Structure of recombinant formate dehydrogenase from Methylobacterium extorquens (MeFDH1).

Formate dehydrogenase (FDH) is critical for the conversion between formate and carbon dioxide. Despite its importance, the structural complexity of FDH and difficulties in the production of the enzyme have made elucidating its unique physicochemical properties challenging. Here, we purified recombinant Methylobacterium extorquens AM1 FDH (MeFDH1) and used cryo-electron microscopy to determine its structure. We resolved a heterodimeric MeFDH1 structure at a resolution of 2.8 Å, showing a noncanonical active site and a well-embedded Fe-S redox chain relay. In particular, the tungsten bis-molybdopterin guanine dinucleotide active site showed an open configuration with a flexible C-terminal cap domain, suggesting structural and dynamic heterogeneity in the enzyme.

Expression of toxic genes in Methylorubrum extorquens with a tightly repressed, cumate-inducible promoter.

Methylorubrum extorquens is an important model methylotroph and has enormous potential for the development of C1-based microbial cell factories. During strain construction, regulated promoters with a low background expression level are important genetic tools for expression of potentially toxic genes. Here we present an accordingly optimised promoter, which can be used for that purpose. During construction and testing of terpene production strains harbouring a recombinant mevalonate pathway, strong growth defects were observed which made strain development impossible. After isolation and characterisation of suppressor mutants, we discovered a variant of the cumate-inducible promoter PQ2148 used in this approach. Deletion of 28 nucleotides resulted in an extremely low background expression level, but also reduced the maximal expression strength to about 30% of the original promoter. This tightly repressed promoter version is a powerful module for controlled expression of potentially toxic genes in M. extorquens.

Biointeraction of cerium oxide and neodymium oxide nanoparticles with pure culture Methylobacterium extorquens AM1.

Rare earth elements (REE) are valuable raw materials in our modern life. Extensive REE application from electronic devices to medical instruments and wind turbines, and non-uniform distribution of these resources around the world, make them strategically and economically important for countries. Current REE physical and chemical mining and recycling methods could have negative environmental consequences, and biologically-mediated techniques could be applied to overcome this issue. In this study, the bioextraction of cerium oxide and neodymium oxide nanoparticles (REE-NP) by a pure culture Methylobacterium extorquens AM1 (ATCC®14718™) was investigated in batch experiments. Results show that adding up to 1000 ppm CeO2 or Nd2O3 nanoparticles (REE-NP) did not seem to affect the bacterial growth over 14-days contact time. Effect of methylamine hydrochloride as an essential electron donor and carbon source for microbial oxidation and growth was also observed inasmuch as there was approximately no growth when it does not exist in the medium. Although very low concentrations of cerium and neodymium in the liquid phase were measured, concentrations of 45 µg/gcell Ce and 154 µg/gcell Nd could be extracted by M. extorquens AM1. Furthermore, SEM-EDS and STEM-EDS confirmed surface and intracellular accumulation of nanoparticles. These results confirmed the ability of M. extorquens to accumulate REE nanoparticles.

Minor Actinides Can Replace Essential Lanthanides in Bacterial Life.

Certain f-block elements-the lanthanides-have biological relevance in the context of methylotrophic bacteria. The respective strains incorporate these 4 f elements into the active site of one of their key metabolic enzymes, a lanthanide-dependent methanol dehydrogenase. In this study, we investigated whether actinides, the radioactive 5 f elements, can replace the essential 4 f elements in lanthanide-dependent bacterial metabolism. Growth studies with Methylacidiphilum fumariolicum SolV and the Methylobacterium extorquens AM1 ΔmxaF mutant demonstrate that americium and curium support growth in the absence of lanthanides. Moreover, strain SolV favors these actinides over late lanthanides when presented with a mixture of equal amounts of lanthanides together with americium and curium. Our combined in vivo and in vitro results establish that methylotrophic bacteria can utilize actinides instead of lanthanides to sustain their one-carbon metabolism if they possess the correct size and a +III oxidation state.

Compartment-related aspects of XoxF protein functionality in Methylorubrum extorquens DM4 analysed using its cytoplasmic targeting.

