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Purpose <p>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 <i>Garcinia</i> plants and could serve as a valuable biosynthetic precursor; however, its uptake systems are poorly known. This study aimed to identify and functionally characterize <i>Bacillus subtilis</i> citrate transporters and their ability to enable HCA uptake in the heterologous <i>Escherichia coli</i> system.</p> Methods <p>Growth of wild-type and transporter-deficient derivatives of <i>B. subtilis</i> (<i>ΔcitM, ΔcimH, ΔcitH,</i> and <i>ΔyraO</i>) was profiled in presence of HCA and citrate using plate and broth assays. Genes encoding these transporters were independently expressed in <i>E. coli</i> to confirm their functional potential, in correlation with structural and topological predictions derived from PSIPRED and MEMSAT-SVM analysis.</p> Results <p>Deletion of <i>citM</i> severely diminished the growth of <i>B. subtilis</i> on HCA and citrate while that of <i>cimH</i> distinctly impaired growth on HCA, unlike <i>citH</i> and <i>yraO</i> knockouts. However, only <i>citM</i> overexpression enabled HCA uptake in <i>E. coli</i>. Favorable membrane-insertion topology predicted by the PSIPRED/MEMSAT analysis further supported CitM’s exclusive functionality as an HCA transporter.</p> Conclusion <p>CitM was identified as primarily necessary and sufficient for HCA uptake in both <i>B. subtilis</i> and <i>E. coli</i>. 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>

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CitM mediates hydroxycitrate (HCA) uptake: insights from heterologous expression in Escherichia coli, Bacillus subtilis mutant analysis and membrane topology prediction

  • 期刊论文

  • 发表日期：

    2026年7月3日

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  • Manali Chandnani,
  • Aditi Buch

摘要

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.