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Bioremediation of complex organic pollutants by engineered Vibrio natriegens

Nature volume 642, pages 1024–1033 (2025)Cite this article

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Abstract

Industrial wastewater, petroleum pollution and plastic contamination are significant threats to global marine biosecurity because of their toxic, mutagenic and persistent nature1. The use of microorganisms in bioremediation has been constrained by the complexity of organic pollutants and limited tolerance to saline stress2. In this study, we used synthetic biology to engineer Vibrio natriegens into a strain capable of bioremediating complex organic pollutants in saline wastewater and soils. The competence master regulator gene tfoX was inserted into chromosome 1 of the V. natriegens strain Vmax and overexpressed to enhance DNA uptake and integration. Degradation gene clusters were chemically synthesized and assembled in yeast. We developed a genome engineering method (iterative natural transformation based on Vmax with amplified tfoX effect) to transfer five gene clusters (43 kb total) into Vmax. The engineered strain has the ability to bioremediate five organic pollutants (biphenyl, phenol, naphthalene, dibenzofuran and toluene) covering a broad substrate range, from monocyclic to multicyclic compounds, in industrial wastewater samples from a chlor–alkali plant and a petroleum refinery.

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Fig. 1: Construction of strains for remediation of complex organic pollutants and selection of chassis cells.
Fig. 2: Development of a helper plasmid-free gene transfer method and selection of promoters for V. natriegens.
Fig. 3: Insertion and in vivo examination of synthetic degradation gene clusters.
Fig. 4: Examining the remediation of complex organic pollutants by VCOD-15 and gene expression levels.
Fig. 5: Bioremediation of complex polluted industrial wastewater samples in activated sludge bioreactor by VCOD-15.

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Data availability

The genome assembly generated in this study was deposited in NCBI under the BioProject PRJNA1240198. All other data are presented in the paper and Supplementary Information. The public data used in this study included function annotations of non-essential genes in the genome of Vmax and degradation gene clusters from the NCBI database (https://www.ncbi.nlm.nih.gov). The accession numbers of the genes are listed in Supplementary Tables 4 and 5.

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Acknowledgements

This study was supported by the National Key Research and Development Program of China (2021YFA0909500), National Natural Science Foundation of China (32030004, 32150025 and 82003626), Guangdong S&T Program (2022B1111080005, 2022A0505090009), Shenzhen Science and Technology Program (KQTD20180413181837372), Innovation Program of Chinese Academy of Agricultural Science and the Shenzhen Outstanding Talents Training Fund. We would like to thank the Core Facility and Service Center for School of Life Sciences and Biotechnology, SJTU for the metabolite analysis data collection. We would also like to thank Y. Li from Shanghai Jiao Tong University for his insightful and valuable assistance in the metabolism testing of stable isotope-labelled compounds and in the analysis of the GC–HRMS data, as well as Q. Wang from Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences for his kind provision of the experimental material IOM.

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Author notes

  1. These authors contributed equally: Cong Su, Haotian Cui, Weiwei Wang

Authors and Affiliations

  1. Shenzhen Key Laboratory of Synthetic Genomics, Guangdong Provincial Key Laboratory of Synthetic Genomics, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China

    Cong Su, Yong Liu, Chen Wang, Liwen Qu & Junbiao Dai

  2. State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, People’s Republic of China

    Haotian Cui, Weiwei Wang, Zhenyu Cheng, Mengqiao Yang, Ye Li, Siyang He, Ping Xu & Hongzhi Tang

  3. Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Shenzhen Key Laboratory of Agricultural Synthetic Biology, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China

    Yuejin Cai, Jiaxin Zheng, Pingping Zhao & Junbiao Dai

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Contributions

H.T. and J.D. designed and supervised the overall research framework and provided acquired funding. C.S., Y. Liu, C.W. and L.Q. conducted molecular biology experiments, including bacterial strain construction, transcriptomic analyses and qPCR gene expression analysis. C.S., H.C. and P.Z. performed bacterial growth characterization and pollutant degradation assays, including optimization of culture conditions, growth curve measurement and degradation testing. H.C., W.W., Z.C., M.Y., Y. Li and S.H. collected and processed industrial wastewater and soil samples and conducted pollutant degradation experiments under practical environmental conditions. C.S., H.C., W.W., Z.C., M.Y., Y. Li, P.X. and H.T. performed chromatographic and mass spectrometric analyses (HPLC, gas chromatography, UPLC–QTOF-MS and HRGC–MS) and conducted data analysis. H.C., W.W., Z.C., M.Y. and Y. Li conducted microbial diversity analyses of environmental samples and performed statistical analyses, data organization and significance testing. C.S., H.C., W.W., Z.C., M.Y., Y. Li, Y.C., S.H., J.Z., P.X., J.D. and H.T. wrote the paper. All authors contributed to reviewing the draft of the paper and approving the final paper.

