Geobacillus Thermoglucosidasius Research Articles

AI-based automated construction of high-precision Geobacillus thermoglucosidasius enzyme constraint model

Geobacillus thermoglucosidasius NCIMB 11955 possesses advantages, such as high-temperature tolerance, rapid growth rate, and low contamination risk. Additionally, it features efficient gene editing tools, making it one of the most promising next-generation cell factories. However, as a non-model microorganism, a lack of metabolic information significantly hampers the construction of high-precision metabolic flux models. Here, we propose a BioIntelliModel (BIM) strategy based on artificial intelligence technology for the automated construction of enzyme-constrained models. 1). BIM utilises the Contrastive Learning Enabled Enzyme Annotation (CLEAN) prediction tool to analyse the entire genome sequence of G. thermoglucosidasius NCIMB 11955, uncovering potential functional proteins in non-model strains. 2). The MetaPatchM module of BIM automates the repair of the metabolic network model. 3). The Tianjin University of Science and Technology-kcat (TUST-kcat) module predicts the kcat values of enzymes within the model. 4). The Enzyme-insert procedure constructs an enzyme-constrained model and performs a global scan to address overconstraint issues. Enzymatic data were automatically integrated into the metabolic flux model, creating an enzyme-constrained model, ec_G-ther11955. To validate model accuracy, we used both the p-thermo and ec_G-ther11955 models to predict riboflavin production strategies. The ec_G-ther11955 model demonstrated significantly higher accuracy. To further verify its efficacy, we employed ec_G-ther11955 to guide the rational design of L-valine-producing strains. Using the Optimisation Procedure for Identifying All Genetic Manipulations Leading to Targeted Overproductions (OptForce), Predictive Knockout Targeting (PKT), and Flux Scanning based on Enforced Objective Flux (FSEOF) algorithms, we identified 24 knockout and overexpression targets, achieving an accuracy rate of 87.5%. Ultimately, this led to an increase of 664.04% in L-valine titre. This study provides a novel strategy for rapidly constructing non-model strain models and demonstrates the tremendous potential of artificial intelligence in metabolic engineering.

Metabolic engineering of Geobacillus thermoglucosidasius for polymer-grade lactic acid production at high temperature

The production and application of biodegradable polylactic acid are still severely hindered by the cost of its polymer-grade lactic acid monomers. High-temperature biomanufacturing has emerged as an increasingly attractive approach to enable low-cost and high-efficiency bulk chemical production. In this study, thermophilic Geobacillus thermoglucosidasius was reprogrammed to obtain optically pure l-lactic acid- and d-lactic acid-producing strains, G. thermoglucosidasius GTD17 and GTD7, by using rational metabolic engineering strategies including pathway construction, by-product elimination, and production enhancing. Moreover, semi-rational adaptive evolution was carried out to further improve their lactic acid synthesis performance. The final strains GTD17-55 and GTD7-144 produce 151.1 g/L of l-lactic acid and 153.1 g/L of d-lactic acid at 60 °C, respectively. In consideration of the high temperature, productive performance of these strains is superior compared to the state-of-the-art industrial strains. This study lays the foundation for the low-cost and efficient production of biodegradable plastic polylactic acid.

Molecular Cloning, Characterization, and Application of Organic Solvent-Stable and Detergent-Compatible Thermostable Alkaline Protease from Geobacillus thermoglucosidasius SKF4.

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80°C. In addition, the enzyme showed a half-life of 15 h at 80°C, and there was a 60% increase in its activity at 10 mM Ca2+ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.

Development of highly efficient electrocompetent cells for electroporation of Geobacillus thermoglucosidasius NCIMB 11955

Geobacillus thermoglucosidasius is a kind of Gram-positive facultative anaerobic bacteria. The fast growth rate under high temperature and less susceptibility to microbial contamination enable G. thermoglucosidasius to be a desirable producer of biofuels and high-value-added chemicals for the next-generation industrial biotechnology. However, compared with the classical model strain Escherichia coli, the applications of G. thermoglucosidasius are hampered by its low transformation efficiency. This study aimed at obtaining competent cells with high transformation efficiency through inactivating restriction enzymes, adding cell membrane inhibitors and cell wall weakening agents. The results showed that the electro-transformation efficiency achieved 1.2×104 CFU/(μg DNA) by knocking out four genes encoding restriction enzymes. Adding a certain amount of tween 80, dl-threonine and glycine further increased the competent efficiency about 22.5, 44, and 334 times, respectively. The electro-transformation efficiency was enhanced to 4.6×106 CFU/(μg DNA) under the optimized conditions, laying a foundation for genetic manipulation and metabolic engineering of G. thermoglucosidasius.

