Corrigendum: Genome-Based Genetic Tool Development for Bacillus methanolicus: Theta- and Rolling Circle-Replicating Plasmids for Inducible Gene Expression and Application to Methanol-Based Cadaverine Production

© 2019 Irla, Heggeset, Nærdal, Paul, Haugen, Le, Brautaset and Wendisch. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these termsBacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the −35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 6.5 to 10.2 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.publishedVersio

Continuous fermentation and kinetic experiments for the conversion of crude glycerol derived from second-generation biodiesel into 1,3 propanediol and butyric acid

This study investigated the performance of different mixed microbial cultures (MMC) able to ferment crude glycerol generated from animal fat-based biodiesel to produce 1,3 propanediol (1,3 PDO) and butyric acid, under non-sterile conditions. Eight different continuous flow stirred-tank reactors (CSTR) were set up with different inoculum types and growth media. The distribution of metabolic products under variable operating conditions was determined. All MMC were characterized from a kinetic point of view and overall stoichiometric reactions were constructed. Changes in the microbial communities were monitored by means of Next Generation Sequencing (NGS). Maximum substrate degradation rate reached approximately 110 g/L/d of glycerol (with a productivity of 38 g/L/d and 11 g/L/d for 1,3 PDO and butyric acid, respectively), obtained with an hydraulic retention time of 12 h and 60 g/L feed. The maximum feed concentration reached almost 90 g/L, leading though to an incomplete substrate degradation.acceptedVersio

Genome-based genetic tool development for Bacillus methanolicus: theta- and rolling circle-replicating plasmids for inducible gene expression and application to methanol-based cadaverine production

Irla M, Heggeset TM, Naerdal I, et al. Genome-based genetic tool development for Bacillus methanolicus: theta- and rolling circle-replicating plasmids for inducible gene expression and application to methanol-based cadaverine production. Frontiers in Microbiology. 2016;7: 1481.Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium

Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity productions from methanol

Müller JEN, Heggeset TMB, Wendisch VF, Vorholt JA, Brautaset T. Methylotrophy in the thermophilic Bacillus methanolicus, basic insights and application for commodity productions from methanol. Applied Microbiology and Biotechnology. 2015;99(2):535-551.Using methanol as an alternative non-food feedstock for biotechnological production offers several advantages in line with a methanol-based bioeconomy. The Gram-positive, facultative methylotrophic and thermophilic bacterium Bacillus methanolicus is one of the few described microbial candidates with a potential for the conversion of methanol to value-added products. Its capabilities of producing and secreting the commercially important amino acids l-glutamate and l-lysine to high concentrations at 50 A degrees C have been demonstrated and make B. methanolicus a promising target to develop cell factories for industrial-scale production processes. B. methanolicus uses the ribulose monophosphate cycle for methanol assimilation and represents the first example of plasmid-dependent methylotrophy. Recent genome sequencing of two physiologically different wild-type B. methanolicus strains, MGA3 and PB1, accompanied with transcriptome and proteome analyses has generated fundamental new insight into the metabolism of the species. In addition, multiple key enzymes representing methylotrophic and biosynthetic pathways have been biochemically characterized. All this, together with establishment of improved tools for gene expression, has opened opportunities for systems-level metabolic engineering of B. methanolicus. Here, we summarize the current status of its metabolism and biochemistry, available genetic tools, and its potential use in respect to overproduction of amino acids

Overexpression of Wild-Type Aspartokinase Increases l-Lysine Production in the Thermotolerant Methylotrophic Bacterium Bacillus methanolicus▿

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 Vmax values (between 47 and 58 μmol/min/mg protein) and Km values for l-aspartate (between 1.9 and 5.0 mM). AKI and AKII were allosterically inhibited by meso-diaminopimelate (50% inhibitory concentration [IC50], 0.1 mM) and by l-lysine (IC50, 0.3 mM), respectively. AKIII was inhibited by l-threonine (IC50, 4 mM) and by l-lysine (IC50, 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

Bacterial strains and plasmids used in this study.

a<p>Ap^r, ampicillin resistance; Cm^r, chloramphenicol resistance.</p

Comparison of the <i>B. methanolicus</i> Mdhs and the <i>K. pneumoniae</i> 1,3-PDH (DhaT^Kp).

<p>The MGA3 Mdhs are denoted Mdh^M, Mdh2^M, and Mdh3^M; the PB1 Mdhs are denoted Mdh^P, Mdh1^P, and Mdh2^P, while the C1 Mdh is denoted Mdh^C1. <b>(</b>A<b>)</b> Primary sequence alignments show a conserved GGGSX₂DX₂K motif involved in NAD⁺ binding (underlined) and residues assumed to be involved in metal ion binding (bold and highlighted). Residues in bold are unique for type III Adhs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059188#pone.0059188-deVries1" target="_blank">[12]</a>. (B<b>)</b> Relationship among the primary protein sequences of the <i>B. methanolicus</i> MGA3, PB1 and C1 Mdhs and the <i>K. pneumoniae</i> 1,3-PDH, represented by a phylogenetic tree.</p

Activation of Mdh by the activator protein ACT.

<p>The catalytic activities of Mdh^M, Mdh2^M and Mdh3^M from MGA3 (upper panel) and of Mdh^P, Mdh1^P and Mdh2^P from PB1 (lower panel) in the absence and presence of ACT. The catalytic activity of the enzymes with methanol (white), methanol+ACT (light grey), ethanol (dark grey), and ethanol+ACT (black) as substrates are shown. The experiments were performed in triplicates with 500 mM alcohols and the mean values with standard errors are indicated.</p

In vitro kinetic constants of Mdhs in the presence and absence of ACT.

a<p>The K_m value for Mdh^M was estimated since the real value is close to the detection limit of the applied method.</p><p>At least three independent experiments were performed, and the mean values with standard errors are given.</p

In vitro substrate specificity of <i>B. methanolicus</i> Mdhs.

<p>MGA3 enzymes are shown in the upper panel, while PB1 enzymes in the lower panel. Catalytic activities of purified Mdh^M/Mdh^P (white), Mdh1^P (light grey), Mdh2^M/Mdh2^P (dark grey) and Mdh3^M (black) on various alcohols (500 mM) is shown. The data were calculated from the mean value from two independent experiments, performed in triplicate, and the standard errors are included.</p