The impact of periplasmic localisation on the functioning of the XoxF protein was evaluated in the well-studied dichloromethane-utilising methylotroph Methylorubrum extorquens DM4, which harbors only one paralogue of the xoxF gene. It was found that the cytoplasmic targeting of XoxF by expression of the corresponding gene without the sequence encoding the N-terminal signal peptide does not impair the activation and lanthanide-dependent regulation of the MxaFI-methanol dehydrogenase genes. Analysis of the viability of ΔxoxF cells complemented with the full-length and truncated xoxF gene also showed that the expression of cytoplasmically targeted XoxF even increases the resistance to acids. These results contradict the proposed function of the XoxF protein as an extracytoplasmic signal sensor. At the same time, the observed dynamics of growth with methanol, as well as with dichloromethane of strains expressing cytoplasmic-targeted XoxF, indicate the probable enzymatic activity of lanthanide-dependent methanol dehydrogenase in this compartment. Herewith, the only available substrate for this enzyme in cells growing with dichloromethane was formaldehyde, which is produced during the primary metabolism of the mentioned halogenated toxicant directly in the cytosol. These findings suggest that the maturation of XoxF-methanol dehydrogenase may occur already in the cytoplasm, while the factors changing affinity of this enzyme for formaldehyde are apparently absent there. Together with the demonstrated functioning of an enhancer-like upstream activating sequence in the promoter region of the xoxF gene in M. extorquens DM4, the obtained information enriches our understanding of the regulation, synthesis and role of the XoxF protein.

Sodium formate redirects carbon flux and enhances heterologous mevalonate production in Methylobacterium extorquens AM1.

Methylobacterium extorquens AM1 (AM1), a model strain of methylotrophic cell factories (MeCFs) could be used to produce fine chemicals from methanol. Synthesis of heterologous products usually needs reducing cofactors, but AM1 growing on methanol lack reducing power. Formate could be used as a reducing agent. In this study, mevalonic acid (MEV) yield of 0.067 gMEV/g methanol was reached by adding 10 mmol L-1 sodium formate in MEV accumulating stage (at 72 h). The yield was improved by 64.57%, and represented the highest yield reported to date. 13 C-labeling experiments revealed global effects of sodium formate on metabolic pathways in engineered Methylobacterium extorquens AM1. Sodium formate significantly increased the ratios of reducing equivalents, enhanced the metabolic rate of pathways demanding reducing cofactors and redirected the carbon flux to MEV synthesis. As a result, coupling formate to methanol-based production provide a promising way for converting C1 substances to useful chemical products.

Integrated rational and evolutionary engineering of genome-reduced Pseudomonas putida strains promotes synthetic formate assimilation.

Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO2-but formatotrophy has been engineered in only a few industrially-relevant microbial hosts. We addressed the challenge of expanding the feedstock range of bacterial hosts by adopting Pseudomonas putida as a robust platform for synthetic formate assimilation. Here, the metabolism of a genome-reduced variant of P. putida was radically rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylobacterium extorquens) and native biochemical reactions. Modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate (predominantly for energy supply), and adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs lead to doubling times of ca. 15 h. We likewise identified emergent metabolic features for assimilation of C1 units in these evolved P. putida populations. Taken together, our results consolidate the landscape of useful microbial platforms that can be implemented for C1-based biotechnological production towards a formate bioeconomy.

Improvement of dicarboxylic acid production with Methylorubrum extorquens by reduction of product reuptake.

The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids. KEY POINTS: • 2,2-Difluorosuccinic acid is used to select for dicarboxylic acid uptake mutations. • Deletion of dctA2 leads to reduction of dicarboxylic acid uptake. • Transporter-deficient strains show improved production of citramalic acid.

Characterization of a Novel Fe2+ Activated Non-Blue Laccase from Methylobacterium extorquens.

