Overexpressing Carotenoid Biosynthetic Genes in Synechocystis sp. PCC 6803 Improved Intracellular Pigments and Antioxidant Activity, Which Can Decrease the Viability and Proliferation of Lung Cancer Cells In Vitro

In the antioxidant system in cyanobacteria, non-enzymatic antioxidants, such as carotenoids, are considered good candidates for coping with oxidative stress, particularly light stress, and pharmaceutical therapeutic applications. A significant amount of carotenoid accumulation has been recently improved by genetic engineering. In this study, to achieve higher carotenoid production with higher antioxidant activity, we successfully constructed five Synechocystis sp. PCC 6803 strains overexpressing (OX) native genes related to the carotenoids biosynthetic pathway, including OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR. All of the engineered strains maintained a significant quantity of myxoxanthophyll, while increasing zeaxanthin and echinenone accumulation. In addition, higher components of zeaxanthin and echinenone were noted in all OX strains, ranging from 14 to 19% and from 17 to 22%, respectively. It is worth noting that the enhanced echinenone component responded to low light conditions, while the increased β-carotene component contributed to a high light stress response. According to the higher antioxidant activity of all OX strains, the carotenoid extracts presented lower IC50 in lung cancer cell lines H460 and A549, with values less than 157 and 139 µg/mL, respectively, when compared with those of WTc, particularly OX_CrtR and OX_CrtQ. A higher proportion of zeaxanthin and β-carotene in OX_CrtR and OX_CrtQ, respectively, may considerably contribute to the ability to treat lung cancer cells with antiproliferative and cytotoxic effects.


Introduction
Carotenoids (Cars), present in all oxygenic photoautotrophic organisms, are intracellular pigments that are typically encased in cellular membranes and assist in the assembly and function of photosynthetic complexes [1][2][3][4][5]. Carotenes and their oxygenated derivatives, i.e., xanthophylls such as echinenone, myxoxanthophyll, nostoxanthine, and zeaxanthin, belong to a group of Cars. Their functions serve as not only accessory light-harvesting pigments but also as photoprotective agents when cells are exposed to light stress, such as high light intensity and ultraviolet radiation [6][7][8]. Regarding the hydrophobic molecules of β-carotene and echinenone, they are able to form complexes with proteins, thereby functioning as a bridge between proteins in photosynthetic systems [1,5], as well as a scavenger of singlet oxygen [9][10][11]. The xanthophylls were found to have a stronger scavenging character than the carotenes [12]. Although there is currently no evidence that the xanthophylls bind to membrane proteins, it was expected that xanthophylls would rigidify membranes, Figure 1. A tentative scheme of carotenoid synthesis and degradation in relation to chlorophyll synthesis and degradation (modified from [1,25,27]). Genes encoding enzymes in each pathway are indicated in italics. The scheme indicates "?" representing enzymes that are encoded by unidentified genes in Synechocystis sp. PCC 6803.
In this study, we improved total Cars and Car components with higher antioxidant activity by constructing five engineered Synechocystis sp. PCC6803 strains with overexpressing genes involved in the carotenoid biosynthetic pathway ( Figure 1). Five engineered strains consisted of (1) Synechocystis sp. PCC6803, overexpressing the slr1255 gene or OX_CrtB, (2) Synechocystis sp. PCC6803, overexpressing the slr1254 gene or OX_CrtP, (3) Synechocystis sp. PCC6803, overexpressing the slr0940 gene or OX_CrtQ, (4) Synechocystis sp. PCC6803, overexpressing the slr0088 gene or OX_CrtO, and (5) Synechocystis sp. PCC6803, overexpressing the sll1468 gene or OX_CrtR. In addition, all engineered stains contained a significant increase in total lipids when compared to the wild-type control. Particularly, the cell viability and proliferation of H460 and A549 lung cancer cell lines could be markedly reduced by carotenoid extracts from OX strains when compared to the wild-type control.

