Specific production of sucrose increases in immobilized Synechocystis S02
In order to enhance sucrose production by the engineered Synechocystis S02 cells and induce its export to the medium, cultures were resuspended in BG11 + NaCl medium (OD750 = 0.5) supplemented with 1 mM IPTG (see the Materials and methods section for more details).
Suspension cultures of sucrose-producing Synechocystis S02 showed almost linear growth during 7 days of incubation reaching optical density at 750 nm (OD750) of ~ 6.8 (Fig. 3A). Simultaneously, chlorophyll a (Chl) concentration steadily increased and saturated at the 6th day (Fig. 3B). Sucrose accumulated in the medium in a linear manner until the 7th day, after which it plateaued at the maximum concentration of 1910 mg l.−1 (Fig. 3C), which corresponds to ~ 70 mg sucrose per mg of Chl (Fig. 3D). These results are comparable to the data obtained by Thiel et al. [14]
The entrapment of algae and cyanobacteria in the polymeric matrix is known to diminish cell division and formation of biomass, thus allowing to divert energy for production of targeted chemicals [23, 25]. Such solid-state photosynthetic production systems transfer cells to long-lived biocatalytic production mode. Therefore, in the next step we immobilized the sucrose-producing cells in 3% (wt/v) alginate beads cross-linked with Ca2+-ions. The beads can endure vigorous shaking, which facilitates the mass transfer between the cells and the medium through the matrix.
Different from suspension cultures, the immobilized cells showed the most pronounced Chl accumulation during the initial phase of the sucrose production (Fig. 4A).
By the 3rd day of the experiment, the color of the beads became much darker than in the beginning due to the Chl accumulation. Then, the Chl content of the beads increased slower until the 5th day and started gradually declining after that (Fig. 4A). The initial rise in the Chl content can be explained by the cell division and the increase of the biomass within the matrix until the point when the immobilized cultures start experiencing light and presumably nutrient limitations, resulting in inhibition of metabolic activity and cell growth by the end of the experiment. It is important to note that cell outgrowth from the beads was hardly noticeable during 7 days of sucrose production.
Ca2+-alginate-entrapped cells produced sucrose similar to suspension cultures (Figs. 4 and 3). The specific productivity of both cultures steadily increased from the beginning of the experiment and reached the maximum on the 7th day, after which production ceased under both setups (Figs. 3C and 4B). It is noteworthy that the immobilized cells showed significantly higher specific production yields compared to suspension cells throughout the experiment reaching the 86% increase (1200 and 700 mg sucrose mg−1 Chl, respectively) by the 7th day (Fig. 4C). The total maximum sucrose yield of immobilized cells was 1150 mg l−1 (Fig. 4B).
It was demonstrated in other works that the immobilization of Synechocystis is an effective way to increase the production yield of the cells. Immobilization in Ca2+-alginate beads was reported to increase succinate [26] and β-phellandrene [27] production, while immobilization in Ca2+-alginate thin film was shown to be effective for the increase of ethylene production [28]. The immobilization was previously reported to be effective to increase sucrose production as well, with engineered Synechococcus elongatus sp. PCC 7942 cells showing a 2- to 3-fold increase in specific sucrose production after their entrapment within Ba2+-alginate beads [8]. Our results obtained with Synechocystis S02 cells immobilized within Ca2+-alginate beads are in line with the above-mentioned studies. The production of sucrose by Synechocystis cells could be further enhanced by genetic engineering towards constitutive sucrose synthesis without application of the high salt stress and by redesigning the photosynthetic electron transport for enhanced carbon partitioning towards the sucrose production [14, 18]. Further technological improvements are also possible, such as the design and use of specialized photobioreactors with the improved light distribution and the application of new immobilization materials with better porosity and mechanical stability.
