Mining Natural Microbial Diversity for Tool-Compatible Chassis Discovery

Mining Natural Microbial Diversity for Tool-Compatible Chassis Discovery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Biological Sciences - Article Mining Natural Microbial Diversity for Tool-Compatible Chassis Discovery Gyoo Yeol Jung, Min Jae Kim, Minsun Kim, Jun Hyeok Choi, Myung Hyun Noh, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6811285/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract The development of microbial bioprocesses for unconventional feedstocks requires chassis organisms capable of efficiently catabolizing complex substrates. However, model organisms, such as Escherichia coli , cannot inherently metabolize non-native compounds, and engineered pathways often result in poor growth on such substrates. Here, we present the Selection of Chassis Organisms Under Target conditions (SCOUT), a strategy for rapidly identifying genetically tractable environmental isolates that are compatible with existing synthetic biology tools. Using SCOUT, we isolated Pseudomonas postechii TPA1, a novel chassis exhibiting the fastest reported growth rate (0.78 h -1 ) on terephthalic acid (TPA) to date. Engineering P. postechii TPA1 enabled direct biosynthesis of 505 mg/L of the natural blue pigment indigoidine from TPA. SCOUT further demonstrates its versatility by enabling the isolation of a ready-to-engineer host for styrene bioconversion. These results establish SCOUT as a powerful platform for expanding the microbial chassis diversity and accelerating the development of sustainable biorefinery processes. Biological sciences/Systems biology/Synthetic biology Biological sciences/Biotechnology/Metabolic engineering Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Biorefineries convert a broad range of feedstocks into commodities and value-added products through microbial processes, thereby improving economic and environmental sustainability. While traditional feedstocks include starch crops, lignocellulosic biomass, and marine biomass 1–3 , recent efforts have expanded to include more non-conventional feedstocks (e.g., plastics and methane), which are abundant in both natural and industrial environments 4,5 . Polyethylene terephthalate (PET) is a widely used plastic with an annual global production exceeding 56 million tons annually 6 . However, more than 40–80% of PET waste is landfilled, creating substantial environmental burdens and emphasizing the need for microbial recycling strategies 7,8 . A major bottleneck in developing such processes lies in the limited catabolic capability of model microbial chassis, such as Escherichia coli , which is poorly equipped to metabolize non-native compounds despite the availability of advanced synthetic biology toolkits 9 . Although the heterologous expression of catabolic pathways can partially address this gap, engineered strains often fail to achieve robust growth on unconventional substrates 10,11 . In contrast, naturally occurring microbes with specialized catabolic capabilities offer attractive alternatives 12–15 . Nonetheless, domestication of these isolates typically requires significant efforts to develop genetic tools and transformation methods. Furthermore, conventional growth-based selection strategies inherently favor fast growers but do not guarantee compatibility with the synthetic biology groundwork, limiting their utility in chassis development. Notably, among the vast diversity of environmental microbes, rare strains that simultaneously possess the ability to catabolize recalcitrant substrates and remain amenable to genetic manipulation tools established for conventional hosts may exist. Although the discovery of dual-capability microorganisms could provide an efficient and elegant solution, their presumed low abundance in natural ecosystems presents a major barrier to their identification and practical use. Here, we present the Selection of Chassis Organisms Under Target conditions (SCOUT) as a strategy designed to streamline the discovery of genetically tractable environmental isolates under defined selection conditions. SCOUT employs the conjugative transfer of a production pathway and biosensor to environmental microbial communities, enabling the fluorescence-based selection of hosts that are catabolically active and compatible with existing synthetic biology toolkits. Using SCOUT, we isolated Pseudomonas postechii TPA1, which exhibited the fastest reported growth rate (0.78 h -1 ) on terephthalic acid (TPA). Subsequent engineering of Pseudomonas postechii TPA1 enabled the direct biosynthesis of the natural blue pigment indigoidine from TPA. Furthermore, SCOUT demonstrated broader applicability by isolating a genetically tractable host capable of styrene bioconversion. These results highlight SCOUT as a powerful approach for bridging natural microbial diversity and synthetic biology, thereby expanding the chassis repertoire available for sustainable biorefinery applications. Results Design of the SCOUT system to select chassis organisms under target conditions We developed a SCOUT system to identify ready-to-use microbial hosts by introducing a genetic circuit on a broad-host-range conjugative plasmid (Fig. 1). The genetic circuit was designed to integrate a chemical production module and biosensor module capable of detecting the synthesized compound and displaying fluorescence as an output. These modules were assembled using Bioparts, which represent current synthetic biology standards. Itaconic acid was selected as the model chemical because it can be synthesized from cis -aconitate, a conserved intermediate in the bacterial TCA cycle. To realize this design, we constructed a pSCOUT plasmid integrating both production and sensor modules, along with features for broad-host-range functionality. Specifically, the production module was constructed by placing two itaconic acid biosynthetic genes, prpD VL and cad 16,17 , under the P tac promoter with customized 5'-untranslated regions for strong expression 18 . For itaconic acid sensing, an expression cassette encoding the itaconic acid-specific transcription factor ItcR 16,19 and superfolder green fluorescent protein (sGFP) was introduced. ItcR was expressed constitutively under the P J23106 promoter, whereas sGFP expression was driven by the P ccl promoter, which was activated upon ItcR binding to itaconic acid. We confirmed that fluorescence could be observed using E. coli as a host in the presence of at least 1 mg/L itaconic acid (Supplementary Fig. 1). To ensure broad host applicability, the plasmid backbone incorporated an RSF origin of replication 20–22 and was engineered for conjugative transfer 23,24 , resulting in the construction of the pSCOUT plasmid (Supplementary Table 1).Screening of a novel chassis with efficient terephthalic acid catabolism using the SCOUT system We used SCOUT to screen for a microbial chassis capable of metabolizing terephthalic acid (TPA) in environmental samples. A soil sample was collected near an industrial plant, where TPA-catabolizing bacteria were likely present (Fig. 2a). The pSCOUT plasmid was introduced into the microbial pool via conjugation using an E. coli S-17 ∆ asd mutant for auxotrophic counterselection on diaminopimelic acid (DAP, see Methods). Following conjugation, fluorescence-positive cells were isolated using fluorescence-activated cell sorting (Fig. 2b). In particular, a gating strategy was applied to exclude cells that displayed background fluorescence with erroneous activation of the biosensor module using a control plasmid lacking the production module (NC). The sorted populations were grown in TPA-supplemented medium, and the cultures were subjected to another round of fluorescence-based sorting. The SCOUT strategy enriched the microbial population with enhanced fluorescence, leading to the isolation of a novel Pseudomonas strain with superior TPA catabolic capability (Fig. 2b). After one round of sorting, the population showed a noteworthy increase in fluorescence (1R) compared with the initial pools (Raw and Conj). Further sorting appeared unnecessary because the fluorescence plateaued in the second round (2R). Sanger sequencing of the 16S rDNA region of 16 random colonies revealed the presence of two Citrobacter and Pseudomonas species. Among these two strains, only the Pseudomonas isolates grew on TPA as the sole carbon source, successfully producing 31 mg/L of itaconic acid (Supplementary Fig. 2). We named this strain Pseudomonas postechii TPA1 (hereafter referred to as TPA1), and determined it to be a novel chassis for TPA conversion. Metagenomic 16S rDNA sequencing tracked changes in microbial diversity during the screening process (Fig. 2c). Bacteria belonging to 14 and 11 genera were detected in the original sample and the conjugant pool, respectively. While TPA1 initially constituted less than 0.4% of the community, its proportion increased to 32.7% after the two rounds of sorting, supporting that the SCOUT strategy is highly effective in isolating a novel chassis. In contrast, although the Citrobacter strain was still dominant in the sorted pools, it did not grow on TPA as the sole carbon source, likely proliferating via yeast extract or metabolic byproducts. We also performed the conventional growth-based enrichment of a potential host from the same sample to compare the two strategies. Interestingly, after multiple serial cultures, we only obtained Acinetobacter species, which took 10.5% of the original pool (Fig. 2c). This strain rapidly dominated on TPA as the sole carbon source, likely because of its slightly faster growth rate than TPA1 (1.1-fold, Supplementary Fig. 3). However, this Acinetobacter strain failed to maintain diverse plasmids or be genetically manipulated via conjugation or transformation, highlighting the advantage of SCOUT in isolating genetically tractable hosts, even when they initially exist at low abundance. Physiological and genomic characterization of P. postechii TPA1 The physiology of TPA1 was further characterized in a TPA-supplemented minimal medium by measuring the growth rate and tolerance levels at different concentrations. When 44 mM of TPA was supplemented, TPA1 displayed an exceptionally high maximum growth rate (μ max = 0.78 h -1 ) and a specific TPA uptake rate of 2.03 g per g dry cell weight per h (g/g DCW/h) (Supplementary Fig. 4). This growth rate is superior to that of previously reported genetically tractable strains (Supplementary Table 3). Although the growth rate tended to decrease as the concentration increased, TPA1 displayed robust growth up to 200 mM TPA, outperforming engineered strains that often show impaired growth at such concentrations 5,25,26 . These observations highlight the potential of microbial diversity as a source of novel chassis strains. Whole-genome sequencing analysis was also performed to investigate the genomic features for efficient TPA conversion. Combined long-read and short-read sequencing analysis revealed that the 6.35-Mb genome comprised a large chromosome and two plasmids (5.74-, 0.34-, 0.26-Mb, Supplementary Fig. 5). Sequence alignment was performed using BLAST against the TPA catabolic genes reported in Comamonas sp. E6, a well-characterized TPA-consuming bacterium 27 (Fig. 2 and Supplementary Fig. 6). It was found that TPA1 possesses similar enzymes (TPA dioxygenase reductase TphA1, TPA dioxygenase component TphA2, TPA dioxygenase component TphA3, TPA dihydrodiol dehydrogenase TphB) for TPA assimilation, with protein identities ranging 40~70% compared to Commamonas sp. E6. Genes encoding these enzymes form a 7.5 kb-long tph operon, together with additional genes putatively encoding the TPA transporter PcaK and transcriptional regulator PcaR. Interestingly, compared to Comamonas species and another TPA-consuming Pseudomonas strain with these genes on their chromosomes, TPA1 possesses these genes in its plasmid, potentially explaining its superior catabolic efficiency for TPA. Furthermore, a copy of tphA1 was detected on the chromosome, potentially augmenting TPA catabolism. Additionally, the TpiAB-TphC transporter system 28 was not detected in TPA1 cells. Although the exact mechanism remains unclear, TPA appears to be efficiently imported into TPA1, possibly via PcaK, other transporters with broad substrate specificity, or as-yet-unidentified novel transporter. Lastly, we analyzed the housekeeping sigma factor σ 70 (encoded by rpoD ) of TPA1. Regions critical for -10 and -35 promoter recognition showed 100% and 96.4% sequence identity, respectively, with those of E. coli (Supplementary Fig. 7), which likely allowed the cross-species compatibility of synthetic promoters. Functional validation with E. coli -optimized inducible systems (P tac , P tet , P lac , P T7 , and P ara ) confirmed robust and controllable gene expression in TPA1 (Supplementary Fig. 8), supporting its