Synechocystis sp. PCC 6803 Mutant Strains and Their Carotenoid Contents under Normal Growth Conditions
Six engineered Synechocystis sp. PCC 6803 strains, including the wild-type control (WTc), OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR (Table 1), were constructed via a double homologous recombination. The wild-type control (WTc) strain was generated by substituting the psbA2 gene with a Cm R cassette (Figure 2A), whereas each OX strain was created by substituting the psbA2 gene with each gene fragment in the Synechocystis wild-type (WT) genome (Figure 2A-E). The complete segregation in Synechocystis genome was confirmed by PCR using UppsbA2 and DSpsbA2 primers (B Primers, Figure   Figure 1. A tentative scheme of carotenoid synthesis and degradation in relation to chlorophyll synthesis and degradation (modified from [1,25,27]). Genes encoding enzymes in each pathway are indicated in italics. The scheme indicates "?" representing enzymes that are encoded by unidentified genes in Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 Mutant Strains and Their Carotenoid Contents under Normal Growth Conditions
Six engineered Synechocystis sp. PCC 6803 strains, including the wild-type control (WTc), OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR (Table 1), were constructed via a double homologous recombination. The wild-type control (WTc) strain was generated by substituting the psbA2 gene with a Cm R cassette (Figure 2A), whereas each OX strain was created by substituting the psbA2 gene with each gene fragment in the Synechocystis wild-type (WT) genome (Figure 2A-E). The complete segregation in Synechocystis genome was confirmed by PCR using UppsbA2 and DSpsbA2 primers (B Primers, Figure 2 and Supplementary Information Table S1). In the WTc strain, PCR products using B Primers showed no band, while it showed a 2.4 kb gene fragment when using A Primers. In Figure 2A-E, PCR products with the A Primers of OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR strains confirmed the correct sizes of 2.2, 2.5, 2.6, 2.7, and 2.0 kb, respectively, whereas PCR products with B Primers confirmed the correct sizes of 3.4, 3.7, 3.7, 3.9, and 3.2 kb, respectively. OX_CrtO, and OX_CrtR strains confirmed the correct sizes of 2.2, 2.5, 2.6, 2.7, and 2.0 kb, respectively, whereas PCR products with B Primers confirmed the correct sizes of 3.4, 3.7, 3.7, 3.9, and 3.2 kb, respectively.  Table S1) were chosen to confirm the integration of each gene into the Synechocystis genome. All strains were constructed by overexpressing each native gene in the wild-type control.   Table S1) were chosen to confirm the integration of each gene into the Synechocystis genome. All strains were constructed by overexpressing each native gene in the wild-type control. In addition, RT-PCR data verified the gene overexpression in all OX strains with a higher transcript amount in comparison with those of WTc ( Figure 3A). For total carotenoid content, the increased carotenoid accumulation in OX strains was noted when the cells were grown before reaching their late-log phase (12 days of cultivation) of growth under normal growth conditions ( Figure 3B). At 12 days of growth, all OX strains cultivated under normal conditions had significantly increased contents of five Cars detected by HPLC, including myxoxanthophyll, zeaxanthin, 3 -hydroxy echinenone, echinenone, and β-carotene, with the exception of OX_CrtQ, OX_CrtO, and OX_CrtR, which had some reduced Car types ( Figure 3C). The zeaxanthin and echinenone levels in all OX strains commonly increased by more than 1.2-fold as compared to WTc ( Figure 3D). The results revealed that each component of the Car types was altered in the OX strains. OX_CrtB contained the increased amounts of all Car types, 1.1-1.3-fold that of WTc, except for 3 -hydroxyechinenone, whereas OX_CrtP contained the significant increases of all Car types, about 1.1-1.4-fold that of WTc, with the exception of β-carotene. For OX_CrtQ, the higher fold increase in each Car type was also observed, 1.2-1.3-fold that of WTc, except for myxoxanthophyll. Meanwhile, 3 -hydroxyechinenone and echinenone contents had a 1.6-fold increase compared with WTc, as noted in OX_CrtO. Interestingly, OX_CrtR had substantial increases in all Car types except for β-carotene; zeaxanthin levels increased by 1.6-fold; and the contents of 3 -hydroxyechinenone and echinenone increased by 1.9-and 1.4-folds, respectively. Although we did not monitor protein levels or enzymatic activity, all OX strains notably showed the altered changes in each Car product in accordance with their biosynthetic pathway. folds, respectively. Although we did not monitor protein levels or enzymatic activity, all OX strains notably showed the altered changes in each Car product in accordance with their biosynthetic pathway.

Growth and Intracellular Pigment Levels of Chlorophyll a and Carotenoids under Various Light Intensity Conditions
After exposing all strains to light stresses (Figure 4), low light (LL) conditions apparently affected the growth reduction in all strains, with the lowest growth rates found when comparing those of normal light (NL) and high light (HL) conditions. When cultivated under NL conditions, all OX strains only displayed a higher growth rate than WTc in the late-log phase ( Figure 4A). However, they had great levels of growth than WTc under both LL and HL conditions throughout a 14-day treatment period ( Figure 4B,C). an asterisk at *p < 0.05. For (A), all cropped gels were taken from the original images of RT-PCR products on agarose gels, as shown in Supplementary Information Figure S2.

