Hybrid metabolic engineering enables xylose-driven co-production of polyhydroxybutyrate and violacein in Escherichia coli.

Hybrid metabolic engineering enables xylose-driven co-production of polyhydroxybutyrate and violacein in Escherichia coli - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice J Biol Eng . 2026 Mar 9;20:66. doi: 10.1186/s13036-026-00653-w Search in PMC Search in PubMed View in NLM Catalog Add to search Hybrid metabolic engineering enables xylose-driven co-production of polyhydroxybutyrate and violacein in Escherichia coli Khoi Nhat Pham Khoi Nhat Pham 1 Department of Chemical Engineering (BK21 FOUR Integrated Engineering), Kyung Hee University, Yongin-si, Gyeonggi-do 17104 Republic of Korea Find articles by Khoi Nhat Pham 1 , Eun Yeol Lee Eun Yeol Lee 1 Department of Chemical Engineering (BK21 FOUR Integrated Engineering), Kyung Hee University, Yongin-si, Gyeonggi-do 17104 Republic of Korea Find articles by Eun Yeol Lee 1, ✉ Author information Article notes Copyright and License information 1 Department of Chemical Engineering (BK21 FOUR Integrated Engineering), Kyung Hee University, Yongin-si, Gyeonggi-do 17104 Republic of Korea ✉ Corresponding author. Received 2026 Jan 27; Accepted 2026 Mar 2; Collection date 2026. © The Author(s) 2026 Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/ . PMC Copyright notice PMCID: PMC13081634  PMID: 41796373 Abstract Background Within lignocellulosic biomass, xylose is the second most abundant sugar after glucose. As a renewable and sustainable substrate, it is gaining attention as a feedstock for microbial bioprocesses. In this study, we demonstrated the co-production of polyhydroxybutyrate (PHB) and violacein from xylose. We initially confirmed the feasibility of co-production through genome-scale metabolic simulations, followed by optimization using a hybrid expression system that combines a conventional tac promoter and synthetic promoter-ribosome-binding site-terminator (semi-endo PRT) elements in dual plasmids. Additionally, we assessed the antimicrobial activity of violacein against type I methanotrophs. Results Recombinant Escherichia coli DH5α harboring a hybrid system produced 111.3 ± 19.7 and 0.88 ± 0.23 mg/L of PHB and violacein, respectively, in M9 using xylose as the sole carbon source, without tryptophan supplementation. Using the synthetic PRT system, 528.9 ± 104 mg/g dry cell weight (DCW) of PHB was obtained. Additionally, violacein inhibits the growth of Methylomicrobium alcaliphilum 20Z at 9 µg/mL in nitrate mineral salt medium containing methanol as the sole carbon source. Conclusions The use of lignocellulose-derived sugars for co-production offers an environmentally sustainable bio-manufacturing approach that contributes to greenhouse gas mitigation and supports the transition toward a circular bioeconomy. Supplementary Information The online version contains supplementary material available at 10.1186/s13036-026-00653-w. Keywords: Lignocellulosic xylose, Co-production, Polyhydroxybutyrate, Violacein, E. coli , Methanotroph Background Xylose is the second most abundant sugar after glucose in plant-derived lignocellulosic biomass and represents a primary constituent of the most plentiful renewable feedstock for microbial bioprocessing [ 1 ]. Lignocellulosic biomass is an eco-friendly non-food-competing alternative to fossil resources because it is synthesized using solar energy and carbon dioxide fixation [ 2 ]. Xylose has been widely used for the microbial production of lactic acid [ 3 ], succinic acid [ 4 ], 2,3-butanediol [ 5 , 6 ], polyhydroxyalkanoate (PHAs) [ 7 ], isobutanol, and other alcohols [ 8 ], and xylitol [ 9 ]. Co-production strategies in bacteria have been demonstrated, such as the production of ethanol and xylitol from sugarcane bagasse as a xylose-containing feedstock [ 10 ], co-production of polyhydroxybutyrate (PHB) and violacein from glucose using the tryptophan-supplying strain Iodobacter sp. PCH194 [ 11 ]. However, the xylose utilization of Iodobacter sp. PCH194 has not mentioned. Although the co-production of PHB and deoxyviolacein has been demonstrated in an Escherichia coli host system [ 12 ], glucose was used as a carbon source in this study. Co-production offers several advantages, including enhanced carbon flux utilization, improved redox balance, and increased economic value per fermentation batch, particularly when high-value compounds such as violacein are produced. As an example, the co-production of cadaverine and succinic acid in Escherichia coli using a dynamic regulation strategy achieved titers of 55.58 g/L and 28.39 g/L, respectively, in a 5-L fermenter [ 13 ]. Co-production facilitates the redistribution of metabolic flux, thereby channeling carbon substrates into two value-added products rather than converting them into CO 2 or overflow metabolites such as acetate. Accumulation of acetate derived from acetyl-CoA is known to reduce growth rates at high concentrations [ 14 ]. Thus, redirecting carbon flux from acetyl-CoA toward PHB biosynthesis may mitigate this effect. Overexpression of NADH oxidase has been shown to decrease acetate formation and improve biomass accumulation, indicating that NADH accumulation promotes acetate overflow and impairs growth efficiency [ 15 ]. Introducing NADH-consuming pathways, such as the violacein biosynthetic pathway, may therefore contribute to improved redox balance while simultaneously enhancing target product formation. Overall, this co-production strategy improves carbon conversion efficiency and increases cumulative product