Journal of Biological Engineering最新文献

{"title":"Engineering FcRn binding kinetics dramatically extends antibody serum half-life and enhances therapeutic potential.","authors":"Sanghwan Ko, Migyeong Jo, Munsu Kyung, Wonju Lee, Woo Hyung Ko, Jung-Hyun Na, Youn Seo Chun, Byoung Joon Ko, Sang Taek Jung","doi":"10.1186/s13036-025-00506-y","DOIUrl":"https://doi.org/10.1186/s13036-025-00506-y","url":null,"abstract":"<p><strong>Background: </strong>Optimizing the IgG Fc domain for neonatal Fc receptor (FcRn) binding is crucial for enhancing antibody pharmacokinetics. The prolonged serum half-life of IgG antibody is governed by its pH-dependent interaction with FcRn, enabling efficient binding at acidic endosomal pH, intracellular trafficking, and release at neutral serum pH. However, a critical yet previously unrecognized challenge in Fc engineering for extending the serum half-life of therapeutic antibodies is the intense competition with endogenous IgG for FcRn binding during intracellular trafficking, which limits FcRn-mediated transport and reduces the serum persistence of therapeutic antibodies. To address this, we developed an Fc variant that precisely modulates pH-dependent FcRn binding kinetics, accelerates FcRn association at acidic pH, and promotes rapid dissociation at neutral pH, thereby enhancing FcRn-driven intracellular transport, outcompeting endogenous IgG, and achieving unprecedented improvement in the serum half-life of therapeutic antibodies.</p><p><strong>Results: </strong>Using comprehensive site-directed saturation mutagenesis coupled with functional screening, we generated a diverse panel of Fc variants and identified two with distinct FcRn binding kinetics: YML (L309Y/Q311M/M428L), which exhibited superior FcRn association at acidic pH and accelerated dissociation at neutral pH, and EML (L309E/Q311M/M428L), which displayed attenuated binding kinetics. In human FcRn transgenic mice, YML extended the serum half-life of clinically used trastuzumab with a wild-type Fc by 6.1-fold, demonstrating a remarkable improvement over previously reported Fc-engineered variants, including PFc29 (Q311R/M428L) and DHS (L309D/Q311H/N434S), which represent the most effective Fc modifications for prolonging serum persistence to date. This in vivo validation underscores the pivotal role of FcRn kinetic tuning in overcoming endogenous IgG competition and maximizing FcRn-mediated antibody transport. Additionally, YML exhibited potent complement-dependent cytotoxicity (CDC) while maintaining favorable physicochemical properties.</p><p><strong>Conclusion: </strong>This study presents a rational Fc engineering framework to optimize FcRn binding kinetics, addressing a previously unconsidered challenge-endogenous IgG competition during intracellular trafficking of therapeutic antibodies. The distinct kinetic behaviors of YML and EML highlight the critical necessity of precise control over pH-dependent association and dissociation rates in FcRn binding. YML represents a next-generation Fc platform, offering enhanced pharmacokinetics and improved effector functions, thus providing a powerful strategy for developing biologics with superior serum persistence and therapeutic efficacy.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"35"},"PeriodicalIF":5.7,"publicationDate":"2025-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12007268/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144000734","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: Microfluidic device for enhancement and analysis of osteoblast differentiation in three-dimensional cell cultures.","authors":"Michael Killinger, Adéla Kratochvilová, Eva Ingeborg Reihs, Eva Matalová, Karel Klepárník, Mario Rothbauer","doi":"10.1186/s13036-025-00501-3","DOIUrl":"https://doi.org/10.1186/s13036-025-00501-3","url":null,"abstract":"","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"33"},"PeriodicalIF":5.7,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12004551/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143966804","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unraveling the unique bioactivities of highly purified C-phycocyanin and allophycocyanin.","authors":"Jimin Na, Soobin Jang, Myeongkwan Song, SeungEun Nam, Woon-Yong Choi, Hwasung Shin, Soonjo Kwon, Youngbin Baek","doi":"10.1186/s13036-025-00496-x","DOIUrl":"https://doi.org/10.1186/s13036-025-00496-x","url":null,"abstract":"<p><strong>Background: </strong>The blue-green microalgae Spirulina, used in human nutrition for centuries, includes phycobiliproteins such as C-phycocyanin (CPC) and allophycocyanin (APC). Assessing their unique bioactivities separately is difficult as they have similar properties, such as molecular weight and isoelectric point. In the present study, we aimed to separate CPC and APC and to evaluate their bioactivities. CPC and APC were separated using a hydrophobic membrane and ammonium sulfate, which promotes reversible and specific protein binding to the membrane. Spectroscopic analysis, HPLC, and SDS-PAGE revealed a successful separation of CPC and APC. Their bioactivities were evaluated through CCK- 8 assays for anticancer activity, radical scavenging assays for antioxidant activity, and albumin denaturation assays for anti-inflammatory activity, respectively.