Growth and Intracellular Pigment Levels of Chlorophyll a and Carotenoids under Various Light Intensity Conditions
After exposing all strains to light stresses (Figure 4), low light (LL) conditions apparently affected the growth reduction in all strains, with the lowest growth rates found when comparing those of normal light (NL) and high light (HL) conditions. When cultivated under NL conditions, all OX strains only displayed a higher growth rate than WTc in the late-log phase ( Figure 4A). However, they had great levels of growth than WTc under both LL and HL conditions throughout a 14-day treatment period ( Figure 4B,C). Intriguingly, all OX strains were promoted to accumulate more chlorophyll a than WTc ( Figure 5). The HL condition resulted in more dark green cell cultures and a higher rise in chlorophyll a content than the NL condition after 6 days of treatment ( Figure 5A,E). Intriguingly, all OX strains were promoted to accumulate more chlorophyll a than WTc ( Figure 5). The HL condition resulted in more dark green cell cultures and a higher rise in chlorophyll a content than the NL condition after 6 days of treatment ( Figure 5A,E). When compared to those under NL conditions, the LL condition undoubtedly lowered the chlorophyll a content and the lighter green types of cell culture after both 6 and 12 days of treatment ( Figure 5C). On the other hand, all OX strains had a considerable increase in total Car content, as anticipated under all light conditions ( Figure 5B,D,F). When compared to those under NL conditions, the LL condition undoubtedly lowered the chlorophyll a content and the lighter green types of cell culture after both 6 and 12 days of treatment ( Figure 5C). On the other hand, all OX strains had a considerable increase in total Car content, as anticipated under all light conditions ( Figure 5B,D,F). Six Car types, including myxoxanthophyll (M), zeaxanthin (Z), 3′-hydroxyechinenone (HE), echinenone (E), β-carotene (bC), and lycopene (L), of all strains were determined under LL and HL conditions ( Figure 6). The increased Car accumulation of OX strains was significantly induced by LL and HL light stresses compared with those of WTc ( Figure 6A-F). All Synechocystis strains apparently contained the highest amount of myxoxanthophyll, followed by β-carotene, echinenone, zeaxanthin, and other minor carotenoids (such as 3′-hydroxyechinenone and lycopene). Although lycopene was accumulated at relatively low levels, all OX strains showed a slight increase as compared to WTc ( Figure  6F). This indicates that total Car accumulation was caused by HL stress rather than LL Six Car types, including myxoxanthophyll (M), zeaxanthin (Z), 3 -hydroxyechinenone (HE), echinenone (E), β-carotene (bC), and lycopene (L), of all strains were determined under LL and HL conditions ( Figure 6). The increased Car accumulation of OX strains was significantly induced by LL and HL light stresses compared with those of WTc ( Figure 6A-F). All Synechocystis strains apparently contained the highest amount of myxoxanthophyll, followed by β-carotene, echinenone, zeaxanthin, and other minor carotenoids (such as 3 -hydroxyechinenone and lycopene). Although lycopene was accumulated at relatively low levels, all OX strains showed a slight increase as compared to WTc ( Figure 6F). This indicates that total Car accumulation was caused by HL stress rather than LL stress. Contrarily, under HL stress, β-carotene levels decreased in OX_CrtO and OX_CrtR, whereas they remained unaltered under NL conditions ( Figure 6E). stress. Contrarily, under HL stress, β-carotene levels decreased in OX_CrtO and OX_CrtR, whereas they remained unaltered under NL conditions ( Figure 6E). In addition, the relative percentage of each Car type was normalized with the total Car content ( Figure 7A-C). Although myxoxanthophyll was mostly preserved as a main component in all Synechocystis cells, light stress caused cells to balance their Car component by slightly reducing the proportion of myxoxanthophyll. Under NL conditions, all OX strains had higher percentages of zeaxanthin and echinenone, approximately 14-19% and 17-22%, respectively, than WTc, with percentages of about 13 and 16%, respectively ( Figure 7A). The HL condition at day 12 of treatment contained a similar pattern of carotenoid composition as those in the NL condition. On the other hand, stronger DPPH radical scavenging activity was shown in all OX strains than WTc under all light conditions (Figure 7D). The highest levels of antioxidant activity were found in the OX_CrtQ and OX_CrtR strains. In addition, the relative percentage of each Car type was normalized with the total Car content ( Figure 7A-C). Although myxoxanthophyll was mostly preserved as a main component in all Synechocystis cells, light stress caused cells to balance their Car component by slightly reducing the proportion of myxoxanthophyll. Under NL conditions, all OX strains had higher percentages of zeaxanthin and echinenone, approximately 14-19% and 17-22%, respectively, than WTc, with percentages of about 13 and 16%, respectively ( Figure 7A). The HL condition at day 12 of treatment contained a similar pattern of carotenoid composition as those in the NL condition. On the other hand, stronger DPPH radical scavenging activity was shown in all OX strains than WTc under all light conditions ( Figure 7D). The highest levels of antioxidant activity were found in the OX_CrtQ and OX_CrtR strains.
Additionally, the amounts of transcripts from genes involved in the biosynthesis and degradation of Cars and chlorophyll were monitored ( Figure 8). The expression of genes involved in Car synthesis was substantially higher on day 12 of treatment under NL and HL conditions than that of genes involved in chlorophyll production and pigment degradation. In all strains cultivated under NL conditions, the transcript levels of the CrtB and CrtQ genes were the highest of all the genes involved in the Car synthetic pathway ( Figure 8A). Furthermore, all five of the genes under investigation, particularly CrtB and CrtR, exhibited higher mRNA levels as a result of HL stress ( Figure 8C). It is curiously interesting that OX strains had higher levels of ChlG and ChlP transcripts than WTc, in relation to chlorophyll synthesis, under all light conditions ( Figure 8). In contrast, there was no difference in the transcript levels of slr1652, a gene involved in chlorophyll breakdown, and Diox1, a gene involved in Car degradation, under all light conditions. Additionally, the amounts of transcripts from genes involved in the biosynthesis and degradation of Cars and chlorophyll were monitored ( Figure 8). The expression of genes involved in Car synthesis was substantially higher on day 12 of treatment under NL and HL conditions than that of genes involved in chlorophyll production and pigment ( Figure 8A). Furthermore, all five of the genes under investigation, particularly CrtB and CrtR, exhibited higher mRNA levels as a result of HL stress ( Figure 8C). It is curiously interesting that OX strains had higher levels of ChlG and ChlP transcripts than WTc, in relation to chlorophyll synthesis, under all light conditions (Figure 8). In contrast, there was no difference in the transcript levels of slr1652, a gene involved in chlorophyll breakdown, and Diox1, a gene involved in Car degradation, under all light conditions.

