Synthetic and Systems Biotechnology

Streptomyces bacteria produce a variety of secondary metabolites that hold clinical and agricultural value, yet their biosynthetic potential remains unrealized as many biosynthetic gene clusters are not expressed under standard laboratory conditions. Expression of these clusters is tightly regulated, often by cluster situated transcription factors. The TetR family are regulators whose activity is modulated by small molecule elicitors. Although many TetRs have been characterized, elicitors have only been identified for a small fraction of them. This lack of data presents a limitation in our ability to exploit elicitor-regulator pairs for activation of silent clusters and underscores the need for predictive and testable strategies for elicitor identification. In this work, we test the use of sequence similarity networks (SSNs) as a predictor of elicitor identity using the well characterized TetR protein, JadR2, that has a known elicitor, chloramphenicol. We utilized SSNs to identify JadR2 homologs that may also be elicited by chloramphenicol. We developed a heterologous Escherichia coli reporter system in which regulator activity was monitored using an EGFP readout of DNA binding activity. Using this system, we screened JadR2 and four homologs for responsiveness to chloramphenicol. We found that 3 homologs were elicited by chloramphenicol, all of which were formerly uncharacterized. These results demonstrate that TetR-family proteins can share elicitor responsiveness and that SSNs can be used to prioritize regulators for functional screening. This work establishes a genomics-informed and bioinformatics-guided framework for linking elicitors to their regulator, expanding the toolkit for natural product discovery by unlocking regulatory information across Streptomyces.

{beta}-galactosidases (BGs) are essential enzymes widely used in the food industry, particularly in the production of lactose-free products. Among them, the BG from Aspergillus oryzae is of industrial relevance due to its activity at acidic pH and moderate thermal tolerance. However, enhancing its catalytic performance remains a key challenge. Traditional enzyme engineering methods are time-consuming and resource-intensive, limiting their scalability. Recent advances in Artificial Intelligence (AI), particularly those based on Natural Language Processing, offer a promising alternative by enabling efficient exploration of protein sequence space and prediction of beneficial mutations. In this study, we introduce an ensemble-based, zero-shot Protein Language Model pipeline that reconciles predictions from six independent models (ESM2 and the five ESM1v variants) combined with a diversity-aware candidate selection strategy. Applied to the BG from A. oryzae, this approach identified beneficial mutations leading to novel enzyme variants with up to a four-fold increase in catalytic efficiency on oNPGal, a two-fold increase on lactose, and, independently, a T338I variant with markedly enhanced thermostability ({approx}80% residual activity after 24 h at 60 {degrees}C), all without requiring supervised fine-tuning on experimental fitness data. Our results demonstrate that consensus across an ensemble of PLMs can efficiently enrich beneficial substitutions in industrially relevant enzymes and substantially reduce the number of wet-lab candidates that need to be screened. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/726739v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@18084f7org.highwire.dtl.DTLVardef@99a102org.highwire.dtl.DTLVardef@19a64forg.highwire.dtl.DTLVardef@1f59cff_HPS_FORMAT_FIGEXP M_FIG C_FIG

Budding yeast Saccharomyces cerevisiae is a workhorse chassis for producing added food and agricultural compounds. However, building multi-enzymatic pathways for these chemicals often requires iterative genomic integration, underscoring the need for efficient, rapid genome-editing tools that can reliably target transcriptionally active chromosomal regions. In this study, to accelerate strain construction, we established a genome-editing toolkit to rapidly engineer eight loci, highly expressed hot-spots, but nonessential genomic sites suitable for stable pathway assembly. Our approach integrates three key design features: (i) selectable markers to enable rapid screening of edited cells, (ii) extended homology arms that leverage the yeast homology-directed repair machinery for robust genomic integration, and (iii) co-delivery of Cas9 and guide RNAs to promote efficient double-stranded DNA breaks at specific integration sites. The sequence independence of FASTOP relies on the release of integration cassettes from integrative vectors, mediated by restriction digestion at two flanking multiple-cutting sites in the integration module to minimize the risk of introducing sequence errors during PCR amplification of the integration cassettes. Following the introduction of a fluorescent reporter cassette, we observed high integration efficiencies across the target sites. We then integrated the biosynthetic pathway of plant-derived flavonoid naringenin into the hot-spots of the yeast genome using the FASTOP toolkit. Our results demonstrated that upon expressing the five essential genes in simple shake flask culture, naringenin production reached 505.7 mg/L, representing a significant (69-fold) increase over previously reported titers for comparable minimal heterologous pathways in S. cerevisiae. Together, the FATSOP toolkit provides a user-friendly platform for reliably modifying hot-spot loci to rapidly construct multi-enzymatic metabolic pathways in S. cerevisiae, while achieving high production levels for high-value food-relevant metabolites.

