Metabolic Engineering of Synechocystis sp. PCC 6803 for Olivetolic acid Production

Metabolic Engineering of Synechocystis sp. PCC 6803 for Olivetolic acid Production | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Metabolic Engineering of Synechocystis sp. PCC 6803 for Olivetolic acid Production E-Bin Gao, Wenrui Ji, YangJie Zhu, Junhua Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9174471/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 9 You are reading this latest preprint version Abstract As a pivotal aromatic precursor for phytocannabinoids, olivetolic acid (OLA) is traditionally sourced from resource-intensive plant extraction or complex chemical synthesis. In this study, we established a sustainable photosynthetic production platform by engineering the model cyanobacterium Synechocystis sp. PCC 6803. We successfully reconstituted the OLA biosynthetic pathway by heterologously expressing Cannabis sativa-derived tetraketide synthase (CsTKS), olivetolic acid cyclase (CsOAC), and acyl-activating enzyme (CsAAE1). To overcome metabolic bottlenecks, the pathway was optimized using the strong promoter P cpc560 and carbon flux redirection. Disruption of the glycogen storage pathway and reinforcement of the malonyl-CoA pool via maeB overexpression significantly enhanced precursor availability. Furthermore, increasing CO 2 supplementation achieved a maximum OLA titer of 9.41 mg/L. This work demonstrates the feasibility of utilizing photosynthetic chassis for the production of olivetolic acid, providing a robust foundation for the sustainable manufacturing of cannabinoid-related metabolites. Synechocystis sp. PCC6803 Metabolic engineering CO2 fixation Olivetolic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Highlights · Initial synthesis of olivetolic acid in PCC6803. · Production titer increased after knocking out the glycogen synthesis pathway. · Overexpression assays optimized the metabolic pathway and increased production. 1. Introduction Phytocannabinoids, such as cannabidiol (CBD) and Δ 9 -tetrahydrocannabinol (THC), represent a class of highly valued meroterpenoids produced by Cannabis sativa [ 1 ]. They exert therapeutic effects through modulation of the endocannabinoid system (CB1/CB2 receptors)[ 2 ], evidenced by FDA-approved drugs for epilepsy (Epidiolex® - CBD) and chemotherapy-induced nausea (Marinol®/Syndros® - synthetic THC)[3; 4]. Typical production relies on plant extraction or chemical synthesis[ 1 ]. However, the sustainable supply of cannabinoid drugs via traditional plant-based extraction is severely bottlenecked. On one hand, production is hindered by the protracted growth cycles of Cannabis sativa (typically 3–4 months) and stringent legal frameworks (generally restricting cultivation to low-THC varieties)[ 5 ]. On the other hand, low specialized metabolite titers and resource-intensive farming render the process both economically and ecologically inefficient[ 6 ]. While chemical synthesis provides a potential route, it remains formidable due to the intricate molecular architecture, necessitating the use of hazardous organic solvents and metal catalysts that not only raise environmental concerns but also pose significant hurdles for the downstream purification of pharmaceutical-grade products[ 7 ]. Consequently, there is an urgent imperative to develop sustainable, carbon-neutral production platforms that leverage green manufacturing principles to circumvent the multifaceted ecological and technical bottlenecks inherent in traditional cannabinoid supply chains. As the major specialized metabolites in Cannabis sativa , phytocannabinoids are predominantly biosynthesized in glandular trichomes and reach their maximum accumulation on the bracts of female inflorescences[ 8 ]. While over 150 different cannabinoids have been identified to date, the biosynthetic pathways of the most extensively studied analogs—including Δ 9 -tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), and cannabigerol (CBG)—all converge at a common core precursor, olivetolic acid (OLA)[9; 10]. In the native Cannabis sativa plant, the formation of this pivotal aromatic intermediate is governed by a three-step enzymatic sequence as follows: The pathway initiates with the activation of the short-chain fatty acid hexanoate into hexanoyl-CoA by the acyl-activating enzyme (CsAAE1) in an ATP-dependent manner. This hexanoyl-CoA then serves as the starter unit for the tetraketide synthase (CsTKS), a type Ⅲ polyketide synthase, which performs the iterative decarboxylative condensation of three molecules of malonyl-CoA to generate a linear triketide intermediate. To prevent the spontaneous formation of metabolic side-products, the olivetolic acid cyclase (CsOAC) acts as a regiospecific scaffold to direct the C2-C7 aldol condensation and subsequent cyclization of the intermediate, ultimately yielding the pivotal aromatic intermediate, olivetolic acid[ 11 ]. The elucidation of this native enzymatic logic has paved the way for synthetic biology to transcend the limitations of traditional plant-based production. Since the production of OLA is a fundamental prerequisite for cannabinoid synthesis, researchers have successfully reconstituted this metabolic pathway in various heterologous microbial hosts. To date, research has predominantly focused on heterotrophic platforms, underscoring the feasibility of OLA production via advanced metabolic engineering strategies. Escherichia coli was among the earliest chassis employed for the biosynthesis of OLA. Tan et al. reconstituted this pathway by integrating Cannabis OLS and OAC with a reversed β-oxidation cycle for de novo hexanoyl-CoA synthesis. Through integrated metabolic optimization and controlled bioreactor cultivation, this strategy ultimately achieved an OLA titer of 80 mg/L[ 12 ].. In the Yarrowia lipolytica chassis, researchers achieved an 83-fold increase in olivetolic acid titers to 9.18 mg/L by optimizing precursor supply, augmenting cofactor regeneration, and integrating metabolic pathways to effectively redirect carbon flux toward polyketide biosynthesis[ 13 ]. Similarly, Schmidt et al.[ 14 ] optimized Saccharomyces cerevisiae by introducing CsAAE1, CsOLS, and CsOAC for olivetolic acid synthesis, alongside a soluble NphB and ERG20 WW fusion to bypass the limitations of membrane-bound prenyltransferases. By further overexpressing the transcription factor HAC1s to boost enzyme activity, they achieved 117 mg/L of olivetolic acid and successfully synthesized CBGA and CBCA. However, cannabinoid biosynthetic enzymes of plant origin are highly susceptible to forming inactive inclusion bodies within E. coli , which limits the yield of soluble active proteins[ 15 ]. While yeast is considered energy-intensive in production due to its reliance on refined organic carbon sources and the requirement for high-density cultivation[ 16 ]. Furthermore, the heavy reliance of these conventional hosts on organic carbon sources necessitates the exploration of more sustainable and cost-efficient production platforms. The model cyanobacterium Synechocystis sp. PCC 6803 represents a versatile photosynthetic chassis exceptionally suited for the sustainable biosynthesis of plant-derived metabolites, including terpenoids, flavonoids, and phenolics[ 17 – 21 ]. Unlike conventional heterotrophic hosts that require costly organic feedstocks, Synechocystis operates photoautotrophically—utilizing ambient CO 2 and solar energy to enable carbon-negative manufacturing[ 22 ]. Benefiting from a rapid doubling time (7–10 h) and a sophisticated carbon-concentrating mechanism (CCM), the organism achieves superior photosynthetic performance and highly efficient nitrogen utilization[23; 24]. Structurally, cyanobacteria share with plant cells thylakoid membranes containing galactolipids, sulfolipids, various photosystem components, and photosynthetic pigments[ 24 – 26 ]. Metabolically, the cyanobacterial photosynthetic apparatus yields substantial reducing equivalents and critical precursor pools (e.g., acetyl-CoA). This dual congruence renders cyanobacteria inherently compatible with the biosynthesis of plant-derived metabolites, such as terpenoids and phenolics[19; 27]. Collectively, these unique features, complemented by the facile genetic engineering of cyanobacteria, position this organism as an optimal photosynthetic platform for the sustainable biosynthesis of cannabinoids and their respective precursors. As a proof of concept, we established a photosynthetic platform for the synthesis of the pivotal precursor, olivetolic acid, in Synechocystis sp. PCC 6803. This was achieved through an integrated metabolic engineering strategy that coupled the heterologous expression of cannabinoid biosynthetic genes (CsTKS, CsOAC, and CsAAE1) with host-specific modifications, including the disruption of carbon storage (ΔglgC) and the reinforcement of the malonyl-CoA pool (maeB). By successfully redirecting photosynthetic carbon flux toward polyketide production, this study lays a robust foundation for sustainable cannabinoid manufacturing aligned with circular economy principles. 2. Materials and methods 2.1. Strains and growth conditions The wild type and genetically engineered Synechocystis strains were cultivated in BG11 medium using an illuminated incubator (GDN-260A, YANGHUI, China) under a light intensity of 28 µmol m − 2 s − 1 and a temperature of 30℃. The cell density of strains was measured at an optical density (OD) of 730 nm using a visible spectrophotometer (YOKE, Shanghai, China). The DH5α Chemically Competent Cell (Vazyme, Nanjing, China) was used for plasmid construction and amplification. The DH5α strain was cultured in LB medium at 37℃ with shaking at 220 rpm (Zhichu, Shanghai, China). Different antibiotic concentrations were employed for various strains during cultivation. For genetically engineered cyanobacteria, spectinomycin was added at a final concentration of 50 µg/mL, while chloramphenicol was added at a final concentration of 34 µg/mL. During the initial selection after cyanobacterial transformation, antibiotics were applied at one-fifth of the regular cultivation concentrations and gradually increased to the required levels for routine. 