Enhanced Intracellular Lipid Accumulation and Lung Cancer Cell Treatment
The cooperative function of lipids and Cars, particularly xanthophylls, has recently been found to stabilize the structure of the membrane and the photosystem in cyanobacteria [2,5,28]. In our study, all OX strains with a higher carotenoid content also had higher total lipid contents under all light conditions (Supplementary Information Figure S1). The Figure 8. The transcript levels and relative intensity ratios of the CrtB, CrtP, CrtQ, CrtO, CrtR, ChlG, ChlP, Slr1652, Diox1, and 16s rRNA genes of Synechocystis sp. PCC6803 WTc and engineered strains, grown in a BG 11 medium under normal light (A), low light (B), and high light (C) conditions for 12 days. The error bars represent the standard deviations of means (mean ± S.D., n = 3). All cropped gels were taken from the original images of RT-PCR products on agarose gels, as shown in Supplementary Information Figures S2-S4.

Enhanced Intracellular Lipid Accumulation and Lung Cancer Cell Treatment
The cooperative function of lipids and Cars, particularly xanthophylls, has recently been found to stabilize the structure of the membrane and the photosystem in cyanobacteria [2,5,28]. In our study, all OX strains with a higher carotenoid content also had higher total lipid contents under all light conditions (Supplementary Information Figure S1). The HL condition at day 12 of treatment significantly increased the total lipid content when compared to the NL and LL conditions on the same day.
Extracted Cars from all strains were evaluated for their cytotoxicity and antiproliferative activities against two lung cancer cell lines (H460 and A549) (Figures 9 and 10). A halfmaximal inhibitory concentration (IC 50 ) was determined in cancer cell lines (H460 and A549) to evaluate the cytotoxic effect of Car extracts at concentrations of 0-400 µg/mL ( Figure 9A,B). The results revealed that the IC 50 Figure 9B). Moreover, the relative cell proliferation level of lung cancer cell lines H460 and A549 after being treated with Car extracts for 72 h also decreased when compared with that of WTc ( Figure 10A,B). The aberrant cancer cell lines with inhibited growth were discernible under a light microscope resulting from treatment with carotenoid extracts when compared to those of the control group without carotenoids ( Figure 10C).
half-maximal inhibitory concentration (IC50) was determined in cancer cell lines (H460 and A549) to evaluate the cytotoxic effect of Car extracts at concentrations of 0-400 µg/mL ( Figure 9A,B). The results revealed that the IC50 values for Car extracts from OX_CrtR, OX_CrtQ, OX_CrtO, OX_CrtB, and OX_CrtP in the H460 cell line, with values of 100.17 ± 2.27, 113.46 ± 4.40, 127.84 ± 4.16, 137.57 ± 4.72, and 137.54 ± 3.76 µg/mL, respectively, were lower than those of Car extracts from WTc (157.60 ± 6.48 µg/mL) ( Figure 9A) Figure 9B). Moreover, the relative cell proliferation level of lung cancer cell lines H460 and A549 after being treated with Car extracts for 72 h also decreased when compared with that of WTc ( Figure 10A,B). The aberrant cancer cell lines with inhibited growth were discernible under a light microscope resulting from treatment with carotenoid extracts when compared to those of the control group without carotenoids ( Figure 10C).

Discussion
In this study, we found promising results of increased carotenoid (Car) production in the cyanobacterium Synechocystis sp. PCC 6803 overexpressing native genes involved in Car biosynthesis with higher antioxidant activity (Figure 1). Five engineered strains, i.e., OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR, significantly promoted Car accumulation and altered Car-type components, particularly zeaxanthin and echinenone, except for OX_CrtQ, which also accumulated the increased β-carotene (Figures 3 and 7).