Optimization of consolidated bioprocessing by response surface methodology in the conversion of corn stover to bioethanol by thermophilic Geobacillus thermoglucosidasius.

The swift depletion of fossil fuels and their associated environment and economic impact has led the world to explore the sustainable alternate fuels. Amidst the available alternatives lignocellulosic bioethanol provides the edge over the exhausting fossil fuels. In this current study, Response surface methodology, a mathematical and statistical tool was used to optimize the fermentation conditions in consolidated bioprocessing of corn stover by Geobacillus thermoglucosidasius. The impact of inoculum concentration, temperature, pH, agitation speed and time in bioethanol fermentation were screened with Plackett-Burman design and it was farther optimized with central composite design. The analysis by PBD confirmed the significant impact of fermentation time, inoculum concentration, and temperature of the fermentation process. Further, it was optimized with CCD. This showed that 15% v/v of Inoculum concentration, 50°C of temperature and fermentation time of 72h increased the bioethanol concentration to a maximum of 9.04g/L with 0.45g/g significant yield and a conversion efficiency of 88%. Thus, the CCD showed a satisfactory result in consolidated bioprocessing of bioethanol from corn stover. Thus, in the future, this approach of optimization will yield a good base for consistent production of bioethanol.

Dynamic Control Strategy to Produce Riboflavin with Lignocellulose Hydrolysate in the Thermophile Geobacillus thermoglucosidasius.

Efficient utilization of both glucose and xylose, the two most abundant sugars in biomass hydrolysates, is one of the main objectives of biofermentation with lignocellulosic materials. The utilization of xylose is commonly inhibited by glucose, which is known as glucose catabolite repression (GCR). Here, we report a GCR-based dynamic control (GCR-DC) strategy aiming at better co-utilization of glucose and xylose, by decoupling the cell growth and biosynthesis of riboflavin as a product. Using the thermophilic strain Geobacillus thermoglucosidasius DSM 2542 as a host, we constructed additional riboflavin biosynthetic pathways that were activated by xylose but not glucose. The engineered strains showed a two-stage fermentation process. In the first stage, glucose was preferentially used for cell growth and no production of riboflavin was observed, while in the second stage where glucose was nearly depleted, xylose was effectively utilized for riboflavin biosynthesis. Using corn cob hydrolysate as a carbon source, the optimized riboflavin yields of strains DSM2542-DCall-MSS (full pathway dynamic control strategy) and DSM2542-DCrib (single-module dynamic control strategy) were 5.3- and 2.3-fold higher than that of the control strain DSM 2542 Rib-Gtg constitutively producing riboflavin, respectively. This GCR-DC strategy should also be applicable to the construction of cell factories that can efficiently use natural carbon sources with multiple sugar components for the production of high-value chemicals in future.

Structure-guided rational design of the Geobacillus thermoglucosidasius feruloyl esterase GthFAE to improve its thermostability

Feruloyl esterases are indispensable biocatalysts catalyzing the cleavage of ester bonds between polysaccharides and their hydroxycinnamoyl cross-links. GthFAE from Geobacillus thermoglucosidasius was identified as a thermophilic alkaline feruloyl esterase with potential applications in paper manufacturing. To improve the enzymatic properties rationally and efficiently, the structure of GthFAE was solved at 1.9 Å, revealing a core domain of classical α/β hydrolase fold and an inserted α/β cap domain. In silico analysis based on it helped us to investigate whether the residues at the active center have positive effects on the stability, and how. Several site-directed mutations were conducted, of which substitutions at residues T41 and T150 apparently improved the thermostability. The combination mutant T41N/T150R exhibited an optimal temperature of 65 °C, a 6.4 °C higher Tm compared to wild type by 80 °C, and a 35-fold longer in half-life (201 min) at 70 °C. Molecular dynamics simulations further illustrated that the structure of T41N/T150R was more stable than the wild type and T150R stabilized the cap domain by introducing salt bridges to the region with E154 and D164. This study not only highlighted residues within the active center on their thermostability improving effects, but also contributed to the prospective industrial application of GthFAE.