yield. Several studies have demonstrated the use of xylose in E. coli for producing value-added biochemicals, including xylonate [ 16 ] and ethanol [ 17 ]. Additional research has elucidated the role of xylR transcription factor in carbon-source adaptation-converting glucose to xylose [ 18 ] and engineering an E. coli strain to co-utilize glucose and xylose simultaneously. E. coli was unable to metabolize both glucose and xylose simultaneously because of carbon catabolite repression [ 19 ]. E. coli remains a preferred microbial host because of its rapid growth in inexpensive media and available genetic tools. Using xylose-assimilating E. coli strains enables the use of extensive genetic engineering toolkits, contributing to sustainable biomanufacturing strategies that mitigate climate change by using renewable biomass resources. Recently, artificial intelligence (AI)-based tools have been developed to predict promoter [ 20 ], ribosome-binding sites (RBS) [ 21 ], and terminator (PRT) system [ 22 ] to enhance protein expression efficiency in bacteria. Optimizing the expression system is essential for co-producing exogenous genes in bacteria. Certain co-production strategies were introduced using dual plasmids—production of virion-like reverse transcriptase [ 23 ], copy-number balancing using low- and high-copy-number plasmids to produce naringenin [ 24 ], a dual antibiotic-free plasmid system based on the toxin-antitoxin system to produce L-fucose [ 25 ], co-expression in one plasmid (pQLink co-expression plasmids) [ 26 ], mono- or bicistronic vector-based strategies for producing cellobiose [ 27 ], pathway optimization by tuning antibiotic concentrations [ 28 ], and two inducible prokaryotic expression promoters, pdMAX [ 29 ]. In this study, we co-produced PHB and violacein, beginning with genome-scale metabolic model (GSMM) predictions to assess production feasibility, followed by cultivation experiments and optimization of the co-expression system using novel sequences for the PRT system. The recombinant strain produced 25% PHB and 0.2% violacein (g product/g DCW), achieving titers of 111.3 ± 19.7 and 0.88 ± 0.23 mg/L for PHB and violacein, respectively, in M9 medium containing xylose as the sole carbon source, without tryptophan supplementation, after 42 h of cultivation. Using the synthesized PRT expression system, over 60% of PHB was produced at 100 µg/mL spectinomycin. Additionally, the antimicrobial activity of violacein was confirmed in type I methanotrophs that used methanol as the sole carbon source at 9 µg/mL. Methanol helps distinguish solvent-induced cytotoxic effects from the antimicrobial activity of violacein because other organisms, such as E. coli , lack this metabolic capability. To our knowledge, this study expands current efforts in microbial co-production by demonstrating the co-production of PHB and violacein from xylose. Furthermore, it offers a preliminary evaluation of violacein activity against methanotrophs. Methods Synthetic semi-endo PRT system The genome sequence of E. coli K12 MG1655 ( NC_000913 ) in FASTA and GBK formats was downloaded from NCBI and used to predict PRT. Promoters and terminators were predicted using PROMOTECH v1.0 [ 30 ] and iTerm-PseKNC [ 22 ], respectively. RBS sequences were selected by 20 nucleotides upstream of the starting codons and subsequently analyzed for conserved motifs. MEME version 5.1.1 was used for motif discovery and sequence searching [ 31 , 32 ]. De Novo DNA [ 33 ] and RBSDesigner [ 34 ] were used to calculate transcription and translation rates, respectively. Seqfold [ 35 ] and Forgi [ 36 ] analyzed RNA secondary structure and predicted the lowest free-energy structure of nucleic acids. The final PRT sequence was synthesized using oligo primers to construct the plasmid. The detailed process was described in a previous study [ 37 ]. GSMM analysis The GSMM iEC1368 model was downloaded from the BiGG database and used for analysis. Violacein and PHB pathway reactions were added to iEC1368 and renamed iEC1368_5alphaXvioPHB. Metabolite data for these reactions were obtained from the SBO, KEGG, ChEBI, PubChem, and MetaCyc databases. The iEC1368 5alphaXvioPHB model was assessed using Memote [ 38 ]. The calculation, simulation, and optimization of carbon flux were performed using COBRApy [ 39 ], Cnapy [ 40 ], and Cameo [ 41 ], respectively. Flux balance analysis [ 42 ] and flux variability analysis (FVA) [ 43 ] were used to simulate the carbon flux in the model. The xylose uptake flux was set to -3 mmol/g DCW/h, based on approximately 50% of wild-type E. coli (6.45 mmol/g DCW/h) [ 44 ] or adaptive-evolved E. coli (0.8 g xylose/g DCW/h; 5.3 mmol/g DCW/h) strain [ 45 ] because the recombinant strain can reduce the uptake rate, such as 28% to 4.65 mmol/g DCW/h [ 44 ]. Chemicals and reagents Sodium (S)-3-hydroxybutyrate (Santa Cruz Biotechnology, Dallas, TX, USA) was used as a high-performance liquid chromatography standard. Xylose was purchased from Glentham (UK). Methanol (molecular biology grade; Merck) was used as the carbon source. Luria-Bertani (LB) powder was provided by FORMEDIUM. Other chemicals were sourced from Sigma-Aldrich (Steinheim, Germany) or TCI (Tokyo, Japan). Oligonucleotides were synthesized by Macrogen (Seoul, South Korea). Polymerase chain reaction (PCR) reagents were obtained from BioFACT (Daejeon, South Korea). The Expin™ Gel SV Kit (GeneAll, Seoul, South Korea) and Exprep Plasmid SV were (GenAll, Seoul, South Korea) were used for gel extraction, DNA purification, and plasmid isolation. Gibson assembly reaction was