 0, 24, 48, and 72 h for OD₇₅₀ and sucrose measurements.

For the experiments specifically evaluating high-light with high-starting density cultivation in the MC-1000 system, overnight cultures (prepared as outlined in section 2.1) were pelleted in a benchtop centrifuge. The supernatant was removed, and the cell pellet was resuspended with fresh BG-11 media supplemented with 10 mM HCO₃ ^- to an OD₇₅₀ of ~1.5 or 2.0 (provided 2000 and 2500 μmol photons m^-2 s^-1, respectively). An additional 10 mM HCO₃ ^- was added at 24 h for vessels that were provided bicarbonate daily. Samples were taken at 0, 24, and 48 h for OD₇₅₀ measurements and sucrose quantification.

The High-Density Cultivation (HDC) 6.10B starter kit (CellDEG GmbH, Germany) was used for high-CO₂ and high-density cultivation. The kit consists of a bottom base with a buffer reservoir and 10 mL cultivators with a gas-permeable bottom membrane. At the start of the experiments, the reservoir was filled with 200 mL of a 3 M KHCO₃/3 M K₂CO₃ solution to provide CO₂. Cultures prepared in BG-11 were centrifuged for 10 min at 3700 xg and 22°C before removing the supernatant and resuspending to an OD₇₅₀ of ~0.3 with CD media. CD media is designed to sustain extended periods of high-density cultivation of photosynthetic microbes, and has higher concentrations of macronutrients (N, P, and K) and micronutrients (Mg, S, Ca, Na, Cl, Fe, Zn, and Mo) relative to BG-11. Due to its enrichment, CD media has a high osmolarity (~140 mOsm) that is approximately three times greater than of BG-11 (~40 mOsm); the exact composition is available on protocols.io (dx.doi.org/10.17504/protocols.io.2bxgapn). Relevant cultures were induced with 1 mM IPTG before transferring 12 mL to the HDC system cultivators. Moderate-light HDC experiments were incubated in a Multitron incubator (Infors HT, Switzerland) providing ~150 rpm shaking and ~250 μmol photons m^-2 s^-1 from Sylvania 15 W Gro-Lux fluorescent bulbs. High-light HDC experiments were incubated in a growth chamber (AL-36L4, Percival Scientific, USA) with either ~500 μmol photons m^-2 s^-1 for 0-120 h, or ~500 μmol photons m^-2 s^-1 for 0-48 h and ~1000 μmol photons m^-2 s^-1 for 48-120 h from a light array fabricated in-house consisting of four equidistant chip-on-board (COB) LED lights (BXRA-56C5300-H, Bridgelux, USA)( Supplementary Figure S1 ). Both incubators provided 2% ambient CO₂, constant illumination, and ~150 rpm shaking. Samples were taken at 0, 24, 48, 72, 96, and 120 h to measure OD₇₅₀ and to collect supernatant for sucrose quantification.

The following experimental configuration was used to measure the relationship between surface irradiance, light penetration, and culture turbidity: a glass graduated cylinder with a clear bottom was clamped onto a ring stand. A Submersible Spherical Micro Quantum Sensor (US-SQS/L; WALZ, Germany) connected to a LI-250A light meter (LI-COR, USA) was clamped above and in-center of the graduated cylinder. A single COB LED from the in-house lighting device was placed below and center of the graduated cylinder. With this setup, 5 cm of liquid (BG-11 or cell culture at the indicated OD₇₅₀) fill the graduated cylinder, and the sensor measures the light that penetrates through the liquid when 0, 1, 2, or 3 cm away from the light interface of the culture (0 cm). A black, opaque sheet was wrapped around the graduated cylinder to cover all but the bottom 4 cm of the culture to block ambient light.

To set the desired irradiance (150, 500, 1000, 2000, 3000, or 4000 µmol photons m^-2 s^-1), the graduated cylinder was filled with 5 cm of BG-11, and the sensor was placed flush to the bottom of the glass to measure the incoming light at 0 cm. WT cultures were prepared as outlined in Section 2.1, pelleted, and supernatants removed. Pellets were resuspended with fresh BG-11 to create a concentrate that was diluted with BG-11 to 0.3, 1.0, 2.0, 5.0, and 10.0 OD₇₅₀. Five mL of each culture filled the graduated cylinder and penetrating irradiances at 1, 2, and 3 cm were recorded at applicable light intensities. For this and setting the irradiance, the average function of the LI-250A was used in which the device averages the values recorded over a 15-second interval.

Samples collected at the time points specified above were pelleted in a microcentrifuge; the supernatants were saved and stored at -20°C until the end of the experiment. Sucrose quantification was performed using the Sucrose/D-Glucose Assay Kit (K-SUCGL; Megazyme, USA).