2.2. Plasmids construction This study constructed a series of integration plasmids based on the pMD18-T backbone (Sangon Biotech Co Ltd., Shanghai, China) to integrate optimized synthetic exogenous genes (Sangon Biotech Co Ltd., Shanghai, China) into the cyanobacterial chromosome. The plasmid construction involved restriction digestion using endonucleases (NEB, Beijing, China), PCR amplification with high-fidelity polymerase (Vazyme, Nanjing, China), and assembly using homologous recombinase (NEB, Beijing, China). DH5α was used for plasmid amplification. Single colonies were screened via colony PCR, and verified plasmids were sent for sequencing. Subsequent experiments proceeded after confirmation of correct sequences. The characteristics of all plasmids used for transformation in this study are summarized in Table 1 . Plasmids construction process and primers are provided in the appendix file. Table 1 Plasmids used for transformation in this study. Plasmids Characteristic Resistance pMD18-T - Amp R pST407-TKS/OAC slr0168up-Spec R -P psbA2s -TKS-OAC-T rbcL -slr0168dw Amp R +Spec R pST02 slr0168up-Spec R -P psbA2s -TKS-OAC-AAE1-T rbcL -slr0168dw Amp R +Spec R pST03 slr0168up-Spec R -P cpc560 -TKS-OAC-AAE1-T rbcL -slr0168dw Amp R +Spec R pST04 slr1176up-Cm R -slr1176dw Amp R + Cm R pST05 slr1176up-Cm R -P cpc560 -maeB-T rbcL -slr1176dw Amp R + Cm R Table notes: slr0168up: upstream homology arm of the slr0168 gene, slr0168dw: downstream homology arm of the slr0168 gene, slr1176up: upstream homology arm of the slr1176 gene, slr1176dw: downstream homology arm of the slr1176 gene, Amp R : ampicillin resistance gene, Spec R : spectinomycin resistance gene, Cm R : chloramphenicol resistance gene, P psbA2s : native light-induced promoter P psbA2s , P cpc560 : native super strong promoter P cpc560 , T rbcL : native terminator T rbcL , TKS: tetraketide synthase, OAC: olivetolic acid cyclase, AAE1: hexanoyl-CoA synthase, maeB: NADP-dependent malic enzyme. 2.3. Transformation of Synechocystis sp. 6803 Transformation of Synechocystis was performed using a method described by John G.K. Williams[ 28 ], with modifications to incubation time, plasmid concentration, and other parameters based on cell conditions and cultivation factors[29; 30]. Cultures in the exponential phase (30mL, OD 730 = 0.6–0.8, cultured at 30℃ with 28 µmol m − 2 s − 1 light) were harvested by centrifugation, washed twice with fresh BG11 medium, and resuspended in 1 mL of antibiotic-free BG11 medium. To the suspension, 10 µg of the target plasmid was added and mixed by gentle pipetting. The mixture was incubated for 18 hours in a low light incubator, with gentle shaking 3–4 times during this period. Following incubation, the mixture was spread onto five antibiotic-free BG11 agar plates, each containing a 0.45 µm hydrophilic cellulose membrane, pretreated by boiling and autoclaving. The plates were incubated for 24 hours, after which the membranes were transferred to BG11 plates supplemented with appropriate antibiotic concentration. Transformants appeared after 2–3 weeks. Individual colonies were isolated and streaked onto BG11 agar plates containing antibiotics for further purification. One colony from each plate was then inoculated into liquid BG11 medium with a low concentration of antibiotics, with the antibiotic concentration gradually increased during serial passaging. After five passages (approximately one month), cells in the exponential phase were harvested for genomic DNA extraction and PCR-based validation. All the strains referred to in this study are presented in Table 2 . Table 2. The strains constructed in this study. Engineered strains Gene cluster transformation SCY01 pST407-TKS/OAC into Synechocystis SCY02 pST02 into Synechocystis SCY03 pST03 into Synechocystis SCY04 pST04 into SCY03 SCY05 pST05 into SCY03 The slr0168 locus is a neutral site on the Synechocystis genome, encoding a hypothetical glycoprotein and forming part of the slr0338-slr0168-slr0169 operon. The functions of this operon and the genes within it remain unclear[ 31 ]. Typically, modifications at neutral sites do not significantly affect cellular functions[ 32 ], which is why this locus is frequently chosen for introducing exogenous genes when Synechocystis is used as a chassis organism. The slr1176 locus ([EC:2.7.7.27]) encodes ADP-Glucose Pyrophosphorylase, referred to as glgC. This gene is involved in the first step of glycogen and starch biosynthesis in Synechocystis , and the activity of glgC regulates the overall rate of glycogen accumulation. If this gene is knocked out, glycogen cannot be synthesized in Synechocystis [ 33 ]. 2.4. Olivetolic acid extraction and measurement All engineered strains were cultured in BG11 medium supplemented with sodium hexanoate at a light intensity of 28 µmol m − 2 s − 1 and incubated at 30℃. Cyanobacterial cells were first disrupted using an ultrasonic cell disruptor, and the resulting mixture was centrifuged at 8000 rpm for 10 minutes. The supernatant was then transferred and extracted with an equal volume of ethyl acetate[ 34 ]. After combining the extracts, the solution was concentrated to 1 mL by rotary evaporation and subsequently dried under a flow of nitrogen gas. The residue was re-suspended in 1 mL methanol and filtered through a 0.22 µm syringe filter before HPLC (high-performance liquid chromatography) analysis. HPLC analysis was performed using a Shimadzu LC-20AT system with a Thermo Scientific Hypersil ODS-2 C18 column (4.6 mm × 250 mm, 5 µm) and detection at 262 nm. 2.5. Improvement of culture conditions Since cyanobacterial growth and olivetolic acid production rely on CO 2 consumption, continuous aeration was employed to enhance CO 2 supply. Logarithmically growing strains were inoculated to an initial OD 730 of 0.50. The CO 2 was mixed with air at a 1:19 ratio (5% CO 2 , 95% air), filtered through a 0.22 µm membrane and continuously supplied to the culture flasks. Cultures were maintained at 30℃ and illuminated at 28 µmol m − 2 s − 1 with continuous aeration for one month. Wild-type Synechocystis grown under the same conditions was used as the control. 2.6. Statistics and reproducibility Statistical analysis was performed using the Tukey test in SPSS statistical software. Statistical significance was defined as a p-value less than 0.05. All experiments were conducted with at least three biological replicates to ensure reproducibility. 3. Results 3.1. Construction of synthetic olivetolic acid pathway To establish a photosynthetic platform for olivetolic acid (OLA) production, we first introduced a heterologous biosynthetic pathway into Synechocystis sp. PCC 6803. The identification results of the relevant plasmids are shown in Appendix Fig. S1 . The pathway comprises type Ⅲ tetraketide synthase (TKS) and olivetolic acid cyclase (OAC) from Cannabis sativa , which together catalyze the transformation of hexanoyl-CoA and malonyl-CoA (Fig. 1 ). In our initial design, the TKS and OAC genes were organized into an operon under the control of the constitutive promoter P psbA2s and integrated into the neutral site slr0168 of the Synechocystis genome (Strain SCY01). However, HPLC analysis revealed that SCY01 failed to produce detectable OLA under routine photoautotrophic conditions (Appendix Fig. S2). We hypothesized that the primary bottleneck was the insufficient endogenous pool of hexanoyl-CoA. To address the precursor limitation and verify the enzymatic functionality, we introduced the hexanoyl-CoA synthetase gene (AAE1) to activate exogenous hexanoic acid into its CoA-thioester form (Strain SCY02). As shown in Fig. 2 A, initial assessments showed that in the absence of hexanoate supplementation, OLA was barely detectable (0.011 mg/L), confirming that hexanoic acid is not naturally accumulated in Synechocystis . To optimize the precursor supply, a range of sodium hexanoate concentrations (2, 4, 6, 8, 10, and 12 mM) was supplemented. OLA titers increased proportionally with exogenous hexanoate, peaking at 0.870 mg/L with an 8 mM supplement. Beyond this threshold (10–12 mM), OLA production declined sharply to 0.137 and 0.097 mg/L, respectively, accompanied by severe inhibition of cyanobacteria growth (Fig. 2 B). Given the lack of growth inhibition at 8 mM, this concentration was utilized for all subsequent experiments. These findings emphasize that the heterologous pathway's productivity is strongly limited by precursor availability. 3.2. Effect of promoter on production Given the functional validation of the OLA pathway in SCY02, we next sought to enhance metabolic flux by optimizing the transcriptional strength of the heterologous genes. We replaced the P psbA2s promoter in SCY02 with the super-strong constitutive promoter P cpc560 to drive the TKS-OAC-AAE1 operon, resulting in strain SCY03. Under the optimized 8 mM sodium hexanoate supplementation, strain SCY03 exhibited a continuous accumulation of OLA throughout the cultivation period. By day 14, the OLA titer in SCY03 reached 1.71 mg/L, representing a significant improvement compared to SCY02 (Fig. 3 ). This enhancement in productivity is consistent with the higher transcription-initiation frequency typically associated with the P cpc560 promoter in Synechocystis . The results indicate that increasing the expression levels of the TKS-OAC-AAE1 operon effectively bolstered the metabolic flux toward OLA. However, the overall yield remained limited, suggesting that the supply of the endogenous precursor, malonyl-CoA, might have become the next bottleneck. 3.3 Deletion of the glgC from the PCC6803 genome While promoter optimization enhanced the pathway efficiency, the availability of malonyl-CoA remained a major constraint for further increasing OLA titers. In Synechocystis , a substantial portion of fixed carbon is diverted toward glycogen as an energy storage molecule. To alleviate this competition, we disrupted the glycogen biosynthetic pathway by knocking out the glgC gene (encoding ADP-glucose pyrophosphorylase) in the SCY03 background, generating strain SCY04. As expected, the glgC deficiency resulted in a notable shift in carbon allocation. Under the 8 mM sodium hexanoate supplementation, SCY04 achieved an OLA titer of 2.43 mg/L by day 14 (Fig. 3 ), representing an approximately 42% increase compared to its parental strain SCY03 (1.71 mg/L). This improvement confirms that blocking the primary carbon sink (glycogen) effectively increases the intracellular malonyl-CoA pool, thereby providing more building blocks for polyketide synthesis. However, the disruption of glycogen synthesis led to a slight impairment in cell growth and biomass accumulation, particularly during the late stationary phase (Fig. 4 D). Despite this minor physiological shift, the overall viability of SCY04 remained robust, and the growth was not severely hindered. This subtle growth-production trade-off suggests that while carbon flux redirection toward OLA synthesis imposes a detectable metabolic load, it does not compromise the fundamental robustness of the Synechocystis chassis. This observation prompted us to explore further strategies to balance carbon fixation and metabolic drainage to achieve even higher titers. 3.4 Effect of maeB overexpression on olivetolic acid production To further enhance OLA productivity, we developed strain SCY05 by integrating the Escherichia coli maeB gene into the slr1176 (glgC) locus of SCY03. This site-specific integration was designed to simultaneously block the major carbon sink (glycogen) and introduce a heterologous malic enzyme to modulate the redirected carbon flux. According to the biochemical properties of maeB, this enzyme is highly specific for NADP⁺ and facilitates the metabolic exchange between TCA cycle intermediates and pyruvate[ 35 ]. In the SCY05 chassis, maeB was expected to function as a metabolic valve to coordinate the distribution of excess carbon resulting from glycogen deficiency, potentially optimizing the NADPH/NADP⁺ ratio to support the energy-demanding OLA pathway. Under 8 mM sodium hexanoate and ambient air, SCY05 achieved an OLA titer of 4.15 mg/L by day 14 (Fig. 3 ). This represents a 2.4-fold increase compared to its parental strain SCY03 (1.71 mg/L), confirming that coupling carbon sink disruption with maeB-mediated metabolic flexibility effectively redirected more carbon flux toward OLA biosynthesis. Furthermore, SCY05 maintained robust growth compared with SCY03 (Fig. 4 E), indicating that the enhancement of central metabolism partially alleviated the physiological stress typically associated with glycogen deficiency. 