Directed Reaction Engineering Boosts Succinate Formation of Synechocystis sp. PCC 6803_Δsll1625

It is known that Synechocystis sp. PCC 6803 carrying a partial deletion of the succinate dehydrogenase (Synechocystis_∆sll1625) secretes succinate during aerobic cultivation with continuous illumination and in the presence of CO2. Maximal succinate titers of 2 mM (236 mg L−1) are reported. CO2 is identified as a crucial parameter for product formation, however, a detailed characterization of different cultivation conditions is still missing. Here the focus is on further reaction engineering to improve the photoautotrophic production of succinate using Synechocystis_∆sll1625. Therefore the impact of light availability, illumination regimes, nutrient availability, and external pH on product formation are investigated. Results obtained in this study reveal the importance of these parameters on the formation of succinate and cultivation with light/dark cycles increases the succinate concentration to 3 mM (354 mg L−1) after 28 days of cultivation. Furthermore, cultivation in unbuffered medium under ambient CO2 conditions even doubled the final succinate titer to 4 mM (472 mg L−1) after 28 days. Taking biomass concentrations into account, a maximal yield of succinate on biomass of 215 mgSucc gCDW−1 is achieved, which is the highest so far reported for the production of succinate utilizing Synechocystis as host organism.


Introduction
Succinic acid was selected as one of the top 12 building block chemicals from biomass by the US Department of Energy. [1] It is a C4-dicarboxylic acid and can be used as a precursor for numerous chemicals such as 1,4-butanediol or tetrahydrofuran. [2] In addition, it has great potential for the production of bio-based polymers like polybutylene succinate. [3] As many of these valuable derivatives were produced directly from petroleum-based chemicals, the current demand for succinic acid of 30 000-50 000 tons per year is rather low. [4] However, the bio-based production of succinic acid has improved during the last years, and the DOI: 10.1002/biot.202000127 market for succinic acid is expected to increase to more than 70 000 tons per year. [3] As succinate is an intermediate of the tricarboxylic acid (TCA) cycle, a wide range of different microorganisms including natural overproducers as well as recombinant production strains was, and is studied to replace the petrochemical process by a biotechnological approach. Currently, four companies (Reverdia, Succinity, BioAmber, and Myriant) have commercialized the production of bio-based succinic acid. However, the large-scale production of bio-succinic acid is based on common feedstocks like sugars, starch, and molasses and these organic carbon-based feedstocks often turn out to be the main cost driver in such bioprocesses. [4,5] In addition, despite being a renewable carbon source, the sustainability of such processes remains questionable. [6] Thus, the use of CO 2 as a cheap, and especially abundant inorganic carbon source moved more and more into the focus of biotech research. In this context cyanobacteria are highly interesting organisms. They are prokaryotes performing an oxygenic photosynthesis utilizing water as electron donor. A number of promising products synthesized by cyanobacteria from CO 2 have been reported in the recent years and it is widely accepted that cyanobacteria are versatile biocatalysts. [7][8][9] Amongst these organisms, Synechocystis sp. PCC 6803 (hereafter Synechocystis) and Synechococcus elongatus PCC 7942 (hereafter Synechococcus) species are the most prominent examples and the photoautotrophic production of succinate was already demonstrated for both strains. The studies for Synechococcus focused on the oxidative branch of the TCA cycle and a final succinate titer of 430 mg L −1 was reported. [10] So far the production of succinate using Synechocystis focused on the reductive arm of the TCA cycle as Synechocystis is known to secrete succinate in the absence of sugars by fermentation under dark anoxic conditions. [11] An additional overexpression of the gene coding for the phosphoenolpyruvate carboxylase and a cultivation temperature of 37°C resulted in the highest reported titer of 1.8 g L −1 (15 mM) under fermentative conditions but at an extraordinary high cell density of 25 g CDW L −1 . [12] All those studies have in common that they usually apply one standard condition for their reaction systems without looking at the impact of the various reaction parameters on productivities. The -ketoglutarate dehydrogenase for the conversion of -ketoglutarate to succinyl-CoA is missing in Synechocystis (dotted, grey arrow) but the TCA cycle is closed by an -ketoglutarate decarboxylase, succinic-semialdehyde dehydrogenase (pink arrows), and the GABA shunt (blue arrows). The succinate dehydrogenase (SDH) catalyzes the conversion of succinate to fumarate and sll1625 encoding subunit B of the SDH is deleted in the strain used in this study (Synechco-cystis_∆sll1625). AcCoA, acetyl coenzyme A; GABA, -aminobutyric acid; GLU, glutamate; KG, -ketoglutarate.
In our study we utilize the mutant strain Synechcocys-tis_∆sll1625 carrying a deletion of sll1625 encoding subunit B of the succinate dehydrogenase (Figure 1). This strain is able to photoautotrophically produce succinate up to 420 mg L −1 (3.5 mM) via the oxidative route of the TCA cycle. [13] Here, we focused on the systematic investigation of reaction parameters, and their impact on the photoautotrophic production of succinate, which can be doubled by simply changing the cultivation conditions.