Modelling the effect of bioreactor height on stripping fermentation products from the engineered bacterium Geobacillus thermoglucosidasius

Mass transfer into microbubbles is important for stripping fermentation products from bioreactors. The effect of bioreactor height on stripping the ethanol produced by the thermophilic bacterium Geobacillus thermoglucosidasius is explored. Being thermophilic, this bacterium allows operation near the boiling point of ethanol. First, a model is developed for a tall bioreactor with a well-mixed liquid phase. An expression relating off-gas concentration, dissolved gas measurements and the mass transfer coefficient is derived. This gives a novel method for estimating the mass transfer coefficient. Second, this approach is adapted to formulate and numerically solve a model for fermentation with Geobacillus thermoglucosidasius in a tall bubble column. For validation, the model is shown to be consistent with an experimentally-validated model of fermentation in a tall bubble column in the literature. The model predicts that under typical operation with 100 µm diameter microbubbles, mass transfer is not limiting: the gas-phase product concentrations are metabolically driven. The model is run with different bubble sizes, obtaining a universal curve for the gas product concentrations. This curve indicates a previously unexplored mass transfer limited regime in which, for a 20 m column, the outlet ethanol gas-phase concentration could be increased by 45% compared to its equilibrium value.

PH-Independent Heat Capacity Changes during Phosphorolysis Catalyzed by the Pyrimidine Nucleoside Phosphorylase from Geobacillus thermoglucosidasius.

Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, the effects of pH and (de)protonation on this phenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilic pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values. Our analysis revealed the presence of a measurable activation heat capacity change ΔCp⧧ in this reaction system, which showed no significant dependence on medium pH or substrate charge. Our results further describe the remarkable effects of a single halide substitution that has a minor influence on ΔCp⧧ but conveys a significant kinetic effect by decreasing the activation enthalpy, causing a >10-fold rate increase. Collectively, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect the rate, affinity, and thermodynamic phenomena independently of one another.

Effect of Cultural Conditions on Protease Production by a Thermophilic Geobacillus thermoglucosidasius SKF4 Isolated from Sungai Klah Hot Spring Park, Malaysia.

Major progress in the fields of agriculture, industry, and biotechnology over the years has influenced the quest for a potent microorganism with favorable properties to be used in scientific research and industry. This study intended to isolate a new thermophilic-protease-producing bacterium and evaluate its growth and protease production under cultural conditions. Protease producing bacteria were successfully isolated from Sungai Klah Hot Spring Park in Perak, Malaysia, and coded as SKF4; they were promising protease producers. Based on microscopic, morphological, and 16S rRNA gene analysis, isolate SKF4 was identified as Geobacillus thermoglucosidasius SKF4. The process of isolating SKF4 to grow and produce proteases under different cultural conditions, including temperature, pH, NaCl concentration, carbon and nitrogen sources, and incubation time, was explored. The optimum cultural conditions observed for growth and protease production were at 60 to 65 °C of temperature, pH 7 to 8, and under 1% NaCl concentration. Further, the use of casein and yeast extract as the nitrogen sources, and sucrose and fructose as the carbon sources enhanced the growth and protease production of isolate SKF4. Meanwhile, isolate SKF4 reached maximum growth and protease production at 24 h of incubation time. The results of this study revealed a new potent strain of thermophilic bacterium isolated from Sungai Klah Hot Spring Park in Perak, Malaysia for the first time. The high production of thermostable protease enzyme by G. thermoglucosidasius SKF4 highlighted the promising properties of this bacterium for industrial and biotechnological applications.

Single pot biovalorization of food waste to ethanol by Geobacillus and Thermoanaerobacter spp.

The current study was focused on developing a green thermophilic bioprocess to produce bioethanol from the food waste. The stored energy in food waste can be used for bioethanol production that can also help in reducing the land and environment impact of current food waste management processes. Geobacillus thermoglucosidasius was used to produce ethanol from food waste in a single pot at 60 °C. Scaling up the single pot bioprocess from 165ml serum bottles (165ml) to 1L bioreactor increased the ethanol yield from 3.03 g/L to 13 g/L. With scaling up to 1L reactor improved the substrate mass recovery from 39.2% to 92.8%. The ethanol production in 1L was further increased to 16.1g/L by the sequential cultivation of Geobacillus thermoglucosidasius and Thermoanaerobacter ethanolicus. Further, scaling up to 40L reactor gave an ethanol yield of 18.4 g/L and improved the ethanol productivity from 0.07 g/L/h (1L reactor) to 0.15 g/L/h (40L reactor). The sequential cultivation of thermophiles was able to produce ethanol from food waste without pretreatment, giving 70.1L of gasoline equivalent per ton of dry food waste. This is the first report of sequential cultivation of thermophiles for ethanol production using food waste and scaling up to pilot plant.