3.5 Enhancement of OLA production via high CO 2 supplementation To maximize photosynthetic carbon fixation and evaluate the long-term production potential of strain SCY05, we monitored its performance under 5% CO 2 over an extended 30-day cultivation period. In this optimized environment, OLA concentrations were measured at three-day intervals. Strikingly, OLA continued to accumulate throughout the stationary phase, ultimately reaching a maximum titer of 9.41 mg/L on day 30 (Fig. 5 ). This represents a substantial enhancement compared to the titer achieved under ambient air at day 14 (4.15 mg/L), suggesting that high CO 2 availability sustained the metabolic activity of the maeB-integrated chassis over a significantly longer duration. The growth profile under these conditions further confirmed the robustness of the engineered chassis. Despite the extended duration and the absence of glycogen as a carbon reserve, SCY05 exhibited stable biomass accumulation under 5% CO 2 , with no significant decline in cell density observed throughout the 30-day period (Fig. 5 ). The ability of SCY05 to sustain OLA production for 30 days indicates that the synergistic effect of glgC disruption, maeB integration, and high CO 2 supplementation successfully balanced the redirection of carbon flux with the physiological requirements for long-term survival and biosynthetic activity. 4. Discussion Over the past years, olivetolic acid (OLA) has been successfully synthesized in several heterotrophic microorganisms, including Escherichia coli [ 12 ], Yarrowia lipolytica [ 13 ] and Saccharomyces cerevisiae [ 14 ]. However, these platforms necessitate the consumption of refined organic carbon sources, posing challenges for large-scale sustainable manufacturing. In this study, we transitioned OLA production to the photoautotrophic chassis Synechocystis sp. PCC 6803. Despite the successful genomic integration of core biosynthetic genes (CsTKS and CsOAC), our initial strain SCY01 failed to accumulate OLA under standard conditions. This absence of production could be tentatively attributed to a limited endogenous pool of hexanoyl-CoA, the essential C6-CoA starter unit. Specifically, while Cannabis sativa utilizes specialized glandular trichomes to pool short-chain fatty acid precursors[ 8 ], Synechocystis presumably directs its fatty acid flux toward long-chain acyl-ACPs for membrane biogenesis rather than short-chain CoA thioesters[ 36 ]. Our findings with SCY01/SCY02 demonstrate that the introduction of CsAAE1 and exogenous hexanoate supplementation are critical for bypassing this endogenous bottleneck. By decoupling precursor supply from primary metabolism, we confirmed the enzymatic functionality of the plant-derived PKS system in a photosynthetic cytosol, establishing a robust foundation for subsequent flux optimization. Beyond the initial pathway assembly, the strategic selection of genetic parts is critical for tailoring the host’s metabolic landscape toward non-native synthesis. In this study, the transition from the P psbA2s promoter to the robust constitutive P cpc560 promoter resulted in a significant elevation of OLA titers (Strain SCY03 vs. SCY02). This marked improvement is likely attributable to the high enzymatic threshold required for the Type III polyketide synthase (TKS) to function effectively within the cyanobacterial chassis. Specifically, TKS must catalyze the iterative condensation of three malonyl-CoA molecules onto a hexanoyl-CoA starter unit—a process that is kinetically slow and must compete directly with the primary fatty acid biosynthetic pathway for the limited intracellular malonyl-CoA pool[ 37 ]. While P psbA2s is a widely utilized light-inducible promoter, its expression strength might be insufficient to maintain the high steady-state concentration of TKS needed to overcome this metabolic competition. Consequently, the superior performance of P cpc560 highlights that ensuring a high-level, consistent transcriptional dosage of the biosynthetic genes is a prerequisite for driving carbon flux toward the kinetically challenging polyketide pathway in Synechocystis . To further elevate the OLA titer, we implemented a coordinated strategy centered on re-routing the global carbon flux toward the heterologous pathway. While the strong P cpc560 promoter provides a robust transcriptional "pull," its efficiency can be hampered by the natural diversion of photosynthetic carbon toward endogenous storage compounds. In this study, strain SCY05 was developed from SCY03 by integrating the Escherichia coli maeB gene into the slr1176 (glgC) locus, a site-specific replacement designed to simultaneously block the primary glycogen biosynthetic sink and introduce a heterologous metabolic valve. The deletion of glgC served to "push" carbon flux away from glycogen synthesis, thereby increasing the availability of central intermediates[20; 38]. Simultaneously, the heterologous expression of maeB was intended to coordinate the distribution of this excess carbon. By facilitating the oxidative decarboxylation of malate to pyruvate, maeB presumably reinforced the intracellular pyruvate pool, a direct precursor for acetyl-CoA, while providing essential NADPH. This elevated NADPH availability not only helps maintain cellular redox homeostasis during high-flux polyketide biosynthesis but also optimizes cyanobacterial physiological conditions, thereby accelerating biomass accumulation[ 39 ]. Such anaplerotic replenishment likely supported the energy-demanding OLA pathway, which requires substantial ATP for precursor activation and NADPH for sustained secondary metabolism[ 40 ]. By effectively blocking the glycogen sink and augmenting these precursor and cofactor nodes, we successfully created a metabolic "surplus" for OLA production. The incremental yield improvements observed from SCY03 to SCY05 underscore that in a photoautotrophic chassis, the systematic elimination of competing carbon sinks, coupled with the reinforcement of precursor supply and redox balance, is a prerequisite for maximizing the partition of fixed CO 2 toward non-native secondary metabolites. Parallel to internal metabolic rewiring, the availability of inorganic carbon in the cultivation environment and the optimization of fermentation parameters play decisive roles in the productivity of photoautotrophic cell factories. Our results demonstrated that increasing the CO 2 concentration from ambient air (0.04%) to 5% significantly boosted the OLA titer in strain SCY05. This response is presumably driven by the native carbon-concentrating mechanism (CCM) of Synechocystis , which actively transports and accumulates HCO 3− within carboxysomes to ensure a high CO 2 concentration around RuBisCO[ 41 ]. Under these elevated CO 2 conditions, the efficiency of this mechanism is expected to be optimized, thereby minimizing the RuBisCO-mediated oxygenation reaction and potentially expanding the carbon flux toward GAP[ 42 ]. As the first stable intermediate of photosynthetic CO 2 fixation and a pivotal metabolic branch point, GAP serves as the foundational precursor for both the storage of glycogen and the initiation of the glycolytic pathway. In this context, the synergy between 5% CO 2 supplementation and the systematic elimination of competing carbon sinks—specifically glycogen synthesis—ensures that the augmented GAP flux is efficiently channeled toward the synthesis of acetyl-CoA and malonyl-CoA. By further extending the cultivation period, we achieved a maximum OLA titer of 9.41 mg/L. While this yield represents a promising start for cannabinoid precursor production in cyanobacteria, further efforts might be required to achieve industrially relevant scales. Nevertheless, this study provides a viable blueprint for the sustainable, light-driven biomanufacturing of complex plant polyketides. 5. Conclusions This study successfully established a green synthesis approach for the cannabinoid precursor olivetolic acid using Synechocystis sp. PCC 6803 as a photosynthetic cell factory. The production of this high-value compound was achieved through the integration of exogenous genes involved in olivetolic acid biosynthesis (TKS, OAC, and AAE1), targeted metabolic pathway optimization (knockout of the glycogen biosynthesis gene and overexpression of the malic enzyme gene), and refinement of cultivation conditions. These advancements provide a robust foundation for the scalable and sustainable biosynthesis of cannabidiol while highlighting the significant potential of cyanobacteria as environmentally friendly and efficient platforms for producing high-value compounds, offering a promising alternative to traditional cannabis cultivation. Declarations Ethics approval and consent to participate No applicable. Consent for publication All authors have participated in the review study and manuscript preparation. Competing interests The authors hereby declare that they have no conflict of interest. Funding declaration This work was supported by the National Natural Science Foundation of China (31200019), Open Fund of Key Laboratory of Tropical Marine Bio-resources and Ecology, Chinese Academy of Sciences (LMB131001), Ningbo Clinical Research Center for Children’s Health and Diseases (2019A21002), Ningbo Top Medical and Health Research Program (No. 2022020405), Ningbo Leading Medical &Health Discipline (No.2026-A34); Ningbo Municipal Bureau of Science and Technology Project (No.2025Z153). Author Contribution E-Bin Gao, Junhua Wu designed the experiment.E-Bin Gao, Wenrui Ji and Yangjie Zhu performed the experiments and collected the data.E-Bin Gao, Wenrui Ji, and Yangjie Zhu analyzed the data and drafted the article.E-Bin Gao and Wenrui Ji critically revised the article.All authors approved the final draft for submission. Data Availability The datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request. 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Switzerland: Springer International Publishing AG; 2017; 1–36. Andre CM, Hausman JF, Guerriero G. Cannabis sativa: The Plant of the Thousand and One Molecules. Front Plant Sci. 2016;7:19. 10.3389/fpls.2016.00019 . van Bakel H, Stout JM, Cote AG, Tallon CM, Sharpe AG, Hughes TR, et al. The draft genome and transcriptome of Cannabis sativa. Genome Biol. 2011;12:R102. 10.1186/gb-2011-12-10-r102 . Tan Z, Clomburg JM, Gonzalez R. Synthetic Pathway for the Production of Olivetolic Acid in Escherichia coli. Acs Synth Biol. 2018;7:1886–96. 10.1021/acssynbio.8b00075 . Ma J, Gu Y, Xu P. Biosynthesis of cannabinoid precursor olivetolic acid in genetically engineered Yarrowia lipolytica. Commun Biol. 2022;5:1239. 