RESUMEN
Zymomonas mobilis is a bacterium of industrial interest due to its high ethanol productivity and high tolerance to stresses. Although the physiological parameters of fermentation are well characterized, there are few studies on the molecular mechanisms that regulate the response to fermentative stress. Z. mobilis ZM4 presents five different sigma factors identified in the genome annotation, but the absence of sigma 38 leads to the questioning of which sigma factors are responsible for its mechanism of fermentative stress response. Thus, in this study, factors sigma 32 and sigma 24, traditionally related to heat shock, were tested for their influence on the response to osmotic and ethanol stress. The overexpression of these sigma factors in Z. mobilis ZM4 strain confirmed that both are associated with heat shock response, as described in other bacteria. Moreover, sigma 32 has also a role in the adaptation to osmotic stress, increasing both growth rate and glucose influx rate. The same strain that overexpresses sigma 32 also showed a decrease in ethanol tolerance, suggesting an antagonism between these two mechanisms. It was not possible to conclude if sigma 24 really affects ethanol tolerance in Z. mobilis, but the overexpression of this sigma factor led to a decrease in ethanol productivity.
Asunto(s)
Fermentación , Presión Osmótica , Factor sigma/genética , Estrés Fisiológico/genética , Zymomonas/genética , Zymomonas/fisiología , ARN Polimerasas Dirigidas por ADN/genética , Etanol/farmacología , Glucosa/metabolismo , Proteínas de Choque Térmico/genética , Zymomonas/efectos de los fármacosRESUMEN
Zymomonas mobilis has long attracted attention owing to its capacity to ferment hexose to ethanol. From a taxonomic viewpoint, Z. mobilis is a unique species of the genus Zymomonas, separated into three subspecies, Z. mobilis subsp. mobilis, Z. mobilis subsp. pomaceae and Z. mobilis subsp. francensis on the basis of physiological tests, which are often unreliable owing to the genetic proximity among these species. Currently, the use of molecular techniques is more appropriate for identification of these bacterial subspecies. In this study, the 32 strains of Z. mobilis present in the UFPEDA bacterial collection were characterized using molecular techniques, such as sequencing of the 16S rDNA gene and its theoretical restriction profile, classifying them as members of the subspecies, Z. mobilis subsp. mobilis. In addition, anaerobic cultivations were performed, which showed the biological diversity of the strains in terms of growth, sugar consumption and ethanol production. From these results, it was possible to identify the strain Z-2-80 as a promising bacterium for use in the fermentation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Zymomonas mobilis is a bacterium of great relevance to biotechnology, owing to its capacity to ferment hexose to ethanol. On a molecular basis, 32 isolates were identified as Z. mobilis subsp. mobilis. However, intraspecific diversity was identified when these were grown under strictly anaerobic conditions. The results obtained from this study suggest a strain of Z. mobilis as an alternative for use in the fermentation process.
Asunto(s)
Reactores Biológicos/microbiología , ADN Bacteriano/genética , Etanol/metabolismo , Zymomonas/clasificación , Zymomonas/metabolismo , Anaerobiosis , Brasil , ADN Ribosómico/genética , Fermentación , Hexosas/metabolismo , ARN Ribosómico 16S/genética , Zymomonas/genética , Zymomonas/aislamiento & purificaciónRESUMEN
Background: Zymomonas mobilis is a Gram-negative microaerophilic bacterium with excellent ethanol-producing capabilities. The RecET recombination system provides an efficient tool for direct targeting of genes in the bacterial chromosome by PCR fragments. Results: The plasmids pSUZM2a-RecET and pSUZM2a-RecE588T were first developed to co-express RecE or RecE588 and RecT for homologous recombination. Thereafter, the PCR fragments of the tetracycline resistance marker gene flanked by 60 bp of adhA (alcohol dehydrogenase I) or adhB (alcohol dehydrogenase II) homologous sequences were electroporated directly into ZM4 cells harboring pSUZM2a-RecET or pSUZM2a-RecE588T. Both adhA and adhB were replaced by the tetracycline resistance gene in ZM4, yielding two mutant strains, Z. mobilis ZM4 ΔadhA and Z. mobilis ZM4 ΔadhB. These two mutants showed varying extent of reduction in ethanol production, biomass generation, and glucose metabolism. Furthermore, enzyme activity of alcohol dehydrogenase II in Z. mobilis ZM4 ΔadhB exhibited a significant reduction compared to that of wild-type ZM4. Conclusion: This approach provided a simple and useful method for introducing mutations and heterologous genes in the Z. mobilis genome.
