470 research outputs found
Hydrogen production by Parageobacillus thermoglucosidasius – Genomic insights and process investigation
Comparative analysis of the Geobacillus hemicellulose utilization locus reveals a highly variable target for improved hemicellulolysis
BACKGROUND: Members of the thermophilic genus Geobacillus can grow at high temperatures and produce a
battery of thermostable hemicellulose hydrolytic enzymes, making them ideal candidates for the bioconversion of
biomass to value-added products. To date the molecular determinants for hemicellulose degradation and utilization
have only been identified and partially characterized in one strain, namely Geobacillus stearothermophilus T-6, where
they are clustered in a single genetic locus.
RESULTS: Using the G. stearothermophilus T-6 hemicellulose utilization locus as genetic marker, orthologous
hemicellulose utilization (HUS) loci were identified in the complete and partial genomes of 17/24 Geobacillus strains.
These HUS loci are localized on a common genomic island. Comparative analyses of these loci revealed extensive
variability among the Geobacillus hemicellulose utilization systems, with only seven out of 41–68 proteins encoded
on these loci conserved among the HUS+ strains. This translates into extensive differences in the hydrolytic enzymes,
transport systems and metabolic pathways employed by Geobacillus spp. to degrade and utilize hemicellulose polymers.
CONCLUSIONS: The genetic variability among the Geobacillus HUS loci implies that they have variable capacities to
degrade hemicellulose polymers, or that they may degrade distinct polymers, as are found in different plant species and
tissues. The data from this study can serve as a basis for the genetic engineering of a Geobacillus strain(s) with an
improved capacity to degrade and utilize hemicellulose.This project was partially funded through the University of Pretoria
Postdoctoral Fellowship Programme and the National Research Foundation
(NRF) Research Career Advancement (RCA) Fellowship Programme.http://www.biomedcentral.com/bmcgenomics/am201
Rapid, Heuristic Discovery and Design of Promoter Collections in Non-Model Microbes for Industrial Applications
This is the author accepted manusript. The final version is available from American Chemical Society via the DOI in this recordAccession Codes:
The sequence data for the four Geobacillus spp. used in this study have been submitted to the NCBI Sequence Read Archive and are available under the accession number PRJNA521450.Well-characterized promoter collections for synthetic biology applications are not always available in industrially relevant hosts. We developed a broadly applicable method for promoter identification in atypical microbial hosts that requires no a priori understanding of cis-regulatory element structure. This novel approach combines bioinformatic filtering with rapid empirical characterization to expand the promoter toolkit and uses machine learning to improve the understanding of the relationship between DNA sequence and function. Here, we apply the method in Geobacillus thermoglucosidasius, a thermophilic organism with high potential as a synthetic biology chassis for industrial applications. Bioinformatic screening of G. kaustophilus, G. stearothermophilus, G. thermodenitrificans, and G. thermoglucosidasius resulted in the identification of 636 100 bp putative promoters, encompassing the genome-wide design space and lacking known transcription factor binding sites. Eighty of these sequences were characterized in vivo, and activities covered a 2-log range of predictable expression levels. Seven sequences were shown to function consistently regardless of the downstream coding sequence. Partition modeling identified sequence positions upstream of the canonical -35 and -10 consensus motifs that were predicted to strongly influence regulatory activity in Geobacillus, and artificial neural network and partial least squares regression models were derived to assess if there were a simple, forward, quantitative method for in silico prediction of promoter function. However, the models were insufficiently general to predict pre hoc promoter activity in vivo, most probably as a result of the relatively small size of the training data set compared to the size of the modeled design space
Overexpression and Characterization of a Laccase from Geobacillus Thermoglucosidasius
The use of enzymes as industrial oxidants has become popular due to their high substrate specificity and mild reaction conditions. Specifically, laccases are multi-copper oxidases that can oxidize a disparate range of organic substrates using oxygen and producing water as a byproduct without requirement for additional reactive compounds. Currently, all laccases used in industrial processes are fungal in origin. Although fungal laccases have high activities under near-ambient conditions, their use is limited at higher temperatures. Also, expression of fungal laccases in heterologous hosts is limited due to incorrect glycosylation. Bacterial laccases are much easier to express heterologously and are more active and stable at high temperatures, pH and salt concentrations.