Herein, a novel laccase gene, Melac13220, was amplified from Methylobacterium extorquens and successfully expressed in Escherichia coli with a molecular weight of approximately 50 kDa. The purified Melac13220 had no absorption peak at 610 nm and remained silent within electron paramagnetic resonance spectra, suggesting that Melac13220 belongs to the non-blue laccase group. Both inductively coupled plasma spectroscopy/optical emission spectrometry (ICP-OES) and isothermal titration calorimetry (ITC) indicated that one molecule of Melac13220 can interact with two iron ions. Furthermore, the optimal temperature of Melac13220 was 65 °C. It also showed a high thermolability, and its half-life at 65 °C was 80 min. Melac13220 showed a very good acid environment tolerance; its optimal pH was 1.5. Cu2+ and Co2+ can slightly increase enzyme activity, whereas Fe2+ could increase Melac13220's activity five-fold. Differential scanning calorimetry (DSC) indicated that Fe2+ could also stabilize Melac13220. Unlike most laccases, Melac13220 can efficiently decolorize Congo Red and Indigo Carmine dyes even in the absence of a redox mediator. Thus, the non-blue laccase from Methylobacterium extorquens shows potential application value and may be valuable for environmental protection, especially in the degradation of dyes at low pH.

Isolation and Characterization of Homologically Expressed Methanol Dehydrogenase from Methylorubrum extorquens AM1 for the Development of Bioelectrocatalytical Systems.

(Ca2+)-dependent pyrroloquinolinequinone (PQQ)-dependent methanol dehydrogenase (MDH) (EC: 1.1.2.7) is one of the key enzymes of primary C1-compound metabolism in methylotrophy. PQQ-MDH is a promising catalyst for electrochemical biosensors and biofuel cells. However, the large-scale use of PQQ-MDH in bioelectrocatalysis is not possible due to the low yield of the native enzyme. Homologously overexpressed MDH was obtained from methylotrophic bacterium Methylorubrum extorquens AM1 by cloning the gene of only one subunit, mxaF. The His-tagged enzyme was easily purified by immobilized metal ion affinity chromatography (36% yield). A multimeric form (α6ß6) of recombinant PQQ-MDH possessing enzymatic activity (0.54 U/mg) and high stability was demonstrated for the first time. pH-optimum of the purified protein was about 9-10; the enzyme was activated by ammonium ions. It had the highest affinity toward methanol (KM = 0.36 mM). The recombinant MDH was used for the fabrication of an amperometric biosensor. Its linear range for methanol concentrations was 0.002-0.1 mM, the detection limit was 0.7 µM. The properties of the invented biosensor are competitive to the analogs, meaning that this enzyme is a promising catalyst for industrial methanol biosensors. The developed simplified technology for PQQ-MDH production opens up new opportunities for the development of bioelectrocatalytic systems.

Role of EPS in mitigation of plant abiotic stress: The case of Methylobacterium extorquens PA1.

Methylobacterium extorquens is a facultative methylotrophic Gram-negative bacterium, often associated with plants, that exhibits a unique ability to grow in the presence of high methanol concentrations, which serves as a single carbon energy source. We found that M. extorquens strain PA1 secretes a mixture of different exopolysaccharides (EPSs) when grown in reference medium or in presence of methanol, that induces the secretion of a peculiar and heterogenous mixture of EPSs, with different structure, composition, repeating units, bulk and a variable degree of methylation. These factors influenced 3D structure and supramolecular assets, diffusion properties and hydrodynamic radius, and likely contribute to increase methanol tolerance and cell stability. No direct methanol involvement in the EPSs solvation shell was detected, indicating that the polymer exposure to methanol is water mediated. The presence of methanol induces no changes in size and shape of the polymer chains, highlighting how water-methanol mixtures are a good solvent for refEPS and metEPS.

Poly-3-hydroxybutyrate production in acetate minimal medium using engineered Methylorubrum extorquens AM1.

Acetate is regarded as a sustainable microbial feedstock that is synthesized from biowastes such as synthesis gas (syngas), carbon dioxide, lignocellulose, or organic waste. In this study, Methylorubrum extorquens AM1 was engineered to improve the production of bioplastic poly-3-hydroxybutyrate (PHB) using acetate as the sole carbon source. To utilize acetate as a carbon source and methanol as an energy source, acs encoding acetyl-CoA synthetase and fdh from Burkholderia stabilis were overexpressed, while ftfL involved in the assimilation of methanol into formyl-tetrahydrofolate was deleted. The yields of biomass and PHB from acetate significantly improved, and the growth rate and PHB content of the bacteria increased. In addition, sustainability of the PHB production was demonstrated using acetate derived from carbon dioxide and syngas. This study shows that biopolymers could be synthesized efficiently using acetate as the sole carbon source through metabolic engineering and the supply of energy cofactors.