To determine the dry cell weight of S. elongatus PCC 7942 strains, cultivation conditions were recreated as outlined in Section 2.2 for low-starting density experiments in the MC-1000, and Section 2.3 for moderate-light experiments in the HDC. Samples were collected at a variety of culture densities (OD₇₅₀) in order to generate a broad distribution for standard curves correlating OD₇₅₀ with dry cell biomass ( Supplementary Figure S2 ). The OD₇₅₀ of collected samples was determined with a visible spectrophotometer (Genesys 20; Thermo Fisher Scientific, USA), washed with Milli-Q water twice, and resuspended into the desired OD₇₅₀ with Milli-Q water. Three to 20 mL of the prepared samples were pelleted in a Falcon tube and most of the supernatant removed; the remaining water was used to create a concentrated resuspension to transfer to a pre-weighed 3 mL glass test tube. Milli-Q water was used as needed to rinse the Falcon tube and added to the glass test tube. Samples were dried in a 90°C oven for 24 h, or until the mass remained stable. The mass of the empty glass test tube was subtracted from the final weight, and the resulting value was divided by the volume used to determine the dry cell weight (g L^-1) for corresponding OD₇₅₀. Python was used to create a linear regression model and derive the function parameters, which was then used to convert OD₇₅₀ into dry cell weight.

We first selected a strain of S. elongatus PCC 7942 that has been engineered for sucrose secretion in a salt-independent manner. Briefly, hyperaccumulation of cytosolic sucrose is a common strategy for osmotic stress protection in many freshwater cyanobacterial species (Klähn and Hagemann, 2011), where this compatible solute can act as a counter osmoticum while also conferring other cellular protective effects. A rate-limiting step of sucrose biosynthesis occurs at the enzyme sucrose phosphate synthase (SPS), that catalyzes the condensation of NDP-glucose with fructose-6-phosphate to form sucrose-6-phosphate. SPS enzymes are frequently encoded as a bi-domanial protein that also contains sucrose-6-phosphate phosphatase (SPP) activity that dephosphorylates this intermediate to sucrose. We and others have found that expression of sucrose permease (cscB), a symporter of sucrose and protons, will lead to the export of cytosolic sucrose to the medium (Ducat et al., 2012; Santos-Merino et al., 2023). In S. elongatus PCC 7942 strains with inducible copies of both sps and cscB, sucrose synthesis and export can be achieved in the absence of external osmotic pressure (Abramson et al., 2016, 2018; Lin et al., 2020; Dan et al., 2022). We therefore selected this strain to minimize the requirement for addition of salt to the growth medium.

We first used the MC-1000 multi-cultivator to evaluate the impact of varied light intensity (150, 250, 500, 1000, 2000 µmol photons m^-2 s^-1) on growth and sucrose production of S. elongatus PCC 7942 cscB/sps strains and to broadly assess light tolerance. The growth of WT strains cultured with 150 and 1000 µmol photons m^-2 s^-1, and non-exporting cscB/sps with 1000 µmol photons m^-2 s^-1 grew at similar rates, reaching a final density of ~2.2 OD₇₅₀ ( Figure 1A , left). Sucrose-exporting strains, however, can partition a large fraction of their fixed carbon to secreted sucrose, leading to less cellular growth and biomass under certain conditions (Ducat et al., 2012)(see also, Supplementary Figure S3 ). The highest OD₇₅₀ among sucrose exporters were from cultures grown with moderate irradiances of 250 and 500 µmol photons m^-2 s^-1 ( Figure 1A , center and right), though differences were not statistically significant from other exporting conditions (p > 0.05). For sucrose-secreting lines in the MC-1000, there was a direct correlation between illumination and both cell growth and sucrose production up to 500 µmol photons m^-2 s^-1; light intensities beyond this threshold led to a decline in productivity ( Figures 1A, B ). Sucrose production over 72 hours peaked at 0.56 g L^-1 for 250 µmol photons m^-2 s^-1 and 0.68 g L^-1 for 500 µmol photons m^-2 s^-1 (p < 0.001 against all other light conditions) ( Figure 1B ). The relationship between irradiance and growth, however, is abolished when strains are not induced to export sucrose, and growth rates were relatively unaffected by illumination between 150 and 1000 µmol photons m^-2 s^-1 ( Figure 1A , left). These data suggest that high light may indeed be harmful, and the increased photosynthetic capacity provided by a heterologous sink (i.e., sucrose export) is not protective against light stress. However, it appears that S. elongatus PCC 7942 can remain resilient when not imposed with the additional burden of producing and secreting sucrose.

To evaluate if the sucrose productivity in these experiments was limited by a low-starting density, we increased the starting inoculation density. Since the effects of self-shading within higher density cultures lead to an effectively lower irradiation under the same outside illumination, we utilized brighter lights for our high-density starter cultures of 1.5 and 2.0 OD₇₅₀, the upper range of OD₇₅₀ the MC-1000 was able to support. To select the irradiances for these dense cultures, we revisited the most successful light intensity in our previous experiment. Cultures of 0.3 OD₇₅₀ exposed to 500 µmol photons m^-2 s^-1 light receive ~300 µmol photons m^-2 s^-1 light when measured 1 cm from the surface ( Supplementary Figure S4 ). Application of 2000 and 2500 µmol photons m^-2 s^-1 to cultures of 1.5 and 2.0 OD₇₅₀, respectively, would achieve similar internal light intensities ( Supplementary Figure S4 ).