Geobacillus is a genus of gram-positive thermophilic bacteria, many of which have been found to naturally secrete proteins at high levels. A novel laccase has been predicted to be present in multiple Geobacillus strains using comparative genomics. This laccase is approximately half the size of those found in other gram-positives or fungi, making it a better candidate for lignocellulosic biomass degradation because of easier access to the substrate. In this work, we seek to isolate and characterize this laccase, and determine the types of substrates it can oxidize. We then want to compare the activity of our laccase with that of a fungal laccase at different temperatures.
A plasmid was successfully constructed for the overexpression of laccase in Geobacillus thermoglucosidasius 95A1 and Escherichia coli DH5á. The novel laccase was isolated and purified from E. coli. The laccase was characterized by determining the activity for 5 substrates at a range of pHs and temperatures. Finally, the thermal stability of our laccase was compared with that from a fungal source, Trametes versicolor. Laccase from G. thermoglucosidasius demonstrated a 20-fold higher initial activity than Trametes laccase at 80°C, and was superior to the latter in terms of thermal stability and activity at high temperatures
Genetic toolbox for controlled expression of functional proteins in Geobacillus spp.
Species of genus Geobacillus are thermophilic bacteria and play an ever increasing role as hosts for biotechnological applications both in academia and industry. Here we screened a number of Geobacillus strains to determine which industrially relevant carbon sources they can utilize. One of the strains, G. thermoglucosidasius C56-YS93, was then chosen to develop a toolbox for controlled gene expression over a wide range of levels. It includes a library of semi-synthetic constitutive promoters (76-fold difference in expression levels) and an inducible promoter from the xylA gene. A library of synthetic in silico designed ribosome binding sites was also created for further tuning of translation. The PxylA was further used to successfully express native and heterologous xylanases in G. thermoglucosidasius. This toolbox enables fine-tuning of gene expression in Geobacillus species for metabolic engineering approaches in production of biochemicals and heterologous proteins
Not All That Glitters Is Gold: The Paradox of CO-dependent Hydrogenogenesis in Parageobacillus thermoglucosidasius
The thermophilic bacterium Parageobacillus thermoglucosidasius has recently gained interest due to its ability to catalyze the water gas shift reaction, where the oxidation of carbon monoxide (CO) is linked to the evolution of hydrogen (H) gas. This phenotype is largely predictable based on the presence of a genomic region coding for a carbon monoxide dehydrogenase (CODH—Coo) and hydrogen evolving hydrogenase (Phc). In this work, seven previously uncharacterized strains were cultivated under 50% CO and 50% air atmosphere. Despite the presence of the coo—phc genes in all seven strains, only one strain, Kp1013, oxidizes CO and yields H. The genomes of the H producing strains contain unique genomic regions that code for proteins involved in nickel transport and the detoxification of catechol, a by-product of a siderophore-mediated iron acquisition system. Combined, the presence of these genomic regions could potentially drive biological water gas shift (WGS) reaction in P. thermoglucosidasius
Isolation, characterization, and hydrolytic activities of Geobacillus species from Jordanian hot springs
The present study was conducted to isolate, identify, characterize and to determine the enzymatic activities of the thermophilic Geobacillus species from five Jordanian hot springs. Based on phenotypic characters, eight thermophilic isolates were identified and belonged to the genus Geobacillus. The Geobacillus isolates were abundant in all investigated hot springs. The optimal temperature for growth of the isolates was 60 to 65°C and the optimal pH was 6 to 8. Colonies were light yellow circular to rhizoid. The bacterial cells were Gram positive rods and endospore forming. All isolates produced amylase, caseinase, alkaline and acid phosphatases, esterase (C4), esterase lipase (C8), α-Galactosidase, β-Glucuronidase, β-Glucosidase, and N-Acetyl-β-glucosaminidase. Seven isolates produced leucine and valine arylamidases and five isolates produced naphthol-AS-B1- phsphohydrolase. Lipase (C14) activity from two isolates and α-chymotrypsin activity from three isolates were also detected. The phenotypic characterization of those isolates was confirmed by genotypic method using 16S rDNA sequence analysis. Maximal homology of all eight isolates to genus Geobacillus was observed. Five of these isolates showed greater than 98% homology with Geobacillus stearothermophilus and one isolate showed 100% homology with Geobacillus thermoglucosidasius. Therefore, 16S rRNA gene sequence analysis can be considered as a valuable genotypic tool for the identification and characterization of thermophilic bacteria at genus level. Moreover, enzymatic products of those isolates could receive considerable attention due to their potential applications in biotechnology.Keywords: Thermophiles, Geobacillus, hydrolytic enzymes, hot spring, 16S rRNA
Petrified Geobacillus thermoglucosidasius colony to strontianite
When biomass of the thermophilic bacteria Geobacillus thermoglucosidasius is brought into contact with a hydrogel containing sodium acetate and strontium, the biomass petrifies and hardens, becoming a mineralized thin film after incubation at 60ËšC for 72 h. Analysis by energy dispersive X-ray and X-ray diffraction shows that the mineralized thin film is strontianite. This is the first report of biomass completely changing to strontianite. Strontianite of thermophilic bacterial origin may be formed in the hydrothermal oligotrophic environment of the deep subsurface.
DOI: http://dx.doi.org/10.5281/zenodo.114670
Relaxed control of sugar utilization in Parageobacillus thermoglucosidasius DSM 2542
Though carbon catabolite repression (CCR) has been intensively studied in some more characterised organisms, there is a lack of information of CCR in thermophiles. In this work, CCR in the thermophile, Parageobacillus thermoglucosidasius DSM 2542 has been studied during growth on pentose sugars in the presence of glucose. Physiological studies under fermentative conditions revealed a loosely controlled CCR when DSM 2542 was grown in minimal medium supplemented with a mixture of glucose and xylose. This atypical CCR pattern was also confirmed by studying xylose isomerase expression level by qRT-PCR. Fortuitously, the pheB gene, which encodes catechol 2, 3-dioxygenase was found to have a cre site highly similar to the consensus catabolite-responsive element (cre) at its 3′ end and was used to confirm that expression of pheB from a plasmid was under stringent CCR control. Bioinformatic analysis suggested that the CCR regulation of xylose metabolism in P. thermoglucosidasius DSM 2542 might occur primarily via control of expression of pentose transporter operons. Relaxed control of sugar utilization might reflect a lower affinity of the CcpA-HPr (Ser46-P) or CcpA-Crh (Ser46-P) complexes to the cre(s) in these operons
Thermophilic Water Gas Shift Reaction at High Carbon Monoxide and Hydrogen Partial Pressures in Parageobacillus thermoglucosidasius KP1013
The facultatively anaerobic Parageobacillus thermoglucosidasius oxidizes carbon monoxide to produce hydrogen via the water gas shift (WGS) reaction. In the current work, we examined the influence of carbon monoxide (CO) and hydrogen (H) on the WGS reaction in the thermophilic P. thermoglucosidasius by cultivating two hydrogenogenic strains under varying CO and H compositions. Microbial growth and dynamics of the WGS reaction were monitored by evaluating parameters such as pressure, headspace composition, metabolic intermediates, pH, and optical density. Our analyses revealed that compared to the previously studied P. thermoglucosidasius strains, the strain KP1013 demonstrated higher CO tolerance and improved WGS reaction kinetics. Under anaerobic conditions, the lag phase before the WGS reaction shortened to 8 h, with KP1013 showing no hydrogen-induced product inhibition at hydrogen partial pressures up to 1.25 bar. The observed lack of product inhibition and the reduced lag phase of the WGS reaction support the possibility of establishing an industrial process for biohydrogen production with P. thermoglucosidasius
- …