Genetic modification of Methylobacterium extorquens G10 producer strain of polyhydroxybutyrate

The effect of the increased copy number of polyhydroxybutyrate (PHB) biosynthesis genes in pink-pigmented methylobacterium Methylobacterium extorquens G10 on properties of the biopolymer was studied. The activity of poly-3-hydroxybutyrate-synthase (PHB-synthase) was shown to increase and the molecular weight of synthesized PHB decreases twofold (150 → 79 kDa) after insertion of extra copies of phaC and phaCAB genes into cells of the producer strain, whereas the physicochemical properties of the plastic changed insignificantly. White mutant M. extorquens G10-W with disrupted synthesis of the carotenoid pigment (defect by the crtI gene, which codes for phytoene desaturase) was established to have the same rate of growth and level of PHB accumulation as the initial strain G10. The G10-W strain is a promising producer of PHB, with decreased expenses for purification and PHB biosynthesis.     

Biosynthesis of polyhydroxybutyrate/valerate with different molecular weights during the growth of Methylobacterium extorquens G-10 on a methanol-pentanol mixture

The influence of the concentration and time of addition of cosubstrate (pentanol) on the molecular weight (MW) of the polyhydroxybutyrate/valerate (PHBV) copolymer synthesized by Methylobacterium extorquens G-10 during cultivation in a methanol-containing medium has been studied. It was shown that an increase in the pentanol concentration to 20% in a mixture with methanol stimulated the biosynthesis of PHBV with a MW of ～1500 kDa and increased the content of valerate up to 50%, especially when pentanol was added to the log phase culture. High pentanol concentrations are toxic for the producer and reduce the total yield of PHBV. An MW increase to 1500 kDa lowers the melting temperature (from 172 to 162°C) and the crystallinity degree (from 63 to 8%) of the biopolymer but increases its elasticity. The revealed variability of PHBV properties extends considerably the potential application areas of synthetic bioplastics.     

Synthesis of high-molecular-mass polyhydroxybutyrate from methanol in <Emphasis Type="Italic">Methyloligella halotolerans</Emphasis> C2

The influence of the concentrations of carbon and energy sources (methanol) and nitrogen (NH ₄ ⁺ ) on the yield and molecular mass (Mm) of poly-β-hydroxybutyrate (PHB) synthesized by the novel methylotroph Methyloligella halotolerans C2 was investigated. It was shown that the maximum concentrations of NH ₄ ⁺ and methanol in the medium for PHB biosynthesis were 0.15 g/L and 1 ± 0.2 mL/L, respectively. A unique high-molecular (8000–10000 kDa) PHB was obtained with NH ₄ ⁺ and methanol concentrations of 0.12 g/L and 1.25 mL/L, respectively, in a fermenter. The main physicochemical and mechanical properties of the obtained bioplastic with a molecular mass of 10000 kDa were determined.     

Biosynthesis of polyhydroxyalkanoate copolymers from methanol by Methylobacterium extorquens AM1 and the engineered strains under cobalt-deficient conditions

Methylobacterium extorquens AM1 has been shown to accumulate polyhydroxyalkanoate (PHA) composed solely of (R)-3-hydroxybutyrate (3HB) during methylotrophic growth. The present study demonstrated that the wild-type strain AM1 grown under Co²⁺-deficient conditions accumulated copolyesters of 3HB and a C₅-monomer, (R)-3-hydroxyvalerate (3HV), using methanol as the sole carbon source. The 3HV unit was supposed to be derived from propionyl-CoA, synthesized via the ethylmalonyl-CoA pathway impaired by Co²⁺ limitation. This assumption was strongly supported by the dominant incorporation of the 3HV unit into PHA when a strain lacking propionyl-CoA carboxylase was incubated with methanol. Further genetic engineering of M. extorquens AM1 was employed for the methylotrophic synthesis of PHA copolymers. A recombinant strain of M. extorquens AM1C_Ac in which the original PHA synthase gene phaC _Me had been replaced by phaC _Ac , encoding an enzyme with broad substrate specificity from Aeromonas caviae, produced a PHA terpolymer composed of 3HB, 3HV, and a C₆-monomer, (R)-3-hydroxyhexanoate, from methanol. The cellular content and molecular weight of the PHA accumulated in the strain AM1C_Ac were higher than those of PHA in the wild-type strain. The triple deletion of three PHA depolymerase genes in M. extorquens AM1C_Ac showed no significant effects on growth and PHA biosynthesis properties. Overexpression of the genes encoding β-ketothiolase and NADPH-acetoacetyl-CoA reductase increased the cellular PHA content and 3HV composition in PHA, although the cell growth on methanol was decreased. This study opens up the possibility of producing practical PHA copolymers with methylotrophic bacteria using methanol as a feedstock.     