Development of Methylorubrum extorquens AM1 as a promising platform strain for enhanced violacein production from co-utilization of methanol and acetate.

Violacein, a blue-violet compound with a wide range of beneficial bioactivities, is an attractive product for microbial production. Currently, violacein production has been demonstrated in several sugar heterotrophs through metabolic engineering; however, the cost of production remains an obstacle for business ventures. To address this issue, the development of host strains that can utilize inexpensive alternative substrates to reduce production costs would enable the commercialization of violacein. In this study, we engineered a facultative methylotroph, Methylorubrum extorquens AM1, to develop a methanol-based platform for violacein production. By optimizing expression vectors as well as inducer concentrations, 11.7 mg/L violacein production was first demonstrated using methanol as the sole substrate. Considering that unidentified bottlenecks for violacein biosynthesis in the shikimate pathway of M. extorquens AM1 would be difficult to address using generic metabolic engineering approaches, random mutagenesis and site-directed mutagenesis were implemented, and a 2-fold improvement in violacein production was achieved. Finally, by co-utilization of methanol and acetate, a remarkable enhancement of violacein production to 118 mg/L was achieved. Our results establish a platform strain for violacein production from non-sugar feedstocks, which may contribute to the development of an economically efficient large-scale fermentation system for violacein production.

Structural insight into a molecular mechanism of methenyltetrahydrofolate cyclohydrolase from Methylobacterium extorquens AM1.

Bioconversion of the C1 compounds into value-added products is one of the CO2-reducing strategies. In particular, because CO2 can be easily converted into formate, the efficient and direct bioconversion of CO2 through formate assimilation is attracting attention. The tetrahydrofolate (THF) cycle is the highly efficient reconstructed formate assimilation pathway, and 5,10-methenyltetrahydrofolate cyclohydrolase (FchA) is an essential enzyme involved in the THF cycle. In this study, a kinetic analysis of FchA from Methylobacterium extorquens AM1 (MeFchA) was performed and revealed that the enzyme has much higher cyclization than hydrolyzation activity, making it an optimal enzyme for formate assimilation. The crystal structure of MeFchA in the apo- and the THF-complexed forms was also determined, revealing that the substrate-binding site of the enzyme has three differently charged regions to stabilize the three differently charged moieties of the formyl-THF substrate. The residues involved in the substrate binding were also verified through site-directed mutagenesis. This study provides a biochemical and structural basis for the molecular mechanism underlying formate assimilation.

Characterization of Americium and Curium Complexes with the Protein Lanmodulin: A Potential Macromolecular Mechanism for Actinide Mobility in the Environment.

Anthropogenic radionuclides, including long-lived heavy actinides such as americium and curium, represent the primary long-term challenge for management of nuclear waste. The potential release of these wastes into the environment necessitates understanding their interactions with biogeochemical compounds present in nature. Here, we characterize the interactions between the heavy actinides, Am3+ and Cm3+, and the natural lanthanide-binding protein, lanmodulin (LanM). LanM is produced abundantly by methylotrophic bacteria, including Methylorubrum extorquens, that are widespread in the environment. We determine the first stability constant for an Am3+-protein complex (Am3LanM) and confirm the results with Cm3LanM, indicating a ∼5-fold higher affinity than that for lanthanides with most similar ionic radius, Nd3+ and Sm3+, and making LanM the strongest known heavy actinide-binding protein. The protein's high selectivity over 243Am's daughter nuclide 239Np enables lab-scale actinide-actinide separations as well as provides insight into potential protein-driven mobilization for these actinides in the environment. The luminescence properties of the Cm3+-LanM complex, and NMR studies of Gd3+-LanM, reveal that lanmodulin-bound f-elements possess two coordinated solvent molecules across a range of metal ionic radii. Finally, we show under a wide range of environmentally relevant conditions that lanmodulin effectively outcompetes desferrioxamine B, a hydroxamate siderophore previously proposed to be important in trivalent actinide mobility. These results suggest that natural lanthanide-binding proteins such as lanmodulin may play important roles in speciation and mobility of actinides in the environment; it also suggests that protein-based biotechnologies may provide a new frontier in actinide remediation, detection, and separations.