performed following the protocol of manufacturer (ClonExpress Ultra One-step Cloning Kit, Vazyme). Bacterial strains The bacterial strains and plasmids used in this study are listed in Tables 1 and 2 . Table 1. Bacterial strains were used for this experiment Strain Relevant characteristics Reference E. coli DH5α Host strain Novagen E. coli p89-empty E. coli DH5α harbored pAWP89 without the gene This research E. coli p89-ptac-sfGFP E. coli DH5α expressed pAWP89 with tac promoter, ribosome binding site of pAWP89, and sfGFP gene This research E. coli p89-PRT-sfGFP E. coli DH5α expressed pAWP89 with a synthetic promoter, ribosome binding site, and terminator (PRT E ) and sfGFP gene This research E. coli p89-PRT-phaCAB E. coli DH5α expressed pAWP89 with a synthetic promoter, ribosome binding site, and terminator (PRT E ) system and the cluster phaCAB gene for PHB biosynthesis This research E. coli Ptac-vio-Kan R E. coli DH5α expressed pAWP89 with tac promoter, ribosome binding site of pAWP89, and vioABCDE cluster gene, growing on Kanamycin This research E. coli Spec R -ptac-CAB E. coli DH5α expressed pAWP89-Spectinomycin with tac promoter and cluster phaCAB gene. This research E. coli Kan R -ptac-CAB E. coli DH5α expressed pAWP89-Kanamycin with tac promoter and cluster phaCAB gene. This research E. coli Amp R -PRT-CAB E. coli DH5α expressed pAWP89-Ampicillin with synthetic PRT and cluster phaCAB gene. This research E. coli Spec R -PRT-CAB E. coli DH5α expressed pAWP89-Spectinomycin with synthetic PRT and cluster phaCAB gene. This research E. coli Ptac-vio-PRT-CAB E. coli DH5α expressed pAWP89-kanamycin with Ptac-p89RBS- vioABCDE and pAWP89-spectinomycin with synthetic PRT and phaCAB cluster gene. This research M. alcaliphilum 20Z Methylotuvimicrobium alcaliphilum 20Z, wild-type strain for antimicrobial test DMSZ Open in a new tab Table 2. Plasmids were used for this experiment Name Relevant characteristics reference pAWP89 Expression vector, tac promoter, kan R This research pAWP89-empty pAWP89 without promoter, RBS, and gene used as control This research pAWP89-Ptac- sfGFP pAWP89 with tac promoter and sfGFP gene in E. coli with Kan R gene for selection, in FACS assay This research pAWP89-PRT E - sfGFP pAWP89 with PRT system to express sfGFP gene in E. coli , construction with spectinomycin R gene for selection, in FACS assay This research pAWP89-ptac- phaCAB-Kan R pAWP89 with tac promoter with phaCAB cluster constructed with Spectinomycin resistance gene, in the optimization of the PHB biosynthesis experiment This research pAWP89-ptac- phaCAB-Spec R pAWP89 with tac promoter with phaCAB cluster constructed with Spectinomycin resistance gene, in the optimization of the PHB biosynthesis experiment This research pAWP89-Amp R -PRT E - phaCAB pAWP89 with a synthetic PRT system with phaCAB cluster constructed with an Ampicillin resistance gene, in the optimization of the PHB biosynthesis experiment. This research pAWP89-Spec R -PRT E - phaCAB pAWP89 with synthetic PRT system to express phaCAB gene in E. coli , construction with Spectinomycin resistance gene for selection, in optimization of PHB biosynthesis experiment, and co-production of violacein and PHB biosynthesis experiment. This research pAWP89-Ptac- vioABCDE-Kan R pAWP89 with tac promoter and available RBS in pAWP89 to express vioABCDE cluster genes in E. coli , construction with Kanamycin resistance gene for selection, in the co-production of violacein and PHB biosynthesis experiment. [ 47 ] Open in a new tab Media and culture conditions E. coli DH5α and recombinant strains were grown in 10 mL Falcon tubes on LB, LB supplemented with kanamycin (50 mg/L), and LB + spectinomycin (final concentration 100 mg/L) for screening and assessment. To screen the recombinant E. coli strain, LB agar supplemented with 1.5% (w/v) agar and appropriate antibiotics (50 or 100 mg/L of kanamycin or spectinomycin, respectively) were used. All cultures were incubated at 37 °C with shaking at 200 rpm. Minimal media consisted of standard M9 media supplemented with 2 mM Mg 2 SO 4 , 0.1 mM CaCl 2 , standard trace elements, and vitamins, including 4.5 µM folic acid, 15 µM thiamin HCl, 10 µM Ca pantothenate, 0.07 µM vitamin B12, 13 µM riboflavin, 41 µM nicotinamide, and 10 g/L xylose. Methylotuvimicrobium alcaliphilum 20Z wildtype was cultured in nitrate mineral salt medium supplemented with 5 mM Na 2 CO 3 , 45 mM NaHCO 3 , phosphate buffer (0.8 mM KH 2 PO 4 /0.8 mM Na 2 HPO 4 .7H 2 O), 1% (v/v) methanol, and 9 µg/mL standard/or extracted violacein at 30 °C and 200 rpm. The wild-type E. coli DH5α strain was cultured in M9 medium containing 10 g/L xylose as the sole carbon source, with 9 µg/mL standard violacein or crude violacein extract added to the antibacterial assay at 37 °C in a shaking incubator at 200 rpm. Cloning, transformation, and expression The synthetic semi-endo PRT system, designed for gene expression in E. coli , was incorporated into an oligonucleotide primer, synthesized, amplified by PCR, and ligated into the vector using standard ligation protocols. Superfolder green fluorescent protein (sfGFP) gene was assembled under the control of the tac promoter and synthetic semi-endo PRT system in the pAWP89 plasmid. The recombinant vector was transformed into E. coli DH5α using a heat shock method. phaCAB genes from Ralstonia. eutropha H16 were cloned into the pAWP89 vector using a synthetic semi-endo PRT system. Semi-endo PRT sequences were synthesized by Macrogen (Seoul, South Korea) and inserted into oligo primers, followed by ligation with the target gene and