Methylotrophic Metabolism Is Advantageous for Methylobacterium extorquens during Colonization of Medicago truncatula under Competitive Conditions

Facultative methylotrophic bacteria of the genus Methylobacterium are commonly found in association with plants. Inoculation experiments were performed to study the importance of methylotrophic metabolism for colonization of the model legume Medicago truncatula. Competition experiments with Methylobacterium extorquens wild-type strain AM1 and methylotrophy mutants revealed that the ability to use methanol as a carbon and energy source provides a selective advantage during colonization of M. truncatula. Differences in the fitness of mutants defective in different stages of methylotrophic metabolism were found; whereas approximately 25% of the mutant incapable of oxidizing methanol to formaldehyde (deficient in methanol dehydrogenase) was recovered, 10% or less of the mutants incapable of oxidizing formaldehyde to CO₂ (defective in biosynthesis of the cofactor tetrahydromethanopterin) was recovered. Interestingly, impaired fitness of the mutant strains compared with the wild type was found on leaves and roots. Single-inoculation experiments showed, however, that mutants with defects in methylotrophy were capable of plant colonization at the wild-type level, indicating that methanol is not the only carbon source that is accessible to Methylobacterium while it is associated with plants. Fluorescence microscopy with a green fluorescent protein-labeled derivative of M. extorquens AM1 revealed that the majority of the bacterial cells on leaves were on the surface and that the cells were most abundant on the lower, abaxial side. However, bacterial cells were also found in the intercellular spaces inside the leaves, especially in the epidermal cell layer and immediately underneath this layer.     

Methylotrophic bacteria on the surfaces of field-grown sunflower plants: a biogeographic perspective

Plant-associated methylobacteria of the genus Methylobacterium colonize the foliage and roots of embryophytes, living on the volatile compound methanol emitted from the cells of their host organism. In this study we analyzed these surface-dwelling pink-pigmented epiphytes in three contrasting habitats of field-grown sunflower plants (Helianthus annuus). Using the methanol–ammonium salts agar surface impression method and a polymerase chain reaction (PCR)-based assay, we document the occurrence and characterize the composition of the methylobacteria in these epiphytic habitats. In both the sun-exposed phylloplane (yellow ligulate florets; green leaves) and the moist, dark rhizoplane pink-pigmented methylobacteria were detected that are assigned to the taxa M. mesophilicum, M. extorquens, M. radiotolerans and M. sp. (un-identifiable by our methods). Considerable differences in relative species compositions were found. These data are discussed with respect to a biogeographic model of the plant surface and microbial population dynamics on leaves. In addition, methylobacteria were analyzed by microscopic techniques. We document that in sedentary colonies extracellular polymers are secreted. However, flagella, which were observed in single cells maintained in liquid cultures, are absent in these bacterial aggregates.     

Effects of DNA-damaging Agents on Aerobic Methylobacteria Capable and Incapable of Utilizing Dichloromethane

Methylobacterium dichloromethanicum DM4, a degrader of dichloromethane (DCM), was more tolerant to the effect of H₂O₂ and UV irradiation than Methylobacterium extorquens AM1, which does not consume DCM. The addition of CH₂Cl₂ to methylobacteria with active serine, ribulose monophosphate, and ribulose bisphosphate pathways of C₁ metabolism, grown on methanol, resulted in a 1.1- to 2.5-fold increase in the incorporation of [α-³²P]dATP into DNA by the Klenow fragment (exo^?). Since DCM dehalogenase was not induced in this process, the increase in the total lengths of DNA gaps resulted from the action of DCM rather than S-chloromethylglutathione (intermediate of primary dehalogenation). The degree of DNA damage in the presence of CH₂Cl₂ was lower in DCM degraders than methylobacteria incapable of degrading this pollutant. This suggests that DCM degraders possess a more efficient mechanism of DNA repair.     