10.1038/s42003-022-04202-1 . Schmidt C, Aras M, Kayser O. Engineering cannabinoid production in Saccharomyces cerevisiae. Biotechnol J. 2024;19:e2300507. 10.1002/biot.202300507 . Bhatwa A, Wang W, Hassan YI, Abraham N, Li X, Zhou T. Challenges Associated With the Formation of Recombinant Protein Inclusion Bodies in Escherichia coli and Strategies to Address Them for Industrial Applications. Front Bioeng Biotechnol. 2021;9:630551. 10.3389/fbioe.2021.630551 . Vásquez Castro E, Memari G, Ata Ö, Mattanovich D. Carbon efficient production of chemicals with yeasts. Yeast (Chichester England). 2023;40:583–93. 10.1002/yea.3909 . Liu Y, Cui Y, Chen J, Qin S, Chen G. Metabolic engineering of Synechocystis sp. PCC6803 to produce astaxanthin. Algal Res. 2019;44:101679. 10.1016/j.algal.2019.101679 . Ni J, Tao F, Xu P, Yang C. Engineering Cyanobacteria for Photosynthetic Production of C3 Platform Chemicals and Terpenoids from CO(2). Adv Exp Med Biol. 2018;1080:239–59. 10.1007/978-981-13-0854-3_10 . Gu F, Li C, Zheng H, Ni J. Engineering cyanobacteria for the production of aromatic natural products. Blue Biotechnol. 2024;1:2. 10.1186/s44315-024-00002-w . Yoo D, Hong S, Yun S, Kang M, Cho B, Lee H, et al. Metabolic Engineering for Redirecting Carbon to Enhance the Fatty Acid Content of Synechocystis sp. PCC6803. Biotechnol Bioproc E. 2023;28:274–80. 10.1007/s12257-020-0386-x . Hidese R, Matsuda M, Osanai T, Hasunuma T, Kondo A. Malic Enzyme Facilitates d–Lactate Production through Increased Pyruvate Supply during Anoxic Dark Fermentation in Synechocystis sp. PCC 6803. Acs Synth Biol. 2020;9:260–8. 10.1021/acssynbio.9b00281 . Veetil VP, Angermayr SA, Hellingwerf KJ. Ethylene production with engineered Synechocystis sp PCC 6803 strains. Microb Cell Fact. 2017;16:34. 10.1186/s12934-017-0645-5 . Mueller TJ, Ungerer JL, Pakrasi HB, Maranas CD, Washington Univ. SLMU. Identifying the Metabolic Differences of a Fast-Growth Phenotype in Synechococcus UTEX 2973. Sci Rep-Uk. 2017;7:41569. 10.1038/srep41569 . VERMAAS W. Molecular genetics of the cyanobacterium Synechocystis sp. PCC 6803: Principles and possible biotechnology applications. In; 1996-01-01; Dordrecht. Springer; 1996. 263 – 73. Kupriyanova EV, Pronina NA, Los DA. Adapting from Low to High: An Update to CO(2)-Concentrating Mechanisms of Cyanobacteria and Microalgae. Plants (Basel). 2023;12. 10.3390/plants12071569 . Sato N. Are Cyanobacteria an Ancestor of Chloroplasts or Just One of the Gene Donors for Plants and Algae? Genes-Basel. 2021;12:823. 10.3390/genes12060823 . Englund E, Andersen-Ranberg J, Miao R, Hamberger B, Lindberg P. Metabolic Engineering of Synechocystis sp. PCC 6803 for Production of the Plant Diterpenoid Manoyl Oxide. Acs Synth Biol. 2015;4:1270–8. 10.1021/acssynbio.5b00070 . Williams JGK. [85] Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. In: 167. Edited by Packer L, Glazer AE: Elsevier Science & Technology; 1988; 766 – 78. Zang X, Liu B, Liu S, Arunakumara KK, Zhang X. Optimum conditions for transformation of Synechocystis sp. PCC 6803. J Microbiol. 2007;45:241–5. Kufryk GI, Sachet M, Schmetterer G, Vermaas WF. Transformation of the cyanobacterium Synechocystis sp. PCC 6803 as a tool for genetic mapping: optimization of efficiency. Fems Microbiol Lett. 2002;206:215–9. 10.1111/j.1574-6968.2002.tb11012.x . Sergeyenko TV, Los DA. The Effect of Various Stresses on the Expression of Genes Encoding the Secreted Proteins of the Cyanobacterium Synechocystis sp. PCC 6803. Russ J Plant Physl+. 2022;49:650–6. Xia PF, Ling H, Foo JL, Chang MW. Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria. Biotechnol J. 2019;14:e1800496. 10.1002/biot.201800496 . Luan G, Zhang S, Wang M, Lu X. Progress and perspective on cyanobacterial glycogen metabolism engineering. Biotechnol Adv. 2019;37:771–86. 10.1016/j.biotechadv.2019.04.005 . Al UH, Bhuyan DJ, Alsherbiny MA, Basu A, Vuong QV. A Comprehensive Review on the Techniques for Extraction of Bioactive Compounds from Medicinal Cannabis. Molecules. 2022;27. 10.3390/molecules27030604 . Bologna FP, Andreo CS, Drincovich MF. Escherichia coli malic enzymes: two isoforms with substantial differences in kinetic properties, metabolic regulation, and structure. J Bacteriol. 2007;189:5937–46. 10.1128/JB.00428-07 . Gong Y, Miao X. Short Chain Fatty Acid Biosynthesis in Microalgae Synechococcus sp. PCC 7942. Mar Drugs. 2019;17. 10.3390/md17050255 . Kearsey LJ, Nicole P. Structure of the Cannabis sativa olivetol-producing enzyme reveals cyclization plasticity in type III polyketide synthases. Febs J. 2019. van der Woude AD, Angermayr SA, Puthan Veetil V, Osnato A, Hellingwerf KJ. Carbon sink removal: Increased photosynthetic production of lactic acid by Synechocystis sp. PCC6803 in a glycogen storage mutant. J Biotechnol. 2014;184:100–2. 10.1016/j.jbiotec.2014.04.029 . Choi Y, Park JM. Enhancing biomass and ethanol production by increasing NADPH production in Synechocystis sp. PCC 6803. Bioresource Technol. 2016;213:54–7. 10.1016/j.biortech.2016.02.056 . Yang X, Liang W, Lin X, Zhao M, Zhang Q, Tao Y, et al. Efficient Escherichia coli Platform for Cannabinoid Precursor Olivetolic Acid Biosynthesis from Inexpensive Inputs. J Agric Food Chem. 2025;73:3611–21. 10.1021/acs.jafc.4c11867 . Liang F, Lindblad P. Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803. Metab Eng. 2016;38:56–64. 10.1016/j.ymben.2016.06.005 . Price GD, Howitt SM. The cyanobacterial bicarbonate transporter BicA: its physiological role and the implications of structural similarities with human SLC26 transporters. Biochem Cell Biol. 2011;89:178–88. 10.1139/O10-136 . Additional Declarations No competing interests reported. Supplementary Files 3.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 06 May, 2026 Reviews received at journal 04 May, 2026 Reviews received at journal 18 Apr, 2026 Reviewers agreed at journal 30 Mar, 2026 Reviewers agreed at journal 28 Mar, 2026 Reviewers invited by journal 26 Mar, 2026 Editor assigned by journal 25 Mar, 2026 Submission checks completed at journal 25 Mar, 2026 First submitted to journal 19 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9174471","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":612514294,"identity":"5c4208f0-0c9d-4649-8a72-3c3acf9e2c3e","order_by":0,"name":"E-Bin Gao","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"E-Bin","middleName":"","lastName":"Gao","suffix":""},{"id":612514295,"identity":"06062cc0-9987-4531-9e5f-b18425338935","order_by":1,"name":"Wenrui Ji","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"Wenrui","middleName":"","lastName":"Ji","suffix":""},{"id":612514297,"identity":"621cbcc9-addb-4098-9530-032209b70552","order_by":2,"name":"YangJie Zhu","email":"","orcid":"","institution":"Jiangsu University","correspondingAuthor":false,"prefix":"","firstName":"YangJie","middleName":"","lastName":"Zhu","suffix":""},{"id":612514299,"identity":"c723f8c0-45f5-41e7-80eb-2404860c29a7","order_by":3,"name":"Junhua Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYHACAyC24eHnbyBFywGGNBnJGQdI03LYxqAhgVj1N5I3fv5Qc54HqJHxw8ccorSkFUscOHabx5y5gVly5jYitJjdyDGQOMB2m8ey4QAbMy+RWox/HPh3jsfgQALxWswkDrYdIEGL/ZlnZRZn+5J5JGccbCbOL5LtyZtvVHyzs+fnbz744SMxWpAAYwNp6kfBKBgFo2AU4AYA1/Y51aHnq58AAAAASUVORK5CYII=","orcid":"","institution":"The affiliated Women and Children's Hospital of Ningbo University","correspondingAuthor":true,"prefix":"","firstName":"Junhua","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2026-03-20 03:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9174471/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9174471/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105574754,"identity":"b2e0be8d-687b-40f1-9e18-ccd9a1d91c38","added_by":"auto","created_at":"2026-03-27 13:36:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":141520,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModifications of metabolic pathway in engineered strains.\u003c/strong\u003e In the engineered cyanobacteria constructed in this study, the heterologous olivetolic acid (OLA) synthesis pathway (Blue part) begins with exogenously supplied sodium hexanoate and endogenously derived malonyl-CoA. Hexanoate is converted to hexanoyl-CoA by the enzyme AAE1. Subsequently, TKS catalyzes three successive two-carbon extensions using malonyl-CoA as the donor via decarboxylative condensation. The intermediate is finally cyclized at the C2-C7 position through an alcohol-aldehyde cyclization catalyzed by OAC, forming OLA. To redirect carbon flux toward product synthesis, the glgC gene was knocked out to block glycogen synthesis (Red part), and the maeB gene was introduced to adjust cellular metabolism (Orange part).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-9174471/v1/a0db23b69b4a6e685fd817df.png"},{"id":105574466,"identity":"a0c31b90-3fe6-4654-b392-04e376260dd3","added_by":"auto","created_at":"2026-03-27 13:35:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":30217,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of hexanoic concentration on the growth and production of the engineered cyanobacterial strain SCY02. \u003c/strong\u003eOlivetolic acid production (A) and cell growth (B) of SCY02 strain at day 14 with the addition of hexanoic acid. The initial cell concentration OD\u003csub\u003e730\u003c/sub\u003e = 0.06.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-9174471/v1/a7efdab4d42e80faf091dca7.png"},{"id":105574976,"identity":"8e413df9-c3aa-47c2-9244-a3a8c224a105","added_by":"auto","created_at":"2026-03-27 13:37:05","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":95890,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferent metabolic modification strategies to promote olivetolic acid production. (\u003c/strong\u003eA) Production of engineered strains at day 14 with the addition of 8 mM sodium hexanoate. (B) Extracts were analyzed by HPLC at a wavelength of 262 nm and signals were compared to authentic OLA standards (Appendix Fig. S3).\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9174471/v1/c473bdd8c96edb976f19f5c9.jpeg"},{"id":105574731,"identity":"63fe0b0b-63cb-461e-ad38-c4396d85a59f","added_by":"auto","created_at":"2026-03-27 13:36:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":116831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOlivetolic acid production of engineered strains in 14 days. \u003c/strong\u003eTimes courses of olivetolic acid production (bars) and cell growth (circles) in the (A)SCY01, (B)SCY02, (C)SCY03, (D)SCY04 and (E)SCY05 cultured with the addition of 8mM hexanoic acid are shown.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-9174471/v1/779adadb3f0674115fd1afcc.png"},{"id":105574887,"identity":"1a4865ba-427d-4a4f-830c-52f8957e56d3","added_by":"auto","created_at":"2026-03-27 13:36:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31758,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of aeration conditions on the growth and production of the engineered cyanobacterial strain SCY05. \u003c/strong\u003eOlivetolic acid production (bars) and cell growth (circles) of SCY05 strain in 14 days with bubbling 5% CO\u003csub\u003e2\u003c/sub\u003e-air (v/v) and adding 8mM hexanoic.