Biostimulation by methanol enables the methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. to reveal high formaldehyde biodegradation potential as well as to adapt to this toxic pollutant

The methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. revealed enhanced biodegradation capability of exogenously applied formaldehyde (Fd) upon biostimulation achieved by the presence of methanol, as compared to glucose. Upon growth on either of the above substrates, the strains proved to produce the activity of glutathione-dependent formaldehyde dehydrogenase—the enzyme known to control the biooxidative step of Fd detoxification. However, in the absence of methanol, the yeasts’ tolerance to Fd was decreased, and the elevated sensitivity was especially pronounced for Trichosporon sp. Both strains responded to the methanol and/or Fd treatment by increasing their unsaturation index (UI) at xenobiotic levels below minimal inhibitory concentrations. This indicated that the UI changes effected from the de novo synthesis of (poly) unsaturated fatty acids carried out by viable cells. It is concluded that the yeast cell response to Fd intoxication involves stress reaction at the level of membranes. Fluidization of the lipid bilayer as promoted by methanol is suggested as a significant adaptive mechanism increasing the overall fitness enabling to cope with the formaldehyde xenobiotic via biodegradative pathway of C₁-compound metabolism.     

Coping with formaldehyde during C1 metabolism of Paracoccus denitrificans

Methylotrophic bacteria are capable of growth using reduced one-carbon (C1) compounds like methanol or methylamine as free energy sources. Paracoccus denitrificans, which is a facultative methylotrophic organism, switches to this type of autotrophic metabolism only when it experiences a shortage of available heterotrophic free energy sources. Since the oxidation of C1 substrates is energetically less favourable than that of the heterotrophic ones, a global regulatory circuit ensures that the enzymes involved in methylotrophic growth are repressed during heterotrophic growth. Once the decision is made to switch to methylotrophic growth, additional regulatory proteins ensure the fine-tuned expression of the participating enzymes such that the steady-state concentration of formaldehyde, the oxidation product of C1 substrates, is kept below cytotoxic levels.     

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.     

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.     

Multiple formaldehyde oxidation/detoxification pathways in Burkholderia fungorum LB400

Burkholderia species are free-living bacteria with a versatile metabolic lifestyle. The genome of B. fungorum LB400 is predicted to encode three different pathways for formaldehyde oxidation: an NAD-linked, glutathione (GSH)-independent formaldehyde dehydrogenase; an NAD-linked, GSH-dependent formaldehyde oxidation system; and a tetrahydromethanopterin-methanofuran-dependent formaldehyde oxidation system. The other Burkholderia species for which genome sequences are available, B. mallei, B. pseudomallei, and B. cepacia, are predicted to contain only the first two of these pathways. The roles of the three putative formaldehyde oxidation pathways in B. fungorum LB400 have been assessed via knockout mutations in each of these pathways, as well as in all combinations of knockouts. The resulting mutants have the expected loss of enzyme activities and exhibit defects of varying degrees of severity during growth on choline, a formaldehyde-producing substrate. Our data suggest that all three pathways are involved in formaldehyde detoxification and are functionally redundant under the tested conditions.     

Reactions of direct formaldehyde oxidation to CO2 are non-essential for energy supply of yeast methylotrophic growth

Mutants of the methylotrophic yeast Hansenula polymorpha deficient in NAD-dependent formaldehyde or formate dehydrogenases have been isolated. They were more sensitive for exogenous methanol but retained the ability for methylotrophic growth. In the medium with methanol the growth yields of the mutant 356–83 deficient in formaldehyde dehydrogenase and of the wild-type strain were identical (0.34 g cells/g methanol) under chemostat cultivation. These results indicate that enzymes of direct formaldehyde oxidation are not indispensable for methylotrophic growth. At the same time inhibition of tricarboxylic acid cycle has resulted in suppression of growth in the media with multicarbon nonfermentable substrates such as glycerol, succinate, ethanol and dihydroxyacetone as well as with methanol, but not with glucose. In the experiments with the wild-type strain H. polymorpha it has been shown that citrate and dihydroxyacetone inhibit the radioactivity incorporation from ¹⁴C-methanol into CO₂. All obtained data indicate that for the dissimilation of methanol and the supplying of energy for methylotrophic growth, the functioning of tricarboxylic acid cycle reactions as oppossed to those of direct formaldehyde oxidation is essential.     

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.     

Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria

Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO(2) for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO(2) have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C(1) unit conversion bound to the cofactors tetrahydrofolate (H(4)F), tetrahydromethanopterin (H(4)MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C(1) carrier, the oxidation of the cofactor-bound C(1) unit and its conversion to formate, and the oxidation of formate to CO(2). However, the H(4)MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C(1) units for biosynthetic reactions (e.g., H(4)F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).     

细菌的核酮糖单磷酸途径与甲醛同化作用

核酮糖单磷酸途径最初在甲基营养菌中发现,现在被认为是在细菌中广泛存在的和甲醛同化作用及脱毒相关的一条途径,该途径的关键酶是6-磷酸己酮糖合成酶和6-磷酸己酮糖异构酶。文章将介绍来源于各种细菌的核酮糖单磷酸途径的生理作用及其两个关键酶基因的组织结构、表达调控机制与应用前景。     

Methylamine Utilization via the N-Methylglutamate Pathway in Methylobacterium extorquens PA1 Involves a Novel Flow of Carbon through C1 Assimilation and Dissimilation Pathways

Methylotrophs grow on reduced single-carbon compounds like methylamine as the sole source of carbon and energy. In Methylobacterium extorquens AM1, the best-studied aerobic methylotroph, a periplasmic methylamine dehydrogenase that catalyzes the primary oxidation of methylamine to formaldehyde has been examined in great detail. However, recent metagenomic data from natural ecosystems are revealing the abundance and importance of lesser-known routes, such as the N-methylglutamate pathway, for methylamine oxidation. In this study, we used M. extorquens PA1, a strain that is closely related to M. extorquens AM1 but is lacking methylamine dehydrogenase, to dissect the genetics and physiology of the ecologically relevant N-methylglutamate pathway for methylamine oxidation. Phenotypic analyses of mutants with null mutations in genes encoding enzymes of the N-methylglutamate pathway suggested that γ-glutamylmethylamide synthetase is essential for growth on methylamine as a carbon source but not as a nitrogen source. Furthermore, analysis of M. extorquens PA1 mutants with defects in methylotrophy-specific dissimilatory and assimilatory modules suggested that methylamine use via the N-methylglutamate pathway requires the tetrahydromethanopterin (H₄MPT)-dependent formaldehyde oxidation pathway but not a complete tetrahydrofolate (H₄F)-dependent formate assimilation pathway. Additionally, we present genetic evidence that formaldehyde-activating enzyme (FAE) homologs might be involved in methylotrophy. Null mutants of FAE and homologs revealed that FAE and FAE2 influence the growth rate and FAE3 influences the yield during the growth of M. extorquens PA1 on methylamine.     

Microbial degradation of formaldehyde in white mineral dispersions preserved with formaldehyde-releasing biocides

Two different formaldehyde-degrading microorganisms, Pseudomonas putida and Methylobacterium extorquens, were isolated from calcium carbonate slurry containing the formaldehyde-releasing biocide (ethylenedioxy) dimethanol. Their relative formaldehyde biodegradation and formic acid production kinetics were studied in broth and in calcium carbonate slurry for each microorganism individually, as well as in mixed cultures. Furthermore, the minimal inhibition concentration (MIC) was determined. The results indicated that in slurry, M. extorquens is more tolerant of formaldehyde than P. putida. In slurry, microbial-induced oxidation of formaldehyde caused a temporary accumulation of formic acid, which is presumed to be responsible for pH drop and destabilisation of the calcium carbonate slurry suspension systems. In addition, the residual formaldehyde concentration was observed to drive dominance and recovery of individual formaldehyde-resistant microorganisms in the slurry. Overall, this investigation indicated that biodegradation of formaldehyde in calcium carbonate slurry is brought about by alternating dominance of bacterial genera of mixed formaldehyde-resistant microbial populations.     

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.     

Formaldehyde Fixation Contributes to Detoxification for Growth of a Nonmethylotroph, Burkholderia cepacia TM1, on Vanillic Acid

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [¹⁴C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.     