Discussion
In this study, we found promising results of increased carotenoid (Car) production in the cyanobacterium Synechocystis sp. PCC 6803 overexpressing native genes involved in Car biosynthesis with higher antioxidant activity (Figure 1). Five engineered strains, i.e., OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR, significantly promoted Car accumulation and altered Car-type components, particularly zeaxanthin and echinenone, except for OX_CrtQ, which also accumulated the increased β-carotene (Figures 3 and 7). All Synechocystis cells mostly contained the highest content of myxoxanthophyll due to its vital roles for cell wall structure and thylakoid membrane stabilization [29]. Zeaxanthin can be produced by Synechocystis sp. PCC 6803, while other cyanobacteria, such as Anabaena sp.
PCC 7120 and Nostoc punctiforme PCC 73102, are able to generate canthaxanthin and astaxanthin from β-carotene [30][31][32]. We demonstrated that a certain increase in total carotenoid accumulation in all OX strains was found during the late-log phase of cell growth, especially under light stress (Figures 3 and 6). At day 12 of normal cultivation, the native gene overexpression of slr0088, encoding CrtO, led to a 1.6-fold increase in echinenone, while an overexpression of sll1468, encoding CrtR, contained a 1.6-fold increase in zeaxanthin in comparison with the WT control (Figure 3). In a previous report, a native overexpression of CrtR in Synechocystis sp. PCC 6803 could induce zeaxanthin with a 2.5-fold increase compared to that in the WT strain [23]. However, we also demonstrated that a higher amount of 3 -hydroxyechinenone, in comparison with WTc, was found in both OX_CrtO and OX_CrtR strains (Figure 3). In particular, the highest level of 3 -hydroxyechinenone was induced by HL stress in OX_CrtR. When cells are exposed to intense light, the photoprotective mechanism at the phycobilisome is induced by the orange carotenoid protein (OCP)-binding hydroxyechinenone [24,33]. The high light intensity stress in this study at 150 µmole photon/m 2 /s was considered a mind photoinhibition inducer, as evidenced by the higher growth and pigment accumulation in Figures 4 and 5. Synechocystis cells only experienced severe photoinhibition above 800 µmole photon/m 2 /s, and they displayed an efficient capacity to recover from the complete state of photoinhibition at 1460 µmole photon/m 2 /s when light exposure was reduced to 200 µmole photon/m 2 /s [34]. In addition to the higher accumulation of Cars, we also showed how the types of Cars were distributed differently. In Synechocystis wild-type control (Figure 7), the major Car components were myxoxanthophyll (35.3%), β-carotene (26.8%), echinenone (16%), zeaxanthin (13.2%), other minor Cars, such as 3 -hydroxyechinenone (1.5%), lycopene (0.02%), and unidentified carotenoids (7.2%). Mostly, high Cars-producing strains had lowered main components of myxoxanthophyll and β-carotene but increased zeaxanthin, 3-hydroxyechinenone, and echinenone compositions. In addition, after exposure to high light conditions in this study, WT control cells were rearranged for each Car type by inducing proportions of zeaxanthin (from 13.2% to 14.6%), 3 -hydroxyechinenone (from 1.5% to 1.6%), and β-carotene (from 26.8% to 29%), while low light conditions were handled with increased proportions of zeaxanthin (from 13.2% to 15.8%), 3 -hydroxyechinenone (from 1.5% to 2.5%), and echinenone (from 16% to 17.9%) (Figure 7). These findings suggest that zeaxanthin and 3 -hydroxyechinenone are the main Car components that respond to light stress. Zeaxanthin was previously found to enhance high light acclimation by acting as an antioxidant and lipid stabilizer in Arabidopsis [35]. High light stress substantially induced the binding of zeaxanthin to specific proteins in photosystems, which enhanced photoprotection and modulated chlorophyll triplet yield [5,36,37]. Both zeaxanthin and echinenone certainly modify photosystem I trimer structure and protect photosystem II repair from singlet oxygen damage in Synechocystis sp. PCC6803 [38][39][40]. It was considerably confirmed by our results that OX_CrtR with a high zeaxanthin component had the highest capacity for DPPH scavenging activity among all the strains studied ( Figure 7D). Additionally, it is worth noting that increased β-carotene composition also helped wild-type cells to cope with high light stress, whereas higher echinenone composition enabled cells to handle low light stress. The native overexpression of CrtQ or slr0940 significantly increased the highest levels of β-carotene and antioxidant activity (Figures 6 and 7). β-carotene accumulation was light-dependently induced, contributing to the acclimation to light stress in Euglena gracilis [41]. On the other hand, the transcript results indicated that light stress had a close relationship with enhanced carotenoid synthesis rather than degradation and chlorophyll metabolism, particularly under high light conditions (Figure 8).
We also demonstrated that carotenoid synthetic gene overexpression could improve intracellular lipid contents (Supplementary Information Figure S1). An increase in the light intensity during cultivation resulted in an increase in lipids and fatty acid production, regulating membrane integrity [42][43][44]. The cooperative function of lipids and carotenoids led cells to acclimate their membrane structure and function against light stress [1,28,45,46]. On the other hand, the carotenoid extracts from all engineered strains were applied to treat lung cancer cell lines H460 and A549. Lung cancer has been the first and foremost leading cause of death around the world. A higher intake of carotenoids could lower the risk of lung cancer [17,47,48]. Our findings demonstrated that the carotenoid extracts from all engineered Synechocystis sp. PCC 6803 strains significantly decreased the viability and proliferation of lung cancer cell lines, particularly OX_CrtR and OX_CrtQ (Figures 9 and 10). Synechocystis sp. PCC 6803 overexpressing CrtR with high antioxidant activity showed the lowest level of IC 50 at 100.17 and 90.32 µg/mL on H460 and A549 cell lines, respectively. The anti-cancer therapeutic targets of Cars include cell cycle arrest, an apoptosis-inducing effect, an anti-metastatic effect, and an anti-angiogenic effect in cancer cells [17], as well as modulating oxidative stress and redox balance [16]. For all Car types, they have synergistic functions of antioxidant activity to modulate ROS levels and have also shown pro-oxidant effects in cancer cells [16,49]. β-carotene and lycopene were also considered potential anti-cancer compounds previously found to inhibit tumor cell growth by interfering at different phases of the cell cycle and generate apoptosis-inducing effects in cancer cells, such as the G1 phase arrest of human promyelocytic leukemia (HL-60) cells and the G0/G1 phase arrest of HL-60 cells, respectively [50,51]. In addition, β-carotene played an effective anti-metastatic role in lung cancer cells B16F-10 by reducing collagen, hydoxylproline, uronic acid, and hexamine contents [52]. On the other hand, zeaxanthin treated uveal melanoma cell lines by inducing apoptosis by activating the intrinsic apoptosis signaling pathway [53]. In accordance with our findings, Synechocystis sp. PCC6803 strains with CrtR and CrtQ overexpression, respectively, had the highest amounts of zeaxanthin and β-carotene. These two OX strains exhibited a certain increase in antioxidant activity, and their carotenoid extracts had the highest capacity to treat lung cancer cells in vitro.