</p><p><strong>Results: </strong>The results revealed that highly purified APC showed 40% higher anticancer activity than the control, whereas CPC increased the viability of cancer cells, resulting in a 30% decrease in anticancer activity compared to the control. In contrast, highly purified CPC showed approximately 25% higher antioxidant activity and twice as much anti-inflammatory activity as APCs; moreover, the presence of both showed higher antioxidant activity.</p><p><strong>Conclusion: </strong>This study provides important insights into the unique bioactivities of CPC and APC for their appropriate application as anticancer, antiphlogistic, and antioxidant agents.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"34"},"PeriodicalIF":5.7,"publicationDate":"2025-04-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12004856/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144010760","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unsilencing a cryptic xylose metabolic pathway in Rhodococcus jostii RHA1 for efficient lipid production from lignocellulosic biomass.","authors":"Miguel G Acedos, Isabel De la Torre, Jorge Barriuso, José L García","doi":"10.1186/s13036-025-00503-1","DOIUrl":"https://doi.org/10.1186/s13036-025-00503-1","url":null,"abstract":"<p><p>Rhodococcus jostii RHA1 is an oleaginous bacterium that has attracted considerable attention due to its capacity to use different carbon sources to accumulate significant levels of triacylglycerols that might be converted into biofuels. However, this strain cannot transform xylose into lipids reducing its potential when growing on saccharified lignocellulosic biomass. In this work, we demonstrate that wild type R. jostii RHA1 can be evolved by adaptive laboratory evolution (ALE) to metabolize xylose without engineering heterologous metabolic pathways in the host. We have generated a phenotypically adapted ALE-xyl strain able to use xylose as the sole carbon and energy source more efficiently that an engineered recombinant strain harbouring heterologous xylA and xylB genes encoding a xylose isomerase metabolic pathway. The R. jostii RHA1 ALE-xyl strain accumulates lipids very efficiently using xylose as substrate, but even more importantly it can consume glucose and xylose at the same time. Transcriptomic analyses of ALE-xyl strain growing with glucose or xylose revealed the existence of a silent pentose metabolizing operon that is overexpressed in the presence of xylose. The detection of a xylose reductase activity together with the presence of xylitol in the cytoplasm of ALE-xyl strain suggests that xylose is consumed by a reductase pathway. This study demonstrates that, in cases where a clear phenotypic selection method is available, ALE can be used to improve very efficiently industrial microbial strains without using genetic engineering tools. Strategies focused to exploit the silent phenotypic flexibility of microorganisms to metabolize different carbon sources are powerful tools for the production of microbial value-added products using saccharified lignocellulosic wastes.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"32"},"PeriodicalIF":5.7,"publicationDate":"2025-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11998424/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144025525","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Biomaterial-based chitosan nanohydrogel films: combination of Bistorta officinalis and Ca-doped carbon dots for improved blood clotting.","authors":"Hassan Tavakoli, Meysam Najaflou, Ahmad Yarikhosroushahi","doi":"10.1186/s13036-025-00498-9","DOIUrl":"https://doi.org/10.1186/s13036-025-00498-9","url":null,"abstract":"<p><strong>Background: </strong>Bleeding and traumatic injuries are still a major issue necessitating the development of advanced hemostatic materials that are economical, biocompatible, and effective. Chitosan's (CS) haemostatic and biocompatible properties make it a promising wound-healing material, however, effective cross-linking is essential for appropriate physiochemical properties. In this study, calcium-doped carbon dots (CDs) produced from coriander leaves were used as cross-linking agents to improve the functional performance and structural integrity of nanohydrogel films. Furthermore, extract of the medicinal plant Bistorta officinalis (BEX), a traditional medicinal plant with strong hemostatic and antibacterial qualities, was incorporated into the hydrogel matrix.</p><p><strong>Results: </strong>Analysis and characterization of the synthesized CDs thoroughly confirmed that they have monodispersed spherical shape, negative zeta potential, and active functional groups which effectively cross-linked the chitosan matrix and increased the mechanical strength and stability of the film. Cytotoxicity and antibacterial results of the final films showed the desired cytocompatibility against Human skin fibroblast (HFF-1 cells) with over 80% viability at the highest concentration and effective antibacterial activity against gram-positive and gram-negative bacteria (further improved by cross-linking with CDs and incorporating BEX), respectively. The incorporation of BEX and CDs in hydrogel films significantly enhanced the film's blood-clotting ability with negligible hemolysis due to blood clotting index and hemolysis tests.</p><p><strong>Conclusions: </strong>The findings of this study highlight the potential of biomaterial-based nano hydrogel film, composed of CS cross-linked with CDs and containing BEX, as a promising wound dressing with outstanding biocompatibility, minimal cytotoxicity, enhanced hemostatic efficacy, and strong antibacterial properties.