Functional diversity of isoprenoid lipids in Methylobacterium extorquens PA1.

Hopanoids and carotenoids are two of the major isoprenoid-derived lipid classes in prokaryotes that have been proposed to have similar membrane ordering properties as sterols. Methylobacterium extorquens contains hopanoids and carotenoids in their outer membrane, making them an ideal system to investigate the role of isoprenoid lipids in surface membrane function and cellular fitness. By genetically knocking out hpnE and crtB we disrupted the production of squalene and phytoene in M. extorquens PA1, which are the presumed precursors for hopanoids and carotenoids respectively. Deletion of hpnE revealed that carotenoid biosynthesis utilizes squalene as a precursor resulting in pigmentation with a C30 backbone, rather than the previously predicted canonical C40 phytoene-derived pathway. Phylogenetic analysis suggested that M. extorquens may have acquired the C30 pathway through lateral gene transfer from Planctomycetes. Surprisingly, disruption of carotenoid synthesis did not generate any major growth or membrane biophysical phenotypes, but slightly increased sensitivity to oxidative stress. We further demonstrated that hopanoids but not carotenoids are essential for growth at higher temperatures, membrane permeability and tolerance of low divalent cation concentrations. These observations show that hopanoids and carotenoids serve diverse roles in the outer membrane of M. extorquens PA1.

Methylotrophic bacterium-based molecular sensor for the detection of low concentrations of methanol.

Methylotrophic bacterium Methylorubrum extorquens is a promising microorganism for the production of value-added compounds from methanol. This study focused on the development of a single-cell level biosensor system that detects methanol by using the intrinsic regulatory machinery which responds to the presence of methanol in this bacterium. A green fluorescent protein (GFP) gene located downstream of the promoter region of the serine glyoxylate aminotransferase gene (Psga) or the methanol dehydrogenase subunit 1 precursor gene (PmxaF) was inserted into the chromosome of M. extorquens wild-type strain AM1. The expression of GFP upon methanol exposure was measured by spectrofluorometer and fluorescence-activated cell sorting (FACS). The strain harboring Psga-gfp emitted fluorescence only when methanol was supplied to the culture medium, while the other strain harboring PmxaF-gfp showed high basal fluorescence even in the absence of methanol. The fluorescence intensity of the Psga-gfp strain depended on a methanol concentration higher than 25 µM, and the sensitivity and dose-dependency of this strain were much higher than previous systems using Escherichia coli. The methanol-sensing properties of the engineered M. extorquens strain were comparable to those of a methylotrophic yeast-based biosensor, suggesting the usefulness of methylotrophic microorganisms as platforms for single-cell sensing of C1 compounds. The constructed methanol sensor strain, coupled with flow cytometry techniques, provides a high-throughput and highly sensitive screening method for the selection of functional methanol-producing enzymes.

EfgA is a conserved formaldehyde sensor that leads to bacterial growth arrest in response to elevated formaldehyde.

Normal cellular processes give rise to toxic metabolites that cells must mitigate. Formaldehyde is a universal stressor and potent metabolic toxin that is generated in organisms from bacteria to humans. Methylotrophic bacteria such as Methylorubrum extorquens face an acute challenge due to their production of formaldehyde as an obligate central intermediate of single-carbon metabolism. Mechanisms to sense and respond to formaldehyde were speculated to exist in methylotrophs for decades but had never been discovered. Here, we identify a member of the DUF336 domain family, named efgA for enhanced formaldehyde growth, that plays an important role in endogenous formaldehyde stress response in M. extorquens PA1 and is found almost exclusively in methylotrophic taxa. Our experimental analyses reveal that EfgA is a formaldehyde sensor that rapidly arrests growth in response to elevated levels of formaldehyde. Heterologous expression of EfgA in Escherichia coli increases formaldehyde resistance, indicating that its interaction partners are widespread and conserved. EfgA represents the first example of a formaldehyde stress response system that does not involve enzymatic detoxification. Thus, EfgA comprises a unique stress response mechanism in bacteria, whereby a single protein directly senses elevated levels of a toxic intracellular metabolite and safeguards cells from potential damage.