pAWP89 vector using a commercial Cloning Kit. The ligated plasmid was amplified in E. coli DH5α, extracted, and re-transformed for further assessment. Violacein cluster genes were obtained from previous studies. Extraction of crude violacein Recombinant E. coli strains were cultured in LB or M9 media containing 10 g/L xylose. The cell pellet was harvested by centrifugation (7,000 × g, 7 min, 4 °C), resuspended in 300–400 µL of 100% methanol, and incubated at 37 °C with shaking on an Eppendorf ThermoMixer C for 30 min. The mixture was centrifuged (7000 × g, 7 min, 4 °C) and transferred to a fresh 1.5 mL microcentrifuge tube. This extraction step was repeated thrice to obtain 1–1.2 mL of crude violacein extract. Subsequently, the extract was dried in an incubator at 50 °C, as previously described [ 46 , 47 ], and freeze-dried for 30–60 min. The dried crude violacein was weighed and redissolved in 100% methanol for subsequent experiments. Flow cytometry analysis Single-cell fluorescence was quantified using an Attune ® NxT flow cytometer (Thermo Fisher Scientific, USA) equipped with a 488 nm blue laser to excite the sfGFP. Positive transformants were cultured in 10 mL of selective medium for 24 h, harvested, washed once with phosphate-buffered saline (PBS), and resuspended in 1 mL of PBS. Cell concentrations were measured and diluted to an optical density (OD600) of 0.5 in 0.5 mL. For each biological replicate, 10,000 events from the defined single-cell population (gate R1) were collected under flow cytometry settings of forward scatter (FSC) at 300 V and side scatter at 300 V. Simultaneously, all other detector voltages were maintained at the default value of 400 V. Signal acquisition was performed at a flow rate of 12.5 µL/min, with a total sampling volume of 40 µL. Data acquisition and gating analyses were performed using the Attune ® Cytometric Software suite. The mean fluorescence intensity of the designated fluorescence-positive subpopulation (gate R2) was quantitatively extracted to represent the sfGFP expression levels in each sample. Analysis and method detection using gas chromatograph (GC) To measure the PHB content, cells were collected after culturing in M9 with xylose as the sole carbon source or in LB media and freeze-dried for 4–12 h. Methanolysis was performed by incubating the dried biomass at approximately 100 °C for 8–12 h with 2 mL chloroform and 2 mL methanol (containing 15% v/v H 2 SO 4 ), with 20 mg of benzoic acid added as an internal standard. After adding 2 mL of H 2 O and vortexing, the organic phase was collected, filtered through a 0.2 μm polytetrafluoroethylene membrane (Agilent), and 1 µL of the sample was injected into a GC for PHA analysis (GC; Agilent 8890). Analysis and method detection using spectrophotometer The extracted crude violacein was measured using a Synergy HTX multi-mode reader at absorbance of 570 nm, as previously described [ 47 ]. The concentration of crude violacein was calculated using a standard curve generated using standard violacein. Visualization of sfGFP using UV light Colonies with sfGFP plasmids were visualized using UV light. Fluorescence was detected using a WiseUV WUV-M20 transilluminator (Daihan, Korea) at 312 nm wavelength. Statistical method In this study, values were calculated as the means of biological triplicates to assess the statistical significance between the control and experimental samples. Error bars represent the standard deviation. Statistical significance was determined using Student’s t-test, with p-values of < 0.05, 0.01, 0.001, and 0.0001 considered significant. Results Confirmation of co-production capability for violacein and PHB using GSMM To assess the feasibility of co-producing the compounds, we used GSMM using the iEC1368_DH5a [ 48 ] model. However, iEC1368_DH5a lacked reactions for violacein and PHB biosynthesis. The metabolites and reactions for these pathways were added to the model (Tables S2 and S3 ) and renamed iEC1368_DH5aXvioPHB. The xylose uptake rate was set at -3 mmol/g DCW/h, corresponding to 50% of the value reported in previous studies, to account for a reduction of approximately 28% caused by the introduction of novel pathways [ 44 ]. All other carbon sources were constrained to zero because xylose was the sole carbon source. FVA revealed that 0–0.222 mmol/g DCW/h PHB can produce and react with tryptophan to form indole-3-pyruvic acid imine at 0–0.517 mmol/g DCW/h, the first step in violacein synthesis. FVA provides the maximum and minimum possible fluxes for each reaction. In this simulation, we assumed that 1–2% of the maximum tryptophan flux can be converted into indole-3-pyruvic acid imine. Additionally, 50% of the maximum 3HB-coA flux can convert 3-hydroxybutanoyl-CoA into PHB. All selected values were calculated from experimental data on the co-production of violacein and PHB using glucose as the sole carbon source [ 11 , 12 ]. These reactions were set to 0.01 and 0.111 mmol/g DCW/h, respectively (Fig. 1 and Fig. S1 ). In silico calculations estimated a growth rate of 0.114 h − 1 , with 0.005 and 0.111 mmol/g DCW/h of violacein and PHB, respectively. This simulation revealed that violacein and PHB can be co-produced in E. coli DH5α using xylose as the sole carbon source, derived from two precursors (acetyl-CoA and tryptophan). However, the growth rate was slower than that with glucose as a carbon source, at 0.982 and 0.114 (h − 1 ) in the iEC1368_DH5a and iEC1368_DH5aXvioPHB models, respectively. In FBA method, biomass flux in GSMM is interpreted as the predicted specific growth rate. Within