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-9174471/v1/1c9b4fa4e339daec1f3f3811.png"},{"id":105575523,"identity":"b8c34a0f-44fe-45a4-95f2-24aff5249473","added_by":"auto","created_at":"2026-03-27 13:39:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1361987,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9174471/v1/14279582-c0a7-4ab5-a58d-a7ef54db1f3b.pdf"},{"id":105574440,"identity":"364eba06-8f82-47af-bbaf-d5d66287b87c","added_by":"auto","created_at":"2026-03-27 13:34:56","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":508993,"visible":true,"origin":"","legend":"","description":"","filename":"3.docx","url":"https://assets-eu.researchsquare.com/files/rs-9174471/v1/213022f2924c986ee05c6e48.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metabolic Engineering of Synechocystis sp. PCC 6803 for Olivetolic acid Production","fulltext":[{"header":"Highlights","content":"\u003cp\u003e\u0026middot; Initial synthesis of olivetolic acid in PCC6803.\u003c/p\u003e\u003cp\u003e\u0026middot; Production titer increased after knocking out the glycogen synthesis pathway.\u003c/p\u003e\u003cp\u003e\u0026middot; Overexpression assays optimized the metabolic pathway and increased production.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003ePhytocannabinoids, such as cannabidiol (CBD) and Δ\u003csup\u003e9\u003c/sup\u003e-tetrahydrocannabinol (THC), represent a class of highly valued meroterpenoids produced by \u003cem\u003eCannabis sativa\u003c/em\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They exert therapeutic effects through modulation of the endocannabinoid system (CB1/CB2 receptors)[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], evidenced by FDA-approved drugs for epilepsy (Epidiolex\u0026reg; - CBD) and chemotherapy-induced nausea (Marinol\u0026reg;/Syndros\u0026reg; - synthetic THC)[3; 4]. Typical production relies on plant extraction or chemical synthesis[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, the sustainable supply of cannabinoid drugs via traditional plant-based extraction is severely bottlenecked. On one hand, production is hindered by the protracted growth cycles of \u003cem\u003eCannabis sativa\u003c/em\u003e (typically 3\u0026ndash;4 months) and stringent legal frameworks (generally restricting cultivation to low-THC varieties)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. On the other hand, low specialized metabolite titers and resource-intensive farming render the process both economically and ecologically inefficient[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. While chemical synthesis provides a potential route, it remains formidable due to the intricate molecular architecture, necessitating the use of hazardous organic solvents and metal catalysts that not only raise environmental concerns but also pose significant hurdles for the downstream purification of pharmaceutical-grade products[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Consequently, there is an urgent imperative to develop sustainable, carbon-neutral production platforms that leverage green manufacturing principles to circumvent the multifaceted ecological and technical bottlenecks inherent in traditional cannabinoid supply chains.\u003c/p\u003e \u003cp\u003eAs the major specialized metabolites in \u003cem\u003eCannabis sativa\u003c/em\u003e, phytocannabinoids are predominantly biosynthesized in glandular trichomes and reach their maximum accumulation on the bracts of female inflorescences[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While over 150 different cannabinoids have been identified to date, the biosynthetic pathways of the most extensively studied analogs\u0026mdash;including Δ\u003csup\u003e9\u003c/sup\u003e-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), and cannabigerol (CBG)\u0026mdash;all converge at a common core precursor, olivetolic acid (OLA)[9; 10]. In the native \u003cem\u003eCannabis sativa\u003c/em\u003e plant, the formation of this pivotal aromatic intermediate is governed by a three-step enzymatic sequence as follows: The pathway initiates with the activation of the short-chain fatty acid hexanoate into hexanoyl-CoA by the acyl-activating enzyme (CsAAE1) in an ATP-dependent manner. This hexanoyl-CoA then serves as the starter unit for the tetraketide synthase (CsTKS), a type Ⅲ polyketide synthase, which performs the iterative decarboxylative condensation of three molecules of malonyl-CoA to generate a linear triketide intermediate. To prevent the spontaneous formation of metabolic side-products, the olivetolic acid cyclase (CsOAC) acts as a regiospecific scaffold to direct the C2-C7 aldol condensation and subsequent cyclization of the intermediate, ultimately yielding the pivotal aromatic intermediate, olivetolic acid[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe elucidation of this native enzymatic logic has paved the way for synthetic biology to transcend the limitations of traditional plant-based production. Since the production of OLA is a fundamental prerequisite for cannabinoid synthesis, researchers have successfully reconstituted this metabolic pathway in various heterologous microbial hosts. To date, research has predominantly focused on heterotrophic platforms, underscoring the feasibility of OLA production via advanced metabolic engineering strategies. \u003cem\u003eEscherichia coli\u003c/em\u003e was among the earliest chassis employed for the biosynthesis of OLA. Tan et al. reconstituted this pathway by integrating Cannabis OLS and OAC with a reversed β-oxidation cycle for de novo hexanoyl-CoA synthesis. Through integrated metabolic optimization and controlled bioreactor cultivation, this strategy ultimately achieved an OLA titer of 80 mg/L[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].. In the \u003cem\u003eYarrowia lipolytica\u003c/em\u003e chassis, researchers achieved an 83-fold increase in olivetolic acid titers to 9.18 mg/L by optimizing precursor supply, augmenting cofactor regeneration, and integrating metabolic pathways to effectively redirect carbon flux toward polyketide biosynthesis[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Similarly, Schmidt et al.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] optimized \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e by introducing CsAAE1, CsOLS, and CsOAC for olivetolic acid synthesis, alongside a soluble NphB and ERG20\u003csup\u003eWW\u003c/sup\u003e fusion to bypass the limitations of membrane-bound prenyltransferases. By further overexpressing the transcription factor HAC1s to boost enzyme activity, they achieved 117 mg/L of olivetolic acid and successfully synthesized CBGA and CBCA. However, cannabinoid biosynthetic enzymes of plant origin are highly susceptible to forming inactive inclusion bodies within \u003cem\u003eE. coli\u003c/em\u003e, which limits the yield of soluble active proteins[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. While yeast is considered energy-intensive in production due to its reliance on refined organic carbon sources and the requirement for high-density cultivation[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Furthermore, the heavy reliance of these conventional hosts on organic carbon sources necessitates the exploration of more sustainable and cost-efficient production platforms.\u003c/p\u003e \u003cp\u003eThe model cyanobacterium \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 represents a versatile photosynthetic chassis exceptionally suited for the sustainable biosynthesis of plant-derived metabolites, including terpenoids, flavonoids, and phenolics[\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Unlike conventional heterotrophic hosts that require costly organic feedstocks, \u003cem\u003eSynechocystis\u003c/em\u003e operates photoautotrophically\u0026mdash;utilizing ambient CO\u003csub\u003e2\u003c/sub\u003e and solar energy to enable carbon-negative manufacturing[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Benefiting from a rapid doubling time (7\u0026ndash;10 h) and a sophisticated carbon-concentrating mechanism (CCM), the organism achieves superior photosynthetic performance and highly efficient nitrogen utilization[23; 24]. Structurally, cyanobacteria share with plant cells thylakoid membranes containing galactolipids, sulfolipids, various photosystem components, and photosynthetic pigments[\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Metabolically, the cyanobacterial photosynthetic apparatus yields substantial reducing equivalents and critical precursor pools (e.g., acetyl-CoA). This dual congruence renders cyanobacteria inherently compatible with the biosynthesis of plant-derived metabolites, such as terpenoids and phenolics[19; 27]. Collectively, these unique features, complemented by the facile genetic engineering of cyanobacteria, position this organism as an optimal photosynthetic platform for the sustainable biosynthesis of cannabinoids and their respective precursors.\u003c/p\u003e \u003cp\u003eAs a proof of concept, we established a photosynthetic platform for the synthesis of the pivotal precursor, olivetolic acid, in \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803. This was achieved through an integrated metabolic engineering strategy that coupled the heterologous expression of cannabinoid biosynthetic genes (CsTKS, CsOAC, and CsAAE1) with host-specific modifications, including the disruption of carbon storage (ΔglgC) and the reinforcement of the malonyl-CoA pool (maeB). By successfully redirecting photosynthetic carbon flux toward polyketide production, this study lays a robust foundation for sustainable cannabinoid manufacturing aligned with circular economy principles.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1. Strains and growth conditions\u003c/h2\u003e\n \u003cp\u003eThe wild type and genetically engineered \u003cem\u003eSynechocystis\u003c/em\u003e strains were cultivated in BG11 medium using an illuminated incubator (GDN-260A, YANGHUI, China) under a light intensity of 28 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a temperature of 30℃. The cell density of strains was measured at an optical density (OD) of 730 nm using a visible spectrophotometer (YOKE, Shanghai, China).\u003c/p\u003e\n \u003cp\u003eThe DH5\u0026alpha; Chemically Competent Cell (Vazyme, Nanjing, China) was used for plasmid construction and amplification. The DH5\u0026alpha; strain was cultured in LB medium at 37℃ with shaking at 220 rpm (Zhichu, Shanghai, China).\u003c/p\u003e\n \u003cp\u003eDifferent antibiotic concentrations were employed for various strains during cultivation. For genetically engineered cyanobacteria, spectinomycin was added at a final concentration of 50 \u0026micro;g/mL, while chloramphenicol was added at a final concentration of 34 \u0026micro;g/mL. During the initial selection after cyanobacterial transformation, antibiotics were applied at one-fifth of the regular cultivation concentrations and gradually increased to the required levels for routine.