immediate compatibility with standard synthetic biology parts. Production of indigoidine from TPA by engineering the TPA1 strain Finally, the potential of TPA1 as a chassis protein was validated by introducing a heterologous biosynthetic pathway. We selected indigoidine as the model chemical because it is an industrially relevant natural blue pigment that can bypass the severe water pollution caused by conventional dye production 29–32 . It is converted from L-glutamine by the apo-form of BpsA, a non-ribosomal peptide synthetase, which is catalytically activated via post-translational modification by Sfp, 4′-phosphopantetheinyl transferase. For efficient indigoidine production, TPA1 was engineered to express the bpsA - sfp operon using strong promoters and synthetic untranslated regions from the plasmid 33 . The resulting TPA-indigo strain (Supplementary Table 1) achieved a notable titer of up to 505 mg/L without an apparent growth burden (Fig. 3c). To our knowledge, this is the first demonstration of the direct bioconversion of TPA to indigoidine, establishing TPA1 as a versatile synthetic biology chassis for plastic upcycling and bioremediation. Extension of the SCOUT system for screening a chassis for styrene conversion To expand the applicability of the SCOUT system, we explored its potential for isolating genetically tractable styrene-converting microorganisms (Fig. 4). Styrene, a monomer widely used in plastic and synthetic rubber industries, poses significant environmental concerns owing to its toxicity and persistence 34 . Although several styrene-converting bacteria and their related pathways have been reported, the strains amenable to genetic engineering are limited to a few Pseudomonas species 35,36 . The SCOUT was applied to the sludge sample collected from propylene oxide/styrene monomer (PO/SM) processing facilities and environments enriched with styrene and its intermediates. The microbial consortia from PO/SM aeration tanks are expected to harbor diverse strains that metabolize styrene. Through the SCOUT sorting process, we identified two Brucella species as promising candidates, exhibiting both styrene-metabolic activity and genetic tractability (Fig. 4a, 4b). Further detailed comparisons revealed that one strain exhibited a superior growth rate, higher styrene conversion efficiency, and greater itaconic acid production than the other strains (Fig. 4c and Supplementary Fig. 9). Therefore, we chose this strain as the chassis and named it Brucella ulsangensis STY1. Whole-genome sequencing yielded a total genome length of 4.83-Mb, comprising two chromosomes (2.81-Mb and 2.02-Mb) (Supplementary Fig. 10 ) . Based on the sequencing data, gene functions were annotated; however, not all genes encoding the full styrene-converting pathway were identified. Instead, KEGG Automatic Annotation Server was used to annotate orthologous genes that were expected to be involved in the conversion. A putative styrene-converting gene, catechol 2,3-dioxygenase, was identified by comparison with 12 other Brucella species (Supplementary Fig. 11). Sequence analysis of the housekeeping sigma factor (σ 70 ) encoded by rpoD showed high conservation with E. coli , with 100% and 89.3% identity at the -10 (region 2.4) and -35 (region 4.2) promoter recognition sites, respectively (Supplementary Fig. 12). This suggests that E. coli -optimized synthetic promoters are functional in STY1. To validate its genetic tractability, we used synthetic genetic parts and production plasmids previously developed for TPA-converting strains. Using these tools, we successfully engineered B. ulsangensis STY1 and confirmed its capacity to induce gene expression and biosynthetic production. Controlled gene expression was achieved by introducing conventional inducible promoters and their cognate transcription factors (Supplementary Fig. 13). Moreover, B. ulsangensis STY1 successfully expressed heterologous pathways, producing indigoidine using styrene as the sole carbon source (Fig. 4d), further confirming its potential as an industrially relevant host. Discussion Despite the superior ability of non-conventional strains to metabolize substrates, such as TPA and styrene, their limited genetic tractability has hindered their widespread use in industrial biorefineries. Therefore, identifying a microbial chassis that combines high metabolic efficiency and genetic accessibility remains a key challenge. Among the diverse microbial species present in natural environments, certain strains may possess the dual capability of utilizing substrates recalcitrant to conventional hosts while remaining compatible with existing genetic engineering tools. However, the presumed low abundance of these strains in nature presents a significant obstacle to their discovery. In this study, we present an efficient high-throughput screening strategy, SCOUT, which integrates biosensor-guided selection with substrate-specific enrichment. By applying fluorescence-based cell sorting and carbon-source-limited growth selection, we successfully isolated two promising chassis strains, P. postechii TPA1 and B. ulsangensis STY1, which exhibited robust assimilation of TPA and styrene, respectively. Notably, TPA1 demonstrated a significantly higher rate of TPA consumption than previously reported wild-type strains, highlighting its potential as an efficient host for plastic-derived carbon bioconversion. Traditional methods for identifying new chassis strains often rely on sequential screening steps, first for substrate assimilation, followed by the validation of genetic compatibility, resulting in time-consuming and low-throughput workflows. In contrast, the SCOUT system streamlines this process by directly linking metabolic activity to genetic responsiveness via a biosensor-based reporting circuit, allowing simultaneous selection of both traits. A key feature of our approach is the use of a broad-host-range conjugative plasmid, which enables efficient horizontal gene transfer to diverse bacterial hosts. We used the RP4 conjugation system, which facilitates plasmid mobilization across diverse microbes. This compatibility minimizes the exclusion of potentially valuable strains that might otherwise be overlooked because of their low transformation efficiency. Furthermore, the plasmid design includes a dual-module circuit, comprising a biosensor and a production pathway that ensures fluorescence is triggered only when both modules are functional, thereby reducing false-positive signals. In this study, itaconic acid production served as a robust readout of the central carbon flux, as it was synthesized from cis -aconitate, a conserved TCA cycle intermediate. Importantly, no known bacterial strains naturally produce itaconic acid, making the detection highly specific to engineered pathways. The biosensor itcR exhibited excellent sensitivity, with a detection threshold in the single-digit micromolar range and high specificity, allowing clear discrimination from related metabolites. The strains isolated using the SCOUT system were compatible with standard synthetic biology components, including inducible promoters and synthetic UTRs, suggesting that widely used regulatory elements in E. coli may also be applicable to these non-model organisms. This compatibility opens the door for the further engineering of these strains with a level of flexibility comparable to that of traditional chassis strains. In addition to identifying metabolically specialized strains, the SCOUT platform has the potential to screen chassis for other industrially relevant traits, such as acid or solvent tolerance and thermophilicity. Moreover, the metabolic pathways and enzymes characterized in this study may provide insights into non-conventional substrate utilization mechanisms and support the discovery of novel regulatory factors. By integrating biosensor-based screening with consortium engineering, our strategy offers a scalable and adaptable platform for discovering novel microbial hosts, thereby accelerating the development of next-generation biorefineries. Declarations Acknowledgments This research was supported by the Korea Institute of Marine Science and Technology Promotion (KIMST), funded by the Ministry of Oceans and Fisheries (grant number RS-2022-KS221581). This work was also supported by the National Research Foundation of Korea (NRF) grants (RS-2024-00334792, RS-2025-02215308, RS-2024-00400033, RS-2024-00453085) funded by the Ministry of Science and ICT. Author Contributions M.J.K, M.K, H.G.L., and G.Y.J. contributed to the conception of the project. M.J.K, M.K. contributed to the design and conduction of the experiments. M.J.K, M.K., J.H.C, M.H.N., H.G.L., and G.Y.J. contributed to data analysis, interpretation and writing of the manuscript. H.G.L. and G.Y.J. supervised the project. All the authors read and approved the final manuscript. Competing Interests The authors declare that they have no competing interests. Additional information Supplementary Information is available for this paper. Correspondence and requests for materials should be addressed to H.G.L and G.Y.J. Peer review information Nature thanks XXX and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Reprints and permissions information is available at http://www.nature.com/reprints. References Long, B. et al. Engineering strategies to optimize lignocellulosic biorefineries. Nat. Rev. Bioeng. (2024). Qiao, J. et al. Integrated biorefinery approaches for the industrialization of cellulosic ethanol fuel. Bioresour. Technol. 360, 127516 (2022). Baghel, R. S. Developments in seaweed biorefinery research: A comprehensive review. Chemical Engineering Journal 140177 (2022). Diao, J., Tian, Y., Hu, Y. & Moon, T. S. Producing multiple chemicals through biological upcycling of waste poly(ethylene terephthalate). Trends Biotechnol. 43, 620–646 (2025). Werner, A. Z. et al. Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440. Metab. Eng. 67, 250–261 (2021). Soong, Y.-H. V., Sobkowicz, M. J. & Xie, D. Recent advances in biological recycling of polyethylene terephthalate (PET) plastic wastes. Bioengineering (Basel) 9, (2022). Duan, C., Wang, Z., Zhou, B. & Yao, X. Global Polyethylene Terephthalate (PET) Plastic Supply Chain Resource Metabolism Efficiency and Carbon Emissions Co-Reduction Strategies. Sustainability 16, 3926 (2024). Brivio, L., Meini, S., Sponchioni, M. & Moscatelli, D. Chemical recycling of polyethylene terephthalate (PET) to monomers: Mathematical modeling of the transesterification reaction of bis(2-hydroxyethyl) terephthalate to dimethyl terephthalate. Chem. Eng. Sci. 284, 119466 (2024). Pontrelli, S. et al. Escherichia coli as a host for metabolic engineering. Metab. Eng. 50, 16–46 (2018). Li, Y. et al. Metabolic engineering of Escherichia coli for upcycling of polyethylene terephthalate waste to vanillin. Sci. Total Environ. 957, 177544 (2024). Sadler, J. C. & Wallace, S. Microbial synthesis of vanillin from waste poly(ethylene terephthalate). Green Chem. 23, 4665–4672 (2021). Liu, X.-H. et al. Perspectives on the microorganisms with the potentials of PET-degradation. Front. Microbiol. 16, 1541913 (2025). Lv, X. et al. C1-based biomanufacturing: Advances, challenges and perspectives. Bioresour. Technol. 367, 128259 (2023). Lim, H. G. et al. Vibrio sp. dhg as a platform for the biorefinery of brown macroalgae. Nat. Commun. 10, 2486 (2019). Hyun, S. W., Krishna, S., Chau, T. H. T. & Lee, E. Y. Methanotrophs mediated biogas valorization: Sustainable route to polyhydroxybutyrate production. Bioresour. Technol. 402, 130759 (2024). Ye, D.-Y. et al. Kinetic compartmentalization by unnatural reaction for itaconate production. Nat. Commun. 13, 5353 (2022). Vuoristo, K. S. et al. Heterologous expression of Mus musculus immunoresponsive gene 1 (irg1) in Escherichia coli results in itaconate production. Front. Microbiol. 6, 849 (2015). Seo, S. W. et al. Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency. Metab. Eng. 15, 67–74 (2013). Hanko, E. K. R., Minton, N. P. & Malys, N. A Transcription Factor-Based Biosensor for Detection of Itaconic Acid. ACS Synth. Biol. 7, 1436–1446 (2018). Meyer, R. Replication and conjugative mobilization of broad host-range IncQ plasmids. Plasmid 62, 57–70 (2009). Ronda, C., Chen, S. P., Cabral, V., Yaung, S. J. & Wang, H. H. Metagenomic engineering of the mammalian gut microbiome in situ. Nat. Methods 16, 167–170 (2019). Bishé, B., Taton, A. & Golden, J. W. Modification of RSF1010-Based Broad-Host-Range Plasmids for Improved Conjugation and Cyanobacterial Bioprospecting. iScience 20, 216–228 (2019). Ferrières, L. et al. Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. J. Bacteriol. 192, 6418–6427 (2010). Simon, R., Priefer, U. & Pühler, A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat. Biotechnol. 