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"31"},"PeriodicalIF":5.7,"publicationDate":"2025-04-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11987453/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144025969","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a CRISPR-based cytosine base editor for restriction-modification system inactivation to enhance transformation efficiency in Vibrio Sp. dhg.","authors":"Yang Jun Shon, Dongyeop Baek, Su Bin Jin, Woo Jae Kim, Gyoo Yeol Jung, Hyun Gyu Lim","doi":"10.1186/s13036-025-00500-4","DOIUrl":"https://doi.org/10.1186/s13036-025-00500-4","url":null,"abstract":"<p><strong>Background: </strong>Vibrio sp. dhg is a fast-growing, alginate-utilizing, marine bacterium being developed as a platform host for macroalgae biorefinery. To maximize its potential in the production of various value-added products, there is a need to expand genetic engineering tools for versatile editing.</p><p><strong>Results: </strong>The CRISPR-based cytosine base editing (CBE) system was established in Vibrio sp. dhg, enabling C: G-to-T: A point mutations in multiple genomic loci. This CBE system displayed high editing efficiencies for single and multiple targets, reaching up to 100%. The CBE system efficiently introduced premature stop codons, inactivating seven genes encoding putative restriction enzymes of the restriction-modification system in two rounds. A resulting engineered strain displayed significantly enhanced transformation efficiency by up to 55.5-fold.</p><p><strong>Conclusions: </strong>Developing a highly efficient CBE system and improving transformation efficiency enable versatile genetic manipulation of Vibrio sp. dhg for diverse engineering in brown macroalgae bioconversion.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"30"},"PeriodicalIF":5.7,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11984283/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144006702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Termination sequence between an inducible promoter and ubiquitous chromatin opening element (UCOE) reduces gene expression leakage and silencing.","authors":"Tomoki Yanagi, Shean Fu Phen, Jonah Ayala, Deniz Ece Aydin, Susanna Jaramillo, David M Truong","doi":"10.1186/s13036-025-00499-8","DOIUrl":"https://doi.org/10.1186/s13036-025-00499-8","url":null,"abstract":"<p><strong>Background: </strong>Inducible gene expression circuits enable precise control over target gene activation and are widely used in direct reprogramming. However, their usability is often compromised by DNA methylation-induced silencing, especially in iPSCs. This deactivates genetic circuits in engineered iPSCs preventing them from being used for long-term scalable expansion of desired cell types. A2-ubiquitous chromatin opening elements (A2UCOE) have been recognized for their anti-silencing properties, but they have not been used in human iPSCs with inducible systems for direct reprogramming. This study investigates the role of A2UCOE in inducible systems and identifies strategies to eliminate associated gene leakage enabling long-term use of engineered human iPSCs.</p><p><strong>Results: </strong>We developed a compact all-in-one gene circuit - containing a doxycycline-inducible Tet-On system, 863 bp of A2UCOE, and FOXN1, a transcription factor critical for thymic epithelial cell (TEC) differentiation - easily deployed to new genomic sites. However, we observed significant FOXN1 gene leakage even without doxycycline, which is a novel limitation of A2UCOE. This leakage resulted in premature differentiation of iPSCs into TECs, limiting its continued use. To further investigate the relationship between A2UCOE and gene leakage, we generated A2UCOE fragments of varying lengths (1337 bp, 749 bp, and 547 bp) and found that all fragments, regardless of length, caused significant gene leakage. To solve this issue, we tested different spacer sequences between A2UCOE and the inducible promoter and found that the SV40 poly-A terminator fully eliminated FOXN1 leakage, and we show this effect is not due to AT- or GC-content. Unexpectedly, this architecture further enhanced anti-silencing effects > 60% providing prolonged stability for at least 30 days.</p><p><strong>Conclusions: </strong>This study reveals a novel limitation of A2UCOE in inducible systems, specifically its contribution to gene leakage, which compromise sensitive systems like direct reprogramming of iPSCs. The inclusion of an SV40 poly-A sequence provides a practical solution and genomic architecture to improve the functionality of A2UCOE-based circuits. It also suggests investigating how termination of transcription modulates gene silencing as a novel design parameter. These findings have significant implications for the design of robust gene circuits, particularly in applications involving iPSCs, regenerative medicine, and cell therapy.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"29"},"PeriodicalIF":5.7,"publicationDate":"2025-04-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11983960/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144006042","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering the T6SS of Pseudomonas for targeted delivery of antibacterial and antifungal effectors.","authors":"Alicia Isabel Pérez-Lorente, Mario Araujo-Garrido, Antonio de Vicente, Diego Romero, Carlos Molina-Santiago","doi":"10.1186/s13036-025-00497-w","DOIUrl":"10.1186/s13036-025-00497-w","url":null,"abstract":"<p><strong>Background: </strong>Bacteria employ diverse molecular systems, such as the type VI secretion system (T6SS) to outcompete other microorganisms and adapt to ecological niches. The T6SS is a versatile nanomachine capable of delivering toxic effectors into neighboring cells, providing advantages in bacterial interactions. In recent years, T6SSs have been proposed as promising tools for engineering selective antimicrobial platforms.