Corresponding authors

Correspondence to Junbiao Dai or Hongzhi Tang.

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Extended data figures and tables

Extended Data Fig. 1 Comparative transcriptomic analysis of Vibrio natriegens Vmax in the presence and absence of complex organic pollutants.

a, Heatmap of differentially expressed genes related to aromatic compound resistance, detected based on RNA-seq of V. natriegens Vmax in the presence or absence of the indicated mixture of complex organic pollutants. Transcription factors are in green; energy metabolism genes are in black; multidrug-resistance-related genes are in purple; ABC transporter genes are in blue. b, qPCR analysis of expression of the indicated genes. Data are represented as the mean of three biological triplicates ± SD.

Extended Data Fig. 2 Natural transformation (NT) efficiency of VCOD-1 (Vmax-pET28a-tfoX).

a, NT efficiency testing between VCOD-1 and the original strain with a linear fragment (xxxx) as the donor. b, NT efficiency testing between VCOD-1 and the original strain with the p15A plasmid. c. NT efficiency of VCOD-1 with the indicated quantity of the ΔwbfF::CmR donor DNA fragment containing the indicated length (in kbp) of homology arms on each side of the mutation. Statistical analysis: a-c, data are represented as the mean ± SD. n = 3 independent experiments. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons tests.

Extended Data Fig. 3 Optimization of NT for VCOD-2 (VmaxΔint::tfoX).

a, NT efficiency of VCOD-2 with the indicated quantities of the ΔwbfF::CmR donor DNA fragment (with 0.5 kbp/0.5 kbp homology arms). b, NT efficiency of VCOD-2 in the indicated bacterial growth states (measured by OD600) with 200 ng ΔwbfF::CmR (with 2 kbp/2 kbp homology arms) of the donor DNA fragment. c, NT efficiency of VCOD-2 induced by the indicated concentrations of IPTG with 200 ng of the ΔwbfF::CmR (2 kbp/2 kbp) donor DNA fragment. d, NT efficiency of VCOD-2 with 200 ng of the ΔwbfF::CmR donor DNA fragment (containing the indicated lengths for homology arms). e, NT efficiency of VCOD-2 with different incubation times after adding 200 ng of the ΔwbfF::CmR (2 kbp/2 kbp) donor DNA fragment. Statistical analysis: a-e, data are represented as the mean ± SD. n = 3 independent experiments. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons tests.

Extended Data Fig. 4 Pathways for complex pollutant degradation by Vmax engineered strains.

a-e, Catabolic pathways and LC-MS spectra for degradation intermediates produced by the VCOD-3 (a), VCOD-4 (b), VCOD-5 (c), VCOD-6 (d), and VCOD-7 (e) strains. The organic pollutants were added to resting cell suspensions in nine-salt solution (see Supplementary Information Table 2 for the detailed composition); metabolites were extracted by ethyl acetate after six-hour incubation of cultures with the pollutants (see Methods and Materials for details). Detected biphenyl degradation intermediates included: biphenyl-2,3-diol (2), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (3), and benzoic acid (4). Catechol was detected as a phenol degradation intermediate (6). Detected naphthalene degradation intermediates included naphthalene-1,2-diol (8) and salicylic acid (9). Detected dibenzofuran degradation intermediates included 2,2’,3-trihydroxybiphenyl (11) and salicylic acid (9). Detected toluene degradation intermediates included benzyl alcohol (13) and benzoic acid (4).

Extended Data Fig. 5 qPCR analysis of selected analyte genes from the complex pollutant degrading gene clusters.

a-e, Cultures of all strains were induced using 1 mM IPTG. The analyte genes (as indicated) were assessed with qPCR for strains VCOD-3 (a), VCOD-4 (b), VCOD-5 (c), VCOD-6 (d), and VCOD-7 (e) strains. Data are represented as the mean ± SD. n = 3 independent experiments.