1, 784–791 (1983). Diao, J., Hu, Y., Tian, Y., Carr, R. & Moon, T. S. Upcycling of poly(ethylene terephthalate) to produce high-value bio-products. Cell Rep. 42, 111908 (2023). Bao, T., Qian, Y., Xin, Y., Collins, J. J. & Lu, T. Engineering microbial division of labor for plastic upcycling. Nat. Commun. 14, 5712 (2023). Sasoh, M. et al. Characterization of the terephthalate degradation genes of Comamonas sp. strain E6. Appl. Environ. Microbiol. 72, 1825–1832 (2006). Hosaka, M. et al. Novel tripartite aromatic acid transporter essential for terephthalate uptake in Comamonas sp. strain E6. Appl. Environ. Microbiol. 79, 6148–6155 (2013). Banerjee, D. et al. Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale. Nat. Commun. 11, 5385 (2020). Wehrs, M. et al. Sustainable bioproduction of the blue pigment indigoidine: Expanding the range of heterologous products in R. toruloides to include non-ribosomal peptides. Green Chem. 21, 3394–3406 (2019). Wehrs, M. et al. Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae. Microb. Cell Fact. 17, 193 (2018). Lim, H. G. et al. Generation of Pseudomonas putida KT2440 Strains with Efficient Utilization of Xylose and Galactose via Adaptive Laboratory Evolution. ACS Sustain. Chem. Eng. 9, 11512–11523 (2021). Ghiffary, M. R. et al. High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes. ACS Sustain. Chem. Eng. 9, 6613–6622 (2021). Brown, N. A., Lamb, J. C., Brown, S. M. & Neal, B. H. A review of the developmental and reproductive toxicity of styrene. Regul. Toxicol. Pharmacol. 32, 228–247 (2000). Alonso‐Campos, V., Covarrubias‐García, I. & Arriaga, S. Styrene bioconversion by utilizing a non‐aqueous phase for polyhydroxyalkanoate production. J. Chem. Technol. Biotechnol. 97, 1424–1435 (2022). Tischler, D. Microbial Styrene Degradation. (Springer International Publishing, 2015). Methods Reagents and oligonucleotides Plasmid and genomic DNA were extracted using the GeneAll R Plasmid SV kit and GeneAll R Exgene TM Cell SV kit (GeneAll Biotechnology, Seoul, Korea), respectively. Q5 R High-Fidelity DNA Polymerase and PrimeSTAR R HS DNA Polymerase were purchased from New England Biolabs (Ipswich, MA, USA) and TaKaRa Bio Inc. (Otsu, Japan) for PCR. NEBuilder R HiFi DNA (Ipswich, MA, USA) assembly reagents were used for the Gibson assembly. The oligonucleotides were synthesized by Cosmo Genetech (Seoul, South Korea). All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Collected soil samples were cultured in a minimal salt medium (MSM) at pH 7.0 containing KH 2 PO 4 2.0 g, K 2 HPO 4 2.0 g, KNO 3 1.0 g, (NH 4 ) 2 SO 4 2.0 g, NaCl 0.4 g, MgSO 4 ·7H 2 O 0.4 g, CaCl 2 ·2H 2 O 0.04 g, FeSO 4 ·7H 2 O 0.01 g, 1.0 mL SL-6 trace element solution 37 , 2.0 mL of MD-VS vitamin solution (Manassas, VA, USA) per 1 L, and carbon source. Luria-Bertani (LB) medium containing 10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract was used to cultivate E. coli and transconjugants. 60 µM DAP was supplemented in the medium when E. coli S17-1 λ pir Δ asd was cultivated (Supplementary Table 1). Plasmid and strain construction All bacterial strains and plasmids used in this study are listed in Supplementary Table 1. E. coli Mach-T1 R strain (Thermo Scientific, Waltham, MA, USA) was used for routine cloning. The synthetic promoters and terminators were obtained from the Registry of Standard Biological Parts (https://parts.igem.org/Main_Page), and synthetic 5′-UTRs were computationally designed using UTR Designer (http://sbi.postech.ac.kr/utr_designer) for expression of target genes (Supplementary Table 2). PCR products treated with DpnI (Enzynomics, Daejeon, Korea) were purified using Expin™ PCR SV purification kit. (GeneAll Biotechnology, Seoul, Korea). Each fragment was assembled using NEBuilder R HiFi DNA assembly mix (New England Biolabs, Ipswich, MA, USA). The constructed plasmids were verified using Sanger sequencing. The S17 Δ asd strain was constructed by deleting the asd gene in the E. coli S17-1 λ pir strain (Supplementary Table 1) using pKD46 plasmid-based λ-red recombineering and FLP/FRT-mediated site-specific recombination techniques 38 . The gentamicin resistance gene was used as a selection marker for recombination because the parental donor strain was resistant to kanamycin and streptomycin. Recombineering products were designed to contain FRT sequences, gentamicin resistance genes, and 250 bp long homology arms on both ends of the genes. The gentamycin gene was removed from the recombinant strain by introducing the pCP20 plasmid carrying FLP recombinase. Screening novel chassis strains using the SCOUT system from environmental samples Environmental samples were collected from soil near an industrial complex in Pohang, Korea, and from a microbial septic tank for treating propylene oxide/styrene in Ulsan, Korea, to isolate TPA and styrene-converting bacteria. Initially, residual compounds were discarded by washing the samples twice with phosphate-buffered saline (PBS) at a pH of 7.0. The pellets were then inoculated in a rich LB medium to grow bacterial communities. The S17 Δ asd strains with the pSCOUT or pSENS plasmid (pSCOUT without the production module) were inoculated in the LB medium containing chloramphenicol and DAP and cultured overnight for conjugation. The donor and microbial community samples were washed twice with PBS, diluted to an optical density at 600 nm (OD 600 ) of 1.0, and mixed at a ratio of 1:1. Subsequently, 30 μL of the mixture was spotted on LB agar plates with DAP and incubated at 30°C for 24 h. The spotted microbes were scraped and washed twice with PBS. Washed pellets were spread on an LB agar plate containing antibiotics and incubated at 30°C for 24 h to remove the donor cells. Transconjugants from the selection plates were scraped off and washed twice with PBS. Washed pellets were first cultured overnight in MSM containing 2 g/L yeast extract and appropriate carbon sources. These cultures were then diluted 100-fold into fresh MSM of identical composition and incubated further until they reached an OD 600 of 1.0, at which point they were refreshed to an OD 600 of 0.05 for the main culture. After 12 h, the cells were harvested, washed twice with PBS, and diluted to an OD 600 of 0.001 for fluorescence-based sorting using a CytoFLEX SRT cell sorter (Beckman Coulter, NFEC-2024-12-301263). Blue (488 nm) laser and FITC-525A filter were used to detect sGFP fluorescence. A total of 10 5 cells displaying high fluorescence compared to the negative control (transconjugant with pSENS) were sorted in purity mode. Raw data were analyzed using the CytExpert SRT 1.2 software. Sorted cells were spread directly on MSM agar plates with TPA or styrene as the sole carbon source. To select the styrene-converting chassis, styrene-filled open-glass bottles and agar plates were sealed in zipper bags to prevent evaporation. Once colonies were visually identified, the cells were collected for the next iteration of the sorting process. Microbial diversity was preliminarily monitored by Sanger sequencing of the 16S rDNA of 16 randomly selected colonies on MSM agar plates with the corresponding carbon source; strains isolated from the same species exhibited identical 16S rDNA sequences. To validate the inducible promoters and production of indigoidine in P. postechii TPA1 and B. ulsangensis STY1, the pSCOUT plasmid was cured by repeating subcultures in antibiotic-free LB medium. Plasmids to validate inducible promoters (pRtac, pRtet, pRlac, pRara, pRT7)and indigoidine production (pIND) were transferred via conjugation to construct engineered strains using the same methods for environmental samples. Cell cultures for physiological characterization and biochemical production All cultures were performed in MSM medium in triplicate at 30°C and 200 rpm of continuous shaking, unless otherwise stated. When needed, appropriate concentrations of antibiotics were added at the start of the culture: 50 µg/mL gentamicin, 50 µg/mL kanamycin, 34 µg/mL chloramphenicol, 100 µg/mL ampicillin, 1 mM IPTG, 100 μg/L anhydrotetracycline, 10 g/L arabinose. For validation of itaconic acid-responsive biosensor, the W-SENS strain was cultured in 300-μL MSM media supplemented with 4 g/L of glucose, contained in 96-well deep well plates with an orbital shaker (900 rpm). Different concentrations of itaconic acid (0, 1, 10, 50, and 100 mg/L) were added to initiate the culture. After 6 hours, fluorescence of 100-μL cell cultures was measured. Fluorescence was measured using a VICTOR3 1420 Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA), and the specific fluorescence was calculated by dividing the calculated fluorescence by the OD 600 . For cell culture with TPA, a single colony was inoculated into 3 mL of MSM supplemented with 66 mM TPA. The overnight seed culture was refreshed with 3 mL of the same medium at an OD 600 of 0.05, and incubated until approximately reaching an OD 600 of 1. Refreshed cells were inoculated into 50 mL of the same medium in a 350 mL Erlenmeyer flask at an OD 600 of 0.05. As the pH gradually increased during the culture supplemented with TPA, 10 M HCl solution was added every 12 h to maintain a pH of 7. Cultures with volatile substrates (i.e., styrene, toluene, ethylbenzene) were performed similarly, except that 175-mL serum bottles sealed with rubber stoppers were used to prevent evaporation. Because of slow growth on the substrate, a seed culture was prepared by inoculating a single colony into 3 mL of LB medium and incubating overnight. The seed culture was inoculated into 10 mL of MSM with 1 g/L of the corresponding substrate for refreshing. The media were pre-incubated in a shaker for 12 h before inoculation to ensure sufficient dissolution. The maximum specific growth rate (μ max , h -1 ) was calculated based on the slope of the natural logarithm (ln) of the OD 600 measurements over time during exponential growth. DCW of P. postechii TPA1 was 0.515 g/L per OD 600 . The specific uptake rate was calculated by dividing the substrate consumption rate (g/L/h) by the corresponding biomass concentration (g/L) and was expressed as g substrate per g dry cell weight per hour (g/g DCW/h). Quantification of substrates and metabolites Itaconic acid titers of the filtered cell samples were measured using an Ultimate 3000 high-performance liquid chromatography system (Dionex, Sunnyvale, CA, USA). The refractive index (RI) and absorbance at a UV wavelength of 210 nm were monitored using a Shodex RI-101 detector (Shodex, Klokkerfaldet, Denmark) and a variable wavelength detector (Dionex) 16 . As a mobile phase, 5 mM H 2 SO 4 was used as the mobile phase at a flow rate of 0.6 mL/min. The temperature of the column oven was maintained at 30 °C To quantify indigoidine production, a previously reported analysis method was used 33 . 300 μL of culture medium was centrifuged at 13,0000 × g for 3 minutes, and the supernatant was removed. 100 µL of acid-washed beads (425-600 μm, Sigma-Aldrich, St. Louis, MO, USA) and 1 mL of 2% Tween 20 in DMSO were added to the tube containing cell pellets and vortexed for 10 minutes to extract indigoidine. Subsequently, the supernatant was collected, and its OD 612 was measured using a UV-1700 spectrophotometer (Shimadzu, Kyoto, Japan). A standard curve was prepared by analyzing high-purity indigoidine (Hangzhou Viablife Biotech, Hangzhou, China) dissolved in the same buffer. To quantify residual styrene, toluene, and ethylbenzene in the culture medium, 400 μL of cell culture was mixed with an equal volume of ethyl acetate containing 1-butanol (30 mg/L) as an internal standard and vortexed at 3,000 rpm for 15 minutes 39 . After centrifugation at 13,000 × g for 10 minutes, 200 μL of the ethyl acetate layer was collected and analyzed using a 6890N gas chromatograph equipped with a flame ionization detector (GC-FID, Agilent, Santa Clara, CA, USA). GC analysis was performed following a previously reported method, using a DB-Wax column (15 × 0.32 mm, 0.25 μm) and helium as the carrier gas, with a temperature gradient optimized for the separation of aromatic compounds 40 . For the HPLC measurement of TPA, the culture samples were diluted 100-fold in water to satisfy the detection limits. The analysis was performed using the Ultimate 3000 high-performance liquid chromatography system (Dionex, Sunnyvale, CA) equipped with a Polaris C18 column (4.6 × 100 mm, 2.7 μm) and UV–Vis diode array detector. TPA quantification was performed by HPLC following a previously reported method using gradient elution with acidified water and acetonitrile as the mobile phases 25 . Microbial consortia population analysis by 16S metagenomic amplicon sequencing Genomic DNA from the microbial pools or isolates was extracted using an Exgene kit (GeneAll Biotechnology, Daejeon, Korea) with lysozyme. For 16S rDNA sequencing, 27F and 1492R primers were used to amplify the V1-V9 regions. The resulting amplicons were sent to Macrogen (Daejeon, Korea) to prepare libraries for subsequent long-read sequencing 41 . The generated