</p><p><strong>Results: </strong>In this study, we successfully engineered Pseudomonas putida KT2440 to heterologously express and release T6SS effectors. The expression of Tse1, an effector from Pseudomonas chlororaphis, induced sporulation in plant-beneficial Bacillus strains via a T6SS-dependent mechanism, particularly when Tse1 was paired with a PAAR protein. Similarly, the engineered strain effectively inhibited Aeromonas hydrophila growth using the phospholipase toxin TplE from Pseudomonas aeruginosa. Furthermore, antifungal activity was achieved by coexpressing Tfe2, an effector from Serratia marcescens, with VgrGs, resulting in increased reactive oxygen species levels and cellular damage in Botrytis cinerea. Importantly, the T6SS was also employed to deliver non-T6SS effectors such as chitosanase, demonstrating its versatility in degrading fungal cell walls.</p><p><strong>Conclusions: </strong>Our findings demonstrate that the T6SS can be engineered to deliver both canonical and noncanonical effectors, providing a robust platform for targeted antibacterial and antifungal applications. The modularity of the system enables precise pairing of effectors with structural components such as VgrG and PAAR proteins, optimizing delivery efficiency. These engineered systems provide new opportunities for the development of biocontrol strategies in agriculture, microbiome modulation, and potential therapeutic applications. Future advancements in bioinformatics and protein engineering will further increase the specificity and functionality of T6SS-based delivery systems, offering innovative tools for managing microbial ecosystems and addressing global challenges in health and agriculture.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"28"},"PeriodicalIF":5.7,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11966926/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143772387","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Manipulating subcellular protein localization to enhance target protein accumulation in minicells.","authors":"Junhyeon Park, Karen M Polizzi, Jongmin Kim, Juhyun Kim","doi":"10.1186/s13036-025-00495-y","DOIUrl":"10.1186/s13036-025-00495-y","url":null,"abstract":"<p><strong>Background: </strong>Minicells are chromosome-free derivatives of bacteria formed through irregular cell division. Unlike simplified structures, minicells retain all cellular components of the parent cell except for the chromosome. This feature reduces immunogenic responses, making them advantageous for various biotechnological applications, including chemical production and drug delivery. To effectively utilize minicells, it is essential to ensure the accumulation of target proteins within them, enhancing their efficiency as delivery vehicles.</p><p><strong>Results: </strong>In this study, we engineered Escherichia coli by deleting the minCD genes, generating minicell-producing strains, and investigated strategies to enhance protein accumulation within the minicells. Comparative proteomic analysis revealed that minicells retain most parent-cell proteins but exhibit an asymmetric proteome distribution, leading to selective protein enrichment. We demonstrated that heterologous proteins, such as GFP and RFP, accumulate more abundantly in minicells than in parent cells, regardless of expression levels. To further enhance this accumulation, we manipulated protein localization by fusing target proteins to polar localization signals. While proteins fused with PtsI and Tsr exhibited 2.6-fold and 2.8-fold increases in accumulation, respectively, fusion with the heterologous PopZ protein resulted in a remarkable 15-fold increase in protein concentration under low induction conditions.</p><p><strong>Conclusions: </strong>These findings highlight the critical role of spatial protein organization in enhancing the cargo-loading capabilities of minicells. By leveraging polar localization signals, this work provides a robust framework for optimizing minicells as efficient carriers for diverse applications, from therapeutic delivery to industrial biomanufacturing.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"19 1","pages":"27"},"PeriodicalIF":5.7,"publicationDate":"2025-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11955136/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143742853","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPRi-assisted metabolic engineering of cyanobacteria for photosynthetic hyaluronic acid from CO₂.","authors":"Jigyeong Son, Hyun Jeong Lee, Han Min Woo","doi":"10.1186/s13036-025-00494-z","DOIUrl":"10.1186/s13036-025-00494-z","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 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.</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 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.</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 CO₂. 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":5.7,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11951839/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}