Under Continuous Illumination Increasing Light Intensities Do Not Impact Final Product Titers
For the cultivation of photoautotrophic organisms light is the central energy source. In general, exponential growth of cyanobacte-ria can only be observed at low cell densities below ≈0.2 g CDW L −1 before the culture enters a light-limited stage indicated by a linear growth behavior. This depends on the path length of the light through the culture and incident illumination intensity. [14] Here, we investigated the influence of light availability on the photoautotrophic formation of succinate. Succinate-producing Synechocystis_∆sll1625 was cultivated at 2% CO 2 in shake flasks and light intensities were stepwise increased. Thereby the light regime described by David et al. for Synechocystis sp. PCC 6803 was applied, starting from 25 µE m −2 s −1 , and increasing the light intensity up to 200 µE m −2 s −1 . [15] Results were compared to standard cultivation conditions applying constant light intensities of 50 µE m −2 s −1 (Figure 2). [13] The stepwise increase of the light intensity to 200 µE m −2 s −1 did not really impact final biomass and product titers. Essentially the same values of 2.6 g CDW L −1 and 2 mM of succinate were reached independent of the applied light regime, with most of the succinate being formed during linear and stationary phases. At 25 µE m −2 s −1 growth was clearly light limited and the exponential growth rate decreased from 0.067 to 0.040 h −1 , while succinate production started with a corresponding time shift of 3 days compared to incubation at 50 µE m −2 s −1 .

Dark/Light Cycles Promote Succinate Production
Many studies on the production of value-added compounds using photoautotrophic organisms are performed with a continuous and constant illumination of the respective culture. However, in nature, cells have to cope with seasonal changes in light availability, and with the diurnal rhythm of day and night. Thus Syne-chocystis_∆sll1625 was cultivated in light/dark cycles with 16 h of continuous illumination (50 µE m −2 s −1 ) and 8 h of complete darkness, representing an average summer day in northern Europe. Switches between both phases were instantaneous and gradual illumination and dimming as typical for sunrise and sunset were neglected. Sampling during this experiment was always done directly before turning off, or turning on the light.
As shown in Figure 3A growth was diminished under these conditions as only a final biomass concentration of 1.8 g CDW L −1 (OD 750 = 9) instead of 2.6 g CDW L −1 (OD 750 = 13) was achieved. In addition, OD values only increased during periods of illumination and stayed constant or even decreased in the darkness. As already observed in reference [13] , product formation mainly occurred in the stationary phase, where cells do not divide but are still metabolically active. Furthermore, results showed that succinate was also formed without light and the final product concentration was increased from 2 to 3 mM and consequently also the yield of succinate on biomass of 197 mg Succ g CDW −1 was higher (continuous illumination: 91 mg Succ g CDW −1 ). The results indicated a positive effect of phases without illumination on the formation of succinate. Therefore the experiment was repeated with altered light/dark periods ( Figure 3B). Periods of illumination were reduced to 8 h and phases of darkness were prolonged to 16 h, representing an average day in winter in northern Europe. However, growth was further diminished under these conditions, and only a final biomass concentration of 1.26 g CDW L −1 (OD 750 = 6) was reached. In addition, the  succinate titer was decreased to 2 mM, but the biomass specific succinate yield was essentially the same, and reached a value of 188 mg Succ g CDW −1 . In summary, it was shown that periods of darkness can have a positive impact on the photoautotrophic formation of succinate but the ratio of dark to illuminated periods plays a crucial role.

