Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph
 Methylobacterium extorquens
 AM1: Identification and Mutation of
 gap11
 ,
 gap20
 , and
 phaR

Korotkova, N. N.; Chistoserdova, Ludmila; Lidstrom, Mary E.

doi:10.1128/jb.184.22.6174-6181.2002

Cited by 49 publications

(57 citation statements)

References 49 publications

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“…From these intermediates, it is theoretically possible to generate net reducing equivalents by oxidation to CO 2 via derepression of the tricarboxylic acid cycle. Deregulation of the tricarboxylic acid cycle on methanol has already been observed in a mutant in which regulation of poly-␤-hydroxybutyrate synthesis is affected (17). Therefore, at least one possibility exists to explain how formate might be consumed in the triple mutant.…”

Section: Discussionmentioning

confidence: 99%

Multiple Formate Dehydrogenase Enzymes in the Facultative Methylotroph Methylobacterium extorquens AM1 Are Dispensable for Growth on Methanol

Chistoserdova¹,

Laukel

Portais

et al. 2004

J Bacteriol

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Formate dehydrogenase has traditionally been assumed to play an essential role in energy generation during growth on C 1 compounds. However, this assumption has not yet been experimentally tested in methylotrophic bacteria. In this study, a whole-genome analysis approach was used to identify three different formate dehydrogenase systems in the facultative methylotroph Methylobacterium extorquens AM1 whose expression is affected by either molybdenum or tungsten. A complete set of single, double, and triple mutants was generated, and their phenotypes were analyzed. The growth phenotypes of the mutants suggest that any one of the three formate dehydrogenases is sufficient to sustain growth of M. extorquens AM1 on formate, while surprisingly, none is required for growth on methanol or methylamine. Further studies of the triple mutant showed that formate was not produced quantitatively and was consumed later in growth. These results demonstrated that all three formate dehydrogenase systems must be inactivated in order to disrupt the formate-oxidizing capacity of the organism but that an alternative formate-consuming capacity exists in the triple mutant.The classic scheme of energy metabolism during methylotrophic growth involves a formate oxidation step except in strains in which all formaldehyde is oxidized in the cyclic ribulose monophosphate cycle (1). Formate dehydrogenase (FDH) activity has been detected in most methylotrophs (3,10,13,15,22,42), and a few FDHs have been purified and analyzed (reviewed in reference 39). In the methylotrophic yeast Candida boidinii, the FDH step was shown not to be essential for methylotrophic growth, but FDH mutants showed reduced growth on methanol (32). However, as the complete C. boidinii genome sequence is not available, the presence of other FDHs is not excluded.Mutant-based analysis of the role of the FDH step in C 1 oxidation has not yet been attempted in methylotrophic bacteria. M. extorquens AM1 offers a convenient model to study this question. It possesses two pathways in which formaldehyde can be oxidized to formate (Fig. 1), one linked to tetrahydromethanopterin (H 4 MPT) and another linked to tetrahydrofolate (H 4 F) (5, 6). The enzymes involved in the two pathways have been studied in detail, and current evidence suggests that the main pathway for oxidizing formaldehyde is the H 4 MPTlinked pathway (reviewed in reference 37). It has been demonstrated recently that this pathway produces formate as an intermediate, a result of a formylmethanofuran transferase/ hydrolase reaction (29), and thus in this pathway one molecule of formate is formed in M. extorquens AM1 per oxidized molecule of a C 1 substrate, such as methanol or methylamine. This formate is subsequently oxidized to CO 2 , presumably by FDH (Fig. 1).An FDH from M. extorquens AM1 has recently been purified and characterized and shown to be a novel, tungsten-containing FDH encoded by two genes, fdh1AB (20). In this study we identified two new regions in the M. extorquens AM1 chromosome coding for two addition...