raw data were analyzed using a Primary Analysis software. Genome sequencing and annotation of the isolated strains Whole-genome sequencing of the isolate was performed using both long- and short-read platforms. High-fidelity (HiFi) long-read data were generated using the PacBio Sequel IIe system (Pacific Biosciences, Menlo Park, CA, USA), following the library preparation protocol provided by Macrogen (Daejeon, Korea). Genomic DNA was sheared into 6–20 kb fragments using g-TUBE (Covaris, Woburn, MA, USA), and SMRTbell libraries were constructed using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA). DNA damage and fragment ends were repaired, and SMRTbell hairpin adapters were ligated. Size selection (>10 kb) was conducted using the BluePippin system (Sage Science, Beverly, MA, USA), and libraries were purified using AMPure PB beads. De novo assembly of HiFi reads was performed using Flye v2.9 42 with the default parameters. Although HiFi reads include intrinsic error correction, further polishing was conducted using Illumina short reads to enhance the base-level accuracy. Illumina paired-end (2×150 bp) sequencing was performed using the HiSeq 2500 platform (Illumina, San Diego, CA, USA) by Macrogen. Adapter trimming and quality filtering were performed using Trimmomatic v0.33 43 , and high-quality reads were aligned to the initial assembly using BWA-MEM 44 . The assembly was iteratively polished using Pilon v1.23 45 for base correction. Assembly completeness and quality were assessed using BUSCO v5.4.3 46 on the bacteria_odb10 dataset. Genome annotation was performed using Prokka 47 for gene prediction and sequence similarity searches were conducted against bacterial databases using BLAST (https://blast.ncbi.nlm.nih.gov/). The KEGG Automatic Annotation Server (https://www.genome.jp/tools/kaas/) was used to identify putative styrene-converting genes 48 . BLAST was employed as the search program, and the bidirectional best hit (BBH) method was used for functional assignment. A comparative analysis was conducted across 12 different Brucella species using the following KEGG organism codes: oan, bmr, bcs, bov, bmt, bms, baa, bmc, bmb, and bmf. Data availability The main data supporting the findings of this study are available within the paper and its Supplementary Information. Additional data are available from the corresponding authors upon reasonable request. Methods References 37. Kim, L.-H. & Lee, S.-S. Isolation and characterization of ethylbenzene degrading Pseudomonas putida E41. J. Microbiol. 49, 575–584 (2011). 38. Murphy, K. C. & Campellone, K. G. Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol. Biol. 4, 11 (2003). 39. Kang, A. et al. Optimization of the IPP-bypass mevalonate pathway and fed-batch fermentation for the production of isoprenol in Escherichia coli. Metab. Eng. 56, 85–96 (2019). 40. Fernández-Segovia, I., Escriche, I., Gómez-Sintes, M., Fuentes, A. & Serra, J. A. Influence of different preservation treatments on the volatile fraction of desalted cod. Food Chem. 98, 473–482 (2006). 41. Rhoads, A. & Au, K. F. PacBio sequencing and its applications. Genomics Proteomics Bioinformatics 13, 278–289 (2015). 42. Kolmogorov, M., Yuan, J., Lin, Y. & Pevzner, P. A. Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 37, 540–546 (2019). 43. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014). 44. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv (2013). 45. Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9, e112963 (2014). 46. Manni, M., Berkeley, M. R., Seppey, M., Simão, F. A. & Zdobnov, E. M. BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes. Mol. Biol. Evol. 38, 4647–4654 (2021). 47. Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068–2069 (2014). 48. Nayak, T., Panda, A. N., Kumari, K., Adhya, T. K. & Raina, V. Comparative Genomics of a Paddy Field Bacterial Isolate Ochrobactrum sp. CPD-03: Analysis of Chlorpyrifos Degradation Potential. Indian J. Microbiol. 60, 325–333 (2020). Additional Declarations There is NO Competing Interest. Supplementary Files SCOUTsupple.docx Supplementary Informaton Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6811285","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Biological Sciences - Article","associatedPublications":[],"authors":[{"id":482867879,"identity":"248f32e2-15a0-44b9-ac71-0f7b568495de","order_by":0,"name":"Gyoo Yeol Jung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIiWNgGAWjYHCCBCC2gTAZG0AkG1Fa0oCYmXgtIHCYBC3yEQnPpAt+nc/j5z9/8MHPHQzy/A1saR/waTG8kZAmPbPvdrHkjGRmw94zDIYzDrAdnoFXywygFt6e24kbbjCzSTO2MTBuYGBvxuswqJZziRvOH2b/DdRiT1CLvARQC8+PA4kbDiSzMQO1JG5gYDuMV4sBz4Nka96GZJBfjCV72ySSZxxmS8ZvS3tO4m2eP3bAEDv48MPPNhvb/vY2Y/y2XMhJYAC6JwHKlwDHD35b+o8fYGD4A9cyCkbBKBgFowATAACQ4Eg3X1kLewAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-9742-3207","institution":"Pohang University of Science and Technology","correspondingAuthor":true,"prefix":"","firstName":"Gyoo","middleName":"Yeol","lastName":"Jung","suffix":""},{"id":482867880,"identity":"2e2d80a6-e64d-45b7-b716-888520942dd7","order_by":1,"name":"Min Jae Kim","email":"","orcid":"","institution":"Pohang University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Min","middleName":"Jae","lastName":"Kim","suffix":""},{"id":482867881,"identity":"4b10133e-4b07-48b9-b12d-8a177a54d7ff","order_by":2,"name":"Minsun Kim","email":"","orcid":"","institution":"Pohang University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Minsun","middleName":"","lastName":"Kim","suffix":""},{"id":482867882,"identity":"0e1a2365-a9d1-43c6-899c-afbb3f1a9064","order_by":3,"name":"Jun Hyeok Choi","email":"","orcid":"","institution":"Inha University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"Hyeok","lastName":"Choi","suffix":""},{"id":482867883,"identity":"5483413b-5699-48af-a64e-cb6ffdb087ec","order_by":4,"name":"Myung Hyun Noh","email":"","orcid":"","institution":"Korea Research Institute of Chemical Technology","correspondingAuthor":false,"prefix":"","firstName":"Myung","middleName":"Hyun","lastName":"Noh","suffix":""},{"id":482867884,"identity":"693c10c7-3638-4364-b5f7-236bc1cac5a0","order_by":5,"name":"Hyun Gyu Lim","email":"","orcid":"","institution":"Inha University","correspondingAuthor":false,"prefix":"","firstName":"Hyun","middleName":"Gyu","lastName":"Lim","suffix":""}],"badges":[],"createdAt":"2025-06-03 12:30:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6811285/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6811285/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87024141,"identity":"84cd0939-c186-4673-ad65-d05c4231c455","added_by":"auto","created_at":"2025-07-18 11:42:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":172728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the sorting process of genetically tractable chassis strain using SCOUT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe SCOUT system transfers a genetic circuit, consisting of biochemical production and sensing modules, via conjugation to screen a chassis organism under target conditions. This circuit is located on a plasmid with a broad host range of origins, selection markers, and \u003cem\u003eoriT\u003c/em\u003e, allowing efficient transfer and stable maintenance of environmental microbial consortia. Chassis candidates with successful target chemical production and the subsequent activation of fluorescent protein gene were screened.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6811285/v1/c86c89e1f86517e82ec74d67.png"},{"id":87024425,"identity":"fefa8299-350a-4de3-80c8-63d6ba503078","added_by":"auto","created_at":"2025-07-18 11:50:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":294200,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening and characterization of TPA-converting bacteria using SCOUT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e The SCOUT system to isolate a novel chassis for efficient TPA conversion. The endogenous and heterogeneous pathways of recipient cells are shown in blue and red, respectively. \u003cstrong\u003eb\u003c/strong\u003e Fluorescence changes in raw and conjugated samples during SCOUT. The fluorescence threshold for sorting is indicated by a dashed line. NC, negative control, conjugated with the pSENS plasmid (pSCOUT without the production module); Raw, initial consortium; Conj, pSCOUT-conjugated initial consortium. \u003cstrong\u003ec\u003c/strong\u003e Fermentation profile of \u003cem\u003eP. postechii \u003c/em\u003eTPA1 with pSCOUT plasmids in minimal medium supplemented with 66 mM (11 g/L) TPA. Symbols: black circle, OD\u003csub\u003e600\u003c/sub\u003e; green square, terephthalic acid (TPA); purple triangle, itaconic acid. \u003cstrong\u003ed\u003c/strong\u003e Population analysis based on 16S rDNA sequencing during chassis screening. Species comprising less than 0.2% of the total population were categorized as others.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6811285/v1/2bcfdceab09fb63aa9a0d1d3.png"},{"id":87024140,"identity":"1d26f01a-d56b-4ee4-9bf9-457038b0abd6","added_by":"auto","created_at":"2025-07-18 11:42:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":87611,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of the novel host for TPA conversion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e A schematic diagram of indigoidine production from TPA. \u003cstrong\u003eb\u003c/strong\u003e Fermentation profile of the TPA-Indigo strain in minimal medium supplemented with 66 mM TPA. Symbols: black circle, OD\u003csub\u003e600\u003c/sub\u003e; green square, terephthalic acid (TPA); blue triangle, indigoidine.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6811285/v1/66124e10c91c9daafbe715e7.png"},{"id":87024144,"identity":"e601a48a-b496-4e96-bef0-e500af37cc43","added_by":"auto","created_at":"2025-07-18 11:42:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":181114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScreening of a novel chassis for styrene conversion by SCOUT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Fluorescence changes in raw and conjugated samples during the SCOUT process. The fluorescence threshold for sorting is indicated by a dashed line. NC, negative control, conjugated with the pSENS plasmid (pSCOUT without the production module); Raw, initial consortium; Conj, pSCOUT-conjugated initial consortium. \u003cstrong\u003eb\u003c/strong\u003e Taxonomic composition of the consortium represented by the relative abundance of bacterial species. Species comprising less than 0.2% of the total population were categorized as others. \u003cstrong\u003ec \u003c/strong\u003eand \u003cstrong\u003ed\u003c/strong\u003e Fermentation profiles of the \u003cstrong\u003ec \u003c/strong\u003eSTY1-ITA and \u003cstrong\u003ed \u003c/strong\u003eSTA1-Indigo for itaconic acid and indigoidine production, respectively. Symbols: black circle, OD\u003csub\u003e600\u003c/sub\u003e; red square, styrene; purple or blue triangle; itaconic acid or indigoidine.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6811285/v1/b8c663b62f926eb118864395.png"},{"id":87025503,"identity":"16fff13c-06b9-4a57-9277-6dc9342b103c","added_by":"auto","created_at":"2025-07-18 11:58:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1562871,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6811285/v1/fb91160b-cbaa-4d49-b7e8-adc19a563bf7.pdf"},{"id":87024143,"identity":"894e3cd3-a654-475e-bd70-4bb7bc7e1b69","added_by":"auto","created_at":"2025-07-18 11:42:18","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2867131,"visible":true,"origin":"","legend":"Supplementary Informaton","description":"","filename":"SCOUTsupple.docx","url":"https://assets-eu.researchsquare.com/files/rs-6811285/v1/d65e73e32a95e025254d3e7c.