Infections caused by multi-drug-resistant organisms (MDROs) pose a significant public health threat, responsible for over 2 million hospitalizations and 23,000 deaths annually in the United States. Microbiome dysbiosis (imbalance) is considered one of the main causes for MDRO colonization and the resulting infections. Rapid detection and intervention of MDRO outbreaks are crucial to alleviating strain on patients and healthcare facilities. Current diagnostic methods for MDRO detection are too slow and costly to provide the rapid MDRO detection necessary for patient care facilities. Here we present a rapid, accurate and cost-effective electrochemical sensor capable of MDRO detection down to [~]104 colony forming units (CFU)/g in mice and human stool samples. Our novel sensor utilizes probe-modified Screen-Printed Electrodes (SPEs) capable of hybridizing target gene sequences associated with MDROs. The resulting probe/target complex generates a unique and highly sensitive signal detectable down to 10 atto molar or 10 CFU/mL of target TEM-1 gene. The use of these pre-functionalized SPEs reduces individual sample analysis time to less than an hour. Several target sequences from two chromosomal target genes (AmpC and AcrB found in E. coli) have been identified and successfully detected in clinical stool samples with results comparable to the standard quantitative PCR method. Additional target genes associated with antibiotic resistance (TEM-1, VanA, KPC and SHV) have also been successfully detected in vitro and are ready for clinical evaluation. Future development includes multiplexing the sensor to simultaneously detect up to three MDROs target genes, including {beta}-lactamases that hydrolyze {beta}-lactams, the most commonly used antibiotics in clinical settings. This novel sensor platform will be a rapid, economical, point-of-care device with little requirement of reagent handling or technical training.

Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of few cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of{Delta} sodB mutant further confirmed its targeted deletion. Overall, CRISRPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. Key PointsO_LIRapid and efficient CRISPR/Cas12a editing established in Cyanothece PCC 7425. C_LIO_LIFully segregated knockout mutants obtained after single selection round. C_LIO_LIPlatform for nuclear waste bioremediation and other biotechnological applications. C_LI

Polysaccharide utilization loci (PULs) have been a goldmine for the characterization of novel carbohydrate active enzymes (CAZymes) and the understanding of their synergistic degradation of complex polysaccharides. We collected PUL predictions containing CAZymes from glycoside hydrolase families GH29, GH50 and GH117, expected to participate in marine polysaccharide breakdown. We explored the evolutionary diversity in these families in terms of sequences and PUL composition, based on sulfatases and CAZymes. From 41 selected PULs, more than 400 putative enzymes were produced, purified and screened on a large collection of carbohydrates. We attributed a function to more than 130 enzymes from five sulfatase subfamilies, 29 known CAZymes families and discovered an activity for 4 families previously of unknown function, including an -L-galactosidase structurally and functionally characterized with mutants. Finally, our detailed analysis of the enzymatic synergies in five PULs, two targeting marine polysaccharides and three targeting eukaryotic polysaccharides, by marine and human gut organisms, highlight the efficiency of our exploratory strategy.

A challenge in using plant biomass is its highly recalcitrant nature, which makes it economically infeasible to utilize. In natural environments, various microbes, including bacteria and fungi, are reported to decompose plant cell wall materials such as cellulose and hemicellulose, and there may be undescribed microbes that contribute to the degradation of plant biomass. We focused on isolating novel plant biomass-degrading bacteria and screened more than 100 isolates from the Tomakomai experimental forest in Hokkaido, Japan. Among them, one novel Bacillus species was chosen for whole-genome sequencing. Comparative genomics and a carbon source utilization assay indicated that the isolate belongs to a subspecies of Bacillus subtilis, which we named B. sp. TTS1. Glucose, cellobiose, xylose, xylan, mannose, or mannan was used as the sole carbon source in the minimum medium, and the growth of this bacterium was determined. Furthermore, a proteomic analysis of B. sp. TTS1 was performed using culture supernatants from various polysaccharide-containing media. In the present study, several key enzymes involved in plant biomass degradation were identified, namely {beta}-1,4-mannanase and xylanase, and they were highly enriched in all tested polysaccharides.