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2. Plasmids construction\u003c/h2\u003e\n \u003cp\u003eThis study constructed a series of integration plasmids based on the pMD18-T backbone (Sangon Biotech Co Ltd., Shanghai, China) to integrate optimized synthetic exogenous genes (Sangon Biotech Co Ltd., Shanghai, China) into the cyanobacterial chromosome. The plasmid construction involved restriction digestion using endonucleases (NEB, Beijing, China), PCR amplification with high-fidelity polymerase (Vazyme, Nanjing, China), and assembly using homologous recombinase (NEB, Beijing, China). DH5\u0026alpha; was used for plasmid amplification. Single colonies were screened via colony PCR, and verified plasmids were sent for sequencing. Subsequent experiments proceeded after confirmation of correct sequences. The characteristics of all plasmids used for transformation in this study are summarized in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Plasmids construction process and primers are provided in the appendix file.\u003c/p\u003e\n\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePlasmids used for transformation in this study.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003ePlasmids\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eCharacteristic\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eResistance\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epMD18-T\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eAmp\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epST407-TKS/OAC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eslr0168up-Spec\u003csup\u003eR\u003c/sup\u003e-P\u003csub\u003epsbA2s\u003c/sub\u003e-TKS-OAC-T\u003csub\u003erbcL\u003c/sub\u003e-slr0168dw\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eAmp\u003csup\u003eR\u003c/sup\u003e+Spec\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epST02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eslr0168up-Spec\u003csup\u003eR\u003c/sup\u003e-P\u003csub\u003epsbA2s\u003c/sub\u003e-TKS-OAC-AAE1-T\u003csub\u003erbcL\u003c/sub\u003e-slr0168dw\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eAmp\u003csup\u003eR\u003c/sup\u003e+Spec\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epST03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eslr0168up-Spec\u003csup\u003eR\u003c/sup\u003e-P\u003csub\u003ecpc560\u003c/sub\u003e-TKS-OAC-AAE1-T\u003csub\u003erbcL\u003c/sub\u003e-slr0168dw\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eAmp\u003csup\u003eR\u003c/sup\u003e+Spec\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epST04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eslr1176up-Cm\u003csup\u003eR\u003c/sup\u003e-slr1176dw\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eAmp\u003csup\u003eR\u003c/sup\u003e+ Cm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colname=\"c1\"\u003e\n \u003cp\u003epST05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c2\"\u003e\n \u003cp\u003eslr1176up-Cm\u003csup\u003eR\u003c/sup\u003e-P\u003csub\u003ecpc560\u003c/sub\u003e-maeB-T\u003csub\u003erbcL\u003c/sub\u003e-slr1176dw\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colname=\"c3\"\u003e\n \u003cp\u003eAmp\u003csup\u003eR\u003c/sup\u003e+ Cm\u003csup\u003eR\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eTable notes: slr0168up: upstream homology arm of the slr0168 gene, slr0168dw: downstream homology arm of the slr0168 gene, slr1176up: upstream homology arm of the slr1176 gene, slr1176dw: downstream homology arm of the slr1176 gene, Amp\u003csup\u003eR\u003c/sup\u003e: ampicillin resistance gene, Spec\u003csup\u003eR\u003c/sup\u003e: spectinomycin resistance gene, Cm\u003csup\u003eR\u003c/sup\u003e: chloramphenicol resistance gene, P\u003csub\u003epsbA2s\u003c/sub\u003e: native light-induced promoter P\u003csub\u003epsbA2s\u003c/sub\u003e, P\u003csub\u003ecpc560\u003c/sub\u003e: native super strong promoter P\u003csub\u003ecpc560\u003c/sub\u003e, T\u003csub\u003erbcL\u003c/sub\u003e: native terminator T\u003csub\u003erbcL\u003c/sub\u003e, TKS: tetraketide synthase, OAC: olivetolic acid cyclase, AAE1: hexanoyl-CoA synthase, maeB: NADP-dependent malic enzyme.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3. Transformation of \u003cem\u003eSynechocystis\u003c/em\u003e sp. 6803\u003c/h2\u003e\n \u003cp\u003eTransformation of \u003cem\u003eSynechocystis\u003c/em\u003e was performed using a method described by John G.K. Williams[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], with modifications to incubation time, plasmid concentration, and other parameters based on cell conditions and cultivation factors[29; 30].\u003c/p\u003e\n \u003cp\u003eCultures in the exponential phase (30mL, OD\u003csub\u003e730\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6\u0026ndash;0.8, cultured at 30℃ with 28 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light) were harvested by centrifugation, washed twice with fresh BG11 medium, and resuspended in 1 mL of antibiotic-free BG11 medium. To the suspension, 10 \u0026micro;g of the target plasmid was added and mixed by gentle pipetting. The mixture was incubated for 18 hours in a low light incubator, with gentle shaking 3\u0026ndash;4 times during this period. Following incubation, the mixture was spread onto five antibiotic-free BG11 agar plates, each containing a 0.45 \u0026micro;m hydrophilic cellulose membrane, pretreated by boiling and autoclaving. The plates were incubated for 24 hours, after which the membranes were transferred to BG11 plates supplemented with appropriate antibiotic concentration.\u003c/p\u003e\n \u003cp\u003eTransformants appeared after 2\u0026ndash;3 weeks. Individual colonies were isolated and streaked onto BG11 agar plates containing antibiotics for further purification. One colony from each plate was then inoculated into liquid BG11 medium with a low concentration of antibiotics, with the antibiotic concentration gradually increased during serial passaging. After five passages (approximately one month), cells in the exponential phase were harvested for genomic DNA extraction and PCR-based validation.\u003c/p\u003e\n \u003cp\u003eAll the strains referred to in this study are presented in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp class=\"gridtable\"\u003e\u003c/p\u003e\n \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;Table 2. The strains constructed in this study.\u003cp\u003e\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eEngineered strains\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003eGene cluster\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003etransformation\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eSCY01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u003cimg width=\"171\" height=\"68\" 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v:shapes=\"图片_x0020_2\" alt=\"image\"\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003epST407-TKS/OAC into \u003cem\u003eSynechocystis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eSCY02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u003cimg width=\"206\" height=\"68\" 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NCS5lfvIjDdJk125lUd5lTL5i6xxj634l+vhl1jhkoBZmIZ5mAITEAA7\" v:shapes=\"图片_x0020_7\" alt=\"image\"\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003epST04 into SCY03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 78px;\"\u003e\n \u003cp\u003eSCY05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 347px;\"\u003e\n \u003cp\u003e\u003cimg width=\"337\" height=\"68\" src=\"data:image/png;base64,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\" v:shapes=\"图片_x0020_9\" alt=\"image\"\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 128px;\"\u003e\n \u003cp\u003epST05 into SCY03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eThe slr0168 locus is a neutral site on the \u003cem\u003eSynechocystis\u003c/em\u003e genome, encoding a hypothetical glycoprotein and forming part of the slr0338-slr0168-slr0169 operon. The functions of this operon and the genes within it remain unclear[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Typically, modifications at neutral sites do not significantly affect cellular functions[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], which is why this locus is frequently chosen for introducing exogenous genes when \u003cem\u003eSynechocystis\u003c/em\u003e is used as a chassis organism.\u003c/p\u003e\n \u003cp\u003eThe slr1176 locus ([EC:2.7.7.27]) encodes ADP-Glucose Pyrophosphorylase, referred to as glgC. This gene is involved in the first step of glycogen and starch biosynthesis in \u003cem\u003eSynechocystis\u003c/em\u003e, and the activity of glgC regulates the overall rate of glycogen accumulation. If this gene is knocked out, glycogen cannot be synthesized in \u003cem\u003eSynechocystis\u003c/em\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4. Olivetolic acid extraction and measurement\u003c/h2\u003e\n \u003cp\u003eAll engineered strains were cultured in BG11 medium supplemented with sodium hexanoate at a light intensity of 28 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and incubated at 30℃. Cyanobacterial cells were first disrupted using an ultrasonic cell disruptor, and the resulting mixture was centrifuged at 8000 rpm for 10 minutes. The supernatant was then transferred and extracted with an equal volume of ethyl acetate[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. After combining the extracts, the solution was concentrated to 1 mL by rotary evaporation and subsequently dried under a flow of nitrogen gas. The residue was re-suspended in 1 mL methanol and filtered through a 0.22 \u0026micro;m syringe filter before HPLC (high-performance liquid chromatography) analysis. HPLC analysis was performed using a Shimadzu LC-20AT system with a Thermo Scientific Hypersil ODS-2 C18 column (4.6 mm \u0026times; 250 mm, 5 \u0026micro;m) and detection at 262 nm.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5. Improvement of culture conditions\u003c/h2\u003e\n \u003cp\u003eSince cyanobacterial growth and olivetolic acid production rely on CO\u003csub\u003e2\u003c/sub\u003e consumption, continuous aeration was employed to enhance CO\u003csub\u003e2\u003c/sub\u003e supply. Logarithmically growing strains were inoculated to an initial OD\u003csub\u003e730\u003c/sub\u003e of 0.50. The CO\u003csub\u003e2\u003c/sub\u003e was mixed with air at a 1:19 ratio (5% CO\u003csub\u003e2\u003c/sub\u003e, 95% air), filtered through a 0.22 \u0026micro;m membrane and continuously supplied to the culture flasks. Cultures were maintained at 30℃ and illuminated at 28 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e with continuous aeration for one month. Wild-type \u003cem\u003eSynechocystis\u003c/em\u003e grown under the same conditions was used as the control.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6. Statistics and reproducibility\u003c/h2\u003e\n \u003cp\u003eStatistical analysis was performed using the Tukey test in SPSS statistical software. Statistical significance was defined as a p-value less than 0.05. All experiments were conducted with at least three biological replicates to ensure reproducibility.