RESUMEN
There has been much effort exerted to reduce one carbon (C1) gas emission to address climate change. As one promising way to more conveniently utilize C1 gas, several technologies have been developed to convert C1 gas into useful chemicals such as formic acid (FA). In this study, systems metabolic engineering was utilized to engineer Mannheimia succiniciproducens to efficiently utilize FA. 13 C isotope analysis of M. succiniciproducens showed that FA could be utilized through formate dehydrogenase (FDH) reaction and/or the reverse reaction of pyruvate formate lyase (PFL). However, the naturally favored forward reaction of PFL was found to lower the SA yield from FA. In addition, FA assimilation via FDH was found to be more efficient than the reverse reaction of PFL. Thus, the M. succiniciproducens LPK7 strain, which lacks in pfl, ldh, pta, and ack genes, was selected as a base strain. In silico metabolic analysis confirmed that utilization of FA would be beneficial for the enhanced production of SA and suggested FDH as an amplification target. To find a suitable FDH, four different FDHs from M. succiniciproducens, Methylobacterium extorquens, and Candida boidinii were amplified in LPK7 strain to enhance FA assimilation. High-inoculum density cultivation using 13 C labeled sodium formate was performed to evaluate FA assimilation efficiency. Fed-batch fermentations of the LPK7 (pMS3-fdh2 meq) strain was carried out using glucose, sucrose, or glycerol as a primary carbon source and FA as a secondary carbon source. As a result, this strain produced 76.11 g/L SA with the yield and productivity of 1.28 mol/mol and 4.08 g/L/h, respectively, using sucrose and FA as dual carbon sources. The strategy employed here will be similarly applicable in developing microorganisms to utilize FA and to produce valuable chemicals and materials from FA.
Asunto(s)
Formiato Deshidrogenasas/genética , Formiatos/metabolismo , Mejoramiento Genético/métodos , Mannheimia/fisiología , Ingeniería Metabólica/métodos , Análisis de Flujos Metabólicos/métodos , Ácido Succínico/metabolismo , Simulación por Computador , Mannheimia/clasificación , Modelos Biológicos , Especificidad de la Especie , Especificidad por Sustrato , Ácido Succínico/aislamiento & purificación , Regulación hacia Arriba/genéticaRESUMEN
Mannheimia granulomatis was first isolated from pneumonic European hares in the 1980s and has since been reported sporadically in pneumonic Swedish roe deer and Australian cattle. Although the pneumonic lesions caused by M. haemolytica in livestock have been extensively studied and reported, little is published with regard to the pneumonic lesions associated with M. granulomatis infection in any species. We describe the histopathology of purulent bronchopneumonia associated with M. granulomatis in a Belgian hare ( Lepus europaeus) resident in British Columbia, Canada, and compare the lesions with those caused by M. haemolytica in livestock.
Asunto(s)
Bronconeumonía/veterinaria , Mannheimia/fisiología , Infecciones por Pasteurellaceae/veterinaria , Conejos , Animales , Colombia Británica , Bronconeumonía/microbiología , Resultado Fatal , Infecciones por Pasteurellaceae/microbiologíaRESUMEN
Succinic acid (SA) is a four carbon dicarboxylic acid of great industrial interest that can be produced by microbial fermentation. Here we report development of a high-yield homo-SA producing Mannheimia succiniciproducens strain by metabolic engineering. The PALFK strain (ldhA-, pta-, ackA-, fruA-) was developed based on optimization of carbon flux towards SA production while minimizing byproducts formation through the integrated application of in silico genome-scale metabolic flux analysis, omics analyses, and reconstruction of central carbon metabolism. Based on in silico simulation, utilization of sucrose would enhance the SA production and cell growth rates, while consumption of glycerol would reduce the byproduct formation rates. Thus, sucrose and glycerol were selected as dual carbon sources to improve the SA yield and productivity, while deregulation of catabolite-repression was also performed in engineered M. succiniciproducens. Fed-batch fermentations of PALFK with low- and medium-density (OD600 of 0.4 and 9.0, respectively) inocula produced 69.2 and 78.4g/L of homo-SA with yields of 1.56 and 1.64mol/mol glucose equivalent and overall volumetric SA productivities of 2.50 and 6.02g/L/h, respectively, using sucrose and glycerol as dual carbon sources. The SA productivity could be further increased to 38.6g/L/h by employing a membrane cell recycle bioreactor system. The systems metabolic engineering strategies employed here for achieving homo-SA production with the highest overall performance indices reported to date will be generally applicable for developing superior industrial microorganisms and competitive processes for the bio-based production of other chemicals as well.
Asunto(s)
Proteínas Bacterianas/genética , Glicerol/metabolismo , Mannheimia/fisiología , Ingeniería Metabólica/métodos , Ácido Succínico/metabolismo , Sacarosa/metabolismo , Reactores Biológicos/microbiología , Vías Biosintéticas/genética , Mejoramiento Genético/métodos , Redes y Vías Metabólicas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ácido Succínico/aislamiento & purificaciónRESUMEN
Mannheimia haemolytica is known to be an important cause of intramammary infection in sheep. It usually causes severe clinical mastitis, followed by toxaemia and gangrenous necrosis of the udder. However there are limited data available on the epidemiology and pathogenesis of mastitis associated with Mannheimia species. These organisms can be more significant as a cause of mastitis than Staphylococcus aureus in some flocks. Some data suggest the possibility of horizontal transmission of Mannheimia species between ewes via lamb sucking. There is no vaccine available for prevention, and the sudden onset of mastitis and its peracute nature renders most treatments unsuccessful. This review examines the significance of the species within this genus in sheep mastitis.
Asunto(s)
Mannheimia/fisiología , Mastitis/veterinaria , Enfermedades de las Ovejas/microbiología , Animales , Femenino , Glándulas Mamarias Animales/patología , Mannheimia/clasificación , Mannheimia/patogenicidad , Mannheimia haemolytica/patogenicidad , Mannheimia haemolytica/fisiología , Mastitis/epidemiología , Mastitis/microbiología , Ovinos , Enfermedades de las Ovejas/epidemiología , Enfermedades de las Ovejas/patología , Oveja Doméstica , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The ArcB/A two-component signal transduction system of Escherichia coli modulates the expression of numerous operons in response to redox conditions of growth. We demonstrate that the putative arcA and arcB genes of Mannheimia succiniciproducens MBEL55E, a capnophilic (CO2-loving) rumen bacterium, encode functional proteins that specify a two-component system. The Arc proteins of the two bacterial species sufficiently resemble each other that they can participate in heterologous transphosphorylation in vitro, and the arcA and arcB genes of M. succiniciproducens confer toluidine blue resistance to E. coli arcA and arcB mutants. However, neither the quinone analogs (ubiquinone 0 and menadione) nor the cytosolic effectors (d-lactate, acetate, and pyruvate) affect the net phosphorylation of M. succiniciproducens ArcB. Our results indicate that different types of signaling molecules and distinct modes of kinase regulation are used by the ArcB proteins of E. coli and M. succiniciproducens.