Strains and Culture Conditions
Cyanobacterium Synechocystis sp. PCC 6803 wild-type (WT) strains and all engineered strains (Table 1) were grown in the BG 11 medium. The growth condition was performed at 27-30 • C under continuous white light illumination with various light intensities, including low light (LL; 10 µmole photon/m 2 /s), normal light (NL; 50 µmole photon/m 2 /s) and high light (HL; 150 µmole photon/m 2 /s) conditions. The cell culture flask with an initial optical density (OD) of about 0.1 at 730 nm was placed on a rotary shaker (160 rpm) for 14 days. Cell growth was spectrophotometrically measured at OD 730 . The host propagation, Escherichia coli DH5α strain, was grown either on a Luria-Bertani (LB) agar plate or its liquid medium at 37 • C.

Constructions of Recombinant Plasmids
The recombinant plasmids (Table 1), including pEERM_CrtB, pEERM_CrtP, pEERM_CrtQ, pEERM_CrtO, and pEERM_CrtR, were constructed to generate OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR strains, respectively. Initially, five target gene fragments involved in the carotenoid biosynthetic pathway were amplified by PCR using each specific pair of primers, as listed in Supplementary Information Table S1, and genomic DNA as a template. Each gene fragment was then ligated into the pEERM vector with the psbA2 promoter, which could practically let the engineered cells grow under high light conditions [54], generating the expected five recombinant plasmids listed in Table 1. pEERM_CrtB was generated via the ligation of the CrtB gene fragment amplified by FW_CrtB and RV_CrtB (Supplementary Information Table S1) with the pEERM vector in the PstI restriction site. For pEERM_CrtP and pEERM_CrtQ, the CrtP and CrtQ gene fragments, amplified by a pair of FW_CrtP and RV_CrtP primers and another pair of FW_CrtQ and RV_CrtQ (Supplementary Information Table S1), respectively, were separately introduced in between XbaI and SpeI restriction sites in the pEERM vector. Moreover, pEERM_CrtO and pEERM_CrtR were generated by ligating CrtO and CrtE gene fragments, amplified by PCR using a pair of FW_CrtO and RV_CrtO and another pair of FW_CrtR and RV_CrtR primers, respectively (Supplementary Information Table S1), in between SpeI and PstI sites in the pEERM vector.

Natural Transformation of Synechocystis sp. PCC 6803 Cells
The recombinant plasmids (Table 1) were transformed into Synechocystis sp. PCC 6803 WT cells by natural transformation, thereby generating OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR. To construct Synechocystis WT control (WTc) or Synechocystis WT containing the Cm R cassettle gene, the empty pEERM vector was transformed into Synechocystis WT cells. First, the host cell suspension was prepared. Then, 50 mL of Synechocystis sp. PCC 6803 WT cell culture with an OD 730 of about 0.5 was harvested via centrifugation at 5500 rpm (2870× g) for 15 min. Cell pellets were resuspended in a fresh BG 11 medium (500 µL). After that, at least 10 µL of the recombinant plasmids were mixed with the Synechocystis host cell suspension and incubated in the culture room overnight. Then, the sample mixture was spread on a BG 11 agar plate containing 10 µg/mL of chloramphenicol. After several weeks, the colonies that survived on antibiotic plates were picked and streaked on a new BG 11 agar plate containing 20, and later 30 µg/mL, of chloramphenicol. The obtained transformants were confirmed for gene size, location, and segregation by PCR using many specific pairs of primers, as shown in the Supplementary Information Table S1.