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Construction of synthetic olivetolic acid pathway\u003c/h2\u003e \u003cp\u003eTo establish a photosynthetic platform for olivetolic acid (OLA) production, we first introduced a heterologous biosynthetic pathway into \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803. The identification results of the relevant plasmids are shown in Appendix Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The pathway comprises type Ⅲ tetraketide synthase (TKS) and olivetolic acid cyclase (OAC) from \u003cem\u003eCannabis sativa\u003c/em\u003e, which together catalyze the transformation of hexanoyl-CoA and malonyl-CoA (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our initial design, the TKS and OAC genes were organized into an operon under the control of the constitutive promoter P\u003csub\u003epsbA2s\u003c/sub\u003e and integrated into the neutral site slr0168 of the \u003cem\u003eSynechocystis\u003c/em\u003e genome (Strain SCY01). However, HPLC analysis revealed that SCY01 failed to produce detectable OLA under routine photoautotrophic conditions (Appendix Fig. S2). We hypothesized that the primary bottleneck was the insufficient endogenous pool of hexanoyl-CoA.\u003c/p\u003e \u003cp\u003eTo address the precursor limitation and verify the enzymatic functionality, we introduced the hexanoyl-CoA synthetase gene (AAE1) to activate exogenous hexanoic acid into its CoA-thioester form (Strain SCY02). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, initial assessments showed that in the absence of hexanoate supplementation, OLA was barely detectable (0.011 mg/L), confirming that hexanoic acid is not naturally accumulated in \u003cem\u003eSynechocystis\u003c/em\u003e. To optimize the precursor supply, a range of sodium hexanoate concentrations (2, 4, 6, 8, 10, and 12 mM) was supplemented. OLA titers increased proportionally with exogenous hexanoate, peaking at 0.870 mg/L with an 8 mM supplement. Beyond this threshold (10\u0026ndash;12 mM), OLA production declined sharply to 0.137 and 0.097 mg/L, respectively, accompanied by severe inhibition of cyanobacteria growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Given the lack of growth inhibition at 8 mM, this concentration was utilized for all subsequent experiments. These findings emphasize that the heterologous pathway's productivity is strongly limited by precursor availability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Effect of promoter on production\u003c/h2\u003e \u003cp\u003eGiven the functional validation of the OLA pathway in SCY02, we next sought to enhance metabolic flux by optimizing the transcriptional strength of the heterologous genes. We replaced the P\u003csub\u003epsbA2s\u003c/sub\u003e promoter in SCY02 with the super-strong constitutive promoter P\u003csub\u003ecpc560\u003c/sub\u003e to drive the TKS-OAC-AAE1 operon, resulting in strain SCY03.\u003c/p\u003e \u003cp\u003eUnder the optimized 8 mM sodium hexanoate supplementation, strain SCY03 exhibited a continuous accumulation of OLA throughout the cultivation period. By day 14, the OLA titer in SCY03 reached 1.71 mg/L, representing a significant improvement compared to SCY02 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This enhancement in productivity is consistent with the higher transcription-initiation frequency typically associated with the P\u003csub\u003ecpc560\u003c/sub\u003e promoter in \u003cem\u003eSynechocystis\u003c/em\u003e. The results indicate that increasing the expression levels of the TKS-OAC-AAE1 operon effectively bolstered the metabolic flux toward OLA. However, the overall yield remained limited, suggesting that the supply of the endogenous precursor, malonyl-CoA, might have become the next bottleneck.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Deletion of the glgC from the PCC6803 genome\u003c/h2\u003e \u003cp\u003eWhile promoter optimization enhanced the pathway efficiency, the availability of malonyl-CoA remained a major constraint for further increasing OLA titers. In \u003cem\u003eSynechocystis\u003c/em\u003e, a substantial portion of fixed carbon is diverted toward glycogen as an energy storage molecule. To alleviate this competition, we disrupted the glycogen biosynthetic pathway by knocking out the glgC gene (encoding ADP-glucose pyrophosphorylase) in the SCY03 background, generating strain SCY04.\u003c/p\u003e \u003cp\u003eAs expected, the glgC deficiency resulted in a notable shift in carbon allocation. Under the 8 mM sodium hexanoate supplementation, SCY04 achieved an OLA titer of 2.43 mg/L by day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), representing an approximately 42% increase compared to its parental strain SCY03 (1.71 mg/L). This improvement confirms that blocking the primary carbon sink (glycogen) effectively increases the intracellular malonyl-CoA pool, thereby providing more building blocks for polyketide synthesis.\u003c/p\u003e \u003cp\u003eHowever, the disruption of glycogen synthesis led to a slight impairment in cell growth and biomass accumulation, particularly during the late stationary phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Despite this minor physiological shift, the overall viability of SCY04 remained robust, and the growth was not severely hindered. This subtle growth-production trade-off suggests that while carbon flux redirection toward OLA synthesis imposes a detectable metabolic load, it does not compromise the fundamental robustness of the \u003cem\u003eSynechocystis\u003c/em\u003e chassis. This observation prompted us to explore further strategies to balance carbon fixation and metabolic drainage to achieve even higher titers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Effect of maeB overexpression on olivetolic acid production\u003c/h2\u003e \u003cp\u003eTo further enhance OLA productivity, we developed strain SCY05 by integrating the \u003cem\u003eEscherichia coli\u003c/em\u003e maeB gene into the slr1176 (glgC) locus of SCY03. This site-specific integration was designed to simultaneously block the major carbon sink (glycogen) and introduce a heterologous malic enzyme to modulate the redirected carbon flux.\u003c/p\u003e \u003cp\u003eAccording to the biochemical properties of maeB, this enzyme is highly specific for NADP⁺ and facilitates the metabolic exchange between TCA cycle intermediates and pyruvate[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the SCY05 chassis, maeB was expected to function as a metabolic valve to coordinate the distribution of excess carbon resulting from glycogen deficiency, potentially optimizing the NADPH/NADP⁺ ratio to support the energy-demanding OLA pathway.\u003c/p\u003e \u003cp\u003eUnder 8 mM sodium hexanoate and ambient air, SCY05 achieved an OLA titer of 4.15 mg/L by day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This represents a 2.4-fold increase compared to its parental strain SCY03 (1.71 mg/L), confirming that coupling carbon sink disruption with maeB-mediated metabolic flexibility effectively redirected more carbon flux toward OLA biosynthesis. Furthermore, SCY05 maintained robust growth compared with SCY03 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), indicating that the enhancement of central metabolism partially alleviated the physiological stress typically associated with glycogen deficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Enhancement of OLA production via high CO\u003csub\u003e2\u003c/sub\u003e supplementation\u003c/h2\u003e \u003cp\u003eTo maximize photosynthetic carbon fixation and evaluate the long-term production potential of strain SCY05, we monitored its performance under 5% CO\u003csub\u003e2\u003c/sub\u003e over an extended 30-day cultivation period. In this optimized environment, OLA concentrations were measured at three-day intervals. Strikingly, OLA continued to accumulate throughout the stationary phase, ultimately reaching a maximum titer of 9.41 mg/L on day 30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This represents a substantial enhancement compared to the titer achieved under ambient air at day 14 (4.15 mg/L), suggesting that high CO\u003csub\u003e2\u003c/sub\u003e availability sustained the metabolic activity of the maeB-integrated chassis over a significantly longer duration.\u003c/p\u003e \u003cp\u003eThe growth profile under these conditions further confirmed the robustness of the engineered chassis. Despite the extended duration and the absence of glycogen as a carbon reserve, SCY05 exhibited stable biomass accumulation under 5% CO\u003csub\u003e2\u003c/sub\u003e, with no significant decline in cell density observed throughout the 30-day period (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The ability of SCY05 to sustain OLA production for 30 days indicates that the synergistic effect of glgC disruption, maeB integration, and high CO\u003csub\u003e2\u003c/sub\u003e supplementation successfully balanced the redirection of carbon flux with the physiological requirements for long-term survival and biosynthetic activity.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOver the past years, olivetolic acid (OLA) has been successfully synthesized in several heterotrophic microorganisms, including \u003cem\u003eEscherichia coli\u003c/em\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], \u003cem\u003eYarrowia lipolytica\u003c/em\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, these platforms necessitate the consumption of refined organic carbon sources, posing challenges for large-scale sustainable manufacturing. In this study, we transitioned OLA production to the photoautotrophic chassis \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803. Despite the successful genomic integration of core biosynthetic genes (CsTKS and CsOAC), our initial strain SCY01 failed to accumulate OLA under standard conditions. This absence of production could be tentatively attributed to a limited endogenous pool of hexanoyl-CoA, the essential C6-CoA starter unit. Specifically, while \u003cem\u003eCannabis sativa\u003c/em\u003e utilizes specialized glandular trichomes to pool short-chain fatty acid precursors[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], \u003cem\u003eSynechocystis\u003c/em\u003e presumably directs its fatty acid flux toward long-chain acyl-ACPs for membrane biogenesis rather than short-chain CoA thioesters[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Our findings with SCY01/SCY02 demonstrate that the introduction of CsAAE1 and exogenous hexanoate supplementation are critical for bypassing this endogenous bottleneck. By decoupling precursor supply from primary metabolism, we confirmed the enzymatic functionality of the plant-derived PKS system in a photosynthetic cytosol, establishing a robust foundation for subsequent flux optimization.