Cyanobacterial natural products are a rich source of bioactive compounds, yet their heterologous production remains challenging. This study investigates the feasibility of expressing the lyngbyatoxin A (LTXA) biosynthetic gene cluster in a fungal host. The lyngbyatoxin biosynthetic genes (ltxA, ltxB, ltxC) were individually cloned and expressed in Aspergillus oryzae NSAR1 under the control of an inducible promoter. Metabolite production was assessed using LC- MS, and transcriptional analysis was performed by RT-PCR. Codon-optimized constructs and precursor feeding experiments were employed to evaluate pathway functionality. No production of LTXA or pathway intermediates was detected upon co-expression of ltxA-C despite confirmed transcription of ltxB and ltxC. RT-PCR analysis revealed truncation of the ltxA transcript, suggesting incompatibility with fungal transcriptional or splicing machinery. In contrast, expression of a codon-optimized ltxC enabled biotransformation of indolactam V to LTXA in A. oryzae, confirming functional expression of the prenyltransferase. These results highlight transcriptional limitations as a key barrier to heterologous expression of cyanobacterial NRPS pathways in fungal hosts, while demonstrating that downstream tailoring enzymes can remain functional. This work provides insights for future engineering of fungal platforms for cyanobacterial natural product biosynthesis.

Wickerhamomyces ciferrii is a non-model diploid yeast that naturally produces tetraacetyl phytosphingosine (TAPS), a sphingoid base used in cosmetic and dermatological applications. However, its strong preference for non-homologous end joining (NHEJ) over homologous recombination (HR) limits conventional genome editing, while disruption of LIG4, a core NHEJ gene, compromises cellular fitness. Here, we repurposed native NHEJ activity to develop a homology-independent multicopy genome integration platform for W. ciferrii. The platform combines three optimized donor-design features: telomeric end-shielding with two tandem copies of an 11 bp repeat to improve linear donor persistence, a defective URA5 auxotrophic marker to enrich multicopy integrants, and 5'-phosphorylated donor termini to enhance transformant recovery and integration output. These features were consolidated into the platform vector pTdmVU5. As a metabolic engineering demonstration, multicopy integration of LCB1 and LCB2, encoding the two subunits of serine palmitoyltransferase, increased TAPS titer by 2.7-fold. This work converts the native NHEJ bias of W. ciferrii from a barrier to precise genome editing into a practical tool for pathway amplification and establishes a framework for engineering NHEJ-dominant non-model yeasts.

Biological metal chelators are of great interest for investigation due to their capacity to retain or mobilize metals from the environment. While some biological and bioinspired chelators find use in medical applications, others are promising platforms for the mining or recycling of technologically important metal ions. In particular, the siderophores, which are primarily iron chelators, have been studied. Four siderophores of relevance are schizokinen and its derivatives, which have been isolated from bacterial and algae cultures, in addition to soil. These siderophores have shown metal chelating activity with different metals such as iron, copper, and aluminum. In the time of metabolomics, it is required to unambiguously determine the identity of the produced siderophores as quickly as possible. Thus, Liquid Chromatography coupled to High Resolution Mass Spectrometry and mass-tandem fragmentation (LC-HRMS-MS) provides a quick and applicable alternative for identification of schizokinen and its derivatives. Here, we report an analytical method for the identification and potential quantification of the schizokinen siderophore series. We developed a working method through LC-HRMS-MS, which provides the unequivocal identification of the four schizokinen derivatives, which has not been reported to date. Additionally, we constructed the molecular network for the four molecules to enable their identification using the Global Natural Products Social Molecular Networking (GNPS) platform. Most importantly, this contribution can help speed up the characterization of schizokinen producers and facilitate the dereplication process of siderophores.

