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Background Hyaluronic acid (HA) is widely used in pharmaceuticals, medicine, and cosmetics. Sustainable production has shifted to microbial fermentation using engineered GRAS strains. Diverse carbon sources and CO 2 conversion via engineered microorganisms enhance HA production. Herein we applied advances in CRISPR technologies and tools to optimize metabolic pathway by redirecting carbon portioning in cyanobacterium Synechoccous elongatus PCC 7942, demonstrating enhanced HA production. Results S. elongatus PCC 7942 lacking hyaluronan synthase (HAS) required pathway engineering for HA production. By expressing heterologous Class I HAS, a modular gene expression system was employed, incorporating hasB and hasC for the HA-GlcA module and glmU , glmM , and glmS for the GlcNAc module. This approach resulted in construction of four engineered cyanobacterial strains. Optimizing metabolic pathway involving the HA-GlcA and GlcNAc modules led to SeHA220 (wild-type with HA-GlcA and GlcNAc modules) producing 2.4 ± 0.85 mg/L HA at 21 d, a 27.5-fold increase compared to the control. Targeting F6P and G6P metabolic nodes via CRISPR interference to repress zwf and pfk genes further improved production, with SeHA226 (SeHA220 with a gene repression module) achieving 5.0 ± 0.3 mg/L HA from CO 2 at 15 d. Notably, SeHA226 produced photosynthetic HA with a molecular weight (Mw) of 4.2 MDa, comparable to native producers, emphasizing the importance of precursor balance and growth conditions. Conclusions This study engineered cyanobacteria for efficient HA biosynthesis using modular gene expression and CRISPR-interference systems. Optimizing heterologous metabolic pathway was key to achieving high-molecular-weight photosynthetic HA production from CO 2 . The findings provide insights into tunable HA production, with future efforts aimed at scaling up photosynthetic HA production for larger-scale applications.

Summary Designing synthetic pathways for efficient CO2 fixation and conversion is essential for sustainable chemical production. Here we have designed a synthetic acetate‐acetyl‐CoA/malonyl‐CoA (AAM) bypass to overcome an enzymatic activity of pyruvate dehydrogenase complex. This synthetic pathway utilizes acetate assimilation and carbon rearrangements using a methyl malonyl‐CoA carboxyltransferase. We demonstrated direct conversion of CO2 into acetyl‐CoA‐derived acetone as an example in photosynthetic Synechococcus elongatus PCC 7942 by increasing the acetyl‐CoA pools. The engineered cyanobacterial strain with the AAM‐bypass produced 0.41 g/L of acetone at 0.71 m/day of molar productivity. This work clearly shows that the synthetic pyruvate dehydrogenase bypass (AAM‐bypass) is a key factor for the high‐level production of an acetyl‐CoA‐derived chemical in photosynthetic organisms.

Hyaluronic acid (HA) is widely used in pharmaceuticals, medicine, and cosmetics. Sustainable production has shifted to microbial fermentation using engineered GRAS strains. Diverse carbon sources and CO conversion via engineered microorganisms enhance HA production. Herein we applied advances in CRISPR technologies and tools to optimize metabolic pathway by redirecting carbon portioning in cyanobacterium Synechoccous elongatus PCC 7942, demonstrating enhanced HA production. S. elongatus PCC 7942 lacking hyaluronan synthase (HAS) required pathway engineering for HA production. By expressing heterologous Class I HAS, a modular gene expression system was employed, incorporating hasB and hasC for the HA-GlcA module and glmU, glmM, and glmS for the GlcNAc module. This approach resulted in construction of four engineered cyanobacterial strains. Optimizing metabolic pathway involving the HA-GlcA and GlcNAc modules led to SeHA220 (wild-type with HA-GlcA and GlcNAc modules) producing 2.4 ± 0.85 mg/L HA at 21 d, a 27.5-fold increase compared to the control. Targeting F6P and G6P metabolic nodes via CRISPR interference to repress zwf and pfk genes further improved production, with SeHA226 (SeHA220 with a gene repression module) achieving 5.0 ± 0.3 mg/L HA from CO at 15 d. Notably, SeHA226 produced photosynthetic HA with a molecular weight (Mw) of 4.2 MDa, comparable to native producers, emphasizing the importance of precursor balance and growth conditions. This study engineered cyanobacteria for efficient HA biosynthesis using modular gene expression and CRISPR-interference systems. Optimizing heterologous metabolic pathway was key to achieving high-molecular-weight photosynthetic HA production from CO . The findings provide insights into tunable HA production, with future efforts aimed at scaling up photosynthetic HA production for larger-scale applications.