Impact of Carbon Fixation, Distribution and Storage on the Production of Farnesene and Limonene in Synechocystis PCC 6803 and Synechococcus PCC 7002.

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803.

Optimization of Recombinant Protein Production in Synechococcus elongatus PCC 7942: Utilizing Native Promoters and Magnetic Fields.

The cyanobacterium Synechococcus elongatus PCC 7942 holds significant potential as a biofactory for recombinant protein (RP) production due to its capacity to harness light energy and utilize CO2. This study aimed to enhance RP production by integration of native promoters and magnetic field application (MF) in S. elongatus PCC 7942. The psbA2 promoter, which responds to stress conditions, was chosen for the integration of the ZsGreen1 gene. Results indicated successful gene integration, affirming prior studies that showed no growth alterations in transgenic strains. Interestingly, exposure to 30 mT (MF30) demonstrated a increase in ZsGreen1 transcription under the psbA2 promoter, revealing the influence of MF on cyanobacterial photosynthetic machinery. This enhancement is likely attributed to stress-induced shifts in gene expression and enzyme activity. MF30 positively impacted photosystem II (PSII) without disrupting the electron transport chain, aligning with the "quantum-mechanical mechanism" theory. Notably, fluorescence levels and gene expression with application of 30 mT were significantly different from control conditions. This study showcases the efficacy of utilizing native promoters and MF for enhancing RP production in S. elongatus PCC 7942. Native promoters eliminate the need for costly exogenous inducers and potential cell stress. Moreover, the study expands the scope of optimizing RP production in photoautotrophic microorganisms, providing valuable insights for biotechnological applications.

A single Prochlorococcus ecotype dominates the tropical Bay of Bengal with ultradian growth.

The Bay of Bengal (BoB) spans >2.2 million km2 in the northeastern Indian Ocean and is bordered by dense populations that depend upon its resources. Over recent decades, a shift from larger phytoplankton to picoplankton has been reported, yet the abundance, activity, and composition of primary producer communities are not well-characterized. We analysed the BoB regions during the summer monsoon. Prochlorococcus ranged up to 3.14 × 105 cells mL-1 in the surface mixed layer, averaging 1.74 ± 0.46 × 105 in the upper 10 m and consistently higher than Synechococcus and eukaryotic phytoplankton. V1-V2 rRNA gene amplicon analyses showed the High Light II (HLII) ecotype formed 98 ± 1% of Prochlorococcus amplicons in surface waters, comprising six oligotypes, with the dominant oligotype accounting for 65 ± 4% of HLII. Diel sampling of a coherent water mass demonstrated evening onset of cell division and rapid Prochlorococcus growth between 1.5 and 3.1 div day-1, based on cell cycle analysis, as confirmed by abundance-based estimates of 2.1 div day-1. Accumulation of Prochlorococcus produced by ultradian growth was restricted by high loss rates. Alongside prior Arabian Sea and tropical Atlantic rates, our results indicate Prochlorococcus growth rates should be reevaluated with greater attention to latitudinal zones and influences on contributions to global primary production.

Proteome trait regulation of marine Synechococcus elemental stoichiometry under global change.

Recent studies have demonstrated regional differences in marine ecosystem C:N:P with implications for carbon and nutrient cycles. Due to strong co-variance, temperature and nutrient stress explain variability in C:N:P equally well. A reductionistic approach can link changes in individual environmental drivers with changes in biochemical traits and cell C:N:P. Thus, we quantified effects of temperature and nutrient stress on Synechococcus chemistry using laboratory chemostats, chemical analyses, and data-independent acquisition mass spectrometry proteomics. Nutrient supply accounted for most C:N:Pcell variability and induced tradeoffs between nutrient acquisition and ribosomal proteins. High temperature prompted heat-shock, whereas thermal effects via the "translation-compensation hypothesis" were only seen under P-stress. A Nonparametric Bayesian Local Clustering algorithm suggested that changes in lipopolysaccharides, peptidoglycans, and C-rich compatible solutes may also contribute to C:N:P regulation. Physiological responses match field-based trends in ecosystem stoichiometry and suggest a hierarchical environmental regulation of current and future ocean C:N:P.

Photosynthetic production of α-farnesene by engineered Synechococcus elongatus UTEX 2973 from carbon dioxide.