the defined constraint boundaries of two reactions, flux distributions were sampled under the condition that the biomass reaction flux was fixed to match the experimentally determined specific growth rate of E. coli grown in minimal medium with xylose as the sole carbon. The simulation with 0.01 and 0.111 mmol/g DCW/h of VIOATRP and PHB rxn in Fig. S1 resulted growth rate of 0.114 h − 1 , consistency to experimental value 0.14 h − 1 in E. coli [ 44 ]. This study also demonstrated that E. coli exhibits a lower specific growth rate under aerobic conditions in M9 minimal medium with xylose as the sole carbon source compared to glucose (0.35 h⁻¹) in the same medium. Simulation results further indicated that the introduction of the violacein and PHB biosynthetic pathways into the E. coli model did not substantially affect the predicted growth rate. Therefore, we constructed and optimized co-expression systems in E. coli DH5α to enable the co-production of violacein and PHB. Fig. 1. Open in a new tab Simulation of carbon flux in co-producing polyhydroxybutyrate (PHB) and violacein using xylose as the sole carbon source. The xylose uptake rate is set to -3.00 mmol/g dry cell weight/h (mmol/gDCW/h). The genome-scale metabolic model iEC1368 and flux balance analysis are used for this simulation. Ru5P: Ribulose-5-phosphate, Xu5P: xylulose-5-phosphate, E4P: erythrose-4-phosphate, 6PGL: 6-phospho-glucono-1,5-lactone, G6P: glucose-6-phosphate, F6P: fructose-6-phosphate, G3P: glyceraldehyde-3-phosphate, S7P: sedoheptulose-7-phosphate, 3PG: 3-phospho-glycerate, 2PG: 2-phospho-glycerate, PEP: phosphoenolpyruvate, 2DDA7P: 2-dehydro-3-deoxy-arabino-heptonate-7-phosphate, 3DHQ: 3-dehydroquinate, 3DHSK: 3-dehydroshikimate, SKM5P: shikimate-5-phosphate, 3PSME: 3-phosphoshikimate, PRAN: 5-phospho-ribosyl-anthranilate, 2CPR5P: 2-carboxyphenylamino-1-deoxy-ribulose-5-phosphate, 3IG3P: 3-indolyl-glycerol-3-phosphate, TRP: tryptophan, IPA-IMINE: indole-3-pyruvic acid imine, PDV-PRE: prodeoxyviolacein precusor, CPA-INT: chromopyrrolate, PROV: proviolacein, AAC-coA: acetoacetyl-coA, 3HB-coA: 3-hydroxylutanoyl-coA, and PHB: polyhydroxybytanoate. The number values responded to simulated metabolic flux (mmol/gDCW/h) Synthesis and functional confirmation of PRT elements in E. coli through sfGFP expression The synthetic semi-endo PRT system was designed using AI-based tools as described in the Methods section. Prediction of ribosome binding sites (RBSs) is relatively straightforward, as they are typically located upstream of the start codon within the 5′ untranslated region (5′ UTR) of each protein-coding gene. In contrast, the number of promoters and terminators may be lower because multiple genes are often organized within a single operon and co-transcribed as polycistronic mRNA. In brief, 40-nucleotide regions corresponding to endogenous promoters and terminators were predicted using AI-based tools. For RBS identification, 20 nucleotides upstream of the start codon were extracted from all coding sequences across the genome. Sequence motifs were subsequently identified and used to construct motif-based elements, which were further fine-tuned to establish a semi-endogenous promoter-RBS-terminator (PRT) system (semi-endo PRT). The promoter sequence was rationally optimized according to the consensus − 35 and − 10 elements of the σ⁷⁰ (RpoD)-dependent promoter motif. During this process, the predicted transcription and translation rates of each genetic component (promoter–RBS–gene–terminator) were evaluated. Additionally, RNA secondary structure and loop free energy were analyzed to optimize terminator stability and efficiency. These sequences were synthesized and initially constructed using sfGFP to assess their expression efficiency before integration into the phaCAB gene cluster. Additionally, a conventional tac promoter was constructed with sfGFP, and fluorescence-activated cell sorting was used for comparison (Fig. 2 ). The synthetic semi-endo PRT system demonstrated a 1.8-fold higher sfGFP signal than that of the tac promoter system (Fig. 2 B). Ten thousand single-cell events of recombinant E. coli DH5α cells were gated for signal measurement. Only a single cell harboring the constructed plasmid expressed sfGFP in region (R2) (Fig. 2 A and D, and 2 E) and under UV light (Fig. 2 C). This demonstrated that the synthetic semi-endo PRT worked with the pAWP89 plasmid and was used for further experiments. We constructed the phaCAB cluster gene and optimized the conditions for PHB biosynthesis in E. coli DH5α. In addition, different antibiotic types and concentrations were assessed to identify the optimal conditions for co-production using a dual-plasmid system. Fig. 2. Open in a new tab Assessment of synthetic system with superfold green fluorescent protein (sfGFP) signal using fluorescence-activated cell sorting (FACS). Promoter, ribosome-binding sites, and terminator are synthesized and constructed using sfGFP compared to conventional tac promoter systems. ( A ) sfGFP signal in certain strains, including wild-type and recombinant Escherichia coli strains with pAWP89 empty, pAWP89-ptac-sfGFP, and pAWP89-PRT-sfGFP. ( B ) Mean fluorescence intensity (MFI) values for four strains—wild-type and plasmid-harboring E. coli strains. ( C ) Visualization of sfGFP in E. coli strain under normal and UV light. Measurement of a single cell using FACS, presenting E. coli wild-type ( D ) and pAWP89-PRT-sfGFP ( E ). R1 represents 10,000 singlet cells gated based on FSC-A versus FSC-H. R2 denotes the sfGFP-positive population derived from R1, and mean fluorescence intensity (MFI) was calculated for cells within R2. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, n = 3 Assessment of PHB production using the semi-endo PRT system across varying antibiotic types and concentrations Co-production using dual plasmids requires different antibiotics. We used the violacein-producing plasmid from a previous study [ 47 ] with kanamycin. Spectinomycin, ampicillin, and kanamycin were tested to optimize the expression of synthetic semi-endo PRT system and phaCAB cluster in pAWP89 (Fig. 3 A and B, and 3 C), respectively. No significant differences in OD600, DCW, PHB titer, or PHB yield were observed across increasing antibiotic concentrations from 0 to 200 µg/mL. Notably, significant fluctuations were observed at 0 µg/mL antibiotics, indicating possible plasmid loss. At 100 µg/mL spectinomycin, PHB production achieved 358.9 ± 9.8 mg/L and 52.89 ± 10.4% (g PHB/g DCW), whereas 50 µg/mL ampicillin yielded 278.6 ± 5.84 mg/L (w/w) and 36.2 ± 0.69% (g PHB/g DCW). Using the same tac promoter–phaCAB construct, spectinomycin produced approximately 1.4-fold higher PHB titer and yield than that of kanamycin, whereas OD600 and DCW were similar (Fig. 3 C). In summary, the synthetic semi-endo PRT that achieved the highest PHB content was constructed using the phaCAB cluster in the pAWP89 vector grown at 100 µg/mL spectinomycin, yielding 528.9 ± 104 mg PHB/g DCW in M9 medium with xylose as the sole carbon source. After establishing the optimal plasmid configuration, we transformed the second violacein-producing plasmid into the host and assessed violacein production. Fig. 3. Open in a new tab Optimization of polyhydroxybutyrate (PHB) production using different types and concentrations of antibiotics. ( A ) PHB production at different spectinomycin concentrations. ( B ) PHB production at different ampicillin concentrations. ( C ) Comparison of PHB production in 100 µg/mL spectinomycin and kanamycin. The concentration range used in figures A and B is 0, 50, 100, and 200 µg/mL Xylose-driven co-production of PHB and violacein in E. coli The conventional tac promoter was used for violacein production in one plasmid, whereas the synthetic semi-endo PRT system driving the phaCAB cluster was present in a second plasmid, with kanamycin and spectinomycin used as selection markers (Fig. 4 C). In this study, we performed co-production experiments using complex and minimal media, LB and M9, respectively. When both plasmids relied solely on the conventional tac promoter, only low amounts of violacein and PHB were produced in LB and M9X (M9 with xylose) media. In M9X, 0.24 ± 0.05 mg/L violacein and 17.1 ± 7.9 mg/L PHB were obtained, whereas in LB medium, 0.51 ± 0.11 mg/L violacein and 10.4 ± 2.1 mg/L PHB were produced. In contrast, the combination of the synthetic semi-endo PRT system with the tac promoter yielded significantly higher production levels. Specifically, 0.88 ± 0.23 mg/L violacein and 111.3 ± 19.7 mg/L PHB were produced in M9X, whereas 0.77 ± 0.2 mg/L violacein and 89.1 ± 11.8 mg/L PHB were obtained in LB medium (Fig. 4 A). The hybrid system (tac promoter and synthetic semi-endo PRT) significantly enhanced co-production, yielding 3.67- and 6.5-fold higher violacein and PHB in M9X, and 1.5- and 8.5-fold higher violacein and PHB in LB media, respectively. Simultaneously, the OD600 and DCW in these samples had no significant differences (Fig. 4 B). The productivity calculations were consistent with the observed titer values (Fig. 4 A and B). The PHB- and violacein-producing cells and their extraction are shown (Fig. 4 D). The recombinant strain exhibiting the highest co-production was selected for cultivation, violacein extraction, and antibacterial activity assays. Fig. 4. Open in a new tab Co-production of polyhydroxybutyrate (PHB) and violacein from xylose using a dual plasmid system. ( A ) Violacein and PHB titers. ( B ) Biomass of two plasmid-harbored strains in M9X and Luria-Bertani media. ( C ) Structures of the two plasmids. One plasmid contained the phaCAB cluster genes, and the other is constructed using the vioABCDE cluster genes. ( D ) Expression and extraction of crude violacein and PHB from recombinant colonies. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, n = 3 Functional validation of violacein through antibacterial activity against M. alcaliphilum 20Z To confirm that the synthesized violacein was biologically active, we evaluated its antibacterial activity against M. alcaliphilum 20Z. Extensive research has demonstrated the antibacterial activity of violacein against various microorganisms [ 46 , 49 , 50 ]. However, this study used ethanol for extraction and experiments, a solvent known to exhibit cytotoxic effects [ 51 , 52 ]. To avoid this issue, we used methanol for violacein extraction and conducted an antibacterial activity assay using the methanotroph type I strain, M. alcaliphilum 20Z, as a model organism. The use of methanotrophs mitigates solvent toxicity because M. alcaliphilum 20Z readily assimilates methanol. The S. aureus strain exhibited 90% mortality at 3.1 mg/L in 6 h [ 50 ]. The minimum inhibitory concentration (MIC) for certain bacteria ranged from 5.7 µg/mL in S. aureus and Salmonella typhi to 20 µg/mL in Vibrio cholerae [ 53 ]. Based on these data, we used 9 µg/mL of violacein—approximately three times 3.1 µg/mL and half the maximum reported MIC (20 µg/mL)—to assess its antibacterial activity against methanotrophs. E. coli DH5α was unaffected by 1% methanol and 9 µg/mL in both M9X and LB media (Fig. 5 B and