\u003c/p\u003e \u003cp\u003eBeyond the initial pathway assembly, the strategic selection of genetic parts is critical for tailoring the host\u0026rsquo;s metabolic landscape toward non-native synthesis. In this study, the transition from the P\u003csub\u003epsbA2s\u003c/sub\u003e promoter to the robust constitutive P\u003csub\u003ecpc560\u003c/sub\u003e promoter resulted in a significant elevation of OLA titers (Strain SCY03 vs. SCY02). This marked improvement is likely attributable to the high enzymatic threshold required for the Type III polyketide synthase (TKS) to function effectively within the cyanobacterial chassis. Specifically, TKS must catalyze the iterative condensation of three malonyl-CoA molecules onto a hexanoyl-CoA starter unit\u0026mdash;a process that is kinetically slow and must compete directly with the primary fatty acid biosynthetic pathway for the limited intracellular malonyl-CoA pool[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. While P\u003csub\u003epsbA2s\u003c/sub\u003e is a widely utilized light-inducible promoter, its expression strength might be insufficient to maintain the high steady-state concentration of TKS needed to overcome this metabolic competition. Consequently, the superior performance of P\u003csub\u003ecpc560\u003c/sub\u003e highlights that ensuring a high-level, consistent transcriptional dosage of the biosynthetic genes is a prerequisite for driving carbon flux toward the kinetically challenging polyketide pathway in \u003cem\u003eSynechocystis\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo further elevate the OLA titer, we implemented a coordinated strategy centered on re-routing the global carbon flux toward the heterologous pathway. While the strong P\u003csub\u003ecpc560\u003c/sub\u003e promoter provides a robust transcriptional \"pull,\" its efficiency can be hampered by the natural diversion of photosynthetic carbon toward endogenous storage compounds. In this study, strain SCY05 was developed from SCY03 by integrating the \u003cem\u003eEscherichia coli\u003c/em\u003e maeB gene into the slr1176 (glgC) locus, a site-specific replacement designed to simultaneously block the primary glycogen biosynthetic sink and introduce a heterologous metabolic valve. The deletion of glgC served to \"push\" carbon flux away from glycogen synthesis, thereby increasing the availability of central intermediates[20; 38]. Simultaneously, the heterologous expression of maeB was intended to coordinate the distribution of this excess carbon. By facilitating the oxidative decarboxylation of malate to pyruvate, maeB presumably reinforced the intracellular pyruvate pool, a direct precursor for acetyl-CoA, while providing essential NADPH. This elevated NADPH availability not only helps maintain cellular redox homeostasis during high-flux polyketide biosynthesis but also optimizes cyanobacterial physiological conditions, thereby accelerating biomass accumulation[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Such anaplerotic replenishment likely supported the energy-demanding OLA pathway, which requires substantial ATP for precursor activation and NADPH for sustained secondary metabolism[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. By effectively blocking the glycogen sink and augmenting these precursor and cofactor nodes, we successfully created a metabolic \"surplus\" for OLA production. The incremental yield improvements observed from SCY03 to SCY05 underscore that in a photoautotrophic chassis, the systematic elimination of competing carbon sinks, coupled with the reinforcement of precursor supply and redox balance, is a prerequisite for maximizing the partition of fixed CO\u003csub\u003e2\u003c/sub\u003e toward non-native secondary metabolites.\u003c/p\u003e \u003cp\u003eParallel to internal metabolic rewiring, the availability of inorganic carbon in the cultivation environment and the optimization of fermentation parameters play decisive roles in the productivity of photoautotrophic cell factories. Our results demonstrated that increasing the CO\u003csub\u003e2\u003c/sub\u003e concentration from ambient air (0.04%) to 5% significantly boosted the OLA titer in strain SCY05. This response is presumably driven by the native carbon-concentrating mechanism (CCM) of \u003cem\u003eSynechocystis\u003c/em\u003e, which actively transports and accumulates HCO\u003csup\u003e3\u0026minus;\u003c/sup\u003e within carboxysomes to ensure a high CO\u003csub\u003e2\u003c/sub\u003e concentration around RuBisCO[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Under these elevated CO\u003csub\u003e2\u003c/sub\u003e conditions, the efficiency of this mechanism is expected to be optimized, thereby minimizing the RuBisCO-mediated oxygenation reaction and potentially expanding the carbon flux toward GAP[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. As the first stable intermediate of photosynthetic CO\u003csub\u003e2\u003c/sub\u003e fixation and a pivotal metabolic branch point, GAP serves as the foundational precursor for both the storage of glycogen and the initiation of the glycolytic pathway. In this context, the synergy between 5% CO\u003csub\u003e2\u003c/sub\u003e supplementation and the systematic elimination of competing carbon sinks\u0026mdash;specifically glycogen synthesis\u0026mdash;ensures that the augmented GAP flux is efficiently channeled toward the synthesis of acetyl-CoA and malonyl-CoA. By further extending the cultivation period, we achieved a maximum OLA titer of 9.41 mg/L. While this yield represents a promising start for cannabinoid precursor production in cyanobacteria, further efforts might be required to achieve industrially relevant scales. Nevertheless, this study provides a viable blueprint for the sustainable, light-driven biomanufacturing of complex plant polyketides.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study successfully established a green synthesis approach for the cannabinoid precursor olivetolic acid using \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 as a photosynthetic cell factory. The production of this high-value compound was achieved through the integration of exogenous genes involved in olivetolic acid biosynthesis (TKS, OAC, and AAE1), targeted metabolic pathway optimization (knockout of the glycogen biosynthesis gene and overexpression of the malic enzyme gene), and refinement of cultivation conditions. These advancements provide a robust foundation for the scalable and sustainable biosynthesis of cannabidiol while highlighting the significant potential of cyanobacteria as environmentally friendly and efficient platforms for producing high-value compounds, offering a promising alternative to traditional cannabis cultivation.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003eNo applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eAll authors have participated in the review study and manuscript preparation.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors hereby declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003edeclaration\u003c/p\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (31200019), Open Fund of Key Laboratory of Tropical Marine Bio-resources and Ecology, Chinese Academy of Sciences (LMB131001), Ningbo Clinical Research Center for Children\u0026rsquo;s Health and Diseases (2019A21002), Ningbo Top Medical and Health Research Program (No. 2022020405), Ningbo Leading Medical \u0026amp;Health Discipline (No.2026-A34); Ningbo Municipal Bureau of Science and Technology Project (No.2025Z153).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eE-Bin Gao, Junhua Wu designed the experiment.E-Bin Gao, Wenrui Ji and Yangjie Zhu performed the experiments and collected the data.E-Bin Gao, Wenrui Ji, and Yangjie Zhu analyzed the data and drafted the article.E-Bin Gao and Wenrui Ji critically revised the article.All authors approved the final draft for submission.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHesami M, Pepe M, Baiton A, Jones A. 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Biochem Cell Biol. 2011;89:178\u0026ndash;88. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1139/O10-136\u003c/span\u003e\u003cspan address=\"10.1139/O10-136\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Synechocystis sp. PCC6803, Metabolic engineering, CO2 fixation, Olivetolic acid","lastPublishedDoi":"10.21203/rs.3.rs-9174471/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9174471/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAs a pivotal aromatic precursor for phytocannabinoids, olivetolic acid (OLA) is traditionally sourced from resource-intensive plant extraction or complex chemical synthesis. In this study, we established a sustainable photosynthetic production platform by engineering the model cyanobacterium Synechocystis sp. PCC 6803. We successfully reconstituted the OLA biosynthetic pathway by heterologously expressing Cannabis sativa-derived tetraketide synthase (CsTKS), olivetolic acid cyclase (CsOAC), and acyl-activating enzyme (CsAAE1). To overcome metabolic bottlenecks, the pathway was optimized using the strong promoter P\u003csub\u003ecpc560\u003c/sub\u003e and carbon flux redirection. Disruption of the glycogen storage pathway and reinforcement of the malonyl-CoA pool via maeB overexpression significantly enhanced precursor availability. Furthermore, increasing CO\u003csub\u003e2\u003c/sub\u003e supplementation achieved a maximum OLA titer of 9.41 mg/L. This work demonstrates the feasibility of utilizing photosynthetic chassis for the production of olivetolic acid, providing a robust foundation for the sustainable manufacturing of cannabinoid-related metabolites.\u003c/p\u003e","manuscriptTitle":"Metabolic Engineering of Synechocystis sp. PCC 6803 for Olivetolic acid Production","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-27 13:10:28","doi":"10.21203/rs.3.rs-9174471/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T06:46:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T09:24:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-18T19:09:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284989003374507284687045109498154562846","date":"2026-03-30T19:47:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"237657591412470994129816205533810749872","date":"2026-03-28T11:30:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-26T08:36:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-25T09:13:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-25T09:13:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Cell Factories","date":"2026-03-20T03:39:46+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-cell-factories","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"micf","sideBox":"Learn more about [Microbial Cell Factories](http://microbialcellfactories.biomedcentral.com/)","snPcode":"12934","submissionUrl":"https://submission.nature.com/new-submission/12934/3","title":"Microbial Cell Factories","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b885ea7-3008-468a-a4f3-e6d8d790b06e","owner":[],"postedDate":"March 27th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T06:46:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-04T09:24:00+00:00","index":16,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T06:54:56+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-27 13:10:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9174471","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9174471","identity":"rs-9174471","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}