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Mining Natural Microbial Diversity for Tool-Compatible Chassis Discovery","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBiorefineries convert a broad range of feedstocks into commodities and value-added products through microbial processes, thereby improving economic and environmental sustainability. While traditional feedstocks include starch crops, lignocellulosic biomass, and marine biomass\u003csup\u003e1–3\u003c/sup\u003e, recent efforts have expanded to include more non-conventional feedstocks (e.g., plastics and methane), which are abundant in both natural and industrial environments\u003csup\u003e4,5\u003c/sup\u003e. Polyethylene terephthalate (PET) is a widely used plastic with an annual global production exceeding 56 million tons annually\u003csup\u003e6\u003c/sup\u003e. However, more than 40–80% of PET waste is landfilled, creating substantial environmental burdens and emphasizing the need for microbial recycling strategies\u003csup\u003e7,8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eA major bottleneck in developing such processes lies in the limited catabolic capability of model microbial chassis, such as \u003cem\u003eEscherichia coli\u003c/em\u003e, which is poorly equipped to metabolize non-native compounds despite the availability of advanced synthetic biology toolkits\u003csup\u003e9\u003c/sup\u003e. Although the heterologous expression of catabolic pathways can partially address this gap, engineered strains often fail to achieve robust growth on unconventional substrates\u003csup\u003e10,11\u003c/sup\u003e. In contrast, naturally occurring microbes with specialized catabolic capabilities offer attractive alternatives\u003csup\u003e12–15\u003c/sup\u003e. Nonetheless, domestication of these isolates typically requires significant efforts to develop genetic tools and transformation methods. Furthermore, conventional growth-based selection strategies inherently favor fast growers but do not guarantee compatibility with the synthetic biology groundwork, limiting their utility in chassis development. Notably, among the vast diversity of environmental microbes, rare strains that simultaneously possess the ability to catabolize recalcitrant substrates and remain amenable to genetic manipulation tools established for conventional hosts may exist. Although the discovery of dual-capability microorganisms could provide an efficient and elegant solution, their presumed low abundance in natural ecosystems presents a major barrier to their identification and practical use.\u003c/p\u003e\n\u003cp\u003eHere, we present the Selection of Chassis Organisms Under Target conditions (SCOUT) as a strategy designed to streamline the discovery of genetically tractable environmental isolates under defined selection conditions. SCOUT employs the conjugative transfer of a production pathway and biosensor to environmental microbial communities, enabling the fluorescence-based selection of hosts that are catabolically active and compatible with existing synthetic biology toolkits. Using SCOUT, we isolated \u003cem\u003ePseudomonas postechii\u003c/em\u003e TPA1, which exhibited the fastest reported growth rate (0.78 h\u003csup\u003e-1\u003c/sup\u003e) on terephthalic acid (TPA). Subsequent engineering of \u003cem\u003ePseudomonas postechii \u003c/em\u003eTPA1 enabled the direct biosynthesis of the natural blue pigment indigoidine from TPA. Furthermore, SCOUT demonstrated broader applicability by isolating a genetically tractable host capable of styrene bioconversion. These results highlight SCOUT as a powerful approach for bridging natural microbial diversity and synthetic biology, thereby expanding the chassis repertoire available for sustainable biorefinery applications.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDesign of the SCOUT system to select chassis organisms under target conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe developed a SCOUT system to identify ready-to-use microbial hosts by introducing a genetic circuit on a broad-host-range conjugative plasmid (Fig. 1). The genetic circuit was designed to integrate a chemical production module and biosensor module capable of detecting the synthesized compound and displaying fluorescence as an output. These modules were assembled using Bioparts, which represent current synthetic biology standards. Itaconic acid was selected as the model chemical because it can be synthesized from \u003cem\u003ecis\u003c/em\u003e-aconitate, a conserved intermediate in the bacterial TCA cycle. To realize this design, we constructed a pSCOUT plasmid integrating both production and sensor modules, along with features for broad-host-range functionality. Specifically, the production module was constructed by placing two itaconic acid biosynthetic genes, \u003cem\u003eprpD\u003c/em\u003e\u003csup\u003eVL\u003c/sup\u003e and \u003cem\u003ecad\u003c/em\u003e\u003cem\u003e\u003csup\u003e16,17\u003c/sup\u003e\u003c/em\u003e, under the P\u003csub\u003etac\u003c/sub\u003e promoter with customized 5\u0026apos;-untranslated regions for strong expression\u003csup\u003e18\u003c/sup\u003e. For itaconic acid sensing, an expression cassette encoding the itaconic acid-specific transcription factor ItcR\u003csup\u003e16,19\u003c/sup\u003e and superfolder green fluorescent protein (sGFP) was introduced. ItcR was expressed constitutively under the P\u003csub\u003eJ23106\u003c/sub\u003e promoter, whereas sGFP expression was driven by the P\u003csub\u003eccl\u003c/sub\u003e promoter, which was activated upon ItcR binding to itaconic acid. We confirmed that fluorescence could be observed using \u003cem\u003eE. coli\u003c/em\u003e as a host in the presence of at least 1 mg/L itaconic acid (Supplementary Fig. 1). To ensure broad host applicability, the plasmid backbone incorporated an RSF origin of replication\u003csup\u003e20\u0026ndash;22\u003c/sup\u003e and was engineered for conjugative transfer\u003csup\u003e23,24\u003c/sup\u003e, resulting in the construction of the pSCOUT plasmid (Supplementary Table 1).Screening of a novel chassis with efficient terephthalic acid catabolism using the SCOUT system\u003c/p\u003e\n\u003cp\u003eWe used SCOUT to screen for a microbial chassis capable of metabolizing terephthalic acid (TPA) in environmental samples. A soil sample was collected near an industrial plant, where TPA-catabolizing bacteria were likely present (Fig. 2a). The pSCOUT plasmid was introduced into the microbial pool via conjugation using an \u003cem\u003eE. coli \u003c/em\u003eS-17 ∆\u003cem\u003easd\u003c/em\u003e mutant for auxotrophic counterselection on diaminopimelic acid (DAP, see Methods). Following conjugation, fluorescence-positive cells were isolated using fluorescence-activated cell sorting (Fig. 2b). In particular, a gating strategy was applied to exclude cells that displayed background fluorescence with erroneous activation of the biosensor module using a control plasmid lacking the production module (NC). The sorted populations were grown in TPA-supplemented medium, and the cultures were subjected to another round of fluorescence-based sorting.\u003c/p\u003e\n\u003cp\u003eThe SCOUT strategy enriched the microbial population with enhanced fluorescence, leading to the isolation of a novel \u003cem\u003ePseudomonas\u003c/em\u003e strain with superior TPA catabolic capability (Fig. 2b). After one round of sorting, the population showed a noteworthy increase in fluorescence (1R) compared with the initial pools (Raw and Conj). Further sorting appeared unnecessary because the fluorescence plateaued in the second round (2R). Sanger sequencing of the 16S rDNA region of 16 random colonies revealed the presence of two \u003cem\u003eCitrobacter\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e species. Among these two strains, only the \u003cem\u003ePseudomonas\u003c/em\u003e isolates grew on TPA as the sole carbon source, successfully producing 31 mg/L of itaconic acid (Supplementary Fig. 2). We named this strain \u003cem\u003ePseudomonas postechii\u003c/em\u003e TPA1 (hereafter referred to as TPA1), and determined it to be a novel chassis for TPA conversion. Metagenomic 16S rDNA sequencing tracked changes in microbial diversity during the screening process (Fig. 2c). Bacteria belonging to 14 and 11 genera were detected in the original sample and the conjugant pool, respectively. While TPA1 initially constituted less than 0.4% of the community, its proportion increased to 32.7% after the two rounds of sorting, supporting that the SCOUT strategy is highly effective in isolating a novel chassis. In contrast, although the \u003cem\u003eCitrobacter \u003c/em\u003estrain was still dominant in the sorted pools, it did not grow on TPA as the sole carbon source, likely proliferating via yeast extract or metabolic byproducts. \u003c/p\u003e\n\u003cp\u003eWe also performed the conventional growth-based enrichment of a potential host from the same sample to compare the two strategies. Interestingly, after multiple serial cultures, we only obtained \u003cem\u003eAcinetobacter\u003c/em\u003e species, which took 10.5% of the original pool (Fig. 2c). This strain rapidly dominated on TPA as the sole carbon source, likely because of its slightly faster growth rate than TPA1 (1.1-fold, Supplementary Fig. 3). However, this \u003cem\u003eAcinetobacter\u003c/em\u003e strain failed to maintain diverse plasmids or be genetically manipulated via conjugation or transformation, highlighting the advantage of SCOUT in isolating genetically tractable hosts, even when they initially exist at low abundance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysiological and genomic characterization of \u003cem\u003eP. postechii \u003c/em\u003eTPA1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe physiology of TPA1 was further characterized in a TPA-supplemented minimal medium by measuring the growth rate and tolerance levels at different concentrations. When 44 mM of TPA was supplemented, TPA1 displayed an exceptionally high maximum growth rate (\u0026mu;\u003csub\u003emax\u003c/sub\u003e = 0.78 h\u003csup\u003e-1\u003c/sup\u003e) and a specific TPA uptake rate of 2.03 g per g dry cell weight per h (g/g DCW/h) (Supplementary Fig. 4). This growth rate is superior to that of previously reported genetically tractable strains (Supplementary Table 3). Although the growth rate tended to decrease as the concentration increased, TPA1 displayed robust growth up to 200 mM TPA, outperforming engineered strains that often show impaired growth at such concentrations\u003csup\u003e5,25,26\u003c/sup\u003e. These observations highlight the potential of microbial diversity as a source of novel chassis strains.\u003c/p\u003e\n\u003cp\u003eWhole-genome sequencing analysis was also performed to investigate the genomic features for efficient TPA conversion. Combined long-read and short-read sequencing analysis revealed that the 6.35-Mb genome comprised a large chromosome and two plasmids (5.74-, 0.34-, 0.26-Mb, Supplementary Fig. 5). Sequence alignment was performed using BLAST against the TPA catabolic genes reported in \u003cem\u003eComamonas\u003c/em\u003e sp. E6, a well-characterized TPA-consuming bacterium\u003csup\u003e27\u003c/sup\u003e (Fig. 2 and Supplementary Fig. 6). It was found that TPA1 possesses similar enzymes (TPA dioxygenase reductase TphA1, TPA dioxygenase component TphA2, TPA dioxygenase component TphA3, TPA dihydrodiol dehydrogenase TphB) for TPA assimilation, with protein identities ranging 40~70% compared to \u003cem\u003eCommamonas\u003c/em\u003e sp. E6. Genes encoding these enzymes form a 7.5 kb-long \u003cem\u003etph\u003c/em\u003e operon, together with additional genes putatively encoding the TPA transporter PcaK and transcriptional regulator PcaR. Interestingly, compared to \u003cem\u003eComamonas\u003c/em\u003e species and another TPA-consuming \u003cem\u003ePseudomonas\u003c/em\u003e strain with these genes on their chromosomes, TPA1 possesses these genes in its plasmid, potentially explaining its superior catabolic efficiency for TPA. Furthermore, a copy of \u003cem\u003etphA1\u003c/em\u003e was detected on the chromosome, potentially augmenting TPA catabolism. Additionally, the TpiAB-TphC transporter system\u003csup\u003e28\u003c/sup\u003e was not detected in TPA1 cells. Although the exact mechanism remains unclear, TPA appears to be efficiently imported into TPA1, possibly via PcaK, other transporters with broad substrate specificity, or as-yet-unidentified novel transporter.