Improving the reconstituted translation system is a key requirement for bottom-up synthetic biology. Here, we developed a two-step in vitro evolutionary method that can be used for improving translational proteins. In this method, two distinct conditions were sequentially applied while maintaining genotype-phenotype linkage in water-in-oil droplets. Using this method, we performed in vitro evolution of four translation factors, IleRS, PheRS, EF-G, and EF-Tu, and identified mutations that modestly enhanced translation activity in in vitro expression assays. One of the EF-G mutations (P610S) increased activity per protein approximately 2-fold for the recombinant protein purified from E. coli. This selection method is useful for improving translational proteins for bottom-up synthetic biology.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, whereas low manufacturing efficiency and a large proportion of empty capsids are major obstacles. This study focused on the Yin Yang 1 (YY1) binding motif (YY1-motif) and investigated the effect of its presence or insertion at upstream of the Replicase (Rep)/Capsid Cap) gene on AAV vector production. We found that the YY1-motif incidentally presented in a Rep/Cap plasmid was associated with high vector production. We then designed several modified Rep/Cap (RC2) constructs. The YY1-motif insertion at the upstream of Rep/Cap gene increased vector yield in a repeat-number-dependent manner, and similar effects were not observed with other promoters insertion. Furthermore, the insertion of the YY1-motif reduced the amount of Cap protein per the same amount of full particle in supernatants on multiple serotypes, indicating the improvement in the empty/full capsid ratio. The YY1-motif insertion did not affect the AAV vector infectivity. These results denote that the YY1-motif has a universal regulatory function that optimizes the Rep/Cap expression balance, and simultaneously improves the production efficiency and full particle formation of AAV vectors. This finding could contribute to the development of highly efficient and high-quality AAV manufacturing processes.

Rapid and specific diagnosis of viral and bacterial infections is a significant challenge in medicine and veterinary science, especially in the case of epidemically dangerous pathogens. The African swine fever virus (ASFV), for example, causes annual outbreaks among livestock, resulting in significant economic losses for farmers. DNA aptamers have been identified as a promising tool for point-of-care diagnostics, being highly specific to the target and stable ambient temperatures during storage. In this study, we describe the selection of DNA aptamers targeting the p54 viral protein using a single-round selection process. These aptamers were able to bind both to recombinant protein and inactivated ASFV viral particles. Analysis of the newly generated aptamers revealed a dependence of affinity and thermal stability on Ni2+ content, which was a dopant in the selection process. In some cases, the affinity increased 100 times, and melting temperature increased by 30{degrees}C. We have identify two novel DNA motifs that bound 2-3 Ni2+ or Zn2+ ions.

Bacteriocins are ribosomally synthesized antimicrobial peptides with promising applications in biotechnology, particularly in food preservation and animal and human health. Circular bacteriocins are especially attractive due to their head-to-tail cyclized structure, which confers enhanced stability and antimicrobial potency relative to linear peptides. Here, we report an in vitro cell-free protein synthesis system coupled with an enhanced Split Intein-Mediated Ligation platform (IV-CFPS/SIML) for the efficient synthesis of circular bacteriocins through systematic evaluation of cyclization sites and alternative split inteins. Using enterocin AS-48 as a model, we systematically evaluated multiple serine-based cyclization sites in combination with three split inteins, NpuDnaE, Gp41-1, and SspGyrB, to identify configurations supporting efficient splicing and high antimicrobial activity. Gp41-1 emerged as the most effective intein and was subsequently applied to the production of garvicin ML, amylocyclicin, and 27 naturally occurring sequence variants, demonstrating that cyclization site selection, intein identity, and minor sequence variations strongly influence antimicrobial potency and target range. Finally, SIML expression cassettes encoded in pUC-derived vectors enabled in vivo production and functional expression of selected circular bacteriocins in recombinant Escherichia coli. Collectively, these results establish SIML as a versatile platform for in vitro and in vivo production, screening, and functional characterization of known and putative circular bacteriocins.