The External pH Influences the Formation of Succinate
The use of buffered YBG11 medium was established as cyanobacteria alkalize their growth medium due to the consumption of HCO 3 leading to the liberation of OH − and thus in an increase in pH. [10] For Synechococcus it was already shown that an adjustment of the pH to a value of 7.5 reduces the formation of succinate and that titers increase with increasing pH. [10] The underlying mechanism of this effect is not known. To check the influence of the external pH on the succinate formation in Syne-chocystis_∆sll1625, shake flask experiments have been carried out in unbuffered YBG11 under constant illumination at 50 µE m −2 s −1 . The initial pH was adjusted to 7.2 (as for the buffered version). It has to be mentioned that the use of unbuffered medium was only possible under ambient CO 2 conditions. Elevated levels  [13] Black symbols correspond to unbuffered, grey symbols correspond to buffered medium. Mean values and standard deviation of biological duplicates are shown. pH was only measured in one of the cultures, respectively. of CO 2 resulted in a significant decrease of pH to a value below 6, which completely prevented growth of Synechocystis (data not shown).
The pH value was determined during the cultivation and compared to the pH of a culture grown in parallel in YBG11 medium that was buffered with 50 mM HEPES ( Figure 4A). In the unbuffered medium, pH increased to a value of 11 during the first 3 days of cultivation, stayed constant for the following 5 days, and afterward decreased again to settle at a level of about 8.6. Growth rates of Synechocystis_∆sll1625 were comparable to the cultivation in buffered medium, only the final biomass concentration was slightly increased to 2.2 g CDW L −1 (OD 750 = 11 for the unbuffered medium instead of 9), and did not decrease after 12 days of cultivation ( Figure 4B). Furthermore, analogous to the results published for Synechococcus, succinate accumulation was higher compared to the cultivation with a constant pH, and the final titer was increased by a factor of nearly 5 to about 4 mM.

Higher Salt Concentrations Increase Biomass and Succinate Concentrations
Besides light and CO 2 , other nutrients/salts in the media can also limit biomass and product formation due to a restricted availability. To evaluate possible limitations due to medium composition, Synechocystis_∆sll1625 was cultivated in 5xYBG11, with a constant illumination of 50 µE m −2 s −1 , and 2% of CO 2 (Figure 5).
Cells grew with a rate of 0.083 h −1 , which is higher compared to the cultivation in normal YBG11 medium where a growth rate of only 0.067 h −1 was reached. Linear growth started already on the second day of the cultivation in 5xYBG11, most likely because the higher cell densities resulted in an earlier light limitation. Nevertheless, the maximal OD 750 was nearly tripled, and Syne- chocystis_∆sll1625 grew to a final OD 750 of 40 which corresponds to a biomass concentration of 8 g CDW L −1 . Also the succinate titer was doubled to 4 mM but taking the high biomass concentration into account, the resulting yield of succinate on biomass was lowered from 91 to 59 mg Succ g CDW −1 compared to the cultivation in normal YBG11 medium. Interestingly, when combining 5xYBG11 with unbuffered conditions or light/dark cycles, the culture barely grew and cells turned yellowish after a short period of time (data not shown). The results of this study clearly showed that reaction engineering which focuses on the investigation of cultivation conditions is very important to optimize the formation and accumulation of succinate in Synechocystis_∆sll1625. While an increase in light intensity did not influence product formation in shake flasks, the application of light/dark cycles and the use of unbuffered culture medium resulted in higher succinate titers and higher biomass specific succinate yields.

Discussion
Here we focused on the impact of cultivation conditions on the phototrophic succinate production by Synechocystis_∆sll1625 via the oxidative branch of the TCA cycle. Especially the influence of the two main substrates CO 2 and light, as well as the impact of the extracellular pH, and the nutrient concentration of the cultivation medium on the accumulation of succinate and cell growth were investigated. All investigated parameters and the impact they had on succinate production are summarized in Figure 6.
The optimal reaction conditions in terms of yield on biomass are achieved in unbuffered medium at ambient CO 2 concentrations and a constant illumination of 50 µE m −2 s −1 where 215 mg succinate g biomass −1 are reached. Furthermore, Figure 6 shows how delicate the balance between reaction conditions, product yields, and titers is and that the tuning of these parameters has a significant influence on the overall productivity of the systems. In the following, the individual parameters are discussed in detail.

Light is a Critical Factor during the Cultivation of Synechocystis
Light is one of the key substrates for photoautotrophic organisms. While growth of heterotrophic organisms in batch cultivations is divided into a log, an exponential, and a stationary growth phase, phototrophic organisms exhibit an additional linear growth phase subsequent to the exponential phase. This phase is most likely due to a limitation of light as a result of self-shading. [14] A stepwise increase of the light intensity during cultivation was shown to prolong exponential growth and also to increase the product titers in case of the photoautotrophic production of propylene glycol with Synechocystis. [14,15] In that case the formation of propylene glycol was shown to be linked to the degradation of the intracellular storage compound glycogen, which was positively affected by increased light intensities. [15] However, succinate excretion by Synechocystis_∆sll1625 is not connected to glycogen titers, and perhaps this is the reason why such a correlation was not observed in the case of succinate. [13] In addition, final OD and product titers were essentially the same as for the cultivation with continuous illumination pointing to another limitation than light in this case.
In contrast to light intensities, the cultivation under a day/night regime had a significant impact on the performance of Synechocystis_∆sll1625. A cycle of 16 h of constant illumination and 8 h of darkness resulted in diminished growth but increased succinate formation to a yield of 197 mg Succ g CDW −1 . OD 750 values always decreased a little during periods of darkness while succinate was still produced. This indicates glycogen conversion in the dark periods, which is connected to a reduction in cell volumes, and thus a decrease in the OD values. [13] Thus, succinate formation during periods of darkness seemed to be linked to glycogen turnover while this is not the case when cells are illuminated. However, extended periods of darkness did not have a positive impact on biocatalyst performance, rather the opposite was observed. Possibly, if illumination periods are too short, the amount of glycogen that can be generated as a storage compound for dark metabolism is too low to maintain the cells.