Extended Data Fig. 6 Examining the remediation of complex organic pollutants by VCOD-12 and expression analysis of selected genes.

a, Schematic for the organization of gene clusters in VCOD-12. Two gene clusters were inserted into the neutral site chr2_297. The screening marker kanamycin resistance gene KanR was present at the end of the dmp gene cluster. b, qPCR analysis of the indicated genes from the complex pollutant degrading gene cluster in VCOD-12, induced with 1 mM IPTG. c-d, Complex organic pollutant remediation efficiency of VCOD-12 in nine-salt solution. e, Growth of the VCOD-2 and VCOD-12 strains in LB3 medium. Statistical analysis: b-e, data are represented as the mean ± SD. n = 3 independent experiments. Statistical significance was assessed using unpaired t-tests with Welch’s correction.

Extended Data Fig. 7 Examining the remediation of complex organic pollutants by VCOD-13 and expression analysis for selected genes.

a, Schematic for the organization of gene clusters in VCOD-13. Three gene clusters were inserted into the neutral site chr2_297. The screening marker chloramphenicol resistance gene CmR was present at the end of the nah gene cluster. b, qPCR analysis of the indicated genes from the complex pollutant degrading gene cluster in VCOD-13, induced with 1 mM IPTG. c-e, Complex organic pollutant remediation efficiency of VCOD-13 in nine-salt solution. f, Growth of the VCOD-2 and VCOD-13 strains in LB3 medium. Statistical analysis: b-f, data are represented as the mean ± SD. n = 3 independent experiments. Statistical significance was assessed using unpaired t-tests with Welch’s correction.

Extended Data Fig. 8 Examining the remediation of complex organic pollutants by VCOD-14 and gene expression levels.

a, Schematic for the organization of gene clusters in VCOD-14. Four gene clusters were inserted into the neutral site chr2_297. The screening marker kanamycin resistance gene KanR was present at the end of the nah gene cluster. b, qPCR analysis of the indicated genes from the complex pollutant degrading gene cluster in transformed V. natriegens cultures, induced with 1 mM IPTG. c-f, Complex organic pollutant remediation efficiency of VCOD-14 in nine-salt solution. g, Bacterial growth of strains VCOD-2 and VCOD-14 in LB3 medium. Statistical analysis: b-g, data are represented as the mean ± SD. n = 3 independent experiments. Statistical significance was assessed using unpaired t-test with Welch’s correction.

Extended Data Fig. 9 Pathways for complex pollutant degradation by VCOD-15.

a-e, Catabolic pathways and LC-MS spectra for degradation intermediates of biphenyl (1) (a), phenol (6) (b), naphthalene (8) (c), dibenzofuran (15) (d), and toluene (18) (e). The organic pollutants were added to resting cell suspensions in nine-salt solution; metabolites were extracted by ethyl acetate after six hours (see Methods and Materials for details). Detected biphenyl degradation intermediates included biphenyl-2,3-diol (3), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (4), and benzoic acid (5). Catechol was a detected degradation intermediate from phenol (7). Detected naphthalene degradation intermediates included naphthalene-1,2-diol (10) and salicylic acid (14). Detected dibenzofuran degradation intermediates included 2,2’,3-trihydroxybiphenyl (16) and salicylic acid (14). Detected toluene degradation intermediates included benzyl alcohol (19) and benzoic acid (5).

Extended Data Fig. 10 Bioremediation of industrial wastewater samples in multi-parallel bioreactors.

a, Photograph of the multi-parallel bioreactors. Industrial wastewater samples were treated with VCOD-15. b-f, Complex organic pollutant bioremediation efficiency of VCOD-15 in industrial wastewater samples. g, The relative abundance of microbial genera in the wastewater samples’ microbial communities was measured at 0, 24, and 48 h during the bioremediation process (n = 3). b-f, Data are represented as the mean of three biological triplicates ± SD.

Supplementary information

Supplementary Information (download PDF )

This file contains Supplementary Figs. 1–21 and Tables 1–10. Supplementary figures: schematic of the workflow of gene cluster assembly and insertion, as well as growth curve, promoter strength, transformation/recombination efficiency, pollutant remediation efficiency and metabolite analysis in this study. Supplementary tables: strains, broths, genes, primers, wastewater samples and selected genome insertion targets used in this study.

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Raw data for Figs. 1–5, Extended Data Figs. 1–10 and Supplementary Figs. 1, 6, 8, 9, 12 and 14–21.

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Su, C., Cui, H., Wang, W. et al. Bioremediation of complex organic pollutants by engineered Vibrio natriegens. Nature 642, 1024–1033 (2025). https://doi.org/10.1038/s41586-025-08947-7

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