This study investigates the potential of the garden rhizosphere as a source of electrochemically active bacteria (EAB) for operating microbial fuel cells (MFCs). We evaluated a diverse array of garden flora, including vegetables (lettuce, Chinese cabbage), flowering plants (August lily, peppermint), and woody species (pine, oak, ginkgo, and bush clover). Among the tested groups, MFCs inoculated with peppermint and ginkgo rhizosphere microbiotas exhibited the highest current densities within their respective categories, significantly outperforming control groups without plant components. 16S rRNA gene microbial community analysis revealed that the initial rhizosphere environment acts as a decisive selective pressure, shaping distinct anode biofilms based on plant types (herbaceous vs. woody). Despite these structural differences in microbial assembly, high current generation was achieved in both peppermint and ginkgo systems, suggesting a high degree of functional redundancy within the rhizosphere-derived consortia. These findings demonstrate that various garden ecosystems can serve as robust biological reservoirs for MFC operation, where diverse microbial configurations are capable of sustaining efficient bio-electrochemical energy conversion.

Black Soldier Fly larvae (BSFL, Hermetia illucens) are highly effective for the bioconversion of food waste. However, their rearing process often produces substantial ammonia emissions, which are malodorous and environmentally concerning. We investigated the co-cultivation of BSFL with the sulfur-oxidizing bacterium Thiobacillus thioparus as a strategy to mitigate ammonia release. Importantly, under conditions where ammonia emissions were significantly reduced, neither larval growth nor bacterial viability was negatively affected. Furthermore, even when the initial bacterial inoculum was reduced to 3.3*105 CFU/g-food wastes, the bacterium rapidly recovered to functional levels and effectively controlled ammonia emissions. This indicates the absence of harmful interaction or nutrient competition between BSFL and T. thioparus. These findings suggest an efficient method for controlling ammonia in large-scale BSFL waste treatment. By reducing the required bacterial inoculum, this approach enables scalable microbial co-culturing with environmental and production benefits.

Organohalide-respiring bacteria (OHRB), such as Dehalobacter, play key roles in the bioremediation of anoxic environments contaminated with chlorinated aromatic compounds. These obligate anaerobes rely on syntrophic interactions to obtain essential resources--hydrogen, acetate, and corrinoid cofactors--from acetogens and fermenters. However, the metabolic interactions enabling complete reductive dehalogenation of compounds like 1,2,4-trichlorobenzene (1,2,4-TCB) to benzene remain incompletely understood. In this study, we asked: (1) What are the key microbial taxa and their functional roles within a Dehalobacter-containing anaerobic microbial community detoxifying chlorinated benzenes? (2) How do syntrophic interactions enable complete dehalogenation of 1,2,4-TCB to benzene under anaerobic conditions? (3) Can genome-resolved metagenomics and genome-scale metabolic modeling elucidate the metabolic dependencies supporting organohalide respiration in complex consortia? To address these questions, we cultivated microbial communities in batch reactors using methanol as electron donor and either 1,2,4-TCB or monochlorobenzene (MCB) as electron acceptor. In active MCB-fed cultures, benzene increased from 0 to 62.3{micro}mol per bottle while MCB decreased from 88.3 to 22.0{micro}mol per bottle over 120 days, with this pattern repeating across multiple substrate additions. Using genome-resolved metagenomics to identify dominant taxa and select 12 high-quality metagenome-assembled genomes (MAGs) for modeling, we reconstructed genome-scale metabolic models (GEMs) to identify candidate metabolic interactions and predict syntrophic dependencies that may support organohalide respiration in these consortia. Community flux sampling predicted that methanol, H2, acetate, and CO2 formed the dominant exchange backbone of the modeled community, while also indicating competition for shared electron donors between the two Dehalobacter populations. Model-guided minimal-community analysis further identified a narrow dechlorinating core in which all feasible minimal consortia retained a Dehalobacter member together with Methanothrix. These results provide a modeling-informed framework for hypothesis generation and future experimental validation of anaerobic consortia relevant to bioremediation.