Cultivation Medium Influences Succinate Titers
Apart from light and CO 2 , pH turned out to be a relevant parameter to increase extracellular succinate concentrations. [13] Cultivation in unbuffered medium boosted succinate titers by a factor of 5. As the final biomass concentration did not change significantly, the biomass specific succinate yield of 215 mg Suc g CDW −1 was the highest achieved with Synechocystis_∆sll1625 ( Figure 6). A positive effect of this approach was already published for the photoautotrophic succinate production via the oxidative route of the TCA cycle with Synechococcus. [10] For Synechococcus, no succinate transporter was annotated. Thus Lan and Wei used BLAST for seven of the succinate transporters described for Escherichia coli against the genome of Synechococcus. [10] The BLAST was conducted on the amino acid level and resulted in one possible hit with an identity of about 30%. The corresponding protein DauA belongs to the superfamily of sulfate transporters and was recently identified to be involved in the succinate import of E. coli. [16] Thereby a pH-dependent activity was shown, as DauA www.advancedsciencenews.com www.biotechnology-journal.com played a more important role in succinate uptake at alkaline pH. [16] For Synechococcus, it was hypothesized that this pH dependency might result in an increased export of succinate at higher pH. However, experimental evidence is still missing. Also for Synechocystis the presence of a specific succinate transporter is unknown. Therefore the protein BLAST of the seven transporters from E. coli was repeated for Synechocystis. Similar to Synechococcus only DauA gave four possible hits. One of the possible ORFs codes for BicA, which is a bicarbonate transporter, and can thus be excluded as a possible succinate transporter. The other three ORFs (slr0096, slr1229, and slr1776, GeneBank accession numbers: AGF52860.1, AGF51203.1, AGF50474.1) had a query cover of 79% or higher, and code for proteins of the sulfate-transporter superfamily and therefore might be identical to DauA. Thus, the hypothesis from Lan and Wei could also explain the results obtained for Synechocystis in this study.
In addition to the pH, the availability of nutrients in the culture medium can also influence biocatalytic performance of Synechocystis. For example, it was stated in the literature that the photoautotrophic formation of ethylene is increased during cultivation in the presence of higher nutrient concentrations. [17] When using 5xYBG11, the biomass concentrations, as well as the final succinate titer, were increased, and Synechocystis_Δsll1625 reached a final succinate concentration of 4 mM (472 mg L −1 ). Nevertheless, taking the high biomass concentration into account, the resulting yield on biomass is lower compared to the cultivation in unbuffered medium or the cultivation with light/dark cycles ( Figure 6). Unfortunately, Synechocystis_Δsll1625 did not grow in 5xYBG11 medium without the addition of HEPES buffer to investigate the impact of combined optimized cultivation conditions.

