Development of an insertional expression vector system for Methylobacterium extorquens AM1 and generation of null mutants lacking mtdA and/or fch

Marx, Christopher J.; Lidstrom, Mary E.

doi:10.1099/mic.0.26587-0

Cited by 49 publications

(56 citation statements)

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“…When compared to the data in the nonredundant sequence database (NCBI), translated orthologs from planctomycetes revealed significant divergence from both MtdA and MtdB polypeptides but possessed higher similarity to the former (43 to 53% and 28 to 32% at the amino acid level, respectively). This finding was surprising, as in methylotrophic Proteobacteria MtdB has been shown to serve as the main enzyme in H 4 MPT-linked C 1 transfer (9,14,15,21,28), while the major function of MtdA seems to be in providing methylene-H 4 F to the serine cycle (24), in addition to its function in general metabolism (7,23). The presence of a single ortholog in planctomycete genomes and the presence of traditional folD orthologs in these genomes (reference 13 and unpublished data) suggested that the function of the MtdA protein orthologs in planctomycetes could be more similar to the function of MtdB than to the function of MtdA.…”

Section: Resultsmentioning

confidence: 71%

“…Mutants of M. extorquens that contain lesions in, respectively, mtdB and mtdA possess characteristic phenotypes: an mtdB mutant is negative for growth on methanol and is highly sensitive to methanol vapors (15,21), while an mtdA mutant is methanol negative and requires an added C 1 substrate (methanol or formate) for growth on succinate but is not sensitive to methanol vapors (7,23). To test for the potential of the proteins encoded by the mtdA orthologs to fulfill either MtdB or MtdA function, genes from Gemmata sp.…”

Section: Resultsmentioning

confidence: 99%

“…A fae gene was amplified from clone LWBAC-L1N9 and cloned into pCM80 in a similar fashion. The Escherichia coli JM109 (Invitrogen) strains harboring the plasmids expressing mtdA-like genes were used as donors in matings with both mtdA and mtdB mutants of M. extorquens (15,23), as previously described (22). The plasmid expressing fae from LWBAC-L1N9 was mated into the fae mutant of M. extorquens (31) in a similar fashion.…”

Section: Methodsmentioning

confidence: 99%

“…MtdA also has a secondary function, in general metabolism (purine biosynthesis, etc.) in the serine cycle methylotrophs that do not possess FolD (7,23), the enzyme fulfilling this function in most bacteria and eukaryotes (20). Heterologously expressed folD was shown to complement the function of mtdA in general metabolism, but not in methylotrophy (23).…”

mentioning

confidence: 99%

“…In some methylotrophs, so far only in methylotrophs employing the serine cycle for formaldehyde assimilation, a paralog of MtdB is present, an NADP-linked methylene-H 4 MPT/methylene-tetrahydrofolate (H 4 F) dehydrogenase (MtdA) (7,10,28,29). Experiments with Methylobacterium extorquens AM1, including protein purification and analysis, mutant analysis, and flux analysis, all suggested that in vivo, the main function of MtdA is in reducing methenyl-H 4 F to methylene-H 4 F, which feeds into the serine cycle (7,23,25,28,29). MtdA also has a secondary function, in general metabolism (purine biosynthesis, etc.)…”

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MtdC, a Novel Class of Methylene Tetrahydromethanopterin Dehydrogenases

