{"title":"CitM mediates hydroxycitrate (HCA) uptake: insights from heterologous expression in Escherichia coli, Bacillus subtilis mutant analysis and membrane topology prediction.","authors":"Manali Chandnani, Aditi Buch","doi":"10.1007/s10529-026-03736-2","DOIUrl":null,"url":null,"abstract":"<p><strong>Purpose: </strong>The biotechnological potential of microbial assimilation of C<sub>6</sub> tricarboxylates relies heavily on the availability of transporters. Hydroxycitrate (HCA), a structural analog of citrate, accumulates abundantly in Garcinia plants and could serve as a valuable biosynthetic precursor; however, its uptake systems are poorly known. This study aimed to identify and functionally characterize Bacillus subtilis citrate transporters and their ability to enable HCA uptake in the heterologous Escherichia coli system.</p><p><strong>Methods: </strong>Growth of wild-type and transporter-deficient derivatives of B. subtilis (ΔcitM, ΔcimH, ΔcitH, and ΔyraO) was profiled in presence of HCA and citrate using plate and broth assays. Genes encoding these transporters were independently expressed in E. coli to confirm their functional potential, in correlation with structural and topological predictions derived from PSIPRED and MEMSAT-SVM analysis.</p><p><strong>Results: </strong>Deletion of citM severely diminished the growth of B. subtilis on HCA and citrate while that of cimH distinctly impaired growth on HCA, unlike citH and yraO knockouts. However, only citM overexpression enabled HCA uptake in E. coli. Favorable membrane-insertion topology predicted by the PSIPRED/MEMSAT analysis further supported CitM's exclusive functionality as an HCA transporter.</p><p><strong>Conclusion: </strong>CitM was identified as primarily necessary and sufficient for HCA uptake in both B. subtilis and E. coli. The potential of CimH untapped under the tested conditions could be determined through host-optimization strategies. Such mechanistic insights into the expanded transporter promiscuity provide an important framework for engineering microbial systems capable of valorizing HCA-rich agrowaste into value-added products.</p>","PeriodicalId":8929,"journal":{"name":"Biotechnology Letters","volume":"48 4","pages":""},"PeriodicalIF":2.5000,"publicationDate":"2026-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biotechnology Letters","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1007/s10529-026-03736-2","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Purpose: The biotechnological potential of microbial assimilation of C6 tricarboxylates relies heavily on the availability of transporters. Hydroxycitrate (HCA), a structural analog of citrate, accumulates abundantly in Garcinia plants and could serve as a valuable biosynthetic precursor; however, its uptake systems are poorly known. This study aimed to identify and functionally characterize Bacillus subtilis citrate transporters and their ability to enable HCA uptake in the heterologous Escherichia coli system.
Methods: Growth of wild-type and transporter-deficient derivatives of B. subtilis (ΔcitM, ΔcimH, ΔcitH, and ΔyraO) was profiled in presence of HCA and citrate using plate and broth assays. Genes encoding these transporters were independently expressed in E. coli to confirm their functional potential, in correlation with structural and topological predictions derived from PSIPRED and MEMSAT-SVM analysis.
Results: Deletion of citM severely diminished the growth of B. subtilis on HCA and citrate while that of cimH distinctly impaired growth on HCA, unlike citH and yraO knockouts. However, only citM overexpression enabled HCA uptake in E. coli. Favorable membrane-insertion topology predicted by the PSIPRED/MEMSAT analysis further supported CitM's exclusive functionality as an HCA transporter.
Conclusion: CitM was identified as primarily necessary and sufficient for HCA uptake in both B. subtilis and E. coli. The potential of CimH untapped under the tested conditions could be determined through host-optimization strategies. Such mechanistic insights into the expanded transporter promiscuity provide an important framework for engineering microbial systems capable of valorizing HCA-rich agrowaste into value-added products.
期刊介绍：
Biotechnology Letters is the world’s leading rapid-publication primary journal dedicated to biotechnology as a whole – that is to topics relating to actual or potential applications of biological reactions affected by microbial, plant or animal cells and biocatalysts derived from them.
All relevant aspects of molecular biology, genetics and cell biochemistry, of process and reactor design, of pre- and post-treatment steps, and of manufacturing or service operations are therefore included.
Contributions from industrial and academic laboratories are equally welcome. We also welcome contributions covering biotechnological aspects of regenerative medicine and biomaterials and also cancer biotechnology. Criteria for the acceptance of papers relate to our aim of publishing useful and informative results that will be of value to other workers in related fields.
The emphasis is very much on novelty and immediacy in order to justify rapid publication of authors’ results. It should be noted, however, that we do not normally publish papers (but this is not absolute) that deal with unidentified consortia of microorganisms (e.g. as in activated sludge) as these results may not be easily reproducible in other laboratories.
Papers describing the isolation and identification of microorganisms are not regarded as appropriate but such information can be appended as supporting information to a paper. Papers dealing with simple process development are usually considered to lack sufficient novelty or interest to warrant publication.
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