Activities of the enzymes of formaldehyde catabolism in recombinant strains of <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Activities of the enzymes of formaldehyde (FA) catabolism in recombinant strains of the methylotrophic yeast Hansenula polymorpha overproducing NAD⁺- and glutathione-dependent formaldehyde dehydrogenase (FADH) were studied under different cultivation conditions and at elevated FA content. Southern dot-blot analysis confirmed the presence of six to eight copies of the target FLD1 gene in stable recombinant clones of H. polymorpha. Under certain cultivation conditions, the transformants resistant to elevated FA concentrations were shown to produce FADH and other bioanalytically important enzymes: formate dehydrogenase, alcohol dehydrogenase, alcohol oxidase, and formaldehyde reductase. The optimal cultivation conditions for recombinants were determined, resulting in maximum synthesis of FADH: methanol as a carbon source, methylamine as a nitrogen source, FA as an inducer, temperature of 37°C, and cells in the early exponential phase of growth.     

Cloning and characterisation of S-formylglutathione hydrolase from Arabidopsis thaliana: a pathway for formaldehyde detoxification

The toxic accumulation of formaldehyde in plant cells can occur from a number of sources: atmospheric pollution, endogenous P450 enzymes de-methylating herbicides and cell wall expansion. Any accumulated formaldehyde is rapidly bound to tetrahydrofolate or coupled to nucleophiles such as glutathione (GSH) resulting in S-hydroxymethylglutathione which is subsequently utilised by a glutathione-NAD-dependent formaldehyde dehydrogenase to form S-formylglutathione. The aim of this study was to examine the little understood pathway of formaldehyde detoxification in Arabidopsis thaliana, by cloning and characterising the key esterase S-formylglutathione hydrolase (FGH). The activity of FGH crucially recycles glutathione and renders formate available to the C1 pathway. Previously identified in bacteria, yeast and humans FGH from A. thaliana was cloned by RT-PCR, with a translated open reading frame that consisted of 284 amino acids and showed appreciable similarity with human esterase D. Over-expression using a pET vector system in combination with Escherichia coli resulted in a protein 30 kDa in size as determined by SDS-PAGE. Soluble A. thaliana FGH extracted from E. coli demonstrated a clear affinity for S-formylglutathione (K_m 0.318 mM) and was inhibited, 48% and 59%, respectively, by the addition of 1 μm 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB) and p-hydroxy-mercuribenzoic acid (PHMB) indicating that an SH group may be essential for hydrolytic activity. The activity of S-formylglutathione hydrolase in the leaves of A. thaliana demonstrated that this enzyme might be part of a universal detoxification pathway shared by a variety of organisms.     

Thiols in formaldehyde dissimilation and detoxification

Glutathione is not a universal coenzyme for formaldehyde oxidation. MySH (mycothiol, 1-O-(2'-[N-acetyl-L-cysteinyl]amido-2'-deoxy-alpha-D-glucopyranosyl)-D-m yo-inositol) is GSH's counterpart as coenzyme in formaldehyde dehydrogenase from certain gram-positive bacteria. However, formaldehyde dissimilation and detoxification not only proceed via thiol-dependent but also via thiol-independent dehydrogenases. The distinct structures and enzymatic properties of MySH-dependent and GSH-dependent formaldehyde dehydrogenases could provide clues for development of selective drugs against pathogenic Mycobacteria. It is to be expected that other new types of thiol-dependent formaldehyde dehydrogenases will be discovered in the future. Indications exist that the product of thiol-dependent formaldehyde oxidation, the thiol formate ester, is not only hydrolytically converted into thiol and formate but can also be oxidatively converted in some cases by a molybdoprotein aldehyde dehydrogenase into the corresponding carbonate ester, decomposing spontaneously into CO2 and the thiol.     

Biodegradation of formaldehyde and its derivatives in industrial wastewater with methylotrophic yeast Hansenula polymorpha and with the yeast-bioaugmented activated sludge

Methylotrophic yeast Hansenula polymorphawere shown to cooperate with activated sludgefrom biological wastewater treatment stations,enhancing substantially its potential tobiodegrade formaldehyde in industrial wastewater. After integration with yeast cells the modified sludge retained its original structure and activity whereas its resistance to elevated formaldehyde concentrations was significantly improved. The applicability of the yeast in the utilization of formaldehyde derivatives, as exemplified by urotropine and trioxane, was also investigated. The treatment of urotropine-containing wastewater with methylotrophic yeast was found to be effective at acidic conditions (pH below 5.5). Trioxane was not degraded due to the stability of an ether bond which made the molecule recalcitrant to oxidation via methylotrophic pathway reactions. It is concluded that the yeast species may be applied to treat wastewater containing formaldehyde and some of its derivatives as either monocultures or as an integrated, specialized element of the activated sludge biocenosis.