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Improvement of dicarboxylic acid production with Methylorubrum extorquens by reduction of product reuptake

ABSTRACT: Improvement of dicarboxylic acid production with Methylorubrum extorquens by reduction of product reuptake

PROVIDER: PRJNA822372 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18584028_1.fastq.gz	Fastqsanger.gz
	SRR18584028_2.fastq.gz	Fastqsanger.gz
	SRR18584029_1.fastq.gz	Fastqsanger.gz
	SRR18584029_2.fastq.gz	Fastqsanger.gz
	SRR18584030_1.fastq.gz	Fastqsanger.gz

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Improvement of dicarboxylic acid production with Methylorubrum extorquens by reduction of product reuptake

Project description:In the past, two dicarboxylic acid products, namely mesaconic acid and 2‑methylsuccinic acid, were successfully produced by Methylorubrum extorquens AM1 with heterologous thioesterase YciA, but the yield was reduced by product reuptake. In our study we conducted comprehensive research on the uptake mechanism of those dicarboxylic acid products. To identify potential import factors, we performed transcriptome analysis of a strain harboring plasmid pCM160_RBS_yciAHI and an empty vector control strain. Samples were taken and analyzed at different timepoints throughout cultivation. However, we could not identify genes that were clearly upregulated in stationary growth phase compared to early sampling points in the exponential growth phase and which were annotated as potential transporters. When specifically analyzing the time-dependent expression levels of dctA homologs and kgtP (suggested as targets in previous studies) and comparing them with a control strain, we found slightly increased levels only for dctA1. After concluding that the transcriptome analysis was obviously no appropriate approach to identify relevant transport factors, we chose a straightforward mutant selection approach using a cytotoxic dicarboxylic acid.

2022-04-04 | GSE199961 | GEO

Improvement of dicarboxylic acid production with Methylorubrum extorquens by reduction of product reuptake.

Project description:The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids. KEY POINTS: • 2,2-Difluorosuccinic acid is used to select for dicarboxylic acid uptake mutations. • Deletion of dctA2 leads to reduction of dicarboxylic acid uptake. • Transporter-deficient strains show improved production of citramalic acid.

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Project description:The meristem-associated endosymbiont M. extorquens DSM13060 significantly increases needle and root growth of Scots pine (Pinus sylvestris L.) seedlings without producing plant hormones, but by aggregating around host nuclei. Here we studied gene expression of the pine host induced by M. extorquens DSM13060 infection. We selected the time point of 90 days post-inoculation for our analysis based, because at this point, Methylorubrum extorquens DSM13060 has systemically colonized the pine seedlings, being found throughout tissues of roots and shoots.

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Project description:Methylorubrum extorquens AM1 is engineered to produce itaconic acid by heterologous expression of cis-aconitic acid decarboxylase. Mutation was also performed on phaR in Methylorubrum extorquens AM1, which regulate poly-beta-hydroxybutyrate accumulation, in attempt to increase carbon flux toward itaconic acid production. However, in our case, itaconic acid production by phaR mutant strain was not higher than that of the wildtype. Transcriptomic analysis was utilized in order to examine the cause for this phenomenon. RNA-seq analysis revealed that phaR mutation in the itaconic acid-producing strain might result in a complex regulatory rewiring at the gene expression level, which could cause a reduced resource flux toward ITA production. Also, RNA profiling gave a hint at the broad regulatory role of PhaR.

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Project description:We report a genetic variant of Methylorubrum extorquens AM1 that hyperaccumulates the heavy lanthanide gadolinium. Using RNA-seq transcriptomics we identified wide-spread metabolic and physiological changes in this strain and experimentally validate several of them, including increased gadolinium transport and storage in an intracellular compartment we name the lanthasome.

2022-01-17 | GSE193171 | GEO

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