Phosphate-solubilizing activity of aerobic methylobacteria

Phosphate-solubilizing activity was found in 14 strains of plant-associated aerobic methylobacteria belonging to the genera Methylophilus, Methylobacillus, Methylovorus, Methylopila, Methylobacterium, Delftia, and Ancyclobacter. The growth of methylobacteria on medium with methanol as the carbon and energy source and insoluble tricalcium phosphate as the phosphorus source was accompanied by a decrease in pH due to the accumulation of up to 7 mM formic acid as a methanol oxidation intermediate and by release of 120–280 μM phosphate ions, which can be used by both bacteria and plants. Phosphate-solubilizing activity is a newly revealed role of methylobacteria in phytosymbiosis.     

Localization of strawberry (Fragaria x ananassa) and Methylobacterium extorquens genes of strawberry flavor biosynthesis in strawberry tissue by in situ hybridization

Strawberry flavor is one of the most popular fruit flavors worldwide, with numerous applications in the food industry. In addition, the biosynthetic origin of the most important strawberry flavor components, such as 2,5-dimethyl-4-hydroxy-2H-furan-3-one (DMHF), is a challenging research area. DMHF's precursor, 2-hydroxy-propanal (or lactaldehyde), is biosynthesized by the endophytic bacterium Methylobacterium extorquens (M. extorquens). In particular, the alcohol dehydrogenase (ADH) enzymes of M. extorquens are involved in the biogenesis of DMHF precursors since they have the capacity to oxidize the strawberry-derived 1,2-propanediol to lactaldehyde. In this study, the expression of the endophytic ADH and the plant DMHF biosynthesis genes was examined in the tissues of raw and ripe strawberry receptacles by in situ hybridization. The presence of endophytic bacteria was studied in the same tissues by probes targeting bacterial 16S ribosomal ribonucleic acid. Hybridization signals of probes specific for endophytic ADH and plant DMHF biosynthesis genes, as well as bacteria-specific probes, were detected in the same locations. The probes were localized near the plasma membranes or intercellular spaces of cortical and vascular tissues of the receptacle, and intracellularly in the tissues of achenes. By localizing the expression of the endophytic methanol ADH and plant DMHF biosynthesis genes to the same tissues, we have reinforced our original hypothesis that an intimate symbiotic relationship between strawberry and endophytic cells exists and leads to the biosynthesis of DMHF.     

The 380 kb pCMU01 Plasmid Encodes Chloromethane Utilization Genes and Redundant Genes for Vitamin B12- and Tetrahydrofolate-Dependent Chloromethane Metabolism in Methylobacterium extorquens CM4: A Proteomic and Bioinformatics Study

Chloromethane (CH₃Cl) is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu) was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B₁₂ are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD), as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B₁₂-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2). In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase) are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex), conversion of tetrahydrofolate-bound one-carbon units, and central metabolism. The mosaic organization of plasmid pCMU01 and the clustering of genes coding for dehalogenase enzymes and for biosynthesis of associated cofactors suggests a history of gene acquisition related to chloromethane utilization.     

Increased Bioplastic Production with an RNA Polymerase Sigma Factor SigE during Nitrogen Starvation in Synechocystis sp. PCC 6803

Because cyanobacteria directly harvest CO₂ and light energy, their carbon metabolism is important for both basic and applied sciences. Here, we show that overexpression of the sigma factor sigE in Synechocystis sp. PCC 6803 widely changes sugar catabolism and increases production of the biodegradable polyester polyhydroxybutyrate (PHB) during nitrogen starvation. sigE overexpression elevates the levels of proteins implicated in glycogen catabolism, the oxidative pentose phosphate pathway, and polyhydroxyalkanoate biosynthesis. PHB accumulation is enhanced by sigE overexpression under nitrogen-limited conditions, yet the molecular weights of PHBs synthesized by the parental glucose-tolerant and sigE overexpression strain are similar. Although gene expression induced by nitrogen starvation is changed and other metabolites (such as GDP-mannose and citrate) accumulate under sigE overexpression, genetic engineering of this sigma factor altered the metabolic pathway from glycogen to PHB during nitrogen starvation.     