Asunto(s)
Zymomonas/genética , Recombinación Homóloga , Plásmidos , Recombinación Genética , Alcohol Deshidrogenasa/metabolismo , Zymomonas/enzimología , Electroporación , Etanol/metabolismo , Técnicas de Inactivación de Genes , MutaciónRESUMEN
Zymomonas mobilis genes encoding INVA and INVB were expressed in Pichia pastoris, under the control of the strong AOX1 promoter, and the recombinant enzymes were named INVAAOX1 and INVBAOX1. The expression levels of INVAAOX1 (1660 U/mg) and INVBAOX1 (1993 U/mg) in P. pastoris were 9- and 7-fold higher than those observed for the native INVA and INVB proteins in Z. mobilis. INVAAOX1 and INVBAOX1 displayed a 2- to 3-fold higher substrate affinity, and a 2- to 200-fold higher catalytic efficiency (kcat/KM) than that observed for native INVA and INVB from Z. mobilis. Positive Schiff staining of INVAAOX1 and INVBAOX1 suggested a glycoprotein nature of both invertases. After deglycosylation of these enzymes, denoted D-INVAAOX1 and D-INVBAOX1, they exhibited a 1.3- and 3-fold lower catalytic efficiency (107 and 164 s(-1) mM(-1), respectively), and a 1.3- to 5-fold lower thermal stability than the glycosylated forms at temperatures of 35-45 °C. After deglycosylation no effect was observed in optimal pH, being of 5.5 for INVAAOX1, INVBAOX1, D-INVAAOX1 and D-INVBAOX1. The invertase activity of both enzymes increased in 80% (INVAAOX1) and 20% (INVBAOX1) in the presence of Mn(2+) at 1 mM and 5 mM, respectively. INVAAOX1 and INVBAOX1 were highly active at sucrose concentrations of up to 400 and 300 mM, respectively; however, the tolerance to sucrose decreased to 300 mM for D-INVAAOX1. Our findings suggest that glycosylation of INVAAOX1 and INVBAOX1 plays an important role in their thermal stability, catalytic efficiency, and tolerance to sucrose. In conclusion, the expression of INVA and INVB from Z. mobilis in P. pastoris yields new catalysts with improved catalytic properties, making them suitable candidates for a number of industrial applications or for the improvement of ethanol production from cane molasses.
Asunto(s)
beta-Fructofuranosidasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Catálisis , Cationes Bivalentes/metabolismo , Estabilidad de Enzimas , Genes Bacterianos , Glicosilación , Concentración de Iones de Hidrógeno , Cinética , Pichia/enzimología , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sacarosa/metabolismo , Temperatura , Zymomonas/enzimología , Zymomonas/genética , beta-Fructofuranosidasa/química , beta-Fructofuranosidasa/genéticaRESUMEN
Background: Zymomonas mobilis, as a novel platform for bio-ethanol production, has been attracted more attention and it is very important to construct vectors for the efficient expression of foreign genes in this bacterium. Results: Three shuttle vectors ( pSUZM 1, pSUZM2 and pSUZM3 ) were first constructed with the origins of replication from the chromosome and two native plasmids (pZZM401 and pZZM402) of Z. mobilis ZM4, respectively. The three shuttle vectors were stable in Z. mobilis ZM4 and have 3,32 and 27 copies, respectively. The promoter Ppdc (a), from the pyruvate decarboxylase gene, was clonedinto the shuttle vectors, generatingthe expressionvectors pSUZM1(2, 3)a. The codon-optimized glucoamylase gene from Aspergillus awamori combined with the signal peptide sequence from the alkaline phosphatase gene of Z. mobilis was cloned into pSUZM1(2, 3)a, resulting in the plasmids pSUZM1a-GA, pSUZM2a-GA and pSUZM3a-GA, respectively. After transforming these plasmids into Z. mobilis ZM4, the host was endowed with glucoamylase activity for starch hydrolysis. Both pSUZM2a-GA and pSUZM3a-GA were more efficientatproducingglucoamylase thanpSUZM1a-GA. Conclusions: These results indicated that these expression vectors are useful tools for gene expression in Z. mobilis and this could provide a solid foundation for further studies of heterologous gene expression in Z. mobilis.