Robust microorganisms for biofuel and chemical production from municipal solid waste

BackgroundWorldwide 3.4 billion tonnes of municipal solid waste (MSW) will be produced annually by 2050, however, current approaches to MSW management predominantly involve unsustainable practices like landfilling and incineration. The organic fraction of MSW (OMSW) typically comprises ~ 50% lignocellulose-rich material but is underexplored as a biomanufacturing feedstock due to its highly inconsistent and heterogeneous composition. This study sought to overcome the limitations associated with studying MSW-derived feedstocks by using OMSW produced from a realistic and reproducible MSW mixture on a commercial autoclave system. The resulting OMSW fibre was enzymatically hydrolysed and used to screen diverse microorganisms of biotechnological interest to identify robust species capable of fermenting this complex feedstock.ResultsThe autoclave pre-treated OMSW fibre contained a polysaccharide fraction comprising 38% cellulose and 4% hemicellulose. Enzymatic hydrolysate of OMSW fibre was high in d-glucose (5.5% w/v) and d-xylose (1.8%w/v) but deficient in nitrogen and phosphate. Although relatively low levels of levulinic acid (30 mM) and vanillin (2 mM) were detected and furfural and 5-hydroxymethylfurfural were absent, the hydrolysate contained an abundance of potentially toxic metals (0.6% w/v). Hydrolysate supplemented with 1% yeast extract to alleviate nutrient limitation was used in a substrate-oriented shake-flask screen with eight biotechnologically useful microorganisms (Clostridium saccharoperbutylacetonicum, Escherichia coli, Geobacillus thermoglucosidasius, Pseudomonas putida, Rhodococcus opacus, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Zymomonas mobilis). Each species’ growth and productivity were characterised and three species were identified that robustly and efficiently fermented OMSW fibre hydrolysate without significant substrate inhibition: Z. mobilis, S. cerevisiae and R. opacus, respectively produced product to 69%, 70% and 72% of the maximum theoretical fermentation yield and could theoretically produce 136 kg and 139 kg of ethanol and 91 kg of triacylglycerol (TAG) per tonne of OMSW.ConclusionsDeveloping an integrated biorefinery around MSW has the potential to significantly alleviate the environmental burden of current waste management practices. Substrate-oriented screening of a representative and reproducible OMSW-derived fibre identified microorganisms intrinsically suited to growth on OMSW hydrolysates. These species are promising candidates for developing an MSW biorefining platform and provide a foundation for future studies aiming to valorise this underexplored feedstock.

Engineering thermophilic Geobacillus thermoglucosidasius for riboflavin production.

SummaryThe potential advantages for fermentation production of chemicals at high temperatures are attractive, such as promoting the rate of biochemical reactions, reducing the risk of contamination and the energy consumption for fermenter cooling. In this work, we de novo engineered the thermophile Geobacillus thermoglucosidasius to produce riboflavin, since this bacterium can ferment diverse carbohydrates at an optimal temperature of 60°C with a high growth rate. We first introduced a heterogeneous riboflavin biosynthetic gene cluster and enabled the strain to produce detectable riboflavin (28.7 mg l−1). Then, with the aid of an improved gene replacement method, we preformed metabolic engineering in this strain, including replacement of ribC Gtg with a mutant allele to weaken the consumption of riboflavin, manipulation of purine pathway to enhance precursor supply, deletion of ccpN Gtg to tune central carbon catabolism towards riboflavin production and elimination of the lactate dehydrogenase gene to block the dominating product lactic acid. Finally, the engineered strain could produce riboflavin with the titre of 1034.5 mg l−1 after 12‐h fermentation in a mineral salt medium, indicating G. thermoglucosidasius is a promising host to develop high‐temperature cell factory of riboflavin production. This is the first demonstration of riboflavin production in thermophilic bacteria at an elevated temperature.

A plasmid vector that directs hyperproduction of recombinant proteins in the thermophiles Geobacillus species.