Thioesterases for ethylmalonyl–CoA pathway derived dicarboxylic acid production in <Emphasis Type="Italic">Methylobacterium extorquens</Emphasis> AM1

The ethylmalonyl–coenzyme A pathway (EMCP) is a recently discovered pathway present in diverse α-proteobacteria such as the well studied methylotroph Methylobacterium extorquens AM1. Its glyoxylate regeneration function is obligatory during growth on C1 carbon sources like methanol. The EMCP contains special CoA esters, of which dicarboxylic acid derivatives are of high interest as building blocks for chemical industry. The possible production of dicarboxylic acids out of the alternative, non-food competing C-source methanol could lead to sustainable and economic processes. In this work we present a testing of functional thioesterases being active towards the EMCP CoA esters including in vitro enzymatic assays and in vivo acid production. Five thioesterases including TesB from Escherichia coli and M. extorquens, YciA from E. coli, Bch from Bacillus subtilis and Acot4 from Mus musculus showed activity towards EMCP CoA esters in vitro at which YciA was most active. Expressing yciA in M. extorquens AM1 led to release of 70 mg/l mesaconic and 60 mg/l methylsuccinic acid into culture supernatant during exponential growth phase. Our data demonstrates the biotechnological applicability of the thioesterase YciA and the possibility of EMCP dicarboxylic acid production from methanol using M. extorquens AM1.     

Co-Consumption of Methanol and Succinate by Methylobacterium extorquens AM1

Methylobacterium extorquens AM1 is a facultative methylotrophic Alphaproteobacterium and has been subject to intense study under pure methylotrophic as well as pure heterotrophic growth conditions in the past. Here, we investigated the metabolism of M. extorquens AM1 under mixed substrate conditions, i.e., in the presence of methanol plus succinate. We found that both substrates were co-consumed, and the carbon conversion was two-thirds from succinate and one-third from methanol relative to mol carbon. ¹³C-methanol labeling and liquid chromatography mass spectrometry analyses revealed the different fates of the carbon from the two substrates. Methanol was primarily oxidized to CO₂ for energy generation. However, a portion of the methanol entered biosynthetic reactions via reactions specific to the one-carbon carrier tetrahydrofolate. In contrast, succinate was primarily used to provide precursor metabolites for bulk biomass production. This work opens new perspectives on the role of methylotrophy when substrates are simultaneously available, a situation prevailing under environmental conditions.     

Adaptation of aerobic methylobacteria to dichloromethane degradation

A shortening of the lag phase in dichloromethane (DCM) consumption was observed in the methylobacteria Methylopila helvetica DM6 and Albibacter methylovorans DM10 after prior growth on methanol with the presence of 1.5% NaCl. Neither heat nor acid stress accelerated methylobacterium adaptation to DCM consumption. Sodium azide (1 mM) and potassium cyanide (1 mM) inhibited consumption of DCM by these degraders but not by transconjugants Methylobacterium extorquens AM1, expressing DCM dehalogenase but unable to grow on DCM. This indicates that the degrader strains possess energy-dependent systems of transport of DCM or chloride anions produced during DCM dehalogenation. Inducible proteins were found in the membrane fraction of A. methylovorans DM10 cells adapted to DCM and elevated NaCl concentration.     