\u003c/p\u003e\n\u003cp\u003eLastly, we analyzed the housekeeping sigma factor \u0026sigma;\u003csup\u003e70\u003c/sup\u003e (encoded by \u003cem\u003erpoD\u003c/em\u003e) of TPA1. Regions critical for -10 and -35 promoter recognition showed 100% and 96.4% sequence identity, respectively, with those of \u003cem\u003eE. coli\u003c/em\u003e (Supplementary Fig. 7), which likely allowed the cross-species compatibility of synthetic promoters. Functional validation with \u003cem\u003eE. coli\u003c/em\u003e-optimized inducible systems (P\u003csub\u003etac\u003c/sub\u003e, P\u003csub\u003etet\u003c/sub\u003e, P\u003csub\u003elac\u003c/sub\u003e, P\u003csub\u003eT7\u003c/sub\u003e, and P\u003csub\u003eara\u003c/sub\u003e) confirmed robust and controllable gene expression in TPA1 (Supplementary Fig. 8), supporting its immediate compatibility with standard synthetic biology parts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduction of indigoidine from TPA by engineering the TPA1 strain\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFinally, the potential of TPA1 as a chassis protein was validated by introducing a heterologous biosynthetic pathway. We selected indigoidine as the model chemical because it is an industrially relevant natural blue pigment that can bypass the severe water pollution caused by conventional dye production\u003csup\u003e29\u0026ndash;32\u003c/sup\u003e. It is converted from L-glutamine by the apo-form of BpsA, a non-ribosomal peptide synthetase, which is catalytically activated via post-translational modification by Sfp, 4\u0026prime;-phosphopantetheinyl transferase. For efficient indigoidine production, TPA1 was engineered to express the \u003cem\u003ebpsA\u003c/em\u003e-\u003cem\u003esfp\u003c/em\u003e operon using strong promoters and synthetic untranslated regions from the plasmid\u003csup\u003e33\u003c/sup\u003e. The resulting TPA-indigo strain (Supplementary Table 1) achieved a notable titer of up to 505 mg/L without an apparent growth burden (Fig. 3c). To our knowledge, this is the first demonstration of the direct bioconversion of TPA to indigoidine, establishing TPA1 as a versatile synthetic biology chassis for plastic upcycling and bioremediation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExtension of the SCOUT system for screening a chassis for styrene conversion \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo expand the applicability of the SCOUT system, we explored its potential for isolating genetically tractable styrene-converting microorganisms (Fig. 4). Styrene, a monomer widely used in plastic and synthetic rubber industries, poses significant environmental concerns owing to its toxicity and persistence\u003csup\u003e34\u003c/sup\u003e. Although several styrene-converting bacteria and their related pathways have been reported, the strains amenable to genetic engineering are limited to a few \u003cem\u003ePseudomonas\u003c/em\u003e species\u003csup\u003e35,36\u003c/sup\u003e. The SCOUT was applied to the sludge sample collected from propylene oxide/styrene monomer (PO/SM) processing facilities and environments enriched with styrene and its intermediates. The microbial consortia from PO/SM aeration tanks are expected to harbor diverse strains that metabolize styrene. Through the SCOUT sorting process, we identified two \u003cem\u003eBrucella\u003c/em\u003e species as promising candidates, exhibiting both styrene-metabolic activity and genetic tractability (Fig. 4a, 4b). Further detailed comparisons revealed that one strain\u003cem\u003e \u003c/em\u003eexhibited a superior growth rate, higher styrene conversion efficiency, and greater itaconic acid production than the other strains (Fig. 4c and Supplementary Fig. 9). Therefore, we chose this strain as the chassis and named it \u003cem\u003eBrucella ulsangensis\u003c/em\u003e STY1.\u003c/p\u003e\n\u003cp\u003eWhole-genome sequencing yielded a total genome length of 4.83-Mb, comprising two chromosomes (2.81-Mb and 2.02-Mb) (Supplementary Fig. 10\u003cstrong\u003e)\u003c/strong\u003e. Based on the sequencing data, gene functions were annotated; however, not all genes encoding the full styrene-converting pathway were identified. Instead, KEGG Automatic Annotation Server was used to annotate orthologous genes that were expected to be involved in the conversion. A putative styrene-converting gene, catechol 2,3-dioxygenase, was identified by comparison with 12 other \u003cem\u003eBrucella\u003c/em\u003e species (Supplementary Fig. 11). Sequence analysis of the housekeeping sigma factor (\u0026sigma;\u003csup\u003e70\u003c/sup\u003e) encoded by \u003cem\u003erpoD\u003c/em\u003e showed high conservation with \u003cem\u003eE. coli\u003c/em\u003e, with 100% and 89.3% identity at the -10 (region 2.4) and -35 (region 4.2) promoter recognition sites, respectively (Supplementary Fig. 12). This suggests that \u003cem\u003eE. coli\u003c/em\u003e-optimized synthetic promoters are functional in STY1.\u003c/p\u003e\n\u003cp\u003eTo validate its genetic tractability, we used synthetic genetic parts and production plasmids previously developed for TPA-converting strains. Using these tools, we successfully engineered \u003cem\u003eB. ulsangensis\u003c/em\u003e STY1 and confirmed its capacity to induce gene expression and biosynthetic production. Controlled gene expression was achieved by introducing conventional inducible promoters and their cognate transcription factors (Supplementary Fig. 13). Moreover, \u003cem\u003eB. ulsangensis\u003c/em\u003e STY1 successfully expressed heterologous pathways, producing indigoidine using styrene as the sole carbon source (Fig. 4d), further confirming its potential as an industrially relevant host.\u003c/p\u003e\n"},{"header":"Discussion","content":"\n\u003cp\u003eDespite the superior ability of non-conventional strains to metabolize substrates, such as TPA and styrene, their limited genetic tractability has hindered their widespread use in industrial biorefineries. Therefore, identifying a microbial chassis that combines high metabolic efficiency and genetic accessibility remains a key challenge. Among the diverse microbial species present in natural environments, certain strains may possess the dual capability of utilizing substrates recalcitrant to conventional hosts while remaining compatible with existing genetic engineering tools. However, the presumed low abundance of these strains in nature presents a significant obstacle to their discovery. In this study, we present an efficient high-throughput screening strategy, SCOUT, which integrates biosensor-guided selection with substrate-specific enrichment. By applying fluorescence-based cell sorting and carbon-source-limited growth selection, we successfully isolated two promising chassis strains, \u003cem\u003eP. postechii\u0026nbsp;\u003c/em\u003eTPA1 and \u003cem\u003eB. ulsangensis\u0026nbsp;\u003c/em\u003eSTY1, which exhibited robust assimilation of TPA\u0026nbsp;and styrene, respectively. Notably, TPA1 demonstrated a significantly higher rate of TPA consumption than previously reported wild-type strains, highlighting its potential as an efficient host for plastic-derived carbon bioconversion. Traditional methods for identifying new chassis strains often rely on sequential screening steps, first for substrate assimilation, followed by the validation of genetic compatibility, resulting in time-consuming and low-throughput workflows. In contrast, the SCOUT system streamlines this process by directly linking metabolic activity to genetic responsiveness via a biosensor-based reporting circuit, allowing simultaneous selection of both traits.\u003c/p\u003e\n\u003cp\u003eA key feature of our approach is the use of a broad-host-range conjugative plasmid, which enables efficient horizontal gene transfer to diverse bacterial hosts. We used the RP4 conjugation system, which facilitates plasmid mobilization across diverse microbes. This compatibility minimizes the exclusion of potentially valuable strains that might otherwise be overlooked because of their low transformation efficiency. Furthermore, the plasmid design includes a dual-module circuit,\u0026nbsp;comprising a biosensor and a production pathway that ensures fluorescence is triggered only when both modules are functional, thereby reducing false-positive signals. In this study, itaconic acid production served as a robust readout of the central carbon flux, as it\u0026nbsp;was synthesized from \u003cem\u003ecis\u003c/em\u003e-aconitate, a conserved TCA cycle intermediate. Importantly, no known bacterial strains naturally produce itaconic acid, making the detection highly specific to engineered pathways. The biosensor \u003cem\u003eitcR\u003c/em\u003e exhibited excellent sensitivity, with a detection threshold in the single-digit micromolar range and high specificity, allowing clear discrimination from related metabolites.\u003c/p\u003e\n\u003cp\u003eThe strains isolated using the SCOUT system were compatible with standard synthetic biology components, including inducible promoters and synthetic UTRs, suggesting that widely used regulatory elements in \u003cem\u003eE. coli\u003c/em\u003e may also be applicable to these non-model organisms. This compatibility opens the door for the further engineering of these strains with a level of flexibility comparable to that of traditional chassis strains. In addition to identifying metabolically specialized strains, the SCOUT platform has the potential\u0026nbsp;to screen chassis for other industrially relevant traits, such as acid or solvent tolerance and thermophilicity. Moreover, the metabolic pathways and enzymes characterized in this study may provide insights into non-conventional substrate utilization mechanisms and support the discovery of novel regulatory factors. By integrating biosensor-based screening with consortium engineering, our strategy offers a scalable and adaptable platform for discovering novel microbial hosts, thereby accelerating the development of next-generation biorefineries.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Korea Institute of Marine Science and Technology Promotion (KIMST), funded by the Ministry of Oceans and Fisheries (grant number RS-2022-KS221581). This work was also supported by the National Research Foundation of\u003c/p\u003e\n\u003cp\u003eKorea (NRF) grants (RS-2024-00334792, RS-2025-02215308, RS-2024-00400033, RS-2024-00453085) funded by the Ministry of Science and ICT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.J.K, M.K,\u0026nbsp;H.G.L., and G.Y.J. contributed to the conception of the project.\u0026nbsp;M.J.K, M.K.\u0026nbsp;contributed to the design and conduction of the experiments.\u0026nbsp;M.J.K, M.K., J.H.C, M.H.N., H.G.L., and G.Y.J. contributed to data analysis, interpretation and writing of the manuscript.\u0026nbsp;H.G.L. and G.Y.J. supervised the project. All the authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Information\u0026nbsp;\u003c/strong\u003eis available for this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrespondence and requests for materials\u0026nbsp;\u003c/strong\u003eshould be addressed to H.G.L and G.Y.J.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeer review information\u0026nbsp;\u003c/strong\u003e\u003cem\u003eNature\u0026nbsp;\u003c/em\u003ethanks XXX and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReprints and permissions information\u0026nbsp;\u003c/strong\u003eis available at http://www.nature.com/reprints.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLong, B. et al. Engineering strategies to optimize lignocellulosic biorefineries. Nat. Rev. Bioeng. (2024).\u003c/li\u003e\n\u003cli\u003eQiao, J. et al. Integrated biorefinery approaches for the industrialization of cellulosic ethanol fuel. Bioresour. Technol. 360, 127516 (2022).\u003c/li\u003e\n\u003cli\u003eBaghel, R. S. Developments in seaweed biorefinery research: A comprehensive review. Chemical Engineering Journal 140177 (2022).\u003c/li\u003e\n\u003cli\u003eDiao, J., Tian, Y., Hu, Y. \u0026amp; Moon, T. S. Producing multiple chemicals through biological upcycling of waste poly(ethylene terephthalate). Trends Biotechnol. 43, 620\u0026ndash;646 (2025).\u003c/li\u003e\n\u003cli\u003eWerner, A. Z. et al. Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to \u0026beta;-ketoadipic acid by Pseudomonas putida KT2440. Metab. Eng. 67, 250\u0026ndash;261 (2021).\u003c/li\u003e\n\u003cli\u003eSoong, Y.-H. V., Sobkowicz, M. J. \u0026amp; Xie, D. Recent advances in biological recycling of polyethylene terephthalate (PET) plastic wastes. Bioengineering (Basel) 9, (2022).\u003c/li\u003e\n\u003cli\u003eDuan, C., Wang, Z., Zhou, B. \u0026amp; Yao, X. Global Polyethylene Terephthalate (PET) Plastic Supply Chain Resource Metabolism Efficiency and Carbon Emissions Co-Reduction Strategies. Sustainability 16, 3926 (2024).\u003c/li\u003e\n\u003cli\u003eBrivio, L., Meini, S., Sponchioni, M. \u0026amp; Moscatelli, D. Chemical recycling of polyethylene terephthalate (PET) to monomers: Mathematical modeling of the transesterification reaction of bis(2-hydroxyethyl) terephthalate to dimethyl terephthalate. Chem. Eng. Sci. 284, 119466 (2024).\u003c/li\u003e\n\u003cli\u003ePontrelli, S. et al. Escherichia coli as a host for metabolic engineering. Metab. Eng. 50, 16\u0026ndash;46 (2018).\u003c/li\u003e\n\u003cli\u003eLi, Y. et al. Metabolic engineering of Escherichia coli for upcycling of polyethylene terephthalate waste to vanillin. Sci. Total Environ. 957, 177544 (2024).\u003c/li\u003e\n\u003cli\u003eSadler, J. C. \u0026amp; Wallace, S. Microbial synthesis of vanillin from waste poly(ethylene terephthalate). Green Chem. 23, 4665\u0026ndash;4672 (2021).\u003c/li\u003e\n\u003cli\u003eLiu, X.-H. et al. Perspectives on the microorganisms with the potentials of PET-degradation. Front. Microbiol. 16, 1541913 (2025).\u003c/li\u003e\n\u003cli\u003eLv, X. et al. C1-based biomanufacturing: Advances, challenges and perspectives. Bioresour. Technol. 367, 128259 (2023).\u003c/li\u003e\n\u003cli\u003eLim, H. G. et al. Vibrio sp. dhg as a platform for the biorefinery of brown macroalgae. Nat. Commun. 10, 2486 (2019).\u003c/li\u003e\n\u003cli\u003eHyun, S. W., Krishna, S., Chau, T. H. T. \u0026amp; Lee, E. Y. Methanotrophs mediated biogas valorization: Sustainable route to polyhydroxybutyrate production. Bioresour. Technol. 402, 130759 (2024).\u003c/li\u003e\n\u003cli\u003eYe, D.-Y. et al. Kinetic compartmentalization by unnatural reaction for itaconate production. Nat. Commun. 