Geobacillus spp. are moderate thermophiles that have great potential for use in diverse applications. For effective utilization of the species, genetic tools have been extensively studied; however, an overexpression vector remains to be developed. Here we constructed a plasmid vector that can shuttle between Escherichia coli and Geobacillus spp., and which contained a maltose-inducible promoter from Geobacillus kaustophilus HTA426. Although the vector (termed pGKE119) was originally designed for basal gene expression, it surprisingly directed robust protein production in G. kaustophilus. Protein production essentially occurred in an auto-inducible manner without maltose; however, some proteins were produced more efficiently in the presence of maltose. Although the productivity was affected by culture conditions, three proteins were successfully produced with abundance ratios of 12-27% (on a total protein basis) and yields of 77-170mg (per L culture). pGKE119 directed substantial protein production even in Geobacillus subterraneus, Geobacillus thermoglucosidasius, and Geobacillus thermoleovorans. This suggests that pGKE119 can use a range of Geobacillus spp. as hosts and widely expand their genetic toolbox. Because Geobacillus spp. are highly proliferative bacteria that are distinct from organisms used as protein production hosts, pGKE119 may also provide a novel platform for hyperproduction of recombinant proteins.

Effect of Target Gene Silencing on Calcite Single Crystal Formation by Thermophilic Bacterium Geobacillus thermoglucosidasius NY05.

Geobacillus thermoglucosidasius NY05 catalyzes calcite single crystal formation at 60°C by using acetate and calcium. Endospores are embedded at the central part of the calcite single crystal and carbon atoms in the calcite lattice are derived from acetate carbon. Here, we synthesized 21-mer antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs and evaluated the effect of these oligonucleotides on calcite formation in G. thermoglucosidasius NY05. G. thermoglucosidasius NY05 cells containing antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs had reduced calcite single crystal formation by 18.7, 50.6, 55.7, and 82.3%, respectively, compared with cells without antisense DNA oligonucleotides. These results support that calcite formation needs endospores as the nucleus to grow, and carbon dioxide generated from acetate, which is metabolized via the glyoxylate pathway and glucogenesis, is supplied to the crystal lattice.

Engineering Geobacillus thermoglucosidasius for direct utilisation of holocellulose from wheat straw

BackgroundA consolidated bioprocessing (CBP), where lignocellulose is converted into the desired product(s) in a single fermentative step without the addition of expensive degradative enzymes, represents the ideal solution of renewable routes to chemicals and fuels. Members of the genus Geobacillus are able to grow at elevated temperatures and are able to utilise a wide range of oligosaccharides derived from lignocellulose. This makes them ideally suited to the development of CBP.ResultsIn this study, we engineered Geobacillus thermoglucosidasius NCIMB 11955 to utilise lignocellulosic biomass, in the form of nitric acid/ammonia treated wheat straw to which expensive hydrolytic enzymes had not been added. Two different strains, BZ9 and BZ10, were generated by integrating the cglT (β-1,4-glucosidase) gene from Thermoanaerobacter brockii into the genome, and localising genes encoding different cellulolytic enzymes on autonomous plasmids. The plasmid of strain BZ10 carried a synthetic cellulosomal operon comprising the celA (Endoglucanase A) gene from Clostridium thermocellum and cel6B (Exoglucanase) from Thermobifida fusca; whereas, strain BZ9 contained a plasmid encoding the celA (multidomain cellulase) gene from Caldicellulosiruptor bescii. All of the genes were successfully expressed, and their encoded products secreted in a functionally active form, as evidenced by their detection in culture supernatants by Western blotting and enzymatic assay. In the case of the C. bescii CelA enzyme, this is one of the first times that the heterologous production of this multi-functional enzyme has been achieved in a heterologous host. Both strains (BZ9 and BZ10) exhibited improved growth on pre-treated wheat straw, achieving a higher final OD600 and producing greater numbers of viable cells. To demonstrate that cellulosic ethanol can be produced directly from lignocellulosic biomass by a single organism, we established our consortium of hydrolytic enzymes in a previously engineered ethanologenic G. thermoglucosidasius strain, LS242. We observed approximately twofold and 1.6-fold increase in ethanol production in the recombinant G. thermoglucosidasius equivalent to BZ9 and BZ10, respectively, compared to G. thermoglucosidasius LS242 strain at 24 h of growth.ConclusionWe engineered G. thermoglucosidasius to utilise a real-world lignocellulosic biomass substrate and demonstrated that cellulosic ethanol can be produced directly from lignocellulosic biomass in one step. Direct conversion of biomass into desired products represents a new paradigm for CBP, offering the potential for carbon neutral, cost-effective production of sustainable chemicals and fuels.