Enhanced Resistance of Pea Plants to Oxidative Stress Caused by Paraquat during Colonization by Aerobic Methylobacteria

The influence of colonization of the pea (Pisum sativum L.) by aerobic methylobacteria of five different species (Methylophilus flavus Ship, Methylobacterium extorquens G10, Methylobacillus arboreus Iva, Methylopila musalis MUSA, Methylopila turkiensis Side1) on plant resistance to paraquat-induced stresses has been studied. The normal conditions of pea colonization by methylobacteria were characterized by a decrease in the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidases) and in the concentrations of endogenous H₂O₂, proline, and malonic dialdehyde, which is a product of lipid peroxidation and indicator of damage to plant cell membranes, and an increase in the activity of the photosynthetic apparatus (the content of chlorophylls а, b and carotenoids). In the presence of paraquat, the colonized plants had higher activities of antioxidant enzymes, stable photosynthetic indices, and a less intensive accumulation of the products of lipid peroxidation as compared to noncolonized plants. Thus, colonization by methylobacteria considerably increased the adaptive protection of pea plants to the paraquat-induced oxidative stress.     

Aerobic Methylobacteria Are Capable of Synthesizing Auxins

Obligately and facultatively methylotrophic bacteria with different pathways of C1 metabolism were found to be able to produce auxins, particularly indole-3-acetic acid (IAA), in amounts of 3–100 g/ml. Indole-3-pyruvic acid and indole-3-acetamide were detected only in methylobacteria with the serine pathway of C1 metabolism (Methylobacterium mesophilicumand Aminobacter aminovorans).The production of auxins by methylobacteria was stimulated by the addition of L-tryptophan to the growth medium and was inhibited by ammonium ions. The methylobacteria under study lacked tryptophan decarboxylase and tryptophan side-chain oxidase. At the same time, they were found to contain several aminotransferases. IAA is presumably synthesized by methylobacteria through indole-3-pyruvic acid.     

Development of an Optimized Medium,Strain and High-Throughput Culturing Methods for Methylobacterium extorquens

Methylobacterium extorquens strains are the best-studied methylotrophic model system, and their metabolism of single carbon compounds has been studied for over 50 years. Here we develop a new system for high-throughput batch culture of M. extorquens in microtiter plates by jointly optimizing the properties of the organism, the growth media and the culturing system. After removing cellulose synthase genes in M. extorquens strains AM1 and PA1 to prevent biofilm formation, we found that currently available lab automation equipment, integrated and managed by open source software, makes possible reliable estimates of the exponential growth rate. Using this system, we developed an optimized growth medium for M. extorquens using response surface methodologies. We found that media that used EDTA as a metal chelator inhibited growth and led to inconsistent culture conditions. In contrast, the new medium we developed with a PIPES buffer and metals chelated by citrate allowed for fast and more consistent growth rates. This new Methylobacterium PIPES (‘MP’) medium was also robust to large deviations in its component ingredients which avoided batch effects from experiments that used media prepared at different times. MP medium allows for faster and more consistent growth than other media used for M. extorquens.     

High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB

Methylobacterium extorquens ATCC 55366 was successfully cultivated at very high cell densities in a fed-batch fermentation system using methanol as a sole carbon and energy source and a completely minimal culture medium for the production of poly--hydroxybutyrate (PHB). Cell biomass levels were between 100 g/l and 115 g/l (dry weight) and cells contained between 40% and 46% PHB on a dry-weight basis. PHB with higher molecular mass values than previously reported for methylotrophic bacteria was obtained under certain conditions. Shake-flask and fermentor experiments showed the importance of adjusting the mineral composition of the medium for improved biomass production and higher growth rates. High-cell-density cultures were obtained without the need for oxygen-enriched air; once the oxygen transfer capacity of the fermentor was reached, methanol was thereafter added in proportion to the amount of available dissolved oxygen, thus preventing oxygen limitation. Controlling the methanol concentration at a very low level (less than 0.01 g/l), during the PHB production phase, led not only to prevention of oxygen limitation but also to the production of very high-molecular-mass PHB, in the 900–1800 kDa range. Biomass yields relative to the total methanol consumed were in the range 0.29–0.33 g/g, whereas PHB yields were in the range 0.09–0.12 g/g. During the active period of PHB synthesis, PHB yields relative to the total methanol consumed were between 0.2 g/g and 0.22 g/g. M. extorquens ATCC 55366 appears to be a promising organism for industrial PHB production.