Glucose, a vital energy source and biorefinery feedstock, is predominantly produced through the plant-biomass-sugar route. This method's efficiency is limited by factors like plant cultivation cycles and biomass processing costs. Developing more efficient and sustainable glucose production methods is crucial given the global climate crisis and food shortages. While chemical-biochemical, electrocatalytical-biological, and in vitro enzymatic routes for direct CO2 conversion to glucose exist, continuous glucose production through photosynthesis remains largely unachieved. This study investigates the possibility of engineering cyanobacteria, specifically Synechococcus elongatus PCC 7942, to directly convert CO2 into glucose via photosynthesis. Cyanobacteria, being oxygenic phototrophs, are promising candidates due to their photosynthetic capabilities. However, their natural metabolic pathways generally prevent glucose accumulation, often redirecting glucose towards other metabolites or using it for cellular processes. This study aims to overcome this limitation by manipulating the cyanobacterial metabolic pathway to enhance glucose production and secretion.

The literature highlights the importance of glucose as a fundamental energy source and building block in all life domains. Its role in various metabolic pathways (EMP, OPP, ED) and macromolecule synthesis is well-established. Current glucose production primarily relies on the processing of plant biomass, a process with inherent limitations in efficiency and sustainability. While some research has demonstrated the potential of cyanobacteria to produce sugars through genetic manipulation, the direct photosynthetic production of glucose has received less attention. Previous studies have shown limited glucose production in recombinant strains, often alongside other sugars, highlighting the need for a deeper understanding of glucose metabolism in phototrophs. Existing methods for direct CO2 conversion to glucose, such as chemical-biochemical, electrocatalytical-biological, and in vitro cascade enzymatic routes, have not yet achieved continuous production, further emphasizing the novelty and importance of the current research.

The study employed a multi-step approach involving genetic engineering and metabolic analysis of Synechococcus elongatus PCC 7942. Initially, two glucokinase genes (glk1 and glk2) were targeted for knockout to prevent intracellular glucose consumption. This was done in parallel in wild-type strains and recombinant strains containing an E. coli-derived glucose transporter (GalP). The knockout process was challenging in wild-type strains, requiring extensive passages to obtain homozygous mutants, suggesting essential physiological functions of glucokinase. Recombinant strains with GalP showed easier isolation of the double-knockout mutants. The resulting strains exhibited significantly reduced glucokinase activity, with residual activity possibly attributed to non-specific kinases. Metabolic profiling of glucokinase-deficient strains revealed glucose accumulation in the culture medium, demonstrating that blocking glucose consumption redirected metabolic flux towards glucose synthesis. Further experiments, including modifications of the culture medium and isotopic labeling, confirmed that the produced glucose primarily originated from photosynthetic CO2 fixation. To investigate the metabolic pathway leading to glucose accumulation, the study examined the sucrose metabolism pathway. Knockout of genes involved in sucrose hydrolysis (invA and sps) significantly reduced glucose secretion, indicating that this pathway contributed significantly to glucose production. The study also involved introducing a heterologous sucrose transporter (Scb) to facilitate sucrose secretion, which further confirmed the sucrose metabolism pathway's role. Whole-genome sequencing was performed on a glucokinase-deficient strain (SZ3) to identify spontaneous mutations facilitating glucose secretion. A specific SNP (G274A) in the syncp7942_1161 gene was identified and confirmed to be essential for efficient glucose secretion. Finally, the study performed RNA-seq analysis to investigate global transcriptional changes in the engineered strains, revealing significant regulation of genes involved in photosynthesis and oxidative phosphorylation. Cultivation optimization, including the use of concentrated medium and a fed-batch strategy, further enhanced and prolonged glucose production.

The key findings include: 1. Knockout of two glucokinase genes (glk1 and glk2) in Synechococcus elongatus PCC 7942 led to glucose accumulation and secretion. 2. The presence of a heterologous glucose transporter facilitated easier isolation of the glucokinase-deficient mutants and enhanced glucose secretion. 3. Glucose secretion was significantly reduced upon knockout of genes involved in sucrose hydrolysis (invA and sps), indicating a crucial role of sucrose metabolism in glucose synthesis. 4. A specific spontaneous mutation (G274A) in the syncp7942_1161 gene was identified and found to be essential for efficient glucose secretion. 5. Metabolic and cultivation engineering increased glucose secretion from 1.5 g/L to 5 g/L. 6. RNA-seq analysis revealed significant transcriptional changes in photosynthesis and oxidative phosphorylation pathways in the engineered strain. 7. Cultivation optimization strategies, such as using concentrated medium and fed-batch cultivation, enhanced and prolonged glucose production. Specific data points include a glucose secretion of 1.5 g/L without heterologous transporters and a further increase to 5 g/L with metabolic and cultivation engineering. Intracellular glucose concentrations increased significantly in the glucokinase-deficient strains. Knockout of invA and sps reduced glucose secretion by about 90%. The G274A mutation in syncp7942_1161 reduced glucose secretion by 78%.

This study successfully demonstrated the feasibility of directly converting CO2 into glucose using engineered cyanobacteria. By targeting glucokinase activity and leveraging spontaneous mutations, the researchers redirected a significant portion of the photosynthetic carbon flux towards glucose production and secretion. The results highlight the remarkable plasticity of cyanobacterial metabolism and offer a novel approach for sustainable glucose production. The findings challenge the conventional understanding of glucose metabolism in cyanobacteria, indicating that glucose synthesis can be significantly enhanced by manipulating key regulatory points. The significant role of the sucrose metabolism pathway in this process is noteworthy, suggesting that further manipulation of this pathway could further improve glucose production. The identification of the spontaneous mutation (G274A) provides valuable insights into the genetic mechanisms underlying glucose secretion. The study's success in enhancing glucose production through metabolic and cultivation engineering offers promising avenues for optimizing this approach for industrial applications.

This research successfully engineered cyanobacteria for direct photosynthetic glucose production, overcoming previous limitations. Knockout of glucokinase genes, combined with a spontaneous mutation and cultivation optimization, resulted in a significant increase in glucose secretion. These findings pave the way for developing sustainable and efficient glucose production systems using solar energy and CO2. Future research could focus on further optimizing the engineered strains, exploring different cyanobacterial species, and scaling up the process for industrial applications. Investigating other potential pathways contributing to glucose synthesis could also yield further improvements.

While the study achieved significant glucose production, several limitations exist. The isolation of homozygous glucokinase-deficient mutants proved challenging in the wild-type strain, indicating potential pleiotropic effects of glucokinase. The study focused on one cyanobacterial species, and the results might not be generalizable to all cyanobacteria. Further research is needed to fully elucidate the mechanisms underlying glucose secretion and to optimize the process for large-scale production. The long cultivation times required may represent a limitation for industrial applications. A deeper investigation into the stability and long-term productivity of the engineered strains is also needed before large-scale implementation.