The engineering of TBBPA-degrading synthetic microbiomes with integrated strategies

Abstract

The capability to understand and construct synthetic microbiomes is crucial in biotechnological innovation and application. Tetrabromobisphenol A (TBBPA) is an emerging pollutant, and the understanding of its biodegradation is very limited. Here, a top-down approach was applied for the enrichment of TBBPA-degrading microbiomes from natural microbiomes. Ten keystone taxa correlated to TBBPA degradation and their co-occurrence interactions were identified by the dissection of the degrading microbiomes. Those keystone taxa were targeted and cultivated, and the genomic information was obtained by genome sequencing of strains and metagenomic binning. The keystone bacterial strains showed efficient degradation of TBBPA, and L-amino acids were important co-metabolic substrates to promote the degradation. Guided by this knowledge, a bottom-up approach was applied to design and construct a simplified synthetic consortium SynCon2, that consisted of four strains. The SynCon2 demonstrated efficient TBBPA degradation activity and soil bioremediation. Our study demonstrates the importance of the application of multiple tools in understanding the functions of microbiomes and provides an integrated top-down and bottom-up strategy for the construction of synthetic microbiomes with various applications.

Fig. 1. TBBPA and BPA-degrading microbiomes are obtained by a top-down strategy.

a Overview of experimental design. Some of the elements were created in BioRender (https://BioRender.com/k0m85rp). Degradation (b) and debromination (c) of TBBPA in GSM medium by TBBPA-degrading microbiomes. M3-1: TBBPA degraded 7.67 μM and Br⁻ released 5.55 μM; M3-2: TBBPA degraded 6.03 μM and Br^- released 4.59 μM; M3-3: TBBPA degraded 16.83 μM and Br⁻ released 24.78 μM; M3-4: TBBPA degraded 12.99 μM and Br⁻ released 13.73 μM. d Scanning electron microscopy of TBBPA-degrading microbiomes. e Degradation of BPA in GSM medium by BPA-degrading microbiomes. The group containing G represents transfer in GSM medium, and the group containing M represents transfer in MSM medium. The error bar represents the Standard Error of the Mean.

Fig. 2. Identification of keystone taxa and co-occurrence networks.

a Heatmap showing correlation between the microbial abundance at genus level and TBBPA degradation. The average relative abundance of each genus was greater than 0.1%. * p < 0.05, ** p < 0.01. b Bacterial taxonomic biomarkers of TBBPA degradation in TBBPA-degrading microbiomes. The biomarkers were identified by Random Forests regression. c Heatmap showing the abundances of biomarkers against TBBPA degradation. The insert represents 10-fold cross-validation error as a function of the number of input genera. The dashed gray line marks the optimal cut-off for biomarker selection. d TBBPA co-occurrence network at the genus level (left), and modularized network (right). Red and green links represent positive and negative interactions, respectively. The color changes depending on the analysis.

Fig. 3. Bacterial strains and genomic resources for TBBPA-degradation.

a Neighbor-joining tree based on the 16S rRNA gene sequence of isolates. b Degradations of TBBPA and BPA with various representative strains in different media. The strains marked in red are keystone taxa. c Phylogenomic tree and COG characteristics of MAGs and complete genomes of strains. The genomes marked in red are keystone taxa. Abbreviations in Metabolism categories, E: Amino acid transport and metabolism; C: Energy production and conversion; P: Inorganic ion transport and metabolism; I: Lipid transport and metabolism; Q: Secondary metabolites biosynthesis, transport and catabolism; H: Coenzyme transport and metabolism; G: Carbohydrate transport and metabolism; F: Nucleotide transport and metabolism. d Degradation activity on TBBPA with three keystone strains in the presence of different L-amino acids. See the Methods for specific L-amino acid groups. e Degradation and growth of TBBPA or BPA by strain WTB6 in the presence of L-alanine (2 g/L), L-valine (1 g/L), and L-leucine (1 g/L).

Fig. 4. Construction of synthetic consortia and proposed degradation of TBBPA.

a The construction of the synthetic consortium SynCon2. b Upstream metabolites of TBBPA degradation and degradation of aromatic organic compounds by the synthetic consortium SynCon2. The circles under the arrows indicate that different strains possess metabolic genes for this step. Gene names are summarized in Table S6. c Degradation of TBBPA by synthetic consortia at low L-alanine concentration (1 g/L). The degradation percentages were determined after incubation at 30 °C for 36 h. d Degradation of TBBPA in soil microcosms with and without SynCon2. Residual TBBPA was extracted from soils at day 7 of treatment. Error bars are the Standard Error of the Mean of triplicate experiments. Significance was analyzed by unpaired, two-tailed Student’s t-test. *p < 0.05, ** p < 0.01, *** p < 0.001.