Dynamic Modelling of Phosphorolytic Cleavage Catalyzed by Pyrimidine-Nucleoside Phosphorylase

Pyrimidine-nucleoside phosphorylases (Py-NPases) have a significant potential to contribute to the economic and ecological production of modified nucleosides. These can be produced via pentose-1-phosphates, an interesting but mostly labile and expensive precursor. Thus far, no dynamic model exists for the production process of pentose-1-phosphates, which involves the equilibrium state of the Py-NPase catalyzed reversible reaction. Previously developed enzymological models are based on the understanding of the structural principles of the enzyme and focus on the description of initial rates only. The model generation is further complicated, as Py-NPases accept two substrates which they convert to two products. To create a well-balanced model from accurate experimental data, we utilized an improved high-throughput spectroscopic assay to monitor reactions over the whole time course until equilibrium was reached. We examined the conversion of deoxythymidine and phosphate to deoxyribose-1-phosphate and thymine by a thermophilic Py-NPase from Geobacillus thermoglucosidasius. The developed process model described the reactant concentrations in excellent agreement with the experimental data. Our model is built from ordinary differential equations and structured in such a way that integration with other models is possible in the future. These could be the kinetics of other enzymes for enzymatic cascade reactions or reactor descriptions to generate integrated process models.

Biochemical characterization of a novel thermostable feruloyl esterase from Geobacillus thermoglucosidasius DSM 2542T.

The ferulic acid esterase (FAE) gene from Geobacillus thermoglucosidasius DSM 2542T was cloned into pET28a(+) expression vector and characterized and is being reported in this study for the first time in Geobacillus. The enzyme, designated as GthFAE, was purified by heat shock and ion-exchange column chromatography. In addition, a second clone containing a Histidine tag was expressed and purified by affinity column chromatography demonstrating future potential for scale-up. FAE gene contains an open reading frame (ORF) of 759-bp encoding a hypothetical 252 amino acid protein, a molecular mass of 28.11kDa and an isoelectric point of 5.53. From this study it was found that GthFAE had optimal activity at 50°C and pH of 8.5. Furthermore, the enzyme has been found to retain 64% of its activity after two days incubation at 50°C and exhibited a high level of functionality with p-nitrophenyl caprylate (C8). Km, Vmax, kcat and kcat/Km values for p-nitrophenyl caprylate were determined as 0.035mM, 11,735µmol/min/mg protein, 5491 (1/s) and 156,885s-1mM-1 respectively. The combination of higher activity and stability compared to previously reported FAEs makes GthFAE a potential candidate for use in the paper manufacturing industry.

Comparative transcriptomic analysis revealed the key pathways responsible for organic sulfur removal by thermophilic bacterium Geobacillus thermoglucosidasius W-2

Biodesulfurization is a promising method to desulfurize sulfur-containing compounds in oil with its unique advantages, such as environment-friendly treatments and moderate reaction conditions. In this study, a thermophilic bacterium Geobacillus thermoglucosidasius W-2 was reported to show nearly 40% and 55% desulfurization rates on heavy oil with 2.81% and 0.46% initial total sulfur content, respectively. Subsequently, comparative transcriptome analysis indicated that several possible key desulfurization-related genes of this strain were found to be differentially up-regulated induced by benzothiophene and dibenzothiophene, respectively. These desulfurization-related genes were considered to conduct key step to convert organic sulfur to inorganic sulfur. Moreover, the characterization of thermophilic alkanesulfonate monooxygenase systems SsuD1/SsuE1 and SsuD2/SsuE2 revealed that the enzymes exhibit considerable thermal and pH stability and wide substrates applicability. These enzymes probably endowed the strain W-2 with the ability to desulfurize oil and eliminate the sulfur-containing surfactants. Thus, this study provides novel alkanesulfonate monooxygenase systems that have the application potential for heavy oil biodesulfurization, oil demulsification and other biocatalytic processes.

Rapid, Heuristic Discovery and Design of Promoter Collections in Non-Model Microbes for Industrial Applications.

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.