Cyanobacterial Routes toward Succinate
In 2015 the first publication on the production of succinate utilizing Synechocystis as host organism was published [11] and it was shown that Synechocystis excretes succinate under dark and anaerobic conditions via the reductive arm of the TCA cycle. This approach was taken up in several other studies and by metabolic engineering and adaptation of cultivation conditions a final succinate titer of 1.8 g L −1 (15 mM) could be achieved, which is the highest titer reported so far for the photoautotrophic production of succinate. [12,18] However, taking the high biomass concentration of 25 g CDW L −1 into account, the approach via the reductive branch of the TCA cycle resulted in a yield of 72 mg Succ g CDW −1 . [12] As an alternative to Synechocystis, Synechococcus was also used to produce succinate from CO 2 . In contrast to the studies on Synechocystis, the oxidative branch of the TCA cycle was utilized in Synechococcus, and a maximal biomass specific succinate yield of 843 mg Succ g CDW −1 is reported. [10] A comparison of the results obtained for Synechocystis under dark and anaerobic conditions with the results obtained in this study clearly shows that the yields of succinate on biomass are higher if succinate is produced via the oxidative route of the TCA cycle although cultivation times are longer. However, the published examples include two steps of cultivation. Biomass is formed in periods of illumination and afterward cells are harvested and concentrated for cultivation under dark and anaerobic conditions. Taking this into account, differences become minor, and the labor input is lower if the oxidative branch is utilized. A comparison with the results published from Lan and Wei using Synechococcus as host organism shows the potential of the phototrophic production of succinate.
Despite being interesting microbial cell factories for biotechnological processes, there are still major bottlenecks to overcome before cyanobacteria will be applied for truly competitive production processes. Especially when considering bulk chemicals like succinate where established production routes are already in place, pending challenges appear huge. In the case of succinate a couple of biotechnological processes have been developed based on heterotrophic organisms and productivities of up to 3 g L −1 h −1 have been achieved with the respective strains. [19] Compared to those examples, rates and titers which we can reach with Synechocystis sp. PCC 6803 are at least factor 1000 too low to reach a competitive level.
Reaction engineering, as performed in this study, illustrates that parameters like light, illumination regimes, and pH have a huge impact on reaction performance and need to be looked at on a case to case basis. Nevertheless, productivities could at best be improved by a factor of five. A better knowledge on the overall cyanobacterial system on all levels is required to enable directed metabolic engineering to enhance formation and secretion of succinate by the whole cell biocatalyst. This includes the interplay of light and dark reactions with storage compound turn over, overall role of the TCA cycle in cyanobacterial metabolism, and the impact of pH on the cellular physiology. Besides this, succinate titers may also be increased by higher biomass concentrations. Typically cell densities are limited to 3 g CDW L −1 in established closed photo-bioreactors like flatpanels and tubular systems. One approach to overcome this limitation could be the cultivation of Synechocystis as a biofilm as recently reported in ref. [20] . Thereby, 40 g L −1 were reached with Synechocystis sp. PCC 6803. Assuming a succinate yield of ≈1 g g CDW −1 as a benchmark, cultivation in a biofilm format would result in a succinate titer of 40 g L −1 , and a productivity of 0.08 g L −1 h −1 (assuming a stable production over 20 days), reducing the necessary improvement factor from 1000 to 40.

Experimental Section
Chemicals: All chemicals used in this work were obtained in the highest purity available from Carl Roth GmbH & Co. KG (Karlsruhe, Germany), Merck KGaA (Darmstadt, Germany), Sigma-Aldrich (St. Louis, USA), and Th. Geyer GmbH & Co. KG (Renningen, Germany).
Cultivation Conditions: If not stated otherwise, wild type and the ∆sll1625 mutant of the substrain Synechocystis sp. PCC 6803_moscow were grown in YBG11 medium containing 50 mM HEPES. For the mutant strain, the medium was supplemented with 50 µg mL −1 kanamycin. [21] For solid medium cultivations, BG11 supplemented with 0.75% agar was used. [22] Liquid pre-cultures were inoculated with Synechocystis from an agar plate. Main cultures were inoculated with liquid pre-cultures to an initial OD 750 of 0.05. Cultivation was carried out in a Multitron shaker (Infors, Bottmingen, Switzerland), equipped with LED panels at 30°C, 150 rpm, and 75% relative humidity. Depending on the experimental set-up, illumination was either kept at a constant value of 50 µE m −2 s −1 , or was increased stepwise from 25 to 200 µE m −2 s −1 , or light/dark cycles were applied. The CO 2 concentration during cultivation was either set to 0.04% (ambient conditions) or was increased to 2% or 5%. During cultivation planktonic cell growth of Synechocystis sp. was followed spectrophotometrically measuring the optical density at a wavelength of 750 nm (OD 750 ), ensuring no overlap with chlorophyll absorptions. For the calculation of the CDW from OD 750 values correlation factors of 0.17 for Synechocystis wild type and 0.21 for the ∆1625 mutant were used. [13] Determination of Extracellular Succinate Levels: Succinate was quantified by HPLC on a Dionex Ultimate 3000 system (Thermo Fisher Scientific, Waltham, USA) equipped with a RI detector and a HyperREZ XP Carbohydrate H + column (300 × 7.7 mm, 5 µM). 5 mM H 2 SO 4 was used as eluent with a flowrate of 0.75 mL min −1 . The column temperature was set to 25°C and a volume of 20 µL was injected. Samples were centrifuged for 10 min at rcf 17 000 and the supernatant was transferred to HPLC vials. Quantification was based on calibration curves prepared with standard concentrations of succinic acid.