Analysis and Manipulation of Aspartate Pathway Genes for
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            -Lysine Overproduction from Methanol by Bacillus methanolicus

Nærdal, Ingemar; Netzer, Roman; Ellingsen, Trond E.; Brautaset, Trygve

doi:10.1128/aem.05093-11

Cited by 28 publications

(36 citation statements)

References 42 publications

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“…We previously presented a total of 14 different wild-type isolates of B. methanolicus (10), and among these, MGA3 has been the major model strain used for metabolic engineering (9,11,12,26,27,32). The majority of these strains-including MGA3-have been isolated from soil samples in the Minnesota area, while one strain, PB1, has been isolated from a sugar beet factory wastewater treatment system in Europe (20).…”

Section: Resultsmentioning

confidence: 99%

“…Some B. methanolicus genes en- (27,39), and hom1 and hom2 (encoding homoserine dehydrogenase [HD I and II, respectively]) (9). The asd (encoding aspartate-semialdehyde dehydrogenase) and dapA (encoding dihydrodipicolinate synthase) genes were also cloned by us (27), and we recently demonstrated that they both encode active enzymes (32). Overexpression of dapG, lysC, and yclM increased L-lysine production in wild-type B. methanolicus MGA3 up to 60-fold (corresponding to 11 g liter Ϫ1 ) in fed-batch methanol cultivation (27), and we showed that hom1 is the major target for S-(2-aminoethyl)-Lcysteine (AEC) resistance in B. methanolicus (9).…”

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confidence: 99%

“…Recombinant B. methanolicus strains producing up to 11 g liter Ϫ1 of L-lysine have been constructed by manipulation with single genes representing the aspartate pathway (9,27). We recently showed that multiple genes of the aspartate pathway can play important roles in L-lysine overproduction by B. methanolicus (32). Manipulation with enzymes representing the phosphoenol pyruvate (PEP) and pyruvate anaplerotic node and the entry into the tricarboxylic acid (TCA) cycle has indicated that the oxaloacetate (OAA) precursor supply is not a major bottleneck for L-lysine overproduction by B. methanolicus (9,12).…”

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confidence: 99%

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Genome Sequence of Thermotolerant Bacillus methanolicus: Features and Regulation Related to Methylotrophy and Production of l -Lysine and l -Glutamate from Methanol

Heggeset

Krog

Balzer

et al. 2012

Appl Environ Microbiol

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140

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bBacillus methanolicus can utilize methanol as its sole carbon and energy source, and the scientific interest in this thermotolerant bacterium has focused largely on exploring its potential as a biocatalyst for the conversion of methanol into L-lysine and L-glutamate. We present here the genome sequences of the important B. methanolicus model strain MGA3 (ATCC 53907) and the alternative wild-type strain PB1 (NCIMB13113). The physiological diversity of these two strains was demonstrated by a comparative fed-batch methanol cultivation displaying highly different methanol consumption and respiration profiles, as well as major differences in their L-glutamate production levels (406 mmol liter ؊1 and 11 mmol liter ؊1 , respectively). Both genomes are small (ca 3.4 Mbp) compared to those of other related bacilli, and MGA3 has two plasmids (pBM19 and pBM69), while PB1 has only one (pBM20). In particular, we focus here on genes representing biochemical pathways for methanol oxidation and concomitant formaldehyde assimilation and dissimilation, the important phosphoenol pyruvate/pyruvate anaplerotic node, the tricarboxylic acid cycle including the glyoxylate pathway, and the biosynthetic pathways for L-lysine and L-glutamate. Several unique findings were made, including the discovery of three different methanol dehydrogenase genes in each of the two B. methanolicus strains, and the genomic analyses were accompanied by gene expression studies. Our results provide new insight into a number of peculiar physiological and metabolic traits of B. methanolicus and open up possibilities for system-level metabolic engineering of this bacterium for the production of amino acids and other useful compounds from methanol. Methylotrophic microorganisms can utilize one-carbon (C 1 ) sources, such as methane and methanol, as their sole sources for energy and biomass generation, and there exist a variety of different enzymes and pathways for C 1 metabolism among methylotrophs (14,15). Bacteria that fix formaldehyde by the ribulose monophosphate (RuMP) pathway belong to three groups: Gramnegative obligate methylotrophs, Gram-positive facultative methylotrophs, and thermotolerant bacilli (3,4,19,38). A number of Gram-positive thermotolerant bacilli with the ability to grow on methanol at temperatures up to 60°C have been isolated, and they were later collectively classified as Bacillus methanolicus (for a review, see reference 11). B. methanolicus is a so-called restricted methylotroph, which means that it can utilize few multicarbon sources for energy and growth. The scientific interest of these organisms has mainly been dedicated to exploring their potential as cell factories for industrial production of L-lysine and L-glutamate from methanol at elevated temperatures. B. methanolicus MGA3 (ATCC 53907) was isolated from soil samples in Minnesota (38), and it has been the major model strain used for metabolic engineering of this bacterium (9,11,27).B. methanolicus has several additional unique traits, including (i) a novel NAD-dependent met...