13, 5353 (2022).\u003c/li\u003e\n\u003cli\u003eVuoristo, K. S. et al. Heterologous expression of Mus musculus immunoresponsive gene 1 (irg1) in Escherichia coli results in itaconate production. Front. Microbiol. 6, 849 (2015).\u003c/li\u003e\n\u003cli\u003eSeo, S. W. et al. Predictive design of mRNA translation initiation region to control prokaryotic translation efficiency. Metab. Eng. 15, 67\u0026ndash;74 (2013).\u003c/li\u003e\n\u003cli\u003eHanko, E. K. R., Minton, N. P. \u0026amp; Malys, N. A Transcription Factor-Based Biosensor for Detection of Itaconic Acid. ACS Synth. Biol. 7, 1436\u0026ndash;1446 (2018).\u003c/li\u003e\n\u003cli\u003eMeyer, R. Replication and conjugative mobilization of broad host-range IncQ plasmids. Plasmid 62, 57\u0026ndash;70 (2009).\u003c/li\u003e\n\u003cli\u003eRonda, C., Chen, S. P., Cabral, V., Yaung, S. J. \u0026amp; Wang, H. H. Metagenomic engineering of the mammalian gut microbiome in situ. Nat. Methods 16, 167\u0026ndash;170 (2019).\u003c/li\u003e\n\u003cli\u003eBish\u0026eacute;, B., Taton, A. \u0026amp; Golden, J. W. Modification of RSF1010-Based Broad-Host-Range Plasmids for Improved Conjugation and Cyanobacterial Bioprospecting. iScience 20, 216\u0026ndash;228 (2019).\u003c/li\u003e\n\u003cli\u003eFerri\u0026egrave;res, L. et al. Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. J. Bacteriol. 192, 6418\u0026ndash;6427 (2010).\u003c/li\u003e\n\u003cli\u003eSimon, R., Priefer, U. \u0026amp; P\u0026uuml;hler, A. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nat. Biotechnol. 1, 784\u0026ndash;791 (1983).\u003c/li\u003e\n\u003cli\u003eDiao, J., Hu, Y., Tian, Y., Carr, R. \u0026amp; Moon, T. S. Upcycling of poly(ethylene terephthalate) to produce high-value bio-products. Cell Rep. 42, 111908 (2023).\u003c/li\u003e\n\u003cli\u003eBao, T., Qian, Y., Xin, Y., Collins, J. J. \u0026amp; Lu, T. Engineering microbial division of labor for plastic upcycling. Nat. Commun. 14, 5712 (2023).\u003c/li\u003e\n\u003cli\u003eSasoh, M. et al. Characterization of the terephthalate degradation genes of Comamonas sp. strain E6. Appl. Environ. Microbiol. 72, 1825\u0026ndash;1832 (2006).\u003c/li\u003e\n\u003cli\u003eHosaka, M. et al. Novel tripartite aromatic acid transporter essential for terephthalate uptake in Comamonas sp. strain E6. Appl. Environ. Microbiol. 79, 6148\u0026ndash;6155 (2013).\u003c/li\u003e\n\u003cli\u003eBanerjee, D. et al. Genome-scale metabolic rewiring improves titers rates and yields of the non-native product indigoidine at scale. Nat. Commun. 11, 5385 (2020).\u003c/li\u003e\n\u003cli\u003eWehrs, M. et al. Sustainable bioproduction of the blue pigment indigoidine: Expanding the range of heterologous products in R. toruloides to include non-ribosomal peptides. Green Chem. 21, 3394\u0026ndash;3406 (2019).\u003c/li\u003e\n\u003cli\u003eWehrs, M. et al. Production efficiency of the bacterial non-ribosomal peptide indigoidine relies on the respiratory metabolic state in S. cerevisiae. Microb. Cell Fact. 17, 193 (2018).\u003c/li\u003e\n\u003cli\u003eLim, H. G. et al. Generation of Pseudomonas putida KT2440 Strains with Efficient Utilization of Xylose and Galactose via Adaptive Laboratory Evolution. ACS Sustain. Chem. Eng. 9, 11512\u0026ndash;11523 (2021).\u003c/li\u003e\n\u003cli\u003eGhiffary, M. R. et al. High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes. ACS Sustain. Chem. Eng. 9, 6613\u0026ndash;6622 (2021).\u003c/li\u003e\n\u003cli\u003eBrown, N. A., Lamb, J. C., Brown, S. M. \u0026amp; Neal, B. H. A review of the developmental and reproductive toxicity of styrene. Regul. Toxicol. Pharmacol. 32, 228\u0026ndash;247 (2000).\u003c/li\u003e\n\u003cli\u003eAlonso‐Campos, V., Covarrubias‐Garc\u0026iacute;a, I. \u0026amp; Arriaga, S. Styrene bioconversion by utilizing a non‐aqueous phase for polyhydroxyalkanoate production. J. Chem. Technol. Biotechnol. 97, 1424\u0026ndash;1435 (2022).\u003c/li\u003e\n\u003cli\u003eTischler, D. Microbial Styrene Degradation. (Springer International Publishing, 2015).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Methods","content":"\u003ch2\u003eReagents and oligonucleotides\u003c/h2\u003e\n\u003cp\u003ePlasmid and genomic DNA were extracted using the GeneAll\u003csup\u003eR\u003c/sup\u003e Plasmid SV kit and GeneAll\u003csup\u003eR\u003c/sup\u003e Exgene\u003csup\u003eTM\u003c/sup\u003e\u003csub\u003e\u0026nbsp;\u003c/sub\u003eCell SV kit (GeneAll Biotechnology, Seoul, Korea), respectively. Q5\u003csup\u003eR\u003c/sup\u003e High-Fidelity DNA Polymerase and PrimeSTAR\u003csup\u003eR\u003c/sup\u003e HS DNA Polymerase were purchased from New England Biolabs (Ipswich, MA, USA) and TaKaRa Bio Inc. (Otsu, Japan) for PCR. NEBuilder\u003csup\u003eR\u003c/sup\u003e HiFi DNA (Ipswich, MA, USA) assembly reagents were used for the Gibson assembly. The oligonucleotides were synthesized by Cosmo Genetech (Seoul, South Korea). All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Collected soil samples were cultured in a minimal salt medium (MSM) at pH 7.0 containing KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 2.0 g, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 2.0 g, KNO\u003csub\u003e3\u003c/sub\u003e 1.0 g, (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u0026nbsp;\u003c/sub\u003e2.0 g, NaCl 0.4 g, MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO 0.4 g, CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO 0.04 g, FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e\u0026nbsp;\u003c/sub\u003e0.01 g, 1.0 mL SL-6 trace element solution\u003csup\u003e37\u003c/sup\u003e, 2.0 mL of MD-VS vitamin solution (Manassas, VA, USA) per 1 L, and carbon source. Luria-Bertani (LB) medium containing 10 g/L tryptone, 10 g/L NaCl, and 5 g/L yeast extract was used to cultivate \u003cem\u003eE. coli\u003c/em\u003e and transconjugants. 60 \u0026micro;M DAP was supplemented in the medium when \u003cem\u003eE. coli\u003c/em\u003e S17-1 \u0026lambda; pir \u0026Delta;\u003cem\u003easd\u0026nbsp;\u003c/em\u003ewas cultivated (Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid and strain construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll bacterial strains and plasmids used in this study are listed in Supplementary Table 1. \u003cem\u003eE. coli\u003c/em\u003e Mach-T1\u003csup\u003eR\u003c/sup\u003e strain (Thermo Scientific, Waltham, MA, USA) was used for routine cloning. The synthetic promoters and terminators were obtained from the Registry of Standard Biological Parts (https://parts.igem.org/Main_Page), and synthetic 5\u0026prime;-UTRs were computationally designed using UTR Designer (http://sbi.postech.ac.kr/utr_designer) for expression of target genes (Supplementary Table 2). PCR products treated with DpnI (Enzynomics, Daejeon, Korea) were purified using Expin\u0026trade; PCR SV purification kit. (GeneAll Biotechnology, Seoul, Korea). Each fragment was assembled using NEBuilder\u003csup\u003eR\u003c/sup\u003e HiFi DNA assembly mix (New England Biolabs, Ipswich, MA, USA). The constructed plasmids were verified using Sanger sequencing.\u003c/p\u003e\n\u003cp\u003eThe S17 \u0026Delta;\u003cem\u003easd\u0026nbsp;\u003c/em\u003estrain was constructed by deleting the \u003cem\u003easd\u003c/em\u003e gene in the \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eS17-1 \u0026lambda; pir strain (Supplementary Table 1) using pKD46 plasmid-based \u0026lambda;-red recombineering and FLP/FRT-mediated site-specific recombination techniques\u003csup\u003e38\u003c/sup\u003e. The gentamicin resistance gene was used as a selection marker for recombination because the parental donor strain was resistant to kanamycin and streptomycin. Recombineering products were designed to contain FRT sequences, gentamicin resistance genes, and 250 bp long homology arms on both ends of the genes. The gentamycin gene was removed from the recombinant strain by introducing the pCP20 plasmid carrying FLP recombinase.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening novel chassis strains using the SCOUT system from environmental samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEnvironmental samples were collected from soil near an industrial complex in Pohang, Korea, and from a microbial septic tank for treating propylene oxide/styrene in Ulsan, Korea, to isolate TPA and styrene-converting bacteria. Initially, residual compounds were discarded by washing the samples twice with phosphate-buffered saline (PBS) at a pH of 7.0. The pellets were then inoculated in a rich LB medium to grow bacterial communities. The S17 \u0026Delta;\u003cem\u003easd\u003c/em\u003e strains with the pSCOUT or pSENS plasmid (pSCOUT without the production module) were inoculated in the LB medium containing chloramphenicol and DAP and cultured overnight for conjugation. The donor and microbial community samples were washed twice with PBS, diluted to an optical density at 600 nm (OD\u003csub\u003e600\u003c/sub\u003e) of 1.0, and mixed at a ratio of 1:1. Subsequently, 30 \u0026mu;L of the mixture was spotted on LB agar plates with DAP and incubated at 30\u0026deg;C for 24 h. The spotted microbes were scraped and washed twice with PBS. Washed pellets were spread on an LB agar plate containing antibiotics and incubated at 30\u0026deg;C for 24 h to remove the donor cells.\u003c/p\u003e\n\u003cp\u003eTransconjugants from the selection plates were scraped off and washed twice with PBS. Washed pellets were first cultured overnight in MSM containing 2 g/L yeast extract and appropriate carbon sources. These cultures were then diluted 100-fold into fresh MSM of identical composition and incubated further until they reached an OD\u003csub\u003e600\u003c/sub\u003e of 1.0, at which point they were refreshed to an OD\u003csub\u003e600\u003c/sub\u003e of 0.05 for the main culture. After 12 h, the cells were harvested, washed twice with PBS, and diluted to an OD\u003csub\u003e600\u003c/sub\u003e of 0.001 for fluorescence-based sorting using a CytoFLEX SRT cell sorter (Beckman Coulter, NFEC-2024-12-301263). Blue (488 nm) laser and FITC-525A filter were used to detect sGFP fluorescence. A total of 10\u003csup\u003e5\u003c/sup\u003e cells displaying high fluorescence compared to the negative control (transconjugant with pSENS) were sorted in purity mode. Raw data were analyzed using the CytExpert SRT 1.2 software.\u003c/p\u003e\n\u003cp\u003eSorted cells were spread directly on MSM agar plates with TPA or styrene as the sole carbon source. To select the styrene-converting chassis, styrene-filled open-glass bottles and agar plates were sealed in zipper bags to prevent evaporation. Once colonies were visually identified, the cells were collected for the next iteration of the sorting process. Microbial diversity was preliminarily monitored by Sanger sequencing of the 16S rDNA of 16 randomly selected colonies on MSM agar plates with the corresponding carbon source; strains isolated from the same species exhibited identical 16S rDNA sequences.\u003c/p\u003e\n\u003cp\u003eTo validate the inducible promoters and production of indigoidine in \u003cem\u003eP. postechii\u0026nbsp;\u003c/em\u003eTPA1 and \u003cem\u003eB. ulsangensis\u0026nbsp;\u003c/em\u003eSTY1, the pSCOUT plasmid was cured by repeating subcultures in antibiotic-free LB medium. Plasmids to validate inducible promoters (pRtac, pRtet, pRlac, pRara, pRT7)and indigoidine production (pIND) were transferred via conjugation to construct engineered strains using the same methods for environmental samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell cultures for physiological characterization and biochemical production\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll cultures were performed in MSM medium in triplicate at 30\u0026deg;C and 200 rpm of continuous shaking, unless otherwise stated. When needed, appropriate concentrations of antibiotics were added at the start of the culture: 50 \u0026micro;g/mL gentamicin, 50 \u0026micro;g/mL kanamycin, 34 \u0026micro;g/mL chloramphenicol, 100 \u0026micro;g/mL ampicillin, 1 mM IPTG, 100 \u0026mu;g/L anhydrotetracycline, 10 g/L arabinose.