{"title":"CRISPRi-assisted metabolic engineering of cyanobacteria for photosynthetic hyaluronic acid from CO<sub>2</sub>.","authors":"Jigyeong Son, Hyun Jeong Lee, Han Min Woo","doi":"10.1186/s13036-025-00494-z","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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<sub>2</sub> 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.</p><p><strong>Results: </strong>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<sub>2</sub> 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.</p><p><strong>Conclusions: </strong>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<sub>2</sub>. The findings provide insights into tunable HA production, with future efforts aimed at scaling up photosynthetic HA production for larger-scale applications.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"26"},"PeriodicalIF":5.7000,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11951839/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Biological Engineering","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1186/s13036-025-00494-z","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
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 CO2 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 CO2 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 CO2. The findings provide insights into tunable HA production, with future efforts aimed at scaling up photosynthetic HA production for larger-scale applications.
期刊介绍:
Biological engineering is an emerging discipline that encompasses engineering theory and practice connected to and derived from the science of biology, just as mechanical engineering and electrical engineering are rooted in physics and chemical engineering in chemistry. Topical areas include, but are not limited to:
Synthetic biology and cellular design
Biomolecular, cellular and tissue engineering
Bioproduction and metabolic engineering
Biosensors
Ecological and environmental engineering
Biological engineering education and the biodesign process
As the official journal of the Institute of Biological Engineering, Journal of Biological Engineering provides a home for the continuum from biological information science, molecules and cells, product formation, wastes and remediation, and educational advances in curriculum content and pedagogy at the undergraduate and graduate-levels.
Manuscripts should explore commonalities with other fields of application by providing some discussion of the broader context of the work and how it connects to other areas within the field.