Gene repression using synthetic small regulatory RNA in Methylorubrum extorquens.

AIM: Genetic tools are a prerequisite for engineering cell factories for synthetic biology and biotechnology. Methylorubrum extorquens is an important platform for a future one-carbon (C1) bioeconomy, but its application is currently limited by the availability of genetic tools. Small regulatory RNA (sRNA) is an important regulatory factor in bacteria and has been applied for gene repression in several strains. This study aimed to construct a synthetic sRNA system based on the MicC scaffold and the chaperone Hfq to control gene expression in M. extorquens. METHODS AND RESULTS: Initially, the exogenous lacZ gene was transposed into the M. extorquens chromosome as a reporter, and corresponding ß-galactosidase was measured to assess the knockdown efficiency of lacZ. A synthetic sRNA containing a 24-nt antisense RNA targeting lacZ and an Escherichia coli MicC scaffold were constructed, and different Hfqs from E. coli, M. extorquens AM1 and PA1 were further identified. The results showed that the expression of endogenous hfqs from the chromosome in M. extorquens strains was inadequate, and only when it was overexpressed via the plasmid did the colonies show a colour change and a corresponding decrease in ß-galactosidase expression. More specifically, M. extorquens strains with overexpressing their own Hfq showed the best gene repression efficiency. Furthermore, this E. coli MicC scaffold and AM1 Hfq system were combined to knock down crtI gene expression in AM1, leading to an 86% decrease in carotenoid production (0·09 mg g-1 ) compared to that (0·65 mg g-1 ) in the wild-type strain. CONCLUSION: A functional synthetic sRNA system combined with E. coli MicC and endogenous Hfq was constructed in M. extorquens strains, which was able to interfere with the target crtI gene and reduce carotenoid production. SIGNIFICANCE AND IMPACT OF THE STUDY: The synthetic sRNA system reported in this study provides a genetic tool for the manipulation of M. extorquens. The present findings might be helpful for achieving high-throughput gene knockdown expression.

Structural diversity of the coenzyme methylofuran and identification of enzymes for the biosynthesis of its polyglutamate side chain.

Methylofuran (MYFR) is a formyl-carrying coenzyme essential for the oxidation of formaldehyde in most methylotrophic bacteria. In Methylorubrum extorquens, MYFR contains a large and branched polyglutamate side chain of up to 24 glutamates. These glutamates play an essential role in interfacing the coenzyme with the formyltransferase/hydrolase complex, an enzyme that generates formate. To date, MYFR has not been identified in other methylotrophs, and it is unknown whether its structural features are conserved. Here, we examined nine bacterial strains for the presence and structure of MYFR using high-resolution liquid chromatography-mass spectrometry (LC-MS). Two of the strains produced MYFR as present in M. extorquens, while a modified MYFR containing tyramine instead of tyrosine in its core structure was detected in six strains. When M. extorquens was grown in the presence of tyramine, the compound was readily incorporated into MYFR, indicating that the biosynthetic enzymes are unable to discriminate tyrosine from tyramine. Using gene deletions in combination with LC-MS analyses, we identified three genes, orf5, orfY, and orf17 that are essential for MYFR biosynthesis. Notably, the orfY and orf5 mutants accumulated short MYFR intermediates with only one and two glutamates, respectively, suggesting that these enzymes catalyze glutamate addition. Upon homologous overexpression of orf5, a drastic increase in the number of glutamates in MYFR was observed (up to 40 glutamates), further corroborating the function of Orf5 as a glutamate ligase. We thus renamed OrfY and Orf5 to MyfA and MyfB to highlight that these enzymes are specifically involved in MYFR biosynthesis.