\u003c/p\u003e\n\u003cp\u003eFor validation of itaconic acid-responsive biosensor, the W-SENS strain was cultured in 300-\u0026mu;L MSM media supplemented with 4 g/L of glucose, contained in 96-well deep well plates with an orbital shaker (900 rpm). Different concentrations of itaconic acid (0, 1, 10, 50, and 100 mg/L) were added to initiate the culture. After 6 hours, fluorescence of 100-\u0026mu;L cell cultures was measured. Fluorescence was measured using a VICTOR3 1420 Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA), and the specific fluorescence was calculated by dividing the calculated fluorescence by the OD\u003csub\u003e600\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eFor cell culture with TPA, a single colony was inoculated into 3 mL of MSM supplemented with 66 mM TPA. The overnight seed culture was refreshed with 3 mL of the same medium at an OD\u003csub\u003e600\u003c/sub\u003e of 0.05, and incubated until approximately reaching an OD\u003csub\u003e600\u0026nbsp;\u003c/sub\u003eof 1. Refreshed cells were inoculated into 50 mL of the same medium in a 350 mL Erlenmeyer flask at an OD\u003csub\u003e600\u003c/sub\u003e of 0.05. As the pH gradually increased during the culture supplemented with TPA, 10 M HCl solution was added every 12 h to maintain a pH of 7. Cultures with volatile substrates (i.e., styrene, toluene, ethylbenzene) were performed similarly, except that 175-mL serum bottles sealed with rubber stoppers were used to prevent evaporation. Because of slow growth on the substrate, a seed culture was prepared by inoculating a single colony into 3 mL of LB medium and incubating overnight. The seed culture was inoculated into 10 mL of MSM with 1 g/L of the corresponding substrate for refreshing. The media were pre-incubated in a shaker for 12 h before inoculation to ensure sufficient dissolution. The maximum specific growth rate (\u0026mu;\u003csub\u003emax\u003c/sub\u003e, h\u003csup\u003e-1\u003c/sup\u003e) was calculated based on the slope of the natural logarithm (ln) of the OD\u003csub\u003e600\u003c/sub\u003e measurements over time during exponential growth. DCW of \u003cem\u003eP. postechii\u0026nbsp;\u003c/em\u003eTPA1 was 0.515 g/L per OD\u003csub\u003e600\u003c/sub\u003e. The specific uptake rate was calculated by dividing the substrate consumption rate (g/L/h) by the corresponding biomass concentration (g/L) and was expressed as g substrate per g dry cell weight per hour (g/g DCW/h).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of substrates and metabolites\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eItaconic acid titers of the filtered cell samples were measured using an Ultimate 3000 high-performance liquid chromatography system (Dionex, Sunnyvale, CA, USA). The refractive index (RI) and absorbance at a UV wavelength of 210\u0026thinsp;nm were monitored using a Shodex RI-101 detector (Shodex, Klokkerfaldet, Denmark) and a variable wavelength detector (Dionex)\u003csup\u003e16\u003c/sup\u003e. As a mobile phase, 5\u0026thinsp;mM H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was used as the mobile phase at a flow rate of 0.6\u0026thinsp;mL/min. The temperature of the column oven was maintained at 30\u0026thinsp;\u0026deg;C\u003c/p\u003e\n\u003cp\u003eTo quantify indigoidine production, a previously reported analysis method was used\u003csup\u003e33\u003c/sup\u003e. 300 \u0026mu;L of culture medium was centrifuged at 13,0000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 3 minutes, and the supernatant was removed. 100 \u0026micro;L of acid-washed beads (425-600 \u0026mu;m, Sigma-Aldrich, St. Louis, MO, USA) and 1 mL of 2% Tween 20 in DMSO were added to the tube containing cell pellets and vortexed for 10 minutes to extract indigoidine. Subsequently, the supernatant was collected, and its OD\u003csub\u003e612\u003c/sub\u003e was measured using a UV-1700 spectrophotometer (Shimadzu, Kyoto, Japan). A standard curve was prepared by analyzing high-purity indigoidine (Hangzhou Viablife Biotech, Hangzhou, China) dissolved in the same buffer.\u003c/p\u003e\n\u003cp\u003eTo quantify residual styrene, toluene, and ethylbenzene in the culture medium, 400 \u0026mu;L of cell culture was mixed with an equal volume of ethyl acetate containing 1-butanol (30 mg/L) as an internal standard and vortexed at 3,000 rpm for 15 minutes\u003csup\u003e39\u003c/sup\u003e. After centrifugation at 13,000 \u0026times; \u003cem\u003eg\u003c/em\u003e for 10 minutes, 200 \u0026mu;L of the ethyl acetate layer was collected and analyzed using a 6890N gas chromatograph equipped with a flame ionization detector (GC-FID, Agilent, Santa Clara, CA, USA). GC analysis was performed following a previously reported method, using a DB-Wax column (15 \u0026times; 0.32 mm, 0.25 \u0026mu;m) and helium as the carrier gas, with a temperature gradient optimized for the separation of aromatic compounds\u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eFor the HPLC measurement of TPA, the culture samples were diluted 100-fold in water to satisfy the detection limits. The analysis was performed using the Ultimate 3000 high-performance liquid chromatography system (Dionex, Sunnyvale, CA) equipped with a Polaris C18 column (4.6 \u0026times; 100 mm, 2.7 \u0026mu;m) and UV\u0026ndash;Vis diode array detector. TPA quantification was performed by HPLC following a previously reported method using gradient elution with acidified water and acetonitrile as the mobile phases\u003csup\u003e25\u003c/sup\u003e.\u003c/p\u003e\n\u003ch2\u003eMicrobial consortia population analysis by 16S metagenomic amplicon sequencing\u003c/h2\u003e\n\u003cp\u003eGenomic DNA from the microbial pools or isolates was extracted using an Exgene kit (GeneAll Biotechnology, Daejeon, Korea) with lysozyme. For 16S rDNA sequencing, 27F and 1492R primers were used to amplify the V1-V9 regions. The resulting amplicons were sent to Macrogen (Daejeon, Korea) to prepare libraries for subsequent long-read sequencing\u003csup\u003e41\u003c/sup\u003e. The generated raw data were analyzed using a Primary Analysis software.\u003c/p\u003e\n\u003ch2\u003eGenome sequencing and annotation of the isolated strains\u003c/h2\u003e\n\u003cp\u003eWhole-genome sequencing of the isolate was performed using both long- and short-read platforms. High-fidelity (HiFi) long-read data were generated using the PacBio Sequel IIe system (Pacific Biosciences, Menlo Park, CA, USA), following the library preparation protocol provided by Macrogen (Daejeon, Korea). Genomic DNA was sheared into 6\u0026ndash;20 kb fragments using g-TUBE (Covaris, Woburn, MA, USA), and SMRTbell libraries were constructed using the SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA). DNA damage and fragment ends were repaired, and SMRTbell hairpin adapters were ligated. Size selection (\u0026gt;10 kb) was conducted using the BluePippin system (Sage Science, Beverly, MA, USA), and libraries were purified using AMPure PB beads.\u003c/p\u003e\n\u003cp\u003eDe novo assembly of HiFi reads was performed using Flye v2.9\u003csup\u003e42\u003c/sup\u003e with the default parameters. Although HiFi reads include intrinsic error correction, further polishing was conducted using Illumina short reads to enhance the base-level accuracy. Illumina paired-end (2\u0026times;150 bp) sequencing was performed using the HiSeq 2500 platform (Illumina, San Diego, CA, USA) by Macrogen. Adapter trimming and quality filtering were performed using Trimmomatic v0.33\u003csup\u003e43\u003c/sup\u003e, and high-quality reads were aligned to the initial assembly using BWA-MEM\u003csup\u003e44\u003c/sup\u003e. The assembly was iteratively polished using Pilon v1.23\u003csup\u003e45\u003c/sup\u003e for base correction.\u003c/p\u003e\n\u003cp\u003eAssembly completeness and quality were assessed using BUSCO v5.4.3\u003csup\u003e46\u003c/sup\u003e on the \u003cem\u003ebacteria_odb10\u003c/em\u003e dataset. Genome annotation was performed using Prokka\u003csup\u003e47\u003c/sup\u003e for gene prediction and sequence similarity searches were conducted against bacterial databases using BLAST (https://blast.ncbi.nlm.nih.gov/).\u003c/p\u003e\n\u003cp\u003eThe KEGG Automatic Annotation Server (https://www.genome.jp/tools/kaas/) was used to identify putative styrene-converting genes\u003csup\u003e48\u003c/sup\u003e. BLAST was employed as the search program, and the bidirectional best hit (BBH) method was used for functional assignment. A comparative analysis was conducted across 12 different \u003cem\u003eBrucella\u003c/em\u003e species using the following KEGG organism codes: oan, bmr, bcs, bov, bmt, bms, baa, bmc, bmb, and bmf.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe main data supporting the findings of this study are available within the paper and its Supplementary Information. Additional data are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods References\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e37. Kim, L.-H. \u0026amp; Lee, S.-S. Isolation and characterization of ethylbenzene degrading Pseudomonas putida E41. J. Microbiol. 49, 575\u0026ndash;584 (2011).\u003c/p\u003e\n\u003cp\u003e38. Murphy, K. C. \u0026amp; Campellone, K. G. Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli. BMC Mol. Biol. 4, 11 (2003).\u003c/p\u003e\n\u003cp\u003e39. Kang, A. et al. Optimization of the IPP-bypass mevalonate pathway and fed-batch fermentation for the production of isoprenol in Escherichia coli. Metab. Eng. 56, 85\u0026ndash;96 (2019).\u003c/p\u003e\n\u003cp\u003e40. Fern\u0026aacute;ndez-Segovia, I., Escriche, I., G\u0026oacute;mez-Sintes, M., Fuentes, A. \u0026amp; Serra, J. A. Influence of different preservation treatments on the volatile fraction of desalted cod. Food Chem. 98, 473\u0026ndash;482 (2006).\u003c/p\u003e\n\u003cp\u003e41. Rhoads, A. \u0026amp; Au, K. F. PacBio sequencing and its applications. Genomics Proteomics Bioinformatics 13, 278\u0026ndash;289 (2015).\u003c/p\u003e\n\u003cp\u003e42. Kolmogorov, M., Yuan, J., Lin, Y. \u0026amp; Pevzner, P. A. Assembly of long, error-prone reads using repeat graphs. Nat. Biotechnol. 37, 540\u0026ndash;546 (2019).\u003c/p\u003e\n\u003cp\u003e43. Bolger, A. M., Lohse, M. \u0026amp; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114\u0026ndash;2120 (2014).\u003c/p\u003e\n\u003cp\u003e44. Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv (2013).\u003c/p\u003e\n\u003cp\u003e45. Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9, e112963 (2014).\u003c/p\u003e\n\u003cp\u003e46. Manni, M., Berkeley, M. R., Seppey, M., Sim\u0026atilde;o, F. A. \u0026amp; Zdobnov, E. M. BUSCO Update: Novel and Streamlined Workflows along with Broader and Deeper Phylogenetic Coverage for Scoring of Eukaryotic, Prokaryotic, and Viral Genomes. Mol. Biol. Evol. 38, 4647\u0026ndash;4654 (2021).\u003c/p\u003e\n\u003cp\u003e47. Seemann, T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 30, 2068\u0026ndash;2069 (2014).\u003c/p\u003e\n\u003cp\u003e48. Nayak, T., Panda, A. N., Kumari, K., Adhya, T. K. \u0026amp; Raina, V. Comparative Genomics of a Paddy Field Bacterial Isolate Ochrobactrum sp. CPD-03: Analysis of Chlorpyrifos Degradation Potential. Indian J. Microbiol. 60, 325\u0026ndash;333 (2020).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6811285/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6811285/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe development of microbial bioprocesses for unconventional feedstocks requires chassis organisms capable of efficiently catabolizing complex substrates. However, model organisms, such as \u003cem\u003eEscherichia coli\u003c/em\u003e, cannot inherently metabolize non-native compounds, and engineered pathways often result in poor growth on such substrates. Here, we present the Selection of Chassis Organisms Under Target conditions (SCOUT), a strategy for rapidly identifying genetically tractable environmental isolates that are compatible with existing synthetic biology tools. Using SCOUT, we isolated \u003cem\u003ePseudomonas postechii\u003c/em\u003e TPA1, a novel chassis exhibiting the fastest reported growth rate (0.78 h\u003csup\u003e-1\u003c/sup\u003e) on terephthalic acid (TPA) to date. Engineering \u003cem\u003eP. postechii\u003c/em\u003e TPA1 enabled direct biosynthesis of 505 mg/L of the natural blue pigment indigoidine from TPA. SCOUT further demonstrates its versatility by enabling the isolation of a ready-to-engineer host for styrene bioconversion. These results establish SCOUT as a powerful platform for expanding the microbial chassis diversity and accelerating the development of sustainable biorefinery processes.\u003c/p\u003e","manuscriptTitle":"Mining Natural Microbial Diversity for Tool-Compatible Chassis Discovery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-18 11:42:14","doi":"10.21203/rs.3.rs-6811285/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-microbiology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nmicrobiol","sideBox":"Learn more about [Nature Microbiology](http://www.nature.com/nmicrobiol/)","snPcode":"","submissionUrl":"","title":"Nature Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"a20b8c74-fff6-4acb-960c-5632aebb5242","owner":[],"postedDate":"July 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":51270480,"name":"Biological sciences/Systems biology/Synthetic biology"},{"id":51270481,"name":"Biological sciences/Biotechnology/Metabolic engineering"}],"tags":[],"updatedAt":"2025-07-18T11:42:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-18 11:42:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6811285","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6811285","identity":"rs-6811285","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}