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Section: Discussionmentioning

confidence: 99%

Multiple Formate Dehydrogenase Enzymes in the Facultative Methylotroph Methylobacterium extorquens AM1 Are Dispensable for Growth on Methanol

Chistoserdova¹,

Laukel

Portais

et al. 2004

J Bacteriol

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“…The organism has been well studied (Anthony 1982;Chistoserdova et al 1998;Korotkova et al 2002;Van Dien et al 2003;Chistoserdova et al 2004), and recently, a stoichiometric model of its central metabolism was published (Van Dien and Lidstrom 2002). Using Step 3 of OptStrain, we identified that only a single reaction needs to be introduced into the metabolic network of M. extorquens to enable hydrogen production.…”

Section: Extorquens Am1mentioning

confidence: 99%

OptStrain: A computational framework for redesign of microbial production systems

Pharkya¹,

Burgard²,

Maranas³

2004

Genome Res.

446

311

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This paper introduces the hierarchical computational framework OptStrain aimed at guiding pathway modifications, through reaction additions and deletions, of microbial networks for the overproduction of targeted compounds. These compounds may range from electrons or hydrogen in biofuel cell and environmental applications to complex drug precursor molecules. A comprehensive database of biotransformations, referred to as the Universal database (with >5700 reactions), is compiled and regularly updated by downloading and curating reactions from multiple biopathway database sources. Combinatorial optimization is then used to elucidate the set(s) of non-native functionalities, extracted from this Universal database, to add to the examined production host for enabling the desired product formation. Subsequently, competing functionalities that divert flux away from the targeted product are identified and removed to ensure higher product yields coupled with growth. This work represents an advancement over earlier efforts by establishing an integrated computational framework capable of constructing stoichiometrically balanced pathways, imposing maximum product yield requirements, pinpointing the optimal substrate(s), and evaluating different microbial hosts. The range and utility of OptStrain are demonstrated by addressing two very different product molecules. The hydrogen case study pinpoints reaction elimination strategies for improving hydrogen yields using two different substrates for three separate production hosts. In contrast, the vanillin study primarily showcases which non-native pathways need to be added into Escherichia coli. In summary, OptStrain provides a useful tool to aid microbial strain design and, more importantly, it establishes an integrated framework to accommodate future modeling developments.[Supplemental material is available online at www.genome.org. The Universal database can be found at http://fenske.che.psu.edu/Faculty/CMaranas/pubs.html.]A fundamental goal in systems biology is to elucidate the complete "palette" of biotransformations accessible to nature in living systems. This goal parallels the continuing quest in biotechnology to construct microbial strains capable of accomplishing an ever-expanding array of desired biotransformations. These biotransformations are aimed at products that range from simple precursor chemicals (Nakamura and Whited 2003;Causey et al. 2004) or complex molecules such as carotenoids (Misawa et al. 1991), to electrons in biofuel cells (Liu et al. 2004) or batteries (Bond et al. 2002;Bond and Lovley 2003), to even microbes capable of precipitating heavy metal complexes in bioremediation applications (Finneran et al. 2002;Lovley 2003;Methe et al. 2003). Recent developments in molecular biology and recombinant DNA technology have ushered in a new era in the ability to shape the gene content and expression levels for microbial production strains in a direct and targeted fashion (Stephanopoulos 2002). The astounding range and diversity of these newly acquired capabili...

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“…However, Dunstan and coworkers (11)(12)(13)(14) showed in 1972 and 1973 that M. extorquens AM1 lacks the key enzyme of the glyoxylate cycle, isocitrate lyase, but has an alternative route involving oxidation of acetate to glyoxylate that functions during growth on both C1 and C2 compounds. Also, other organisms, including the photosynthetic Rhodobacter sphaeroides are known to require an alternative to the glyoxylate cycle when growing on C2 substrates or on substrates that are converted into acetyl-CoA to enter central metabolism (15-18).Recent studies, including mutant analyses, gene predictions, enzyme assays, and metabolite studies in M. extorquens AM1, have led to the observation that a complex sequence of CoA thioester derivatives is involved in glyoxylate regeneration, resulting in the hypothesis of the so-called glyoxylate regeneration cycle (GRC) (19,20) [ Fig. 1 and supporting information (SI) Table S1].…”

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confidence: 99%

“…Recent studies, including mutant analyses, gene predictions, enzyme assays, and metabolite studies in M. extorquens AM1, have led to the observation that a complex sequence of CoA thioester derivatives is involved in glyoxylate regeneration, resulting in the hypothesis of the so-called glyoxylate regeneration cycle (GRC) (19,20) [ Fig. 1 and supporting information (SI) Table S1].…”