E). However, the methanotrophic strain exhibited significant sensitivity to 9 µg/mL of both standard and crude violacein (Fig. 5 A and C, and 5 D). Specific growth rates were similar within the first 24 h. However, after 24 h, they were significantly reduced upon treatment with standard and crude violacein, by 57.5- and 6.3-fold, respectively. Compared to the control sample, the specific growth rate of M. alcaliphilum 20Z grown in methanol only after 24 h was reduced by 10.2-fold and 1.1-fold upon violacein addition. Crude violacein exhibited significantly weaker antibacterial activity than that of the purified standard, indicating the need for further optimization of the extraction procedure. In E. coli , the calculated growth rate value in M9X 0.115 h − 1 was consistent with simulated value 0.114 in GSMM model and the experimental value in previous research 0.14 h − 1 [ 44 ]. E. coli tolerated well methanol in the presence of 4% (v/v) in complexed LB media but totally inhibited growth at 10% (v/v) [ 54 ]. These results were compatible with this research with no effect in LB media plus 1% (v/v) methanol. Notably, similar result was shown in M9 with xylose as sole carbon source. This showed the advantages of using xylose as carbon source such as supporting NADPH generation [ 55 ]. In engineered E. coli , enhancing redox enzymes during xylose fermentation improved tolerance to furfural and increases ethanol production under stress compared with non-engineered strains, demonstrating the potential for xylose-linked metabolism to intersect with stress resistance mechanisms during toxic compound exposure [ 56 ]. In summary, our findings demonstrate that violacein exhibits antibacterial activity against methanotrophs at 9 µg/mL. Fig. 5. Open in a new tab Antibacterial activity of violacein on Methylotuvimicrobium alcaliphilum 20Z. ( A ) Optical density of M. alcaliphilum 20Z over time in nitrate mineral salt media supplemented with 1% methanol, 9 µg/mL vio/extracted vio. Optical density of Escherichia coli over time in M9X media ( B ) and Luria-Bertani media ( E ) with 1% methanol, 9 µg/mL vio/extracted vio dissolved in 100% methanol. ( C ) and ( D ) Specific growth rates and ratio fold-change of the specific growth rate before/after 24 h, respectively. µ: specific growth rate. vio: standard violacein. extr-vio or extracted vio: extracted violacein Discussion Compared to previous studies, the co-production of violacein and PHB was demonstrated in the native strain Iodobacter sp . PCH194 was cultivated in a 22 L reactor using glucose and tryptone as substrates [ 11 ]. In contrast, a small amount was obtained from E. coli BL21 (DE3) grown in M9 medium with glucose and tryptophan [ 12 ] (Table 3 ). These studies used glucose as a carbon source supplemented with tryptone or tryptophan to enhance violacein content. This study used an environmentally friendly and sustainable resource (xylose) as a carbon source for co-producing violacein and PHB. For PHB production alone in the E. coli strain, 528.9 ± 104 mg PHB/g DCW was produced in M9 with xylose as the sole carbon source, a 4.4-fold higher than that in M9 with glucose as the sole carbon source, 120 ± 1 mg PHB/g DCW [ 12 ]. Several studies have reported high PHB production in E. coli . For example, 6.82 g/L PHB (0.36 g/g glucose yield) was achieved through threonine bypass using MS medium supplemented with 1 g/L yeast extract and 20 g/L glucose [ 57 ], 5.31 g PHB/L was obtained in E. coli cultured in LB medium supplemented with 30 g/L glucose [ 58 ], and 243 and 318 mg/L PHB production was achieved with E. coli in LB medium supplemented with 1% glucose [ 59 ]. A common feature of these studies is the use of complex media, nutrient supplementation, and glucose as the carbon source. In other studies, 840 mg/L PHB was produced by E. coli (DE3) in LB media supplemented with 20 g/L xylose after extensive metabolic engineering [ 60 ]. Additionally, 2.58 g/L PHB was produced in E. coli BL21 (DE3) using Miscanthus and pine-tree hydrolysate as carbon sources [ 61 ]. Moreover, 1.7 g/L PHB was produced by E. coli BL21 (DE3) in R medium containing 20 g/L xylose, yeast extract, bacto peptone, and other nutrients [ 62 ]. Collectively, these findings suggest that PHB production from xylose as the sole carbon source may be limited without supplemental nutrients. Table 3. Comparison of the co-production of polyhydroxybutyrate (PHB) and violacein in bacteria Strain Source Violacein PHB Vol. Substrate Ref. Iodobacter sp . PCH194 Native strain 1.5 ± 0.08 g/L 11 ± 1 g/L 22 L +Glucose +tryptone [ 11 ] E. coli BL21(DE3) Recombinant strain 90 ± 3 mg/L 270 ± 2 mg/g DCW NA +Glucose +tryptophan [ 12 ] E. coli DH5α Recombinant strain 0.88 ± 0.23 mg/L 111.3 ± 19.7 mg/L = 195 ± 5.5 mg/g DCW 10 ml +Xylose This research Open in a new tab The violacein biosynthesis was tightly regulated and controlled by multiple regulatory mechanisms including feedback inhibition of enzymes and transcriptional repression /attenuation of the trp operon, significantly impacting precursor availability for violacein formation [ 63 ]. In the present of tryptophan, the interaction of a repressive regulator trpR protein and tryptrophan will occur, then tryp-trpR protein binds to operator of tryptophan operon blocking translation of genes for biosynthesis. This may lead to low titer of violacein biosynthesis with 0.88 ± 0.23 mg/L. Supplementation with L-tryptophan directly enhances violacein production by increasing precursor availability, as demonstrated in E. coli cultures where external tryptophan feeding improved