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Genome Sequence of Thermotolerant Bacillus methanolicus: Features and Regulation Related to Methylotrophy and Production of l -Lysine and l -Glutamate from Methanol

Heggeset

Krog

Balzer

et al. 2012

Appl Environ Microbiol

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140

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“…mxaFI genes have been found in some Actinobacteria and methylotrophy are widely distributed in this taxon (Anesti et al, 2004;Hung et al, 2011). Biochemically and evolutionary distinct methanol oxidoreductases of Gram positives cannot be detected with currently available primers (for example, those of B. methanolicus; Hektor et al, 2002;Naerdal et al, 2011). Thus, future efforts are needed to improve the assessment of Gram-positive methylotrophs.…”

Section: Diversity Of Soil Methylotrophsmentioning

confidence: 99%

Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs

et al. 2012

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The role of soil methylotrophs in methanol exchange with the atmosphere has been widely overlooked. Methanol can be derived from plant polymers and be consumed by soil microbial communities. In the current study, methanol-utilizing methylotrophs of 14 aerated soils were examined to resolve their comparative diversities and capacities to utilize ambient concentrations of methanol. Abundances of cultivable methylotrophs ranged from 10 6 -10 8 g soilDW. Methanol dissimilation was measured based on conversion of supplemented 14 C-methanol, and occurred at concentrations down to 0.002 lmol methanol g soilDW À 1. Tested soils exhibited specific affinities to methanol (a 0 s ¼ 0.01 d À 1 ) that were similar to those of other environments suggesting that methylotrophs with similar affinities were present. Two deep-branching alphaproteobacterial genotypes of mch responded to the addition of ambient concentrations of methanol (p0.6 lmol methanol g soilDW) in one of these soils. Methylotroph community structures were assessed by amplicon pyrosequencing of genes of mono carbon metabolism (mxaF, mch and fae). Alphaproteobacteria-affiliated genotypes were predominant in all investigated soils, and the occurrence of novel genotypes indicated a hitherto unveiled diversity of methylotrophs. Correlations between vegetation type, soil pH and methylotroph community structure suggested that plant-methylotroph interactions were determinative for soil methylotrophs.