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confidence: 99%

Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

Peyraud

Kiefer

Christen

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

217

242

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The assimilation of one-carbon (C1) compounds, such as methanol, by serine cycle methylotrophs requires the continuous regeneration of glyoxylate. Instead of the glyoxylate cycle, this process is achieved by a not yet established pathway where CoA thioesters are known to play a key role. We applied state-of-the-art metabolomics and 13 C metabolomics strategies to demonstrate how glyoxylate is generated during methylotrophic growth in the isocitrate lyase-negative methylotroph Methylobacterium extorquens AM1. High-resolution mass spectrometry showed the presence of CoA thioesters specific to the recently proposed ethylmalonyl-CoA pathway. The operation of this pathway was demonstrated by short-term 13 C-labeling experiments, which allowed determination of the sequence of reactions from the order of label incorporation into the different CoA derivatives. Analysis of 13 C positional enrichment in glycine by NMR was consistent with the predicted labeling pattern as a result of the operation of the ethylmalonyl-CoA pathway and the unique operation of the latter for glyoxylate generation during growth on methanol. The results also revealed that 2 molecules of glyoxylate were regenerated in this process. This work provides a complete pathway for methanol assimilation in the model methylotroph M. extorquens AM1 and represents an important step toward the determination of the overall topology of its metabolic network. The operation of the ethylmalonyl-CoA pathway in M. extorquens AM1 has major implications for the physiology of these methylotrophs and their role in nature, and it also provides a common ground for C1 and C2 compound assimilation in isocitrate lyase-negative bacteria.13 C labeling ͉ CoA ester ͉ methylotroph ͉ one-carbon metabolism ͉ glyoxylate regeneration M ethylotrophic bacteria are organisms capable of using reduced carbon compounds, such as methanol or methane, as sole sources of carbon and energy, and they play a key role in carbon cycling in their environment. They also represent promising organisms in biotechnology for the conversion of one-carbon (C1) substrates to value-added products (1). The elucidation of the mechanisms enabling growth on reduced C1 compounds of Methylobacterium extorquens AM1, one of the most studied methylotrophs, has been a longstanding goal, and although great progress has been made (2-5), it is still not fully achieved. A key point has been to understand how the bacterium incorporates C1 units into cell material. The serine cycle was elucidated in this organism during the early 1960s by Quayle and coworkers (6-9). The assimilation of C1 units by this pathway requires continuous regeneration of glyoxylate from acetyl-CoA and can be achieved, in principle, via the well-known glyoxylate cycle (10). However, Dunstan and coworkers (11)(12)(13)(14) showed in 1972 and 1973 that M. extorquens AM1 lacks the key enzyme of the glyoxylate cycle, isocitrate lyase, but has an alternative route involving oxidation of acetate to glyoxylate that functions during growth on both C1 and C2 co...

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Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11 , gap20 , and phaR

Cited by 49 publications

References 49 publications

Multiple Formate Dehydrogenase Enzymes in the Facultative Methylotroph Methylobacterium extorquens AM1 Are Dispensable for Growth on Methanol

Multiple Formate Dehydrogenase Enzymes in the Facultative Methylotroph Methylobacterium extorquens AM1 Are Dispensable for Growth on Methanol

OptStrain: A computational framework for redesign of microbial production systems

Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics

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Poly-β-Hydroxybutyrate Biosynthesis in the Facultative Methylotroph Methylobacterium extorquens AM1: Identification and Mutation of gap11 , gap20 , and phaR

Cited by 49 publications

References 49 publications

Multiple Formate Dehydrogenase Enzymes in the Facultative Methylotroph Methylobacterium extorquens AM1 Are Dispensable for Growth on Methanol

Multiple Formate Dehydrogenase Enzymes in the Facultative Methylotroph Methylobacterium extorquens AM1 Are Dispensable for Growth on Methanol

OptStrain: A computational framework for redesign of microbial production systems

Demonstration of the ethylmalonyl-CoA pathway by using 13 C metabolomics

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Demonstration of the ethylmalonyl-CoA pathway by using ¹³ C metabolomics