Determinations of Chlorophyll a and Total Carotenoid Contents
The chlorophyll a and total carotenoid contents were analyzed using the modified method from [55]. Next, 1 mL of cell culture was extracted using the acetone:water mixture (4:1) as a solvent and incubated for 2 min. After centrifugation at 5500 rpm (2870× g) for 5 min, the supernatant was spectrophotometrically measured at wavelengths of 470.0, 646.6, and 663.6 nm, respectively. Contents of chlorophyll a and carotenoids were calculated using the following equations:

Quantitative Analysis of Carotenoids by HPLC
Then, 50 mL of Synechocystis cell culture was harvested by centrifugation at 5500 rpm (2870× g) for 10 min. Before starting solvent extraction, the cell suspension of all samples was diluted to an OD 730 of about 0.5. Then, cell pellets from 3 mL of diluted cell suspension were collected after centrifugation again. The intracellular pigments were then extracted by absolute methanol (1 mL), and centrifuged at 12,000 rpm (21,009× g) and 4 • C for 5 min. After that, the supernatant was collected for further analysis. Samples of extracted intracellular pigments were detected using high-performance liquid chromatography (HPLC) (Shimadzu HPLC LGE System, Kyoto, Japan) using a C18 column, 150 × 46 mm (GL-Sciences, Tokyo, Japan). The HPLC condition was set at 15 • C, with a flow rate of 1 mL/min using the solvent system described by [1]. This solvent system consisted of an isocratic elution with a mixture of solvent A, acetonitrile: methanol: Tris (0.1 M, pH 8.0) (89/9.5/1.5, v/v), for 4 min; a linear gradient from solvent A to solvent B, methanol: hexane (4:1, v/v), for 2.5 min; an isocratic elution with solvent B for 11.5 min; a linear gradient of solvent B to solvent A for 1 min; and a final isocratic elution with solvent A for 9 min. The separated pigments were detected at 440 nm using a UV/VIS detector. The carotenoid contents, including myxoxanthophyll, zeaxanthin, 3 -hydroxy echinenone, echinenone, and β-carotene, were calculated using the following equations from [23]: C car = C chl × [(ε chl × A car )/(ε car × A chl )], where C chl = the chlorophyll concentration in the pigments extracts was determined by the extinction coefficient of chlorophyll a using 820, and the absorbance of the pigment extract at 663 nm. ε chl and ε car = the specific extinction coefficients of the chlorophyll a and the carotenoids at 440 nm, respectively, were taken from [56,57]. A chl and A car = the chlorophyll a and the carotenoid peak areas on the chromatogram, respectively, were detected at 440 nm. The relative amount of each carotenoid type was calculated as a percentage to the total carotenoid content under each light condition.

Determination of Lycopene Content
Lycopene content was determined using the modified method from [58]. Then, 1 mL of cell culture was extracted using the acetone:hexane solvent mixture (2:3) and incubated at room temperature for 2 min. After that, the supernatant, obtained after centrifugation at 5500 rpm (2870× g) for 10 min, was spectrophotometrically measured at four wavelengths of 453, 505, 645, and 663 nm, respectively. Lycopene content was calculated using the following equation The total RNAs were extracted from all strains using the TRIzol ® reagent (Invitrogen, Life Technologies Corporation, Carlsbad, CA, USA). The purified RNAs (1 µg) were converted to cDNA using the SuperScript III First-Strand Synthesis Kit (Invitrogen, Carlsbad, CA, USA). Then, the obtained cDNA was used as a template for PCR with various pairs of specific primers, as listed in Supplementary Information Table S1. We monitored the CrtB, CrtP, CrtQ, CrtR, and CrtO genes in relation to the carotenoid biosynthetic pathway; the Diox1 gene in carotenoid degradation or retinal biosynthesis; the ChlP and ChlG genes in chlorophyll biosynthesis; and the Slr1652 gene in chlorophyll degradation. The 16s rRNA was used as a reference. The PCR reaction mixture using KOD OneTM Master Mix (Toyobo, Osaka, Japan) and its conditions were performed by suitable cycles of each gene at 98 • C for 10 s, at both a primer melting temperature (Tm, Supplementary Information Table S1) for 5 s and 68 • C for 5 s. Then, the PCR products were checked by 1% (w/v) agarose gel electrophoresis. The quantification of band intensity was detected by Syngene ® Gel Documentation (Syngene, Frederick, MD, USA).

Determination of DPPH Radical Scavenging Activity
The free radical scavenging activity of pigment extracts from all strains was measured using a DPPH assay (modified from [59]). Then, 1 mL of cell culture was harvested by centrifugation at 5500 rpm (2870× g) for 10 min. Pigment extracts were obtained from cell pellets after they were incubated with a solvent mixture of acetone and water (4:1) at room temperature for 10 min. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) concentration was prepared as 0.1 mM in ethanol. The DPPH solution (500 µL) was mixed with the cell-free pigment extract (500 µL). The reaction mixture was incubated at room temperature for 30 min. After that, the absorbance at 517 nm was spectrophotometrically measured. An ascorbic acid was used as a standard. The antioxidant activity was calculated using the equation; DPPH radical scavenging activity (%) = A 0 − A 1 /A 0 × 100, where A 0 is the absorbance of the control reaction and A 1 is the absorbance of the sample.