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“…We previously showed that overexpression experiments can be used to efficiently analyze the role and regulation of a number of genes in the L-lysine biosynthetic pathway in B. methanolicus (30,38). Here, we used an analogous approach to map the roles of the gltAB, gltA, gltA2, glnA, and yweB genes.…”

Section: Geobacillus Thermodenitrificans (Yp_001125407) Bacillus Licmentioning

confidence: 99%

Functional Characterization of Key Enzymes involved in l -Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

Krog

Heggeset

Ellingsen

et al. 2013

Appl Environ Microbiol

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bBacillus methanolicus wild-type strain MGA3 secretes 59 g/liter ؊1 of L-glutamate in fed-batch methanol cultivations at 50°C. We recently sequenced the MGA3 genome, and we here characterize key enzymes involved in L-glutamate synthesis and degradation. One glutamate dehydrogenase (GDH) that is encoded by yweB and two glutamate synthases (GOGATs) that are encoded by the gltAB operon and by gltA2 were found, in contrast to Bacillus subtilis, which has two different GDHs and only one GOGAT. B. methanolicus has a glutamine synthetase (GS) that is encoded by glnA and a 2-oxoglutarate dehydrogenase (OGDH) that is encoded by the odhAB operon. The yweB, gltA, gltB, and gltA2 gene products were purified and characterized biochemically in vitro. YweB has a low K m value for ammonium (10 mM) and a high K m value for L-glutamate (250 mM), and the V max value is 7-fold higher for L-glutamate synthesis than for the degradation reaction. GltA and GltA2 displayed similar K m values (1 to 1.4 mM) and V max values (4 U/mg) for both L-glutamate and 2-oxoglutarate as the substrates, and GltB had no effect on the catalytic activities of these enzymes in vitro. Complementation assays indicated that GltA and not GltA2 is dependent on GltB for GOGAT activity in vivo. To our knowledge, this is the first report describing the presence of two active GOGATs in a bacterium. In vivo experiments indicated that OGDH activity and, to some degree, GOGAT activity play important roles in regulating L-glutamate production in this organism.T he production of L-glutamate is a fast-growing industry, and approximately 2 million tons are produced annually, almost exclusively by Corynebacterium glutamicum fermentation using sugar-based raw materials (1). Genetic and biochemical insight into L-glutamate synthesis and degradation is of fundamental importance to understand nitrogen metabolism in the cells, as well as for metabolic engineering of L-glutamate overproduction. In C. glutamicum, L-glutamate is synthesized by both glutamate dehydrogenase (GDH) and glutamate synthase (GOGAT) (2), and L-glutamate overproduction can be achieved by downregulating the 2-oxoglutarate dehydrogenase (OGDH) activity (3, 4).In Bacillus subtilis, the gltAB operon encodes the large (GltA) and the small (GltB) subunit of GOGAT. GOGAT catalyzes a reaction where the NH 2 group from the amide group in L-glutamine is transferred to 2-oxoglutarate, forming two molecules of L-glutamate. Glutamine synthetase (GS) is encoded by glnA and catalyzes the synthesis of L-glutamine from L-glutamate and ammonia. Together, these two enzymes form the GOGAT/GS cycle ( Fig. 1) that is responsible for ammonium assimilation and L-glutamate degradation and synthesis, and B. subtilis mutants deficient in GOGAT are L-glutamate auxotrophs (5, 6). To our knowledge, the kinetic properties of GOGAT in B. subtilis have not been reported in the scientific literature.B. subtilis harbors two genes, rocG and gudB, that both encode GDHs. In the most-studied B. subtilis strain, strain 168, GudB is found to be...

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Analysis and Manipulation of Aspartate Pathway Genes for l -Lysine Overproduction from Methanol by Bacillus methanolicus

Cited by 28 publications

References 42 publications

Genome Sequence of Thermotolerant Bacillus methanolicus: Features and Regulation Related to Methylotrophy and Production of l -Lysine and l -Glutamate from Methanol

Genome Sequence of Thermotolerant Bacillus methanolicus: Features and Regulation Related to Methylotrophy and Production of l -Lysine and l -Glutamate from Methanol

Methanol oxidation by temperate soils and environmental determinants of associated methylotrophs

Functional Characterization of Key Enzymes involved in l -Glutamate Synthesis and Degradation in the Thermotolerant and Methylotrophic Bacterium Bacillus methanolicus

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