RESUMO
Reinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. Here, we report on adaptive laboratory evolution (ALE) of the amino acid-producing bacterium Corynebacterium glutamicum under thermal stress. After 65 days of serial passage of the transgenic strain GLY3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were isolated, and all the mutations they acquired were identified by whole-genome resequencing. Of the 21 mutations common to the three strains, one large deletion and two missense mutations were found to promote growth of the parental strain under thermal stress. Additive effects on thermotolerance were observed among these mutations, and the combination of the deletion with the missense mutation on otsA, encoding a trehalose-6-phosphate synthase, allowed the parental strain to overcome the upper limit of growth temperature. Surprisingly, the three evolved strains acquired cross-tolerance for isobutanol, which turned out to be partly attributable to the genomic deletion associated with the enhanced thermotolerance. The deletion involved loss of two transgenes, pfk and pyk, encoding the glycolytic enzymes, in addition to six native genes, and elimination of the transgenes, but not the native genes, was shown to account for the positive effects on thermal and solvent stress tolerance, implying a link between energy-producing metabolism and bacterial stress tolerance. Overall, the present study provides evidence that ALE can be a powerful tool to refine the phenotype of C. glutamicum and to investigate the molecular bases of stress tolerance.
Assuntos
Adaptação Biológica , Corynebacterium glutamicum/efeitos dos fármacos , Corynebacterium glutamicum/efeitos da radiação , Temperatura Alta , Solventes/toxicidade , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/fisiologia , DNA Bacteriano/química , DNA Bacteriano/genética , Perfilação da Expressão Gênica , Genoma Bacteriano , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/fisiologia , Análise de Sequência de DNA , Deleção de Sequência , Inoculações SeriadasRESUMO
Corynebacterium glutamicum can consume glucose to excrete glycerol under oxygen deprivation. Although glycerol synthesis from 1,3-dihydroxyacetone (DHA) has been speculated, no direct evidence has yet been provided in C. glutamicum. Enzymatic and genetic investigations here indicate that the glycerol is largely produced from DHA and, unexpectedly, the reaction is catalyzed by (S,S)-butanediol dehydrogenase (ButA) that inherently catalyzes the interconversion between S-acetoin and (S,S)-2,3-butanediol. Consequently, the following pathway for glycerol biosynthesis in the bacterium emerges: dihydroxyacetone phosphate is dephosphorylated by HdpA to DHA, which is subsequently reduced to glycerol by ButA. This study emphasizes the importance of promiscuous activity of the enzyme in vivo.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/metabolismo , Corynebacterium glutamicum/enzimologia , Glicerol/metabolismo , Oxigênio/metabolismo , Oxirredutases do Álcool/genética , Proteínas de Bactérias/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Di-Hidroxiacetona/metabolismoRESUMO
The activity of Rieske non-heme iron oxygenases (aromatic hydrocarbon dioxygenases, AhDOs) is important for the bacterial degradation of aromatic pollutants such as polycyclic aromatic hydrocarbons and dioxins. During our analysis of the role of AhDOs in dioxin bioremediation, some enzymes derived from high G + C Gram-positive actinomycetes were difficult to produce in active form in the Escherichia coli protein expression system. In this study, we constructed a heterologous expression system for AhDOs in Rhodococcus species using a constitutive expression promoter, P(dfdB), and a shuttle vector, pRK401, and analyzed the ability of these enzymes to degrade dibenzofuran and deplete several chlorinated dioxins. Three active AhDOs expressed in Rhodococcus strains that were difficult to obtain by the E. coli system showed different regiospecificities for dibenzofuran bioconversion as well as different substrate depletion specificities for chlorinated dioxins. Moreover, AhDO derived from R. erythropolis TA421 showed relatively diverse depletion-substrate specificity for chlorinated dioxins.
Assuntos
Actinomycetales/enzimologia , Benzofuranos/metabolismo , Dioxinas/metabolismo , Dioxigenases/biossíntese , Dioxigenases/metabolismo , Poluentes Ambientais/metabolismo , Rhodococcus/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzofuranos/isolamento & purificação , Biodegradação Ambiental , Clonagem Molecular , Dioxinas/isolamento & purificação , Dioxigenases/genética , Poluentes Ambientais/isolamento & purificação , Regiões Promotoras Genéticas , Rhodococcus/metabolismoRESUMO
Suppression of immune function during long spaceflights is an issue that needs to be overcome. The well-established probiotic Lactobacillus casei strain Shirota (LcS) could be a promising countermeasure, and we have launched a project to investigate the efficacy of its use on the International Space Station (ISS). As a first step, we developed a specialist probiotic product for space experiments, containing freeze-dried LcS in capsule form (Probiotics Package), and tested its stability through 1 month of storage on the ISS. The temperature inside the ISS ranged from 20.0 to 24.5 °C. The absorbed dose rate of the flight sample was 0.26 mGy/day and the dose equivalent rate was 0.52 mSv/day. The number of live LcS was 1.05 × 1011 colony-forming units/g powder (49.5% of the initial value) 6 months after the start of the study; this value was comparable to those in the two ground controls. Profiles of randomly amplified polymorphic DNA, sequence variant frequency, carbohydrate fermentation, reactivity to LcS-specific antibody, and the cytokine-inducing ability of LcS in the flight sample did not differ from those of the ground controls. We can therefore maintain the viability and basic probiotic properties of LcS stored as a Probiotics Package on the ISS.