We observed limited growth within the first 24 h of these cultures, but the density of the culture plateaued or declined in the following day to values statistically similar to starting turbidities (p > 0.05) ( Figure 1C ). Cultures grown with 2000 µmol photons m^-2 s^-1 produced 0.23 g L^-1 sucrose within the first day, and had a final sucrose content of 0.35 g L^-1 (p > 0.4); cultures grown with 2500 µmol photons m^-2 s^-1 produced 0.22 and 0.28 g L^-1 sucrose at 24 and 48 h (p = 1.0), respectively ( Figure 1D ). Notably, the level of sucrose production from high density cultures was only marginally improved (~15%) at 48 h relative to cultures seeded at a lower density grown with 2000 µmol photons m^-2 s^-1 (p = 0.002) ( Figures 1B, D ).

Curiously, both the cellular growth rate and sucrose productivity were relatively modest in the MC-1000 in comparison to values reported for similar strains by our group and others (Santos-Merino et al., 2023), suggesting that these cultures might be limited by another environmental variable ( Figures 1A, B ). Although 3% CO₂ was bubbled into the cultivation tubes for these experiments, the method of delivery (sparging via a large-bore needle) may not be the most efficient compared to other methods for enriching inorganic carbon. Consistent with a possible inorganic carbon limitation, sucrose productivity was slightly, but significantly, enhanced in MC-1000 cultures supplemented with 10 mM HCO₃ ^- ( Figure 1B ) relative to cultures without bicarbonate when grown with 500, 1000, and 2000 µmol photons m^-2 s^-1 (p = 0.038, 0.020, 0.028, respectively) ( Supplementary Figure S5 ). However, we were unable to overcome any potential limitation of inorganic carbon in dense cultures through the supplementation of additional HCO₃ ^- ( Supplementary Figure S6 ).

To more directly evaluate the hypothesis that carbon limitation was limiting for growth and sucrose productivity in the MC-1000, we utilized the High-Density Cultivation (HDC) system (CellDEG GmbH, Germany). These cultivation vessels are designed to deliver a saturating level of inorganic carbon through a permeable membrane with a large surface area (Lippi et al., 2018). Using an identical growth medium (BG-11), a significant enhancement of cell growth and sucrose production was observed in the CellDeg HDC system relative to the MC-1000 with comparable illumination (250 μmol photons m^-2 s^-1). Strains that were not induced to export sucrose grew to 5.3 OD₇₅₀ over 72 h while producing a marginal amount of sucrose (0.02 g L^-1). Strains induced to secrete sucrose grew to 4.6 OD₇₅₀ and produced 1.0 g L^-1 sucrose ( Figures 2A, B ). Curiously, under these conditions of high inorganic carbon availability, we observed little, but significant, difference in growth rate in the sucrose-exporting strains despite a significant rate of carbon export in the form of sucrose ( Figures 2A, B, E ).

Since nutrients in BG-11 medium can become limiting at higher density, we next evaluated an enriched medium (CD) designed to support high-density cultures, such as those achievable in the HDC system (Dienst et al., 2020). When cultures were grown in CD media, non-exporting strains reached 11.1 OD₇₅₀ over the course of 5 days ( Figure 2C ), while also secreting a disproportionally high level of sucrose for an uninduced culture ( Figure 2D ). Strains induced to export sucrose were significantly stunted in growth after 120 h (3 OD₇₅₀; Figure 2C ) and secreted sucrose at a higher rate.

Environmental conditions enriched with nutrients and inorganic carbon supported substantially higher cyanobacterial production with increased irradiation. Cultivating cscB/sps strains with high CO₂ (HDC) and with 500 μmol photons m^-2 s^-1, non-exporting and sucrose-exporting strains reached OD₇₅₀ of 15.8 and 5.2, respectively ( Figure 3A ). Furthermore, strains induced to secrete sucrose accumulated up to 3.3 g L^-1 over the course of 5 days, while uninduced cultures accumulated 1.15 g L^-1 ( Figure 3B ). Further enhancements in bioproduction could be achieved by further increasing light intensity to 1000 μmol photons m^-2 s^-1 at 48 h when cultures were more turbid: sucrose-secreting strains reached an OD₇₅₀ of 6.0 and sucrose yields of 3.8 g L^-1 ( Figures 3C, D ). Uninduced strains under these growth conditions achieved 18.4 OD₇₅₀ and secreted a surprisingly high amount of sucrose, reaching 2.3 g/L sucrose at the end of 120 h.