Sucrose production stimulates photosynthetic O2 evolution and CO2 fixation
The real-time gas fluxes were monitored in immobilized cells and in cells grown in suspension by membrane inlet mass spectrometry (MIMS) to investigate the correlation between sucrose production and photosynthetic activity. After 3 days of sucrose production, net photosynthetic O2 evolution both in suspension and immobilized cultures was significantly higher (P = 0.02–0.04) in cultures producing sucrose (+ NaCl) compared to non-producing ones (-NaCl) (Fig. 5A). Net CO2 yield rates were also higher in the sucrose-producing cells, albeit the difference was statistically significant only in suspension cultures (P = 0.006) (Fig. 5B). These results suggest that sucrose production acts as a strong sink for photosynthetic CO2 fixation, which increases the overall photosynthetic activity of the cells. The increase in net O2 evolution and net CO2 fixation yield in suspension cultures of sucrose-exporting cells was also described previously [14, 29]. It is important to note that we observed higher gas-flux rates in suspension cultures compared to bead-immobilized cells, which can be attributed to the low gas permeability of the alginate matrix and other effects of immobilization on the cell metabolism [25]. By the 7th day, the photosynthetic O2 evolution and carbon fixation rates dropped both in the suspension cultures and immobilized cells, demonstrating a decrease in photosynthetic activity. In line with the real-time gas exchange results the effective photosynthetic yield Y(II) in both cultures dropped from 0.37 to 0.08, which was accompanied by the decline of sucrose production. A possible reason for the decline in photosynthetic activity is the accumulation of sucrose, which switches the cell metabolism into photomixotrophy, when the cells start metabolizing sucrose as a carbon source while performing photosynthesis and CO2 fixation [30]. This form of metabolism, which provides phototrophic cells with extra energy and carbon, often occurs when organic carbon sources are available in the environment, for example during phytoplankton blooms. Besides decreased photosynthetic activity, the transition to photomixotrophic growth is accompanied by increased cell respiration [31]. Indeed, we observed a tendency to enhanced respiration by the end of the experiment almost under all conditions, except in unstressed suspension cultures (-NaCl Susp) where respiration did not change (Fig. 5C). However, based on this data we could not clearly state if enhanced respiration is linked to the transition to mixotrophic growth. Net CO2 yield, which represents a difference between CO2 consumption in the CBB cycle and CO2 release in respiration, was low after 3 days in all cases and dropped below the respiration compensation point by the 7th day. The latter indicates on a significant drop in CO2 fixation capacity and correlates well with high respiration rates (Fig. 5C), which finally affect the sucrose productivity. The further studies are needed for understanding mechanisms leading to the transition to the photomixotrophic growth and the declined sucrose production capacity. In the long-term production process, photomixotrophy can be abolished by periodically refreshing the medium throughout.
The cultivation, thus preventing nutrient limitation and sucrose accumulation in the cultures leading to the end-product inhibition effect and further metabolization of sucrose by the cells.
Semi-continuous production mode leads to prolongation of the sucrose production in beads
To remove secreted sucrose, the medium was refreshed every 3–4 days after sampling throughout the experiment. By employing this method, the period of efficient sucrose production was considerably prolonged. At day 10, the cumulative production yield was close to 2200 mg sucrose l−1 (Fig. 6, black squares) and then, the production activity started to decline gradually (Fig. 6, yellow bars). As a result, the production yield dropped considerably by the 17th day, but even after that point negligible amount of sucrose was detected in the medium until the end of the experiment (Fig. 6). The beads remained stable during the 27 days of incubation and outgrowth from the beads remained inconsequential in the beginning of production but increased after 2 weeks of cultivation (up to OD750 1.0 on the 13th day), and then continued to increase until the end of the experiment reaching the maximum OD750 1.5 (Fig. 6). This can be attributed to the slow degradation and the gradual breach of the surface of the beads. Bead immobilization is a practical and effective method for semi-continuous cultivation since no energy-intensive centrifugation is needed and even on an industrial scale, the medium can be easily changed through a valve system. The cumulative maximum sucrose yield during the semi-continuous cultivation reached 3000 mg l−1 after 17 days (Fig. 6), which is almost three times higher compared to the amount obtained during 7 days of batch production. This clearly demonstrates that semi-continuous cultivation is a better alternative than batch cultivation to maximize the amount of harvested sucrose by prolonging the duration of the production period of Synechocystis S02 immobilized in Ca2+-alginate beads.