Cell Line Culture
NSCLC cell lines A549 (CCL-185) and H460 (HTB-177) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The H460 cell line was obtained from the pleural fluid of a male patient with large cell lung cancer, while the A549 cell line was obtained from an explant culture of lung carcinomatous tissue from an old male patient. The A549 cells were cultured in Dulbecco's modified Eagle medium (DMEM), while the H460 cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640). Both media were supplemented with 10% fetal bovine serum albumin, 2 mM L-glutamine, and 100 U/mL of penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA). Cell culture was maintained in a humidified atmosphere containing 5% CO 2 at 37 • C. The culture medium and trypsinization were renewed every 2-3 days. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Invitrogen, Carlsbad, CA, USA) solution (5 mg/mL) was freshly prepared in phosphate-buffered saline (PBS).

Cytotoxicity and Cell Proliferative Assay
Total carotenoids were extracted from all strains by a solvent mixture of hexane: acetone:ehanol (50:25:25, v/v/v) and a subsequent 1 M KOH solution to remove chlorophyll (modified from [60]). After the centrifugation was performed at 5500 rpm (2870× g), 4 • C for 10 min, the upper fraction layer of hexane containing total carotenoid extracts was carefully collected, and the solvent was evaporated under nitrogen gas at room temperature. Extracted carotenoid powder was dissolved in a PBS buffer with 0.25% (v/v) dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA), the maximum DMSO concentration that did not interfere with the assays in various concentrations (0-400 µg/mL).
The cytotoxicity of the extracts was determined by the MTT assay (modified from [14,61]). Cell lines were seeded into 96-well plates at a density of 10,000 cells per well and incubated at 37 • C overnight for cell attachment purposes. The cells were then treated with various concentrations of carotenoid extracts ranging from 0 to 400 µg/mL for 24 h. After the incubation period of 24 h, 10 µL of MTT solution (5 mg/mL) was added to each well and incubated at 37 • C for 4 h. After the incubation period, the resulting formazan crystals were formed, and dissolved in 100 µL of DMSO. After that, the colored product was spectrophotometrically measured at 570 nm. The results from the MTT assay represented the IC 50 value of the different carotenoid extracts from all strains by analyzing their effect on cell viability.
Cell proliferative assay was determined by the MTT assay (modified from [62]). Cell lines were seeded at a density of 3000 cells per well into 96-well plates. Cells were treated with non-toxic concentrations of the carotenoid extracts (12.5 µg/mL) and incubated at 37 • C for 24, 48, and 72 h. After the incubation at each elapsed time, 10 µL of MTT (5 mg/mL) was added into each well and incubated at 37 • C for 4 h. The formazan crystals were formed and later dissolved by 100 µL of DMSO. After that, the sample was spectrophotometrically measured at 570 nm. Relative cell proliferation was calculated by dividing the OD 570 of the treated group by the OD 570 of the control.

Statistical Analysis
Statistical analyses were performed using SPSS version 29.0, licensed by Chulalongkorn University. The differences in biochemical parameters and molecular responses among the treatments were analyzed using one-way ANOVA, followed by Tukey's HSD test for the multiple comparison tests. The t-test was performed for an independent twopair comparison test. Non-parametric tests were used for the data which did meet the assumption of normality. The significant differences were accepted at p < 0.05.

Conclusions
Five engineered Synechocystis sp. PCC 6803 strains with high carotenoid accumulation and antioxidant activity were achieved by overexpressing five carotenoid biosynthetic genes, thereby generating OX_CrtB, OX_CrtP, OX_CrtQ, OX_CrtO, and OX_CrtR. The increased levels of zeaxanthin and echinenone were mostly found in all engineered strains, whereas myxoxanthophyll was maintained as the main carotenoid type. An increase in carotenoid content was especially induced under high light stress at 150 µmole photon/m 2 /s, except for β-carotene. Changes in the components of all carotenoid types were considered a marker that responded to light stress. Additionally, all modified strains were more susceptible to high light-stress-induced lipid accumulation. It is worth noting that the extracted carotenoids with high antioxidant activity from all engineered strains efficiently treated lung cancer cells H460 and A549 by decreasing their cell viability and proliferation, particularly OX_CrtR and OX_CrtQ, with high levels of zeaxanthin and β-carotene, respectively. Although the mechanisms of all carotenoid types treating cancer cells are still not completely elucidated, this study highlights a promising step towards the cyanobacte-rial application on health and cancer treatment aspects from renewable biosources. It would be useful to characterize and gain more understanding about carotenoid mechanisms or perform animal model testing on the carotenoid extracts in vivo.

Conflicts of Interest:
The authors declare no conflict of interest.