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Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus.

ABSTRACT: Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this organism. Overexpression of dapG, lysC, and yclM increased l-lysine production in wild-type B. methanolicus strain MGA3 2-, 10-, and 60-fold (corresponding to 11 g/liter), respectively, without negatively affecting the specific growth rate. The production levels of l-methionine (less than 0.5 g/liter) and l-threonine (less than 0.1 g/liter) were low in all recombinant strains. The AK proteins were purified, and biochemical analyses demonstrated that they have similar V(max) values (between 47 and 58 micromol/min/mg protein) and K(m) values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC(50)], 0.1 mM) and by l-lysine (IC(50), 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC(50), 4 mM) and by l-lysine (IC(50), 5 mM), and this enzyme was synergistically inhibited in the presence of both of these amino acids at low concentrations. The correlation between the impact on l-lysine production in vivo and the biochemical properties in vitro of the individual AK proteins is discussed. This is the first example of improving l-lysine production by metabolic engineering of B. methanolicus and also the first documentation of considerably increasing l-lysine production by overexpression of a wild-type AK.

SUBMITTER: Jakobsen OM

PROVIDER: S-EPMC2632123 | biostudies-literature | 2009 Feb

REPOSITORIES: biostudies-literature

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Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus.

Jakobsen Oyvind M OM Brautaset Trygve T Degnes Kristin F KF Heggeset Tonje M B TM Balzer Simone S Flickinger Michael C MC Valla Svein S Ellingsen Trond E TE

Applied and environmental microbiology 20081205 3

Aspartokinase (AK) controls the carbon flow into the aspartate pathway for the biosynthesis of the amino acids l-methionine, l-threonine, l-isoleucine, and l-lysine. We report here the cloning of four genes (asd, encoding aspartate semialdehyde dehydrogenase; dapA, encoding dihydrodipicolinate synthase; dapG, encoding AKI; and yclM, encoding AKIII) of the aspartate pathway in Bacillus methanolicus MGA3. Together with the known AKII gene lysC, dapG and yclM form a set of three AK genes in this or ...[more]

PMID: 19060158

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Project description:Bacillus methanolicus MGA3 is a thermophilic, facultative ribulose monophosphate (RuMP) cycle methylotroph. Together with its ability to produce high yields of amino acids, the relevance of this microorganism as a promising candidate for biotechnological applications is evident. The B. methanolicus MGA3 genome consists of a 3,337,035 nucleotides (nt) circular chromosome, the 19,174 nt plasmid pBM19 and the 68,999 nt plasmid pBM69. 3,218 protein-coding regions were annotated on the chromosome, 22 on pBM19 and 82 on pBM69. In the present study, the RNA-seq approach was used to comprehensively investigate the transcriptome of B. methanolicus MGA3 in order to improve the genome annotation, identify novel transcripts, analyze conserved sequence motifs involved in gene expression and reveal operon structures. For this aim, two different cDNA library preparation methods were applied: one which allows characterization of the whole transcriptome and another which includes enrichment of primary transcript 5'-ends.Analysis of the primary transcriptome data enabled the detection of 2,167 putative transcription start sites (TSSs) which were categorized into 1,642 TSSs located in the upstream region (5'-UTR) of known protein-coding genes and 525 TSSs of novel antisense, intragenic, or intergenic transcripts. Firstly, 14 wrongly annotated translation start sites (TLSs) were corrected based on primary transcriptome data. Further investigation of the identified 5'-UTRs resulted in the detailed characterization of their length distribution and the detection of 75 hitherto unknown cis-regulatory RNA elements. Moreover, the exact TSSs positions were utilized to define conserved sequence motifs for translation start sites, ribosome binding sites and promoters in B. methanolicus MGA3. Based on the whole transcriptome data set, novel transcripts, operon structures and mRNA abundances were determined. The analysis of the operon structures revealed that almost half of the genes are transcribed monocistronically (940), whereas 1,164 genes are organized in 381 operons. Several of the genes related to methylotrophy had highly abundant transcripts.The extensive insights into the transcriptional landscape of B. methanolicus MGA3, gained in this study, represent a valuable foundation for further comparative quantitative transcriptome analyses and possibly also for the development of molecular biology tools which at present are very limited for this organism.

Project description:The thermotolerant methylotroph Bacillus methanolicus MGA3 was originally isolated from freshwater marsh soil. Due to its ability to use methanol as sole carbon and energy source, B. methanolicus is increasingly explored as a cell factory for the production of amino acids, fine chemicals, and proteins of biotechnological interest. During high cell density fermentation in industrial settings with the membrane-permeable methanol as the feed, the excretion of low molecular weight products synthesized from it will increase the osmotic pressure of the medium. This in turn will impair cell growth and productivity of the overall biotechnological production process. With this in mind, we have analyzed the core of the physiological adjustment process of B. methanolicus MGA3 to sustained high osmolarity surroundings. Through growth assays, we found that B. methanolicus MGA3 possesses only a restricted ability to cope with sustained osmotic stress. This finding is consistent with the ecophysiological conditions in the habitat from which it was originally isolated. None of the externally provided compatible solutes and proline-containing peptides affording osmostress protection for Bacillus subtilis were able to stimulate growth of B. methanolicus MGA3 at high salinity. B. methanolicus MGA3 synthesized the moderately effective compatible solute L-glutamate in a pattern such that the cellular pool increased concomitantly with increases in the external osmolarity. Counterintuitively, a large portion of the newly synthesized L-glutamate was excreted. The expression of the genes (gltAB and gltA2) for two L-glutamate synthases were upregulated in response to high salinity along with that of the gltC regulatory gene. Such a regulatory pattern of the system(s) for L-glutamate synthesis in Bacilli is new. Our findings might thus be generally relevant to understand the production of the osmostress protectant L-glutamate by those Bacilli that exclusively rely on this compatible solute for their physiological adjustment to high osmolarity surroundings.

Dataset Information

Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus.

Publications

Overexpression of wild-type aspartokinase increases L-lysine production in the thermotolerant methylotrophic bacterium Bacillus methanolicus.

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