Cyanobacteria are the prospective biosolar cell factories to produce a range of bioproducts through CO2 sequestration. Farnesene is a sesquiterpene with an array of applications in biofuels, pest management, cosmetics, flavours and fragrances. This is the first time a codon-optimized farnesene synthase (AFS) gene is engineered into the genomic neutral site of Synechococcus elongatus UTEX 2973 for farnesene synthesis through its endogenous methylerythritol phosphate (MEP) pathway, rendering UTEX AFS strain. Similarly, bottleneck gene(s) of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase (dxs) and/or fusion of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase (idispA) were engineered engendering UTEX AFS::dxs, UTEX AFS::idispA and UTEX AFS::dxs::idispA strains. UTEX AFS::dxs::idispA achieves farnesene productivity of 2.57 mg/L/day, the highest among engineered cyanobacterial strains studied so far. It demonstrates farnesene production, which is 31.3-times higher than the UTEX AFS strain. Moreover, the engineered strains show similar productivity over a three-month period, stipulating the genetic stability of the strains.

Cell morphology engineering enhances grazing resistance of Synechococcus elongatus PCC 7942 for non-sterile large-scale cultivation.

In the practical scale of cyanobacterial cultivation, the golden algae Poterioochromonas malhamensis is a well-known predator that causes devastating damage to the culture, referred to as pond crash. The establishment and maintenance of monoculture conditions are ideal for large-scale cultures. However, this is a difficult challenge because microbial contamination is unavoidable in practical-scale culture facilities. In the present study, we unexpectedly observed the pond crash phenomenon during the pilot-scale cultivation of Synechococcus elongatus PCC 7942 using a 100-L photobioreactor. This was due to the contamination with P. malhamensis, which probably originated from residual fouling. Interestingly, we found that S.elongatus PCC 7942 can alter its morphological structure when subjected to continuous grazing pressure from predators, resulting in cells that were more than 100 times longer than those of the wild-type strain. These hyper-elongated S.elongatus PCC 7942 cells had mutations in the genes encoding FtsZ or Ftn2 which are involved in bacterial cell division. Importantly, the elongated phenotype remained stable during cultivation, enabling S.elongatus PCC 7942 to thrive and resist grazing. The cultivation of the elongated S.elongatus PCC 7942 mutant strain in a 100-L pilot-scale photobioreactor under non-sterile conditions resulted in increased cyanobacterial biomass without encountering pond crash. This study demonstrates an efficient strategy for cyanobacterial cell culture in practical-scale bioreactors without the need for extensive decontamination or sterilization of the growth medium and culture facility, which can contribute to economically viable cultivation and bioprocessing of microalgae.

Genome streamlining to improve performance of a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973.

Cyanobacteria are photosynthetic organisms that have garnered significant recognition as potential hosts for sustainable bioproduction. However, their complex regulatory networks pose significant challenges to major metabolic engineering efforts, thereby limiting their feasibility as production hosts. Genome streamlining has been demonstrated to be a successful approach for improving productivity and fitness in heterotrophs but is yet to be explored to its full potential in phototrophs. Here, we present the systematic reduction of the genome of the cyanobacterium exhibiting the fastest exponential growth, Synechococcus elongatus UTEX 2973. This work, the first of its kind in a photoautotroph, involved an iterative process using state-of-the-art genome-editing technology guided by experimental analysis and computational tools. CRISPR-Cas3 enabled large, progressive deletions of predicted dispensable regions and aided in the identification of essential genes. The large deletions were combined to obtain a strain with 55-kb genome reduction. The strains with streamlined genome showed improvement in growth (up to 23%) and productivity (by 22.7%) as compared to the wild type (WT). This streamlining strategy not only has the potential to develop cyanobacterial strains with improved growth and productivity traits but can also facilitate a better understanding of their genome-to-phenome relationships.IMPORTANCEGenome streamlining is an evolutionary strategy used by natural living systems to dispense unnecessary genes from their genome as a mechanism to adapt and evolve. While this strategy has been successfully borrowed to develop synthetic heterotrophic microbial systems with desired phenotype, it has not been extensively explored in photoautotrophs. Genome streamlining strategy incorporates both computational predictions to identify the dispensable regions and experimental validation using genome-editing tool, and in this study, we have employed a modified strategy with the goal to minimize the genome size to an extent that allows optimal cellular fitness under specified conditions. Our strategy has explored a novel genome-editing tool in photoautotrophs, which, unlike other existing tools, enables large, spontaneous optimal deletions from the genome. Our findings demonstrate the effectiveness of this modified strategy in obtaining strains with streamlined genome, exhibiting improved fitness and productivity.