Sucrose produced by Synechocystis drives biotransformation in E. coli
The next step was to verify the capability of sucrose produced by immobilized Synechocystis S02 cells to sustain the biotransformation of cyclohexanone to ε-caprolactone in recombinant E. coli. For this purpose, we engineered E. coli WΔcscR Inv, which is capable of effectively utilizing even low amounts of sucrose due to the deactivation of sucrose catabolism repressor gene (cscR) and heterologously expressed invertase enzyme with an N-terminal pelB leader sequence for export to the periplasm [32]. Expression of a heterologous Baeyer–Villiger monooxygenase from Parvibaculum lavamentivorans (BVMOParvi) in the WΔcscR Inv background enabled the biotransformation of cyclohexanone, exogenously added substrate, to ε-caprolactone by utilizing the sucrose produced by immobilized Synechocystis S02 (Fig. 7).
Sucrose was produced in BG11 + NaCl by alginate immobilized Synechocystis S02 cells over a 7-day period (1150 mg l−1). Then the medium was removed from the beads and used as culture medium for the biotransformation by the E. coli WΔcscR Inv:Parvi strain. We monitored the biotransformation by the engineered E. coli cells to evaluate the potential of coupled production. Under these conditions, we observed full biotransformation of cyclohexanone to ε-caprolactone within three hours (Fig. 7A) with the average transformation rate of 0.9 mM h−1. M9 minimal medium used for the cultivation of E. coli was supplemented with 10 mM sucrose and used as a positive control for the biotransformation. The control reaction in M9 was faster, and proceeded to completion within 2 h, with the average rate of 2.3 mM h−1 (Fig. 7B). As a negative control fresh BG11 + NaCl medium without any additional sucrose was used to ascertain that the E. coli is not capable of performing the biotransformation reaction without sucrose, utilizing for example, energy stored during previous cultivation steps. Only minimal amount of cyclohexanone was converted to ε-caprolactone in the absence of sucrose over the 24-h time period (Fig. 7C). Since both the substrate (cyclohexanone) and the product (ε-caprolactone) are semi-volatile, the final concentration of the product can deviate from the initial substrate concentration. From these results it is evident that the E. coli WΔcscR Inv:Parvi strain is capable of fast conversion of cyclohexanone to ε-caprolactone without the formation of side-product, cyclohexanol. The conversion rate is comparable to other E. coli strains harboring BVMOs when utilizing rich TB-medium [9]. This also confirms that an organic carbon source, in our case sucrose, is essential for the cyclohexanone biotransformation in E. coli, as expected based on the BVMO dependence on NADPH, which is generated via the glycolytic pentose phosphate pathway in this host. [33]. However, other components of the previously used TB-medium seem to be less important, while the high salt concentration is no hindrance for cells to perform the biotransformation under the used conditions. Altogether, the data unambiguously demonstrate that sucrose produced by Synechocystis S02 over a 1-week batch culture, is sufficient to drive the conversion of 5 mM cyclohexanone to ε-caprolactone by E. coli WΔcscR Inv:Parvi without any downstream modification or manipulation of the medium.
Our findings support the general concept of utilizing carbohydrates synthesized from CO2 by engineered cyanobacteria as a source of energy for biotransformation catalyzed by heterotrophic microbes and lay the foundation for an alternative sustainable ε-caprolactone production platform. To effectively couple the photoautotrophic and heterotrophic production systems in actual co-cultures where the two process is simultaneously ongoing in one bioreactor, however, further optimization is needed. In contrast to the published co-cultures [8, 19,20,21,22] biotransformation offers ‘the substrate-in-product-out’ concept, where besides the sucrose produced by cyanobacteria also the external substrate has to be fed to the E. coli cells. In the published examples the compounds produced by the heterotrophs are products derived from the carbon fixed by the phototroph from CO2 and no compound is fed to the strain to be transformed. The CO2 that the phototroph converts to sucrose is either utilized for the synthesis of compounds or for the growth of the heterotroph [8, 19,20,21,22]. The issues that need to be addressed to establish a working co-culturing system are (i) the considerable difference between the rate of sucrose production and the biotransformation process. This challenge could be overcome by introducing the E. coli performing the biotransformation together with cyclohexanone at later stages of the sucrose production to ensure sufficient amounts of sucrose to sustain the biotransformation; (ii) the overall slow sucrose production rate. This could be addressed by further engineering the sucrose producer or by exploring different strains, such as the promising Synechococcus elongatus UTEX 2973 [18]; (iii) the effective collecting of the biotransformation product without interrupting the sucrose production and co-culturing as well as the (iv) ways to avoid losses of the substrate and the product of the biotransformation in a prolonged setup remain challenges, that need experimental trials to find ways to overcome them.