{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T03:13:31Z","timestamp":1788146011704,"version":"build-2803163510"},"reference-count":23,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T00:00:00Z","timestamp":1787961600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Polymers"],"abstract":"<jats:p>Komagataeibacter species are among the highest-yielding bacterial cellulose producers and offer a promising platform for the genetic engineering of functionalized bacterial cellulose. However, routine strain engineering remains limited by inefficient screening of genomic integrants and acidic culture conditions that inhibit acid-sensitive cellulose modifications. Here, we exploited the reduced-acidification phenotype of a Komagataeibacter sucrofermentans glucose dehydrogenase deletion mutant (\u0394gdh) to overcome both limitations. We developed a simple phenotypic screen based on reduced acidification to identify candidate colonies for subsequent molecular confirmation. We further exploited this phenotype by constructing a \u0394gdh::tyr1 strain that, after optimizing culture conditions, produced melanated bacterial cellulose in a single step, without the manual pH neutralization required by previous methods. Together, these results establish reduced acidification as a practical engineering phenotype that simplifies strain engineering and enables acid-sensitive modification of bacterial cellulose, thereby expanding the range of bacterial cellulose modifications achievable in Komagataeibacter.<\/jats:p>","DOI":"10.3390\/polym18172099","type":"journal-article","created":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T02:08:58Z","timestamp":1788142138000},"page":"2099","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Reduced-Acidification Phenotype Simplifies Strain Engineering in Komagataeibacter and Enables One-Step Production of Melanated Bacterial Cellulose"],"prefix":"10.3390","volume":"18","author":[{"given":"Eric","family":"Handy","sequence":"first","affiliation":[{"name":"Johns Hopkins Applied Physics Laboratory, Laurel, MD 20723, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"}]},{"given":"Andrea","family":"Shepard","sequence":"additional","affiliation":[{"name":"Johns Hopkins Applied Physics Laboratory, Laurel, MD 20723, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3307-7197","authenticated-orcid":false,"given":"Cecelia","family":"Kinane","sequence":"additional","affiliation":[{"name":"Johns Hopkins Applied Physics Laboratory, Laurel, MD 20723, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"given":"Tanner","family":"Hamann","sequence":"additional","affiliation":[{"name":"Johns Hopkins Applied Physics Laboratory, Laurel, MD 20723, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"}]},{"given":"Megan","family":"Hannegan","sequence":"additional","affiliation":[{"name":"Johns Hopkins Applied Physics Laboratory, Laurel, MD 20723, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Julie","family":"Gleason","sequence":"additional","affiliation":[{"name":"Johns Hopkins Applied Physics Laboratory, Laurel, MD 20723, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Zhong, C. (2020). Industrial-Scale Production and Applications of Bacterial Cellulose. Front. Bioeng. Biotechnol., 8.","DOI":"10.3389\/fbioe.2020.605374"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"124826","DOI":"10.1016\/j.carbpol.2025.124826","article-title":"Antibacterial Functionalization of Cellulose and Cotton: A Comprehensive Review of Cellulose-Based Strategies","volume":"379","author":"Kudzin","year":"2026","journal-title":"Carbohydr. Polym."},{"key":"ref_3","first-page":"52951","article-title":"Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles","volume":"111","author":"Arias","year":"2016","journal-title":"JoVE"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"723","DOI":"10.1021\/acspolymersau.5c00074","article-title":"Bacterial Nanocellulose Functionalization for Smart Bioelectronics: Integration into Biosensing, Neural Interfaces, and Tissue Engineering","volume":"5","author":"Cabo","year":"2025","journal-title":"ACS Polym. AU"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"18697","DOI":"10.1007\/s13399-023-03983-7","article-title":"Separation of Bioethanol Using in Situ Composite Membrane of Bacterial Cellulose\/Poly (2-Acrylamido-2-Methylpropane Sulfonic Acid) (AMPS) and Their Characterization","volume":"14","author":"Mansy","year":"2024","journal-title":"Biomass Conv. Bioref."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Singh, A., Walker, K.T., Ledesma-Amaro, R., and Ellis, T. (2020). Engineering Bacterial Cellulose by Synthetic Biology. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21239185"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"129552","DOI":"10.1016\/j.ijbiomac.2024.129552","article-title":"Enhanced Bacterial Cellulose Production in Gluconacetobacter xylinus by Overexpression of Two Genes (bscC and bcsD) and a Modified Static Culture","volume":"260","author":"Yang","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"5339","DOI":"10.1007\/s00253-019-09846-4","article-title":"Structural Changes of Bacterial Nanocellulose Pellicles Induced by Genetic Modification of Komagataeibacter hansenii ATCC 23769","volume":"103","author":"Jacek","year":"2019","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6257","DOI":"10.1128\/AEM.00698-10","article-title":"Novel In Vivo-Degradable Cellulose-Chitin Copolymer from Metabolically Engineered Gluconacetobacter xylinus","volume":"76","author":"Yadav","year":"2010","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"E3431","DOI":"10.1073\/pnas.1522985113","article-title":"Engineering Control of Bacterial Cellulose Production Using a Genetic Toolkit and a New Cellulose-Producing Strain","volume":"113","author":"Florea","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3422","DOI":"10.1021\/acssynbio.1c00358","article-title":"Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria","volume":"10","author":"Goosens","year":"2021","journal-title":"ACS Synth. Biol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1007\/s12257-014-0316-x","article-title":"Knock-out of Glucose Dehydrogenase Gene in Gluconacetobacter xylinus for Bacterial Cellulose Production Enhancement","volume":"20","author":"Kuo","year":"2015","journal-title":"Biotechnol. Bioprocess E"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1186\/s13068-024-02482-9","article-title":"Enhanced Bacterial Cellulose Production in Komagataeibacter sucrofermentans: Impact of Different PQQ-Dependent Dehydrogenase Knockouts and Ethanol Supplementation","volume":"17","author":"Ellis","year":"2024","journal-title":"Biotechnol. Biofuels"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"345","DOI":"10.1038\/s41587-024-02194-3","article-title":"Self-Pigmenting Textiles Grown from Cellulose-Producing Bacteria with Engineered Tyrosinase Expression","volume":"43","author":"Walker","year":"2024","journal-title":"Nat. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1159\/000233506","article-title":"Isolation, Cloning and Characterization of a Tyrosinase with Improved Activity in Organic Solvents from Bacillus megaterium","volume":"17","author":"Shuster","year":"2009","journal-title":"Microb. Physiol."},{"key":"ref_16","unstructured":"(2025, September 18). SnapGene Software. Available online: www.snapgene.com."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"786","DOI":"10.1177\/004051755902901003","article-title":"An Empirical Method for Estimating the Degree of Crystallinity of Native Cellulose Using the X-Ray Diffractometer","volume":"29","author":"Segal","year":"1959","journal-title":"Text. Res. J."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"125193","DOI":"10.1016\/j.ijbiomac.2023.125193","article-title":"In-Situ CBM3-Modified Bacterial Cellulose Film with Improved Mechanical Properties","volume":"243","author":"Liu","year":"2023","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"760","DOI":"10.1021\/ma970768c","article-title":"FT-IR Microscopic Analysis of Changing Cellulose Crystalline Structure during Wood Cell Wall Formation","volume":"31","author":"Kataoka","year":"1998","journal-title":"Macromolecules"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"9739","DOI":"10.1007\/s10570-021-04152-7","article-title":"Cellulose Nanofiber\/Melanin Hybrid Aerogel Supported Phase Change Materials with Improved Photothermal Conversion Efficiency and Superior Energy Storage Density","volume":"28","author":"Chen","year":"2021","journal-title":"Cellulose"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Qiu, Z., Wang, S., Zhao, J., Cui, L., Wang, X., Cai, N., Li, H., Ren, S., Li, T., and Shu, L. (2023). Synthesis and Structural Characteristics Analysis of Melanin Pigments Induced by Blue Light in Morchella sextelata. Front. Microbiol., 14.","DOI":"10.3389\/fmicb.2023.1276457"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Pralea, I.-E., Moldovan, R.-C., Petrache, A.-M., Ilie\u0219, M., Heghe\u0219, S.-C., Ielciu, I., Nicoar\u0103, R., Moldovan, M., Ene, M., and Radu, M. (2019). From Extraction to Advanced Analytical Methods: The Challenges of Melanin Analysis. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20163943"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"U\u011furel, C., and \u00d6\u011f\u00fct, H. (2024). Optimization of Bacterial Cellulose Production by Komagataeibacter rhaeticus K23. Fibers, 12.","DOI":"10.3390\/fib12030029"}],"container-title":["Polymers"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4360\/18\/17\/2099\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,31]],"date-time":"2026-08-31T02:16:53Z","timestamp":1788142613000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4360\/18\/17\/2099"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,29]]},"references-count":23,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["polym18172099"],"URL":"https:\/\/doi.org\/10.3390\/polym18172099","relation":{},"ISSN":["2073-4360"],"issn-type":[{"value":"2073-4360","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,29]]}}}