Taxonomic, Phylogenomic and Bioactivity Profiling of Novel Phycosphere Bacterium from Model Cyanobacterium Synechococcus elongatus PCC 7942.

Phycosphere niches host rich microbial consortia that harbor dynamic algae-bacteria interactions with fundamental significance in varied natural ecosystems. Hence, culturing the uncultured microbial majority of the phycosphere microbiota is vital for deep understanding of the intricate mechanisms governing the dynamic interactions, and also to provide novel and rich microbial resources, and to discover new natural bioactive metabolites. Synechococcus elongatus PCC 7942 is a robust model cyanobacterium widely used in environment, synthesis biology, and biotechnology research. To expand the number of novel phycosphere species that were brought into culture and to discover the natural bioactivities, we presented a new yellow-pigmented bacterium named ABI-127-1, which was recovered from the phycosphere of PCC 7942, using an optimized bacterial isolation procedure. Combined polyphasic taxonomic and phylogenomic characterization was performed to confidently identify the new isolate as a potential novel species belonging to the genus Qipengyuania. The observed bioactivity of strain ABI-127-1 with promoting potential towards the growth and CO2 fixation efficiency of the host microalgae was measured. Additionally, the bacterial production of active bioflocculant exopolysaccharides was evaluated after culture optimization. Thus, these findings revealed the potential environmental and biotechnological implications of this new microalgae growth-promoting bacterium isolated from the phycosphere microenvironment.

Oscillation of type IV pili regulated by the circadian clock in cyanobacterium Synechococcus elongatus PCC7942.

Type IV pili (TFP) are known to be functionally related to cell motilities and natural transformation in many bacteria. However, the molecular and ecological functions of the TFP have rarely been reported for photosynthetic cyanobacteria. Here, by labeling pili in model cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942), we have quantitatively characterized the TFP and its driven twitching motility in situ at the single-cell level. We found an oscillating pattern of TFP in accordance with the light and dark periods during light-dark cycles, which is correlated positively to the oscillating pattern of the natural transformation efficiency. We further showed that the internal circadian clock plays an important role in regulating the oscillating pattern of TFP, which is also supported by evidences at the molecular level by tracking the expression of 16 TFP-related genes. This study adds a detailed picture toward the gap between TFP and its relations to circadian regulations in Syn7942.

Identification of acidic stress-responsive genes and acid tolerance engineering in Synechococcus elongatus PCC 7942.

Cyanobacteria are excellent autotrophic photosynthetic chassis employed in synthetic biology, and previous studies have suggested that they have alkaline tolerance but low acid tolerance, significantly limiting their productivity as photosynthetic chassis and necessitating investigations into the acid stress resistance mechanism. In this study, differentially expressed genes were obtained by RNA sequencing-based comparative transcriptomic analysis under long-term acidic stress conditions and acidic shock treatment, in the model cyanobacterium Synechococcus elongatus PCC 7942. A pathway enrichment analysis revealed the upregulated and downregulated pathways during long-term acidic and shock stress treatment. The subsequent single gene knockout and phenotype analysis showed that under acidic stress conditions, the strains with chlL, chlN, pex, synpcc7942_2038, synpcc7942_1890, or synpcc7942_2547 knocked out grew worse than the wild type, suggesting their involvement in acid tolerance. This finding was further confirmed by introducing the corresponding genes back into the knockout mutant individually. Moreover, individual overexpression of the chlL and chlN genes in the wild type successfully improved the tolerance of S. elongatus PCC 7942 to acidic stress. This work successfully identified six genes involved in acidic stress responses, and overexpressing chIL or chIN individually successfully improved acid tolerance in S. elongatus PCC 7942, providing valuable information to better understand the acid resistance mechanism in S. elongatus PCC 7942 and novel insights into the robustness and tolerance engineering of cyanobacterial chassis. KEY POINTS: • DEGs were identified by RNA-seq based transcriptomics analysis in response to acidic stress in S. elongatus PCC 7942. • Six genes were identified to be involved in acid tolerance in S. elongatus PCC 7942. • Overexpression of chIL or chIN individually successfully improved the acid tolerance of S. elongatus PCC 7942.