product titers [ 64 ]. In contrast, nutrient-rich supplements such as tryptone or yeast extract primarily enhance overall biomass and growth, which can indirectly affect violacein yields without specifically increasing tryptophan precursor supply [ 65 , 66 ]. To preserve the antibacterial activity of violacein, overheating during the redissolution step should be avoided, and the purification process could be improved by employing silica gel column chromatography [ 64 ]. Additionally, PHB biosynthesis requires acetyl-CoA as the precursor and NADPH as the reducing cofactor, and limitations in acetyl-CoA and NADPH pools can restrict 3HB-CoA/PHB formation, making cofactor and precursor engineering essential for improved synthesis [ 67 , 68 ]. Escherichia coli has been subjected to genome-wide screening to enhance tolerance toward a broad range of toxic chemicals, including organic solvents, acids, and biofuel intermediates. These studies identified stress response regulators and adaptive mechanisms that improve growth performance and increase the potential for higher product titers [ 69 ]. Enhancing tolerance to toxic compounds enables E. coli to maintain cellular growth and metabolic activity under stressful conditions [ 70 ]. Collectively, these findings demonstrate that E. coli is a robust host capable of responding to diverse toxic chemicals through specific stress response pathways. In the present study, E. coli demonstrated tolerance to 9 µg/mL violacein in both M9X and LB media. The strain was able to produce 0.88 ± 0.23 mg/L violacein in M9 medium supplemented with xylose. In contrast, Methylomicrobium alcaliphilum 20Z, a type I methanotroph, was negatively affected by violacein at the same concentration. Therefore, E. coli should be considered a more suitable host strain for violacein production under these conditions. This study has not yet assessed large-scale co-production of violacein and PHB, nor has it investigated the effect of tryptophan supplementation on violacein yield. Furthermore, the antibacterial activity against Methylomicrobium alcaliphilum 20Z should be investigated over a broader concentration range. In this study, we used AI-based tools to design and synthesize a semi-endo PRT system that increased the PHB content in M9 medium with xylose as the sole carbon source. Finally, xylose offers a renewable non-food-competing resource alternative to glucose. Conclusions This study demonstrates that renewable xylose supports high PHB accumulation in E. coli DH5α through AI-assisted semi-endo PRT design, achieving 528.9 ± 104 mg/g DCW in M9. Xylose-enabled co-production of PHB and violacein was achieved, and the growth-inhibitory activity of violacein against methanotrophs at 9 µg/mL. To the best of our knowledge, this study reports the co-production of PHB and violacein using xylose as the sole carbon source. Additionally, it provides an initial assessment of the antibacterial activity of violacein against methanotrophs. These findings advance sustainable metabolic engineering strategies for biodegradable polymer production and support efforts toward reducing plastic waste and achieving global carbon neutrality. Supplementary Information Below is the link to the electronic supplementary material. 13036_2026_653_MOESM1_ESM.zip (3.1MB, zip) Supplementary Material 1: Map and information of GSMM (word file), Genome-scale metabolic model with SBML format, Map of Genome-scale metabolic model with json format, Genome-scale metabolic model with json format, Core reaction with carbon flux redox cofactor ratio (Excel file), Supplement of FACS measurement (pdf file). Author contributions KNP developed and performed the experiments and wrote and drafted the manuscript. EYL coordinated the study and edited, reviewed, and finalized the manuscript. Funding This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00466473), and by the Technology Innovation Program (Industrial Strategic Technology Development Program) (RS-2023-00265608 & 1415188462) funded by the Ministry of Trade, Industry and Energy (MOTIE), South Korea. Data availability No datasets were generated or analysed during the current study. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Footnotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. References 1. Zhao Z, Xian M, Liu M, Zhao G. Biochemical routes for uptake and conversion of xylose by microorganisms. Biotechnol Biofuels. 2020;13:21. 10.1186/s13068-020-1662-x. [ DOI ] [ PMC free article ] [ PubMed ] [ Google Scholar ] 2. Kim JS, Lee YY, Kim TH. A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresour Technol. 2016;199. 10.1016/j.biortech.2015.08.085. :42 – 8. [ DOI ] [ PubMed ] 3. Abdel-Rahman MA, Tashiro Y, Zendo T, Hanada K, Shibata K, Sonomoto K. 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Growth of Escherichia coli, Pichia pastoris and Bacillus cereus. Biotechnol Lett. 2006;28(7):465–9. 10.1007/s10529-006-0006-7. in the presence of the ionic liquids [BMIM][BF4] and [BMIM][PF6] and Organic Solvents. [ DOI ] [ PubMed ] Supplementary Materials 13036_2026_653_MOESM1_ESM.zip (3.1MB, zip) Supplementary Material 1: Map and information of GSMM (word file), Genome-scale metabolic model with SBML format, Map of Genome-scale metabolic model with json format, Genome-scale metabolic model with json format, Core reaction with carbon flux redox cofactor ratio (Excel file), Supplement of FACS measurement (pdf file). Data Availability Statement No datasets were generated or analysed during the current study. 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