et al. 2005

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Novel methylene tetrahydromethanopterin (H 4 MPT) dehydrogenase enzymes, named MtdC, were purified after expressing in Escherichia coli genes from, respectively, Gemmata sp. strain Wa1-1 and environmental DNA originating from unidentified microbial species. The MtdC enzymes were shown to possess high affinities for methylene-H 4 MPT and NADP but low affinities for methylene tetrahydrofolate or NAD. The substrate range and the kinetic properties revealed by MtdC enzymes distinguish them from the previously characterized bacterial methylene-H 4 MPT dehydrogenases, MtdA and MtdB. While revealing higher sequence similarity to MtdA enzymes, MtdC enzymes appear to fulfill a function homologous to the function of MtdB, as part of the H 4 MPT-linked pathway for formaldehyde oxidation/detoxification.Formaldehyde detoxification is a metabolic function essential to all of life, due to the extreme toxicity of formaldehyde (4, 12). However, in methylotrophic bacteria formaldehyde oxidation is a part of their central metabolism (1). One of the most widespread modes of formaldehyde oxidation in methylotrophs is the pathway that involves tetrahydromethanopterin (H 4 MPT) as a cofactor (28,30). Most of the enzymes involved in this pathway are homologous to the enzymes involved in methanogenesis by the Archaea (6, 9). However, one enzyme in the pathway, an NAD(P)-linked methylene-H 4 MPT dehydrogenase (MtdB), is unique to Bacteria (14, 15). MtdB has evolved independently of the archaeal functional counterparts that are linked to H 2 or cofactor F 420 (26), based on the lack of sequence similarity (9, 15). Enzyme properties and mutant analyses have demonstrated that MtdB fulfills a dual physiological role in methylotrophic metabolism, in energy generation (in the form of NADH) and in formaldehyde detoxification (9,14,15,21,28). In some methylotrophs, so far only in methylotrophs employing the serine cycle for formaldehyde assimilation, a paralog of MtdB is present, an NADP-linked methylene-H 4 MPT/methylene-tetrahydrofolate (H 4 F) dehydrogenase (MtdA) (7,10,28,29). Experiments with Methylobacterium extorquens AM1, including protein purification and analysis, mutant analysis, and flux analysis, all suggested that in vivo, the main function of MtdA is in reducing methenyl-H 4 F to methylene-H 4 F, which feeds into the serine cycle (7,23,25,28,29). MtdA also has a secondary function, in general metabolism (purine biosynthesis, etc.) in the serine cycle methylotrophs that do not possess FolD (7, 23), the enzyme fulfilling this function in most bacteria and eukaryotes (20). Heterologously expressed folD was shown to complement the function of mtdA in general metabolism, but not in methylotrophy (23).The origin and the evolutionary history of MtdA and MtdB remain poorly understood. While MtdA reveals low levels of sequence similarity to FolD enzymes (15% identity at the amino acid level [7,29]), MtdB shares no similarity with FolD (15). However, the two paralogs reveal a significant level of similarity to each other (about 30% at the a...

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Section: Resultsmentioning

confidence: 71%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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MtdC, a Novel Class of Methylene Tetrahydromethanopterin Dehydrogenases

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Methylotrophs, Industrial Applications

Leak

2010

Encyclopedia of Industrial Biotechnology

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Early investigations into the potential applications of methylotrophs focused mainly on the use of methane and methanol as cheap feedstocks for the production of single cell protein, and until recently this possibility was still being investigated. Despite the decline of this initial interest, the spin‐offs in terms of fundamental biochemical and genetic information, fermentation systems, and bioreactor design have opened up other avenues, either for products where substrate costs are paramount (e.g. amino acids), products originating from some of the unique features of C 1 metabolism (e.g. enzymes) or fortuitous discoveries (e.g. the Pichia pastoris expression system). After considering fundamental aspects of metabolism and energetics of methylotrophs and their implications for product formation, this review follows these subdivisions in turn, endeavoring to assess their future potential.

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Chemical Production from Methanol Using Natural and Synthetic Methylotrophs

Chen

Lan

2020

Biotechnology Journal

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Methanol as a chemical feedstock is becoming increasingly important as it is derived from natural gas and is a feasible end-product for captured carbon dioxide. Biological conversion of methanol through natural and synthetic methylotrophs increases the chemical repertoire and is an important direction for one carbon (C1) based chemical economy. Advances in the metabolic engineering and synthetic biology enable development of microbial cell factories for converting methanol into various platform chemicals. In this review, the current status of methanol utilizing microbial factory development is summarized. Also the development of synthetic methylotrophy and methanol-augmented bioproductions is discussed.

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Development of an insertional expression vector system for Methylobacterium extorquens AM1 and generation of null mutants lacking mtdA and/or fch

Cited by 49 publications

References 34 publications

MtdC, a Novel Class of Methylene Tetrahydromethanopterin Dehydrogenases

MtdC, a Novel Class of Methylene Tetrahydromethanopterin Dehydrogenases

Methylotrophs, Industrial Applications

Chemical Production from Methanol Using Natural and Synthetic Methylotrophs

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