Coordination of carbon partitioning and photosynthesis by a two-component signaling network in Synechococcus elongatus PCC 7942.

Photosynthetic organisms need to balance the rate of photosynthesis with the utilization of photosynthetic products by downstream reactions. While such "source/sink" pathways are well-interrogated in plants, analogous regulatory systems are unknown or poorly studied in single-celled algal and cyanobacterial species. Towards the identification of energy/sugar sensors in cyanobacteria, we utilized an engineered strain of Synechococcus elongatus PCC 7942 that allows experimental manipulation of carbon status. We conducted a screening of all two-component systems (TCS) and serine/threonine kinases (STKs) encoded in S. elongatus PCC 7942 by analyzing phenotypes consistent with sucrose-induced relaxation of sink inhibition. We narrowed the candidate sensor proteins by analyzing changes observed after sucrose feeding. We show that a clustered TCS network containing RpaA, CikB, ManS and NblS are involved in the regulation of genes related to photosynthesis, pigment synthesis, and Rubisco concentration in response to sucrose. Altogether, these results highlight a regulatory TCS group that may play under-appreciated functions in carbon partitioning and energy balancing in cyanobacteria.

DnaK2 Mediates a Negative Feedback Regulation of the Heat Shock Responsive Hik2-Rre1 Two-Component System in the Cyanobacterium Synechococcus Elongatus PCC 7942.

The two-component system (TCS) is a conserved signal transduction module in bacteria. The Hik2-Rre1 system is responsible for transcriptional activation upon high-temperature shift as well as plastoquinone-related redox stress in the cyanobacterium Synechococcus elongatus PCC 7942. As heat-induced de novo protein synthesis was previously shown to be required to quench the heat-activated response, we investigated the underlying mechanism in this study. We found that the heat-inducible transcription activation was alleviated by the overexpression of dnaK2, which is an essential homolog of the highly conserved HSP70 chaperone and whose expression is induced under the control of the Hik2-Rre1 TCS. Phosphorylation of Rre1 correlated with transcription of the regulatory target hspA. The redox stress response was found to be similarly repressed by dnaK2 overexpression. Considered together with the previous information, we propose a negative feedback mechanism of the Hik2-Rre1-dependent stress response that maintains the cellular homeostasis mediated by DnaK2.

Deciphering the structure, function, and mechanism of lysine acetyltransferase cGNAT2 in cyanobacteria.

Lysine acetylation is a conserved regulatory posttranslational protein modification that is performed by lysine acetyltransferases (KATs). By catalyzing the transfer of acetyl groups to substrate proteins, KATs play critical regulatory roles in all domains of life; however, no KATs have yet been identified in cyanobacteria. Here, we tested all predicted KATs in the cyanobacterium Synechococcus sp. PCC 7002 (Syn7002) and demonstrated that A1596, which we named cyanobacterial Gcn5-related N-acetyltransferase (cGNAT2), can catalyze lysine acetylation in vivo and in vitro. Eight amino acid residues were identified as the key residues in the putative active site of cGNAT2, as indicated by structural simulation and site-directed mutagenesis. The loss of cGNAT2 altered both growth and photosynthetic electron transport in Syn7002. In addition, quantitative analysis of the lysine acetylome identified 548 endogenous substrates of cGNAT2 in Syn7002. We further demonstrated that cGNAT2 can acetylate NAD(P)H dehydrogenase J (NdhJ) in vivo and in vitro, with the inability to acetylate K89 residues, thus decreasing NdhJ activity and affecting both growth and electron transport in Syn7002. In summary, this study identified a KAT in cyanobacteria and revealed that cGNAT2 regulates growth and photosynthesis in Syn7002 through an acetylation-mediated mechanism.

Uncovering the diversity and distribution of biosynthetic gene clusters of prochlorosins and other putative RiPPs in marine Synechococcus strains.

