Unknown,Transcriptomics,Genomics,Proteomics

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Methanol and Methylamine Utilization Pathways in Methyloversatilis universalis FAM5: Genome Wide Gene Expression and Mutagenesis Studies

ABSTRACT: Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylated amines as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methanol vs methylamine. Growth on methanol results in activation of mdh2 and a number of known accessory proteins. As expected, all genes for N-methylglutamate pathway were induced during growth on methylated amine. Among other functions, responding to a switch from methanol to methylated amines, are a heme-containing amine dehydrogenase (QHNDH), a PQQ-dependent methanol dehydrogenase homologue, a distant homologue of formaldehyde activating enzyme (fae3), molybdenum containing formate dehydrogenase, a set of transporters homologues to urea/ammonium transporters and amino-acid permeases. Genes encoding the PQQ-dependent methanol dehydrogenase and associated cytochrome, the enzymes from the assimilatory H4F-dependent pathway, and the tungsten-containing aldehyde oxidoreductase were down-regulated during growth on methylamine. Genes essential for carbon assimilation (serine cycle) and H4MTP-pathway for formaldehyde oxidation show similar level of expression on both C1-carbon sources. Phenotypic analysis of mutants lacking functional QHNDH had no growth defect on C1-compounds. M. universalis FAM5 strain with the methylene-tetrahydrofolate dehydrogenase lesion, a key enzyme of the H4-folate pathway, were not able to use any C1-compound, methanol or methylated amines. Methyloversatilis universalis FAM5 possesses three homologs of the formaldehyde activating enzymes. The relative expression of two of the formaldehyde activating enzyme (fae1) and fae2 did not change after the shift from methanol to methylamine growth. The relative expression of the third homologs, fae3, was significantly upregulated by methylamine. Single and double fae 2 and fae 3 mutants display similar to wild type growth M-BM- on methanol or methylamine. Strains lacking fae1 lost the ability to grow on both C1-compounds. However upon incubation on methylated amines the fae1-mutant produce revertants (fae1R ). The revertant strains displayed an impaired growth on methylamine but were not able to use methanol. Double mutations in fae1RM-BM- / fae3 or fae1R/fae2 and triple mutant fae1R/fae2/fae3 showed similar to fae1R phenotype. The metabolic pathways for utilization methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed. Methyloversatilis universalis FAM5 grown on methanol and methylamine with two biologial replicates for each condition. RNA-Seq was used for transcriptomics.

ORGANISM(S): Methyloversatilis universalis FAM5

SUBMITTER: David Beck

PROVIDER: E-GEOD-63822 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Methyloversatilis universalis FAM5 utilizes single carbon compounds such as methanol or methylated amines as a sole source of carbon and energy. Expression profiling reveals distinct sets of genes altered during growth on methanol vs methylamine. Growth on methanol results in activation of mdh2 and a number of known accessory proteins. As expected, all genes for N-methylglutamate pathway were induced during growth on methylated amine. Among other functions, responding to a switch from methanol to methylated amines, are a heme-containing amine dehydrogenase (QHNDH), a PQQ-dependent methanol dehydrogenase homologue, a distant homologue of formaldehyde activating enzyme (fae3), molybdenum containing formate dehydrogenase, a set of transporters homologues to urea/ammonium transporters and amino-acid permeases. Genes encoding the PQQ-dependent methanol dehydrogenase and associated cytochrome, the enzymes from the assimilatory H4F-dependent pathway, and the tungsten-containing aldehyde oxidoreductase were down-regulated during growth on methylamine. Genes essential for carbon assimilation (serine cycle) and H4MTP-pathway for formaldehyde oxidation show similar level of expression on both C1-carbon sources. Phenotypic analysis of mutants lacking functional QHNDH had no growth defect on C1-compounds. M. universalis FAM5 strain with the methylene-tetrahydrofolate dehydrogenase lesion, a key enzyme of the H4-folate pathway, were not able to use any C1-compound, methanol or methylated amines. Methyloversatilis universalis FAM5 possesses three homologs of the formaldehyde activating enzymes. The relative expression of two of the formaldehyde activating enzyme (fae1) and fae2 did not change after the shift from methanol to methylamine growth. The relative expression of the third homologs, fae3, was significantly upregulated by methylamine. Single and double fae 2 and fae 3 mutants display similar to wild type growth on methanol or methylamine. Strains lacking fae1 lost the ability to grow on both C1-compounds. However upon incubation on methylated amines the fae1-mutant produce revertants (fae1R ). The revertant strains displayed an impaired growth on methylamine but were not able to use methanol. Double mutations in fae1R / fae3 or fae1R/fae2 and triple mutant fae1R/fae2/fae3 showed similar to fae1R phenotype. The metabolic pathways for utilization methanol and methylamine in Methyloversatilis universalis FAM5 are reconstructed.

Project description:Previously, it has been demonstrated that formate can be utilized by Saccharomyces cerevisiae as additional energy source using cells grown in a glucose-limited chemostat. Here, we investigated utilization of formaldehyde as co-substrate. Since endogenous formaldehyde dehydrogenase activities were insufficient to allow co-feeding of formaldehyde, the Hansenula polymorpha FLD1, encoding formaldehyde dehydrogenase, was introduced in S. cerevisiae. Chemostat cultivations revealed that formaldehyde was co-utilized with glucose, but the yield was lower than predicted. Moreover, formate was secreted by the cells. Upon co-expression of the H. polymorpha gene encoding formate dehydrogenase, FMD, the levels of secreted formate decreased, but the biomass yield was still lower than anticipated. Transcriptome comparisons of cells of the engineered FLD1/FMD-expressing S. cerevisiae strain grown with or without formaldehyde feed, suggested that the cells experienced biotin limitation, possibly due to inactivation of biotin by formaldehyde in the feed. When separate feeds were used for formaldehyde and biotin, the engineered S. cerevisiae strain was able to efficiently utilize formaldehyde as additional energy source. Experiment Overall Design: In industrial biotechnology, the cost of feedstocks (often sugars) are crucial for production of both biomass related products, such as proteins, or for the production of commodity chemicals. In many such processes a large fraction of the sugars is dissimilated to either provide free-energy (in the form of ATP) or to provide electrons (in the form of NADH). Co-consumption of methanol, which can be derived from fossil sources (via natural gas) or from biomass (via syngas) , via the above mentioned route can provide NADH and/or ATP, thereby creating the possibility to utilise the sugars more efficiently. It would therefore be advantageous to co-feed S. cerevisiae with methanol during fermentations since it is a cheap alternative to sugars as energy source. Experiment Overall Design: Efficient dissimilation of the intermediate formaldehyde is a crucial first step for utilisation of methanol by S. cerevisiae. Therefore, his study analyses the effect of co-expression of H. polymorpha FLD1 encoding NAD+-dependent formaldehyde and FMD encoding formate dehydrogenases, on tolerance to formaldehyde and co-consumption of formaldehyde by S. cerevisiae.

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Methanol and Methylamine Utilization Pathways in Methyloversatilis universalis FAM5: Genome Wide Gene Expression and Mutagenesis Studies

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