1.Abiotic stresses like nitrogen deficiency and soil salinity are major factors contributing to low crop yields. The use of selective biofertilizers alleviates both types of stress. In this study, we investigated the biofertilizer activity and plant growth-promoting properties (PGP) of Rhodococcus jialingiae RS1 through cytosolic proteome remodelling. We cultured RS1 under two conditions, i) without and ii) with 6% NaCl, in nitrogen-deficient defined Burks medium. Under dual stress of nitrogen limitation and salt stress, Orbitrap LC-MS/MS proteomics revealed one-quarter of the proteome remodelling, particularly the upregulation of ribosomal synthesis and protein repair systems. As expected, we found high expression of EctC, an ectoine synthase, a key enzyme in osmolyte biosynthesis. Additionally, ribosomal and translational-associated factors, including RpsL, RpsS, RpsT, RpsR1, RplV, RplL, RplA, and elongation factor Tuf, were highly expressed, suggesting enhanced translational fidelity under dual stress. High levels of DNA protection protein, Dps suggest dual stress may lead to DNA damage. Upregulation of chaperones, environmental sensors (KinE), and redox transcriptional factors like WhiB3, Hsp18, AhpC, and MetE suggests protein misfolding and oxidative stress. Metabolic modulations were evident through high expression of IlvA, NAD-dependent glutamate dehydrogenase, lipid/envelope-remodelling enzymes, cutinase/esterases, lipases, endopeptidases like NlpC/P60 and transport systems. In contrast, proteins involved in urease structural components (urea-G), nitrogen regulators and ammonium transporters (GlnK and Amt) were downregulated. Dual stress may lead to an energy crisis, prompting strategic shifts away from high-ATP-dependent ureolytic nitrogen-scavenging pathways towards lower-energy nitrogen-assimilating routes, such as IlvA-mediated deamination and NAD-dependent glutamate dehydrogenation. Genetic manipulations of the above-mentioned genes or their homologues across the genera of microbes, plants, and crops may enhance resilience to abiotic stresses. Our studies reveal stress-responsive genes and biochemical pathways that could be used to improve transgenic efficacy in nitrogen-limited, saline soil and other (a)biotic stresses. Global Proteome Profiling of Rhodococcus jialingiae RS1 to Develop Transgenics O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/724437v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1719d80org.highwire.dtl.DTLVardef@1b6b59org.highwire.dtl.DTLVardef@24d367org.highwire.dtl.DTLVardef@1b33224_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hydrogenophilus thermoluteolus TH-1 is a thermophilic hydrogen-oxidizing bacterium capable of producing poly(3-hydroxybutyrate) (PHB) from CO2. To redirect carbon flux for producing other useful biomaterials, we disrupted the acetoacetyl-CoA reductase genes (phaB1 and phaB2), which are central to the primary PHB synthesis pathway. Unexpectedly, the resulting {Delta}phaB1B2 mutant still accumulated PHB under autotrophic conditions, reaching approximately 25-35 % of the wild-type level. Furthermore, PHB accumulation in the mutant was significantly restored when fatty acids (butyrate and oleate) were used as carbon sources, whereas acetate and malate resulted in reduced accumulation. These results suggest the existence of a PhaB-independent PHB synthesis pathway. We propose that intermediates from the {beta}-oxidation of fatty acids are converted to (R)-3-hydroxybutyryl-CoA, bypassing the disrupted PhaB enzymes. Additionally, the basal PHB production from non-fatty acid sources implies the involvement of a reverse {beta}-oxidation pathway. This study highlights the metabolic versatility of strain TH-1 for future metabolic engineering.

The facultative methylotroph model organism Methylorubrum extorquens AM1 is a known lanthanide user, which has shed light on the role of rare-earth metals in biochemistry. The characterization of a methanol dehydrogenase (MDH) protein which requires lanthanides as an enzymatic cofactor outlined the question of how these metals are acquired from the environment. It has been proposed that mesophilic organisms as M. extorquens AM1 can produce siderophore-like molecules, which chelate, transport and traffic rare-earth elements into the microbial cell. Therefore, we performed the bioinformatic and chemical investigation of M. extorquens AM1 by using genome mining, the CAS and arsenazo assay, molecular networking and chemical analytical techniques. Our results showed that indeed Methylorubrum extorquens AM1 harbored a gene cluster to produce metal chelators. The chemical analysis confirmed the production of the known hybrid hydroxamate-citrate siderophores schizokinen A and N-deoxyschizokinen A, which are very likely the side products of the transformation of schizokinen and N-deoxyschizokinen. The determination of the lanthanide chelation activity of the schizokinen siderophores series against three different lanthanides (La, Eu and Lu) showed no coordination activity, thus ruling out the involvement of schizokinen siderophores in rare-earth metal transport.