Asunto(s)
Expresión Génica/genética , Zymomonas/genética , Zymomonas/metabolismo , Vectores Genéticos/genética , Plásmidos , Glucano 1,4-alfa-Glucosidasa , Fermentación , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Zymomonas mobilis has aroused considerable interest owing to its rapid metabolism and efficiency in producing ethanol and by-products such as levans, sorbitol, and gluconic acid from simple sugars. We performed a proteomic analysis of Z. mobilis UFPEDA241 to provide a global profile of regulatory proteins. The choice of the methods of extraction and cell lysis are fundamental steps and of great importance for the detection and identification of intra- and extracellular proteins of a proteome. Strains were subjected to protein extraction methods using three different reagents: TRIzol, lysis buffer, and phenol. The optimum method was taken to be the one that produced the greatest quantity and quality of proteins in one dimension for further analysis in two dimensions during the production of ethanol and levans over 72 h. The results showed that the greatest amount of protein was obtained by the phenol method (1.44 ± 0.07 mg/mL), which was significantly different (P < 0.05) to the TRIzol (1.11 ± 0.01 mg/mL), and lysis buffer (0.93 ± 0.01 mg/mL) methods (both with P > 0.05). Fermentation at 20°C produced the highest level of levans, and using two-dimensional electrophoresis and mass spectrometry it was possible to identify 34 differentially expressed spots.
Asunto(s)
Etanol/química , Fructanos/química , Proteínas/aislamiento & purificación , Proteómica , Etanol/metabolismo , Fermentación , Fructanos/metabolismo , Glucosa/química , Glucosa/metabolismo , Proteínas/genética , Zymomonas/genética , Zymomonas/metabolismoRESUMEN
Zymomonas mobilis is a Gram-negative bacterium that has drawn attention in the bioethanol industry. Besides bioethanol, this bacterium also produces other biotechnological products such as levans, which show antitumor activity. Molecular studies involving Z. mobilis have advanced to the point that allows us to characterize interspecies genetic diversity and understand their metabolism, and these data are essential for better utilization of this species. In this study, the genetic diversity of 24 strains from the Microorganisms Collection of Departamento de Antibióticos (UFPEDA) from Universidade Federal de Pernambuco were characterized. The methods used were amplified ribosomal DNA restriction analysis and diversity analysis of the internally transcribed 16S-23S rDNA spacer region (ISR). These analyses revealed low genetic variability of the 16S rDNA gene. These data confirm that these isolates are, or are closely related to, Z. mobilis. Moreover, the analysis of the ISR confirmed the genetic variability of strains deposited in the UFPEDA collection of microorganisms and grouped these strains into ten ribotypes, which can be used in the future for breeding programs and for the preservation of biodiversity. Furthermore, this study characterized the genetic variability between the UFPEDA 205/ ZAP, UFPEDA 98/AG11, and ZAG strains, which were obtained by spheroplast fusion among them. The data also indicate that there is genetic variability among the UFPEDA 202/CP4 and UFPEDA 633/ ZM4 strains, demonstrating that these important Z. mobilis strains are distinct, as suggested in previous studies.
Asunto(s)
Análisis del Polimorfismo de Longitud de Fragmentos Amplificados/métodos , ADN Ribosómico/genética , Variación Genética , Zymomonas/genética , Biocombustibles/microbiología , Etanol/metabolismo , Zymomonas/metabolismoRESUMEN
Zymomonas mobilis ATCC 10988 is the type strain of the Z. mobilis subsp. mobilis taxon, members of which are some of the most rigorous ethanol-producing bacteria. Isolated from Agave cactus fermentations in Mexico, ATCC 10988 is one of the first Z. mobilis strains to be described and studied. Its robustness in sucrose-substrate fermentations, physiological characteristics, large number of plasmids, and overall genomic plasticity render this strain important to the study of the species. Here we report the finishing and annotation of the ATCC 10988 chromosomal and plasmid genome.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Genoma Bacteriano , Análisis de Secuencia de ADN , Zymomonas/genética , Agave/microbiología , Etanol/metabolismo , Fermentación , Microbiología de Alimentos , México , Datos de Secuencia Molecular , Plásmidos , Sacarosa/metabolismo , Zymomonas/aislamiento & purificación , Zymomonas/metabolismo , Zymomonas/fisiologíaRESUMEN
An integrative shuttle vector, pZMOCP1, was constructed by ligating EcoRV digests of the plasmid cloning vector pBluescript and pZMP1, a cryptic plasmid of Zymomonas mobilis PROIMI A1. The 7.2-kb plasmid pZMOCP1 replicated in Escherichia coli and could also be transferred from this host by electroporation to Z. mobilis ATCC 29191. The transformants were selected by ampicillin resistance. The integrative characteristic was detected by hybridization in situ. The vector was stably maintained in Z. mobilis after 200 generations without selective pressure.