IMPORTANCE: Genome mining studies have revealed the remarkable combinatorial diversity of ribosomally synthesized and post-translationally modified peptides (RiPPs) in marine bacteria, including prochlorosins. However, mining strategies also prove valuable in investigating the genomic landscape of associated genes within biosynthetic gene cluster (BGC) specific to targeted RiPPs of interest. Our study contributes to the enrichment of knowledge regarding prochlorosin diversity. It offers insights into potential mechanisms involved in their biosynthesis and modification, such as hyper-modification, which may give rise to active lantibiotics. Additionally, our study uncovers putative novel promiscuous post-translational enzymes, thereby expanding the chemical space explored within the Synechococcus genus. Moreover, this research extends the applications of mining techniques beyond the discovery of new RiPP-like clusters, allowing for a deeper understanding of genomics and diversity. Furthermore, it holds the potential to reveal previously unknown functions within the intriguing RiPP families, particularly in the case of prochlorosins.

Role of ERA protein in enhancing glyceroglycolipid synthesis and phosphate starvation tolerance in Synechococcus elongatus PCC 7942.

Glyceroglycolipids are the primary thylakoid membrane lipids in cyanobacteria. Their diverse bioactivities have led to extensive utilization in the biomedical industry. In this study, we elucidated the role of ERA (E. coli Ras-like protein) in augmenting glyceroglycolipid synthesis and bolstering stress resilience in Synechococcus elongatus PCC 7942 during phosphate starvation. Notably, the ERA overexpression strain (ERA OE) outperformed the wild-type (WT) strain under phosphate-starved conditions, displaying an average 13.9 % increase in biomass over WT during the entire growth period, peaking at 0.185 g L-1 of dry cell weight on day 6. Lipidomic analysis using UHPLC-MS/MS techniques revealed that ERA OE exhibited a higher total glyceroglycolipid content compared to WT under phosphate starvation, representing a 7.95 % increase over WT and constituting a maximum of 5.07 % of dry cell weight on day 6. Transcriptomic analysis identified a significant up-regulation of the gldA gene (encoding glycerol dehydrogenase) involved in glycerolipid metabolism due to overexpression of ERA during phosphate starvation. These findings suggest a potential mechanism by which ERA regulates glyceroglycolipid synthesis through the up-regulation of GldA, thereby enhancing phosphate starvation tolerance in S. elongatus PCC 7942. Furthermore, lipidomic analysis revealed that ERA facilitated the production of glyceroglycolipid molecules containing C16:1 and C18:1 fatty acids. Additionally, ERA redirected lipid flux and promoted glyceroglycolipid accumulation while attenuating triacylglycerol production under phosphate starvation. This study represents the first demonstration of pivotal role of ERA in enhancing glyceroglycolipid synthesis and phosphate starvation tolerance in cyanobacteria, offering new insights into the effective utilization of glyceroglycolipids in various applications.

Chitin degradation by Synechococcus WH7803.

Chitin is an abundant, carbon-rich polymer in the marine environment. Chitinase activity has been detected in spent media of Synechococcus WH7803 cultures-yet it was unclear which specific enzymes were involved. Here we delivered a CRISPR tool into the cells via electroporation to generate loss-of-function mutants of putative candidates and identified ChiA as the enzyme required for the activity detected in the wild type.

Structural insights into the functions of Raf1 and Bsd2 in hexadecameric Rubisco assembly.

Hexadecameric form I Rubisco, which consisting consists of eight large (RbcL) and eight small (RbcS) subunits, is the most abundant enzyme on earth. Extensive efforts to engineer an improved Rubisco to speed up its catalytic efficiency and ultimately increase agricultural productivity. However, difficulties with correct folding and assembly in foreign hosts or in vitro have hampered the genetic manipulation of hexadecameric Rubisco. In this study, we reconstituted Synechococcus sp. PCC6301 Rubisco in vitro using the chaperonin system and assembly factors from cyanobacteria and Arabidopsis thaliana (At). Rubisco holoenzyme was produced in the presence of cyanobacterial Rubisco accumulation factor 1 (Raf1) alone or both AtRaf1 and bundle-sheath defective-2 (AtBsd2) from Arabidopsis. RbcL released from GroEL is assembly capable in the presence of ATP, and AtBsd2 functions downstream of AtRaf1. Cryo-EM structures of RbcL8-AtRaf18, RbcL8-AtRaf14-AtBsd28, and RbcL8 revealed that the interactions between RbcL and AtRaf1 are looser than those between prokaryotic RbcL and Raf1, with AtRaf1 tilting 7° farther away from RbcL. AtBsd2 stabilizes the flexible regions of RbcL, including the N and C termini, the 60s loop, and loop 6. Using these data, combined with previous findings, we propose the possible biogenesis pathways of prokaryotic and eukaryotic Rubisco.

Synechococcus elongatus Argonaute reduces natural transformation efficiency and provides immunity against exogenous plasmids.

IMPORTANCE: S. elongatus is an important cyanobacterial model organism for the study of its prokaryotic circadian clock, photosynthesis, and other biological processes. It is also widely used for genetic engineering to produce renewable biochemicals. Our findings reveal an SeAgo-based defense mechanism in S. elongatus against the horizontal transfer of genetic material. We demonstrate that deletion of the ago gene facilitates genetic studies and genetic engineering of S. elongatus.

Molecular insights into nitrogen constraint for niche partitioning and physiological adaptation of coastal Synechococcus assemblages.

Coastal nitrogen input has substantially increased due to human activity. However, much remains unknown about the nitrogen-driven patterns and the underlying genetic basis of coastal picoplankton. To investigate the response and mechanisms of picoplankton induced by nitrogen variation, we conducted in-situ investigations using high-throughput sequencing in the Bohai Sea and performed laboratory nitrogen simulation experiments accompanied by physiological, genomic, and transcriptomic analyses, with Synechococcus as a representative. The results of in-situ investigation revealed that Synechococcus clades I, III, WPC1, and VI of subcluster 5.1 (S5.1) are prevalent in strait areas characterized by robust water exchange with the North Yellow Sea, while clades II, VIII, and IX of S5.1, as well as subcluster 5.2 (S5.2) and subcluster 5.3 (S5.3) are more abundant in central and bay areas experiencing elevated nitrate and nitrite loads. The laboratory experiments further confirmed that inorganic nitrogen is a crucial determinant of diversity and niche partitioning of Synechococcus lineages. Besides, the raising inorganic nitrogen concentration within the current in-situ range (0.1-10 µmol L-1) enhances photosynthesis and carbon fixation of Synechococcus, however further escalation of inorganic nitrogen (100 µmol L-1) may hinder these processes instead. The phenomenon could be associated with the differential expression of genes in metabolic pathways regulating nitrogen metabolism, photosynthetic system II, and photosynthesis-antenna proteins in response to nitrogen concentration and type variation. These findings expand our understanding of the impact of macronutrient variation resulting from human activities on marine picoplankton and biogeochemical cycles.

Metabolic engineering of Synechococcus elongatus 7942 for enhanced sucrose biosynthesis.

The capability of cyanobacteria to produce sucrose from CO2 and light has a remarkable societal and biotechnological impact since sucrose can serve as a carbon and energy source for a variety of heterotrophic organisms and can be converted into value-added products. However, most metabolic engineering efforts have focused on understanding local pathway alterations that drive sucrose biosynthesis and secretion in cyanobacteria rather than analyzing the global flux re-routing that occurs following induction of sucrose production by salt stress. Here, we investigated global metabolic flux alterations in a sucrose-secreting (cscB-overexpressing) strain relative to its wild-type Synechococcus elongatus 7942 parental strain. We used targeted metabolomics, 13C metabolic flux analysis (MFA), and genome-scale modeling (GSM) as complementary approaches to elucidate differences in cellular resource allocation by quantifying metabolic profiles of three cyanobacterial cultures - wild-type S. elongatus 7942 without salt stress (WT), wild-type with salt stress (WT/NaCl), and the cscB-overexpressing strain with salt stress (cscB/NaCl) - all under photoautotrophic conditions. We quantified the substantial rewiring of metabolic fluxes in WT/NaCl and cscB/NaCl cultures relative to WT and identified a metabolic bottleneck limiting carbon fixation and sucrose biosynthesis. This bottleneck was subsequently mitigated through heterologous overexpression of glyceraldehyde-3-phosphate dehydrogenase in an engineered sucrose-secreting strain. Our study also demonstrates that combining 13C-MFA and GSM is a useful strategy to both extend